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S c i e nt i f ic Co ntri b u t i o n 31 s t Co ngr e ss o f t h e Eu ro p ea n Ac a d e m y o f Al l e rg y a n d C l inic a l Im mu n o l o g y 2012, E A AC I , 16 - 20 Ju n e 2012, G e n eva
D ear Co ngr e ss d e l e ga t e,
On behalf of HAL Allergy we would like to welcome you to the 31st EAACI congress in Geneva. The theme of this year’s congress is “At the crossroads of research, practice and education”. HAL Allergy is one of Europe’s top players in the field of allergen vaccination and devotes a major part of its resources and investments to the development of allergen specific immunotherapy. We are proud to present our ongoing research and clinical practice work during the biggest European congress on allergy and clinical immunology.
In this abstract book you will find fifteen abstracts dealing with clinical, pre-clinical and development work, including information on specific dates and times of presentations.
HAL Allergy’s clinical development program is committed to meeting today’s requirements for gaining registration for specific immunotherapy products. The clinical development program is represented with two abstracts related to the dose range finding study with PURETHAL® Mites which was initiated in the fall of 2011 and will be completed in spring 2013. In addition we also present you with clinical data on the use of PURETHAL®, SUBLIVAC® and VENOMENHAL® in daily practice.
At this year’s meeting there will be two oral presentations dealing with our latest pre-clinical work on a novel vaccine for peanut allergy. We are on track with our research program to develop a subcutaneous immunotherapy product to safely treat peanut-allergic patients and intend to complete all preclinical studies this year.
Furthermore, we present various abstracts on the characterisation and quantification of our allergen products, in particular our PURETHAL® range which involve chemically modified allergens, so-called allergoids. HAL Allergy is progressing with the development and implementation of new antibody based and physicochemical methodologies to characterise pharmaceutical allergoid preparations and to assure their quality.
We hope you will have a successful congress. Please visit our booth in the exhibition area should you require further information on our R&D program or on our products.
Kind regards,
Diderik Boot, PhD Dirk-Jan Opstelten, PhDMedical Director Research & Development Director
Diderik Boot, PhD
Medical Director
Dirk-Jan Opstelten, PhD
Research & DevelopmentDirector
Co nt e nt s
Scientific Contribution
ClinicalEvaluation of the cytokine response induced by allergen-specific immunotherapy with a modified allergoid in patients with allergic rhinits with or without asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Application of a standardised titrated nasal provocation test with HDM extract in a multicentre clinical study in allergic rhinitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Patient knowledge, perceptions, expectations, and satisfaction, on subcutaneous and sublingual allergen-specific immunotherapy: a real life survey . . . . . . . . . . . . . . . . . . . . . . . . . . 10Influence of co-morbidities and co-medications on safety and tolerability in the buildup phase of venom immunotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Design of dose range finding study with allergen specific immunotherapy in patients with house dust mite induced allergic rhinitis/rhinoconjunctivitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Safety of pre- and coseasonal start of sublingual immunotherapy treatment in patients with pollen allergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Pre-clinicalChemical modification of a peanut extract decreases IgE binding while the immunogenicity is maintained . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Allergenic potency of chemically modified Ara h2/h6 evaluated with basophil activation, RBL-SX38 activation, and solid phase IgE-immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20The content of allergens Ara h1, Ara h2, Ara h3 and Ara h6 in different peanut cultivars commonly consumed in Europe and the USA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
DevelopmentComparison of antibody-based assay with physicochemical assays for monitoring the stability of alum-adsorbed mite allergoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Characterisation of pollen allergoids with physicochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Characterisation of alum-adsorbed pollen allergoids with physicochemical techniques . . . . . . . . . . . . . . . . . . . . . . . . 28An inhibition ELISA method for the determination of relative potency of mite preparations . . . . . . . . . . . . . . . . . . . . 30An antibody-based technique for stability studies on alum adsorbed birch pollen allergoid preparations . . . . . . . 32Biochemical characterisation of chemically modified Ara h2 isoforms; structure-function relationships . . . . . . . . . 34
Eva l u a t i o n o f t h e c y t o kin e r e s p o n s e in d u ce d by a l l e rge n - s p e c i f ic im mu n o t h e rap y w i t h a m o d i f i e d a l l e rg o i d in p a t i e nt s w i t h a l l e rgic r h ini t is w i t h o r w i t h o u t a s t h m a
A. Malet-Casajuana (1), A. Roger (2), P. Amat-Par (1), N. Depreux (2), T. Gonzalez-Quevedo (3), M.J. Cruz (4).
(1) Alergocentre, Barcelona, Spain, (2) Allergy Unit. Germans Trias Pujol University Hospital. Badalona, Spain. (3) Allergy Unit. Virgen del Rocío
University Hospital, Sevilla, Spain. (4) HAL Allergy, Barcelona, Spain.
BackgroundAllergy is characterized by a T helper type 2 (Th2) cell-mediated immune response. It has been demonstrated that after allergen-specific immunotherapy (SIT) there is a change in the balance between Th1/Th2 responses and the induction of a more allergen tolerant state. The aim of the present study was to investigate in patients with house dust mite (HDM) indu-ced allergic rhinitis/rhinoconjunctivitis the changes in Th1, Th2 and T regulatory (Treg) cytokines following SIT treatment
MethodsTwenty-nine adult patients (14F/15M, 33 yrs range 16-65 years) with a history of HDM induced allergic rhinitis/rhinocon-junctivitis with or without clinically stable mild persistent asthma were treated with subcutaneous SIT, containing 20,000 AUeq/ml of modified mites allergen extract adsorbed onto aluminium hydroxide. Blood samples were collected before and 3 and 6 months after SIT treatment. In serum samples IFN-γ, IL-10, IL-4, IL-5, and Il-13 levels were determined by flow cytometry using a commercial multiplexing kit (Milliplex Human Cytokine Kit, Millipore Corporation) according to the in-structions provided by the manufacturer. The samples were analyzed using a Fortessa flow cytometer.
Results Following SIT, a significant increase in the level of IFN-γ was found after 3 and 6 months of treatment (p = 0.004 and 0.005 respectively). This increase was also observed in the levels of IL-10 after 6 months of treatment (p = 0.019). Moreover, a tendency towards significant decrease of the levels of IL-5 were observed after 6 months, compared with the basal values (p = 0.088). No significant differences were found in the levels of IL-4 and IL13 after SIT.
ConclusionsThe results obtained in the present study suggest that SIT triggers a Th2 to Th1 shift and an induction of IL-10 secreting Treg cells. These events occur early and suggest a SIT tolerance induction in these patients.
6
EAACI, 16-20 June 2012, GenevaAbstract number: 2, Session date and time: Sunday 17 June; 10:30 - 12:00 Session title: OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment.
OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment.
Eva l u a t i o n o f t h e c y t o kin e r e s p o n s e in d u ce d by a l l e rge n - s p e c i f ic im mu n o t h e rap y w i t h a m o d i f i e d a l l e rg o i d in p a t i e nt s w i t h a l l e rgic r h ini t is w i t h o r w i t h o u t a s t h m a .
7
Bac
kgro
und
& Ai
m:
Alle
rgy
is c
hara
cter
ized
by
a T
help
er t
ype
2 (T
h2)
cell-
med
iate
d im
mun
e re
spon
se.
It ha
s be
en d
emon
stra
ted
that
afte
r al
lerg
en-s
peci
fic
imm
unot
hera
py (S
IT) t
here
is a
mod
ulat
ion
of a
llerg
en-s
peci
fic a
ntib
ody
resp
onse
s, a
cha
nge
in th
e ba
lanc
e be
twee
n Th
1/Th
2 re
spon
ses
and
the
indu
ctio
n of
a m
ore
alle
rgen
tole
rant
sta
te.T
he in
duct
ion
of a
llerg
en s
peci
fic s
erum
IgG
antib
odie
s ha
s pr
evio
usly
bee
n de
mon
stra
ted.
The
ai
m o
f the
pre
sent
stu
dy w
as to
inve
stig
ate
the
chan
ges
in T
h1, T
h2 a
nd T
regu
lato
ry (T
reg)
cyt
okin
es fo
llow
ing
the
initi
atio
n of
SIT
trea
tmen
t in
patie
nts
with
hou
se d
ust m
ite (H
DM
) ind
uced
alle
rgic
rhin
itis/
rhin
ocon
junc
tiviti
s
1 Ale
rgoc
entre
, Bar
celo
na, S
pain
, 2 Alle
rgy
Unit.
Ger
man
s Tr
ias
Pujo
lUni
vers
ity H
ospi
tal.
Bad
alon
a, S
pain
. 3 A
llerg
y Un
it. V
irgen
del R
ocío
Univ
ersi
ty H
ospi
tal,
Sevi
lla, S
pain
, 4 HAL
Alle
rgy,
Bar
celo
na, S
pain
.
2 -E
valu
atio
n of
the
cyto
kine
resp
onse
indu
ced
by a
llerg
en-s
peci
fic
imm
unot
hera
py w
ith a
mod
ified
alle
rgoi
din
pat
ient
s w
ith a
llerg
ic
rhin
itis
with
or w
ithou
t ast
hma
Figu
re 2
: Cyt
okin
e co
ncen
trat
ion
befo
re a
nd a
fter t
reat
men
t with
imm
unot
hera
py: A
= b
efor
e tr
eatm
ent;
B =
3 m
onth
s af
ter t
reat
men
t; C
= 6
mon
ths
afte
r tr
eatm
ent
Tabl
e 1:
Dem
ogra
phic
char
acte
ristic
san
dva
lues
ofsp
ecifi
can
tibod
ies
in th
est
udy
popu
latio
n.
Con
clus
ion:
The
resu
lts o
btai
ned
in th
e pr
esen
t stu
dy s
ugge
st th
at S
IT tr
igge
rs a
Th2
to T
h1 s
hift
and
an in
duct
ion
of IL
-10
secr
etin
g Tr
egce
lls. T
hese
eve
nts
occu
r ear
ly a
nd s
ugge
st a
SIT
tole
ranc
e in
duct
ion
in th
ese
patie
nts.
The
se c
hang
es a
re p
art o
f the
mec
hani
sm o
f act
ion
of im
mun
othe
rapy
and
is
prob
ably
rela
ted
to th
e cl
inic
al e
ffica
cy o
f the
trea
tmen
t.
Met
hods
:Ad
ult p
atie
nts
with
a h
isto
ry o
f HD
M in
duce
d al
lerg
ic rh
initi
s/rh
inoc
onju
nctiv
itis
with
or w
ithou
t con
com
itant
clin
ical
ly s
tabl
e m
ild p
ersi
sten
t ast
hma
wer
e sc
reen
ed.
Elig
ible
pat
ient
s w
ere
treat
ed w
ith s
ubcu
tane
ous
SIT
(PU
RET
HAL
®,
HAL
Alle
rgy
BV,
Leid
en,
The
Net
herla
nds)
, co
ntai
ning
20
,000
AU
eq/m
l of m
odifi
ed m
ites
alle
rgen
ext
ract
ads
orbe
d on
to a
lum
iniu
mhy
drox
ide.
Blo
od s
ampl
es w
ere
colle
cted
bef
ore,
3 a
nd 6
mon
ths
afte
r SIT
trea
tmen
t. In
ser
um s
ampl
es IF
N-
, IL-
10, I
L-4,
IL-5
, and
Il-1
3 le
vels
wer
e de
term
ined
by
flow
cyt
omet
ryus
ing
a co
mm
erci
al m
ultip
lexi
ng
kit (
Milli
plex
Hum
an C
ytok
ine
Kit,
Milli
pore
Cor
pora
tion)
. The
det
ectio
n sy
stem
was
a s
andw
ich
imm
unoa
ssay
per
form
ed o
n un
ique
fluo
resc
ent
bead
s, w
hich
con
tain
ed a
pre
cise
dis
tribu
tion
of t
wo
fluor
esce
ntdy
es,
and
a sp
ecifi
c cy
toki
ne c
aptu
re a
ntib
ody
linke
d to
its
sur
face
. Th
e flu
ores
cent
bea
d se
ts w
ere
incu
bate
d w
ith a
bio
logi
cal s
ampl
e. A
biot
inyla
ted
cyto
kine
repo
rter a
ntib
ody
was
then
use
d to
iden
tify
the
capt
ured
cy
toki
ne b
ound
to
the
spec
ific
fluor
esce
nt b
ead.
The
rea
ctio
n w
as d
etec
ted
by a
ddin
g a
stre
ptav
idin
-phy
coer
ythr
inco
njug
ate
to t
he r
eact
ion
mix
ture
. The
sam
ples
wer
e an
alyz
ed u
sing
a F
orte
ssa
flow
cyt
omet
er.
Res
ults
:Tw
enty
nin
e pa
tient
s (T
able
1, F
igur
e 1)
wer
e in
clud
ed. S
IT in
duce
d a
sign
ifica
nt in
crea
se in
ser
um le
vels
of
and
IL-1
0 an
d a
sign
ifica
nt
decr
ease
in IL
-5 (F
igur
e 2)
. No
sign
ifica
nt d
iffer
ence
s w
ere
foun
d in
the
leve
ls o
f IL-
4 an
d IL
13 a
fter S
IT.
P =
0.00
4
P =
0.03
3
P =
0.00
5
IFNγ A
IFNγ B
IFNγ C
0.1110100
P =
0.02
5
P =
0.37
4
P =
0.01
9
IL10 A
IL10 B
IL10 C
0.1110100
IL10 (pg/ml)
P =
0.27
1
P =
0.00
1
P =
0.08
8
IL5 A
IL5 B
IL5 C
0.010.
1110100
IL5 (pg/ml)
Figu
re 1
: Dis
trib
utio
n of
resp
irato
ry d
isea
se.
4.3
(3.0
-6.7
L)M
edia
n FV
C 1(L
) (ra
nge)
3.7
(2.4
-5.3
L)M
edia
n FE
V 1(L
) (ra
nge)
Patie
nts
n =
29G
ende
r (M
(%) /
F (%
))15
(51.
7%)/
14(4
8.3%
)
Mea
n ag
e in
yea
rs (r
ange
)33
(16-
65)
Med
ian
seru
m s
peci
fic Ig
E(IU
/ml)
to m
ites
(rang
e)17
.3 (1
.3-5
7.5)
Asth
ma
23%
Asth
ma
+ Rh
initi
s23
%
Rhin
itis
54%
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
Bac
kgro
und
& Ai
m:
Alle
rgy
is c
hara
cter
ized
by
a T
help
er t
ype
2 (T
h2)
cell-
med
iate
d im
mun
e re
spon
se.
It ha
s be
en d
emon
stra
ted
that
afte
r al
lerg
en-s
peci
fic
imm
unot
hera
py (S
IT) t
here
is a
mod
ulat
ion
of a
llerg
en-s
peci
fic a
ntib
ody
resp
onse
s, a
cha
nge
in th
e ba
lanc
e be
twee
n Th
1/Th
2 re
spon
ses
and
the
indu
ctio
n of
a m
ore
alle
rgen
tole
rant
sta
te.T
he in
duct
ion
of a
llerg
en s
peci
fic s
erum
IgG
antib
odie
s ha
s pr
evio
usly
bee
n de
mon
stra
ted.
The
ai
m o
f the
pre
sent
stu
dy w
as to
inve
stig
ate
the
chan
ges
in T
h1, T
h2 a
nd T
regu
lato
ry (T
reg)
cyt
okin
es fo
llow
ing
the
initi
atio
n of
SIT
trea
tmen
t in
patie
nts
with
hou
se d
ust m
ite (H
DM
) ind
uced
alle
rgic
rhin
itis/
rhin
ocon
junc
tiviti
s
1 Ale
rgoc
entre
, Bar
celo
na, S
pain
, 2 Alle
rgy
Unit.
Ger
man
s Tr
ias
Pujo
lUni
vers
ity H
ospi
tal.
Bad
alon
a, S
pain
. 3 A
llerg
y Un
it. V
irgen
del R
ocío
Univ
ersi
ty H
ospi
tal,
Sevi
lla, S
pain
, 4 HAL
Alle
rgy,
Bar
celo
na, S
pain
.
2 -E
valu
atio
n of
the
cyto
kine
resp
onse
indu
ced
by a
llerg
en-s
peci
fic
imm
unot
hera
py w
ith a
mod
ified
alle
rgoi
din
pat
ient
s w
ith a
llerg
ic
rhin
itis
with
or w
ithou
t ast
hma
Figu
re 2
: Cyt
okin
e co
ncen
trat
ion
befo
re a
nd a
fter t
reat
men
t with
imm
unot
hera
py: A
= b
efor
e tr
eatm
ent;
B =
3 m
onth
s af
ter t
reat
men
t; C
= 6
mon
ths
afte
r tr
eatm
ent
Tabl
e 1:
Dem
ogra
phic
char
acte
ristic
san
dva
lues
ofsp
ecifi
can
tibod
ies
in th
est
udy
popu
latio
n.
Con
clus
ion:
The
resu
lts o
btai
ned
in th
e pr
esen
t stu
dy s
ugge
st th
at S
IT tr
igge
rs a
Th2
to T
h1 s
hift
and
an in
duct
ion
of IL
-10
secr
etin
g Tr
egce
lls. T
hese
eve
nts
occu
r ear
ly a
nd s
ugge
st a
SIT
tole
ranc
e in
duct
ion
in th
ese
patie
nts.
The
se c
hang
es a
re p
art o
f the
mec
hani
sm o
f act
ion
of im
mun
othe
rapy
and
is
prob
ably
rela
ted
to th
e cl
inic
al e
ffica
cy o
f the
trea
tmen
t.
Met
hods
:Ad
ult p
atie
nts
with
a h
isto
ry o
f HD
M in
duce
d al
lerg
ic rh
initi
s/rh
inoc
onju
nctiv
itis
with
or w
ithou
t con
com
itant
clin
ical
ly s
tabl
e m
ild p
ersi
sten
t ast
hma
wer
e sc
reen
ed.
Elig
ible
pat
ient
s w
ere
treat
ed w
ith s
ubcu
tane
ous
SIT
(PU
RET
HAL
®,
HAL
Alle
rgy
BV,
Leid
en,
The
Net
herla
nds)
, co
ntai
ning
20
,000
AU
eq/m
l of m
odifi
ed m
ites
alle
rgen
ext
ract
ads
orbe
d on
to a
lum
iniu
mhy
drox
ide.
Blo
od s
ampl
es w
ere
colle
cted
bef
ore,
3 a
nd 6
mon
ths
afte
r SIT
trea
tmen
t. In
ser
um s
ampl
es IF
N-
, IL-
10, I
L-4,
IL-5
, and
Il-1
3 le
vels
wer
e de
term
ined
by
flow
cyt
omet
ryus
ing
a co
mm
erci
al m
ultip
lexi
ng
kit (
Milli
plex
Hum
an C
ytok
ine
Kit,
Milli
pore
Cor
pora
tion)
. The
det
ectio
n sy
stem
was
a s
andw
ich
imm
unoa
ssay
per
form
ed o
n un
ique
fluo
resc
ent
bead
s, w
hich
con
tain
ed a
pre
cise
dis
tribu
tion
of t
wo
fluor
esce
ntdy
es,
and
a sp
ecifi
c cy
toki
ne c
aptu
re a
ntib
ody
linke
d to
its
sur
face
. Th
e flu
ores
cent
bea
d se
ts w
ere
incu
bate
d w
ith a
bio
logi
cal s
ampl
e. A
biot
inyla
ted
cyto
kine
repo
rter a
ntib
ody
was
then
use
d to
iden
tify
the
capt
ured
cy
toki
ne b
ound
to
the
spec
ific
fluor
esce
nt b
ead.
The
rea
ctio
n w
as d
etec
ted
by a
ddin
g a
stre
ptav
idin
-phy
coer
ythr
inco
njug
ate
to t
he r
eact
ion
mix
ture
. The
sam
ples
wer
e an
alyz
ed u
sing
a F
orte
ssa
flow
cyt
omet
er.
Res
ults
:Tw
enty
nin
e pa
tient
s (T
able
1, F
igur
e 1)
wer
e in
clud
ed. S
IT in
duce
d a
sign
ifica
nt in
crea
se in
ser
um le
vels
of
and
IL-1
0 an
d a
sign
ifica
nt
decr
ease
in IL
-5 (F
igur
e 2)
. No
sign
ifica
nt d
iffer
ence
s w
ere
foun
d in
the
leve
ls o
f IL-
4 an
d IL
13 a
fter S
IT.
P =
0.00
4
P =
0.03
3
P =
0.00
5IFNγ
A
IFNγ B
IFNγ C
0.1110100
P =
0.02
5
P =
0.37
4
P =
0.01
9
IL10 A
IL10 B
IL10 C
0.1110100
IL10 (pg/ml)
P =
0.27
1
P =
0.00
1
P =
0.08
8
IL5 A
IL5 B
IL5 C
0.010.
1110100
IL5 (pg/ml)
Figu
re 1
: Dis
trib
utio
n of
resp
irato
ry d
isea
se.
4.3
(3.0
-6.7
L)M
edia
n FV
C 1(L
) (ra
nge)
3.7
(2.4
-5.3
L)M
edia
n FE
V 1(L
) (ra
nge)
Patie
nts
n =
29G
ende
r (M
(%) /
F (%
))15
(51.
7%)/
14(4
8.3%
)
Mea
n ag
e in
yea
rs (r
ange
)33
(16-
65)
Med
ian
seru
m s
peci
fic Ig
E(IU
/ml)
to m
ites
(rang
e)17
.3 (1
.3-5
7.5)
Asth
ma
23%
Asth
ma
+ Rh
initi
s23
%
Rhin
itis
54%
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
Bac
kgro
und
& Ai
m:
Alle
rgy
is c
hara
cter
ized
by
a T
help
er t
ype
2 (T
h2)
cell-
med
iate
d im
mun
e re
spon
se.
It ha
s be
en d
emon
stra
ted
that
afte
r al
lerg
en-s
peci
fic
imm
unot
hera
py (S
IT) t
here
is a
mod
ulat
ion
of a
llerg
en-s
peci
fic a
ntib
ody
resp
onse
s, a
cha
nge
in th
e ba
lanc
e be
twee
n Th
1/Th
2 re
spon
ses
and
the
indu
ctio
n of
a m
ore
alle
rgen
tole
rant
sta
te.T
he in
duct
ion
of a
llerg
en s
peci
fic s
erum
IgG
antib
odie
s ha
s pr
evio
usly
bee
n de
mon
stra
ted.
The
ai
m o
f the
pre
sent
stu
dy w
as to
inve
stig
ate
the
chan
ges
in T
h1, T
h2 a
nd T
regu
lato
ry (T
reg)
cyt
okin
es fo
llow
ing
the
initi
atio
n of
SIT
trea
tmen
t in
patie
nts
with
hou
se d
ust m
ite (H
DM
) ind
uced
alle
rgic
rhin
itis/
rhin
ocon
junc
tiviti
s
1 Ale
rgoc
entre
, Bar
celo
na, S
pain
, 2 Alle
rgy
Unit.
Ger
man
s Tr
ias
Pujo
lUni
vers
ity H
ospi
tal.
Bad
alon
a, S
pain
. 3 A
llerg
y Un
it. V
irgen
del R
ocío
Univ
ersi
ty H
ospi
tal,
Sevi
lla, S
pain
, 4 HAL
Alle
rgy,
Bar
celo
na, S
pain
.
2 -E
valu
atio
n of
the
cyto
kine
resp
onse
indu
ced
by a
llerg
en-s
peci
fic
imm
unot
hera
py w
ith a
mod
ified
alle
rgoi
din
pat
ient
s w
ith a
llerg
ic
rhin
itis
with
or w
ithou
t ast
hma
Figu
re 2
: Cyt
okin
e co
ncen
trat
ion
befo
re a
nd a
fter t
reat
men
t with
imm
unot
hera
py: A
= b
efor
e tr
eatm
ent;
B =
3 m
onth
s af
ter t
reat
men
t; C
= 6
mon
ths
afte
r tr
eatm
ent
Tabl
e 1:
Dem
ogra
phic
char
acte
ristic
san
dva
lues
ofsp
ecifi
can
tibod
ies
in th
est
udy
popu
latio
n.
Con
clus
ion:
The
resu
lts o
btai
ned
in th
e pr
esen
t stu
dy s
ugge
st th
at S
IT tr
igge
rs a
Th2
to T
h1 s
hift
and
an in
duct
ion
of IL
-10
secr
etin
g Tr
egce
lls. T
hese
eve
nts
occu
r ear
ly a
nd s
ugge
st a
SIT
tole
ranc
e in
duct
ion
in th
ese
patie
nts.
The
se c
hang
es a
re p
art o
f the
mec
hani
sm o
f act
ion
of im
mun
othe
rapy
and
is
prob
ably
rela
ted
to th
e cl
inic
al e
ffica
cy o
f the
trea
tmen
t.
Met
hods
:Ad
ult p
atie
nts
with
a h
isto
ry o
f HD
M in
duce
d al
lerg
ic rh
initi
s/rh
inoc
onju
nctiv
itis
with
or w
ithou
t con
com
itant
clin
ical
ly s
tabl
e m
ild p
ersi
sten
t ast
hma
wer
e sc
reen
ed.
Elig
ible
pat
ient
s w
ere
treat
ed w
ith s
ubcu
tane
ous
SIT
(PU
RET
HAL
®,
HAL
Alle
rgy
BV,
Leid
en,
The
Net
herla
nds)
, co
ntai
ning
20
,000
AU
eq/m
l of m
odifi
ed m
ites
alle
rgen
ext
ract
ads
orbe
d on
to a
lum
iniu
mhy
drox
ide.
Blo
od s
ampl
es w
ere
colle
cted
bef
ore,
3 a
nd 6
mon
ths
afte
r SIT
trea
tmen
t. In
ser
um s
ampl
es IF
N-
, IL-
10, I
L-4,
IL-5
, and
Il-1
3 le
vels
wer
e de
term
ined
by
flow
cyt
omet
ryus
ing
a co
mm
erci
al m
ultip
lexi
ng
kit (
Milli
plex
Hum
an C
ytok
ine
Kit,
Milli
pore
Cor
pora
tion)
. The
det
ectio
n sy
stem
was
a s
andw
ich
imm
unoa
ssay
per
form
ed o
n un
ique
fluo
resc
ent
bead
s, w
hich
con
tain
ed a
pre
cise
dis
tribu
tion
of t
wo
fluor
esce
ntdy
es,
and
a sp
ecifi
c cy
toki
ne c
aptu
re a
ntib
ody
linke
d to
its
sur
face
. Th
e flu
ores
cent
bea
d se
ts w
ere
incu
bate
d w
ith a
bio
logi
cal s
ampl
e. A
biot
inyla
ted
cyto
kine
repo
rter a
ntib
ody
was
then
use
d to
iden
tify
the
capt
ured
cy
toki
ne b
ound
to
the
spec
ific
fluor
esce
nt b
ead.
The
rea
ctio
n w
as d
etec
ted
by a
ddin
g a
stre
ptav
idin
-phy
coer
ythr
inco
njug
ate
to t
he r
eact
ion
mix
ture
. The
sam
ples
wer
e an
alyz
ed u
sing
a F
orte
ssa
flow
cyt
omet
er.
Res
ults
:Tw
enty
nin
e pa
tient
s (T
able
1, F
igur
e 1)
wer
e in
clud
ed. S
IT in
duce
d a
sign
ifica
nt in
crea
se in
ser
um le
vels
of
and
IL-1
0 an
d a
sign
ifica
nt
decr
ease
in IL
-5 (F
igur
e 2)
. No
sign
ifica
nt d
iffer
ence
s w
ere
foun
d in
the
leve
ls o
f IL-
4 an
d IL
13 a
fter S
IT.
P =
0.00
4
P =
0.03
3
P =
0.00
5
IFNγ A
IFNγ B
IFNγ C
0.1110100
P =
0.02
5
P =
0.37
4
P =
0.01
9
IL10 A
IL10 B
IL10 C
0.1110100
IL10 (pg/ml)
P =
0.27
1
P =
0.00
1
P =
0.08
8
IL5 A
IL5 B
IL5 C
0.010.
1110100
IL5 (pg/ml)
Figu
re 1
: Dis
trib
utio
n of
resp
irato
ry d
isea
se.
4.3
(3.0
-6.7
L)M
edia
n FV
C 1(L
) (ra
nge)
3.7
(2.4
-5.3
L)M
edia
n FE
V 1(L
) (ra
nge)
Patie
nts
n =
29G
ende
r (M
(%) /
F (%
))15
(51.
7%)/
14(4
8.3%
)
Mea
n ag
e in
yea
rs (r
ange
)33
(16-
65)
Med
ian
seru
m s
peci
fic Ig
E(IU
/ml)
to m
ites
(rang
e)17
.3 (1
.3-5
7.5)
Asth
ma
23%
Asth
ma
+ Rh
initi
s23
%
Rhin
itis
54%
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12In
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er is
an
empl
oyee
of H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
Ap p l ic a t i o n o f a s t a n d ard i z e d t i tra t e d n a s a l p ro v o c a t i o n t e s t w i t h H D M ex tra c t in a mu l t ice ntr e cl inic a l s tu d y in a l l e rgic r h ini t is
C. Bachert (1), O. Pfaar (2), M.J. Nell (3), J.D. Boot (3), H. Nienhuis (3), Z. Diamant (4,5).
(1) UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium, (2) Center for Rhinology and Allergology Wiesbaden, Germany, (3) HAL Allergy BV,
Leiden, The Netherlands, (4) Skane University Hospital, Dept of Respir Med & Allergol, Lund, Sweden and (5) QPS-NL, Groningen, The Netherlands.
BackgroundTitrated nasal provocation test (TNPT) can be used in the diagnosis of allergic rhinitis (AR) and as a disease model in clinical trials evaluating the efficacy of treatment. However, the techniques for provocation and readouts vary, requiring standardization to enable the use of TNPT as a tool in multicentre trials. We performed a standardized TNPT and documented the upper airway response by symptom scores and peak nasal inspiratory flow (PNIF) measurements. In addition, we compared the upper airway responses across the different allergen concentrations and investigated a possible correlation.
Methods Patients with suspected HDM-induced AR were screened prior to enrollment into a multicentre efficacy study with HDM immunotherapy. Patients with a positive medical history and a positive skin prick test to HDM underwent a standardized TNPT starting with the diluent and up to 3 serial concentrations of a standardized allergen extract (D. Pter 100, 1,000 and 10,000 AU/ml, 1 puff per nostril) at 20 minutes intervals. The upper airway response was quantified by a composite symptom score according to Lebel and a PNIF measurement 15 minutes after each administration. TNPT was considered positive once a score ≥6 was reached. Symptom scores and PNIF were repeated 1 hour after the provocative allergen concentration.
Results 150 patients were screened. 103 patients (54M/49F, mean age 32 years, mean wheal size HDM 6mm) had a positive TNPT, the most prominent symptoms being nasal blockage and rhinorrhoea, followed by pruritus and sneezing. A consistent dose-response relationship was found between serial allergen concentrations and increasing symptom scores, matching a gradual decrease in PNIF. One hour after the provocative allergen concentration, both measurements had not returned to baseline values (Table). There was a moderate though highly significant correlation between symptom scores and PNIF (r=0.54; p<0.01).
ConclusionWhen applied in a multicentre setting in patients with suspected HDM-induced AR, a standardized TNPT with a HDM extract induces a consistent gradual increase in allergic symptoms and signs that can be reliably quantified by well-defined composite symptom scores and PNIF.
8
EAACI, 16-20 June 2012, GenevaAbstract number: 37, Session date and time: Sunday 17 June; 13:30 - 15:00 Session title: OAS 7 - Diagnosis and treatment of allergic rhinoconjunctivitis: recent development
Table: Symptom scores and PNIF (mean±SD) following TNPT with HDM allergen.
Timepoint Pre Post Diluent 100 AU/ml 1,000 AU/ml 10,000 AU/ml 1 hour Diluent Diluent post TNPT
Lebel Score 0.8±0.8 0.9±1.0 3.7±2.1 5.5±2.2 6.5±2.9 3.2±1.7
PNIF (L/min) 135±45 132±47 113±48 93±44 66±42 109±43
9
OAS 7 - Diagnosis and treatment of allergic rhinoconjunctivitis: recent development
Ap p l ic a t i o n o f a s t a n d ard i z e d t i tra t e d n a s a l p ro v o c a t i o n t e s t w i t h H D M ex tra c t in a mu l t ice ntr e cl inic a l s tu d y in a l l e rgic r h ini t is
1 UZ
Gen
t, Ea
r-, N
ose
and
Thro
at D
epar
tmen
t, G
ent,
Belg
ium
; 2 Cen
ter f
or R
hino
logy
and
Alle
rgol
ogy
Wie
sbad
en, G
erm
any;
3 H
AL A
llerg
y BV
, Le
iden
, The
Net
herla
nds;
4 Ska
ne U
nive
rsity
Hos
pita
l, D
ept o
f Res
pir M
ed &
Alle
rgol
, Lun
d, S
wed
en
and
5 QPS
-NL,
Gro
ning
en, T
he N
ethe
rland
s
37 -
Appl
icat
ion
of a
sta
ndar
dize
d tit
rate
d na
sal
prov
ocat
ion
test
with
HD
M e
xtra
ct in
a
mul
ticen
tre
clin
ical
stu
dy in
alle
rgic
rhin
itis
Bac
kgro
und
& Ai
m:
Titra
ted
nasa
l pro
voca
tion
test
s (T
NPT
) with
a re
leva
nt a
llerg
enca
n be
use
d in
the
diag
nosi
s of
alle
rgic
rhin
itis
(AR
) and
as
a di
seas
e m
odel
in
clin
ical
tria
ls e
valu
atin
g th
e ef
ficac
y of
tre
atm
ent.
How
ever
, bo
th t
he t
echn
ique
s us
ed f
or p
rovo
catio
n an
d th
e re
adou
ts o
f th
e ev
oked
upp
erai
rway
resp
onse
vary
, req
uirin
g st
anda
rdiz
atio
n to
ena
ble
the
use
of T
NPT
as
a to
ol in
mul
ticen
tre tr
ials
. We
perfo
rmed
a s
tand
ardi
zed
TNPT
an
d do
cum
ente
d th
e up
per a
irway
resp
onse
by
sym
ptom
sco
res
and
peak
nas
al in
spira
tory
flow
(PN
IF) m
easu
rem
ents
. In
addi
tion,
we
com
pare
d th
e up
per a
irway
resp
onse
s ac
ross
the
diffe
rent
alle
rgen
con
cent
ratio
ns a
nd in
vest
igat
ed a
pos
sibl
e co
rrela
tion
betw
een
both
read
outs
.
Con
clus
ion:
Whe
n ap
plie
d in
a m
ultic
entre
set
ting
in p
atie
nts
with
sus
pect
ed H
DM
-indu
ced
AR, a
sta
ndar
dize
d TN
PT w
ith a
HD
M e
xtra
ct in
duce
sa
cons
iste
nt g
radu
al in
crea
se in
alle
rgic
sym
ptom
s an
d si
gns
that
can
be
relia
bly
quan
tifie
d by
a w
ell-d
efin
ed c
ompo
site
sym
ptom
sc
ore
and
PNIF
mea
sure
men
ts.
Met
hods
:Pa
tient
s: M
/F a
ged
18-6
0 ye
ars
with
HD
M-re
late
d AR
, w
ith o
r w
ithou
t co
ncom
itant
, cl
inic
ally
sta
ble
asth
ma
(FEV
1 >7
0% p
redi
cted
) w
ere
scre
ened
prio
r to
enro
llmen
t int
o a
mul
ticen
tre e
ffica
cy s
tudy
with
HD
M im
mun
othe
rapy
. Pat
ient
s w
ith a
pos
itive
AR
-his
tory
and
a p
ositi
ve s
kin
pric
k te
st to
HD
M, u
nder
went
a s
tand
ardi
zed
TNPT
. M
etho
ds:
The
TNPT
sta
rted
with
the
alle
rgen
’s d
iluen
t fo
llow
ed b
y m
axim
al3
seria
l co
ncen
tratio
ns o
f a
stan
dard
ized
alle
rgen
ext
ract
(D
. Pt
eron
yssi
nus
100,
1,0
00 a
nd 1
0,00
0 AU
/ml,
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s), a
dmin
iste
red
by a
nas
al s
pray
ing
devi
ce (
1 pu
ff pe
r no
stril
) at
20
min
utes
int
erva
ls.
The
uppe
r ai
rway
res
pons
e w
as q
uant
ified
by
a co
mpo
site
sym
ptom
sco
re a
ccor
ding
to
Lebe
l an
d PN
IF
mea
sure
men
ts (h
ighe
st v
alue
of 3
mea
sure
d by
In-c
heck
nas
al in
spira
tory
flow
met
er [C
lem
ent C
lark
e, H
arlo
w, U
K])a
t bas
elin
e (p
re-d
iluen
t), 1
5 m
inut
es a
fter e
ach
adm
inis
tratio
n an
d 60
min
utes
pos
t-cha
lleng
e. S
ympt
oms
wer
e re
cord
ed u
sing
the
follo
win
g sc
orin
g sy
stem
: sne
ezes
2
= 0,
sn
eeze
s 3-
4 =
1 po
int,
snee
zes
5 =
3 po
ints
, ant
erio
r rh
inor
rhoe
a =
1 po
int,
post
erio
r rh
inor
rhoe
a =
1 po
int,
diffi
cult
brea
thin
g =
1 po
int,
1 bl
ocke
d no
stril
= 2
poi
nts,
2 b
lock
ed n
ostri
ls =
3 p
oint
s, n
asal
prur
itus
= 1
poin
t, pr
uritu
s in
pal
ate
or e
ar =
1 p
oint
, con
junc
tiviti
s =
1 po
int (
tota
l sc
ore
rang
e: 0
-11
poin
ts).
The
pre-
dilu
ent w
as n
ot a
llow
ed to
be
3. T
he T
NPT
was
con
side
red
posi
tive
once
a to
tal s
core
6
was
reac
hed.
Res
ults
: 15
0 pa
tient
s w
ere
scre
ened
. 103
pat
ient
s (T
able
1)h
ad a
pos
itive
TN
PT. T
he T
NPT
was
wel
l tol
erat
ed a
nd n
one
of th
epa
tient
s ex
perie
nced
any
cl
inic
ally
sig
nific
ant a
dver
se e
vent
s. T
he m
ost p
rom
inen
t sym
ptom
s w
ere
nasa
l blo
ckag
e an
d rh
inor
rhoe
a, fo
llow
ed b
y pr
uritu
s an
dsn
eezi
ng. A
co
nsis
tent
con
cent
ratio
n-re
spon
se r
elat
ions
hip
was
foun
d be
twee
n se
rial a
llerg
en c
once
ntra
tions
and
incr
easi
ng s
ympt
om s
core
s, m
atch
ing
a gr
adua
l dec
reas
e in
PN
IF. O
ne h
our a
fter t
he p
rovo
cativ
e al
lerg
en c
once
ntra
tion,
bot
h pa
ram
eter
s ha
d no
t com
plet
ely
retu
rned
to b
asel
ine
valu
es
(Fig
ure
1). T
here
was
a m
oder
ate
thou
gh h
ighl
y si
gnifi
cant
cor
rela
tion
betw
een
sym
ptom
sco
res
and
PNIF
mea
sure
men
ts (r
=0.5
4; p
<0.0
1). T
he
maj
ority
of p
atie
nts
had
a po
sitiv
e TN
PT-re
spon
se fo
llow
ing
adm
inis
tratio
n of
the
10,0
00 A
U/m
l con
cent
ratio
n (T
able
2).
Patie
nts
with
a p
ositi
ve
TNPT
-resp
onse
at a
low
er a
llerg
en c
once
ntra
tion
tend
ed to
hav
e a
larg
er w
heal
siz
e al
thou
gh d
iffer
ence
s w
ere
not s
tatis
tical
ly s
igni
fican
t (Ta
ble
2).
Tabl
e 2:
Pos
itive
TNP
T-re
spon
se a
nd c
orre
spon
ding
SP
T-w
heal
siz
es
Con
cent
ratio
n10
0 A
U/m
l1,
000
AU/
ml
10,0
00
AU/
ml
Posi
tive
resp
onse
(n)
1034
59M
ean
SPT-
whea
lsi
ze (m
m)
76
5Fi
gure
1: C
once
ntra
tion-
resp
onse
cur
ves
with
mea
n (
95%
CI)
chan
ges
in c
ompo
site
sym
ptom
sc
ores
(red
circ
les)
and
PNI
F m
easu
rem
ents
(blu
e ci
rcle
s) fo
llow
ing
TNPT
with
HDM
alle
rgen
.
Tabl
e 1:
Pat
ient
s’de
mog
raph
ic c
hara
cter
istic
s
Patie
nts
n =
103
Gen
der (
M (%
) / F
(%))
54(5
2.4%
)/ 49
(47.
6%)
Mea
n ag
e ±
SD in
yea
rs (r
ange
)32
±11
(18-
57)
SPT
to H
DM e
xtra
ct:
Mea
n wh
eal s
ize
in m
m ±
SD
(rang
e)6
±2
(3-1
2)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
C. B
ache
rt1 , O. P
faar
2 , M.J
. Nel
l3 , J.D
. Boo
t3 , H.E
. Nie
nhui
s3 , Z. D
iam
ant4,
5 1 U
Z G
ent,
Ear-,
Nos
e an
d Th
roat
Dep
artm
ent,
Gen
t, Be
lgiu
m; 2 C
ente
r for
Rhi
nolo
gy a
nd A
llerg
olog
y W
iesb
aden
, Ger
man
y;
3 HAL
Alle
rgy
BV,
Leid
en, T
he N
ethe
rland
s; 4 S
kane
Uni
vers
ity H
ospi
tal,
Dep
t of R
espi
r Med
& A
llerg
ol, L
und,
Sw
eden
an
d 5 Q
PS-N
L, G
roni
ngen
, The
Net
herla
nds
37 -
Appl
icat
ion
of a
sta
ndar
dize
d tit
rate
d na
sal
prov
ocat
ion
test
with
HD
M e
xtra
ct in
a
mul
ticen
tre
clin
ical
stu
dy in
alle
rgic
rhin
itis
Bac
kgro
und
& Ai
m:
Titra
ted
nasa
l pro
voca
tion
test
s (T
NPT
) with
a re
leva
nt a
llerg
enca
n be
use
d in
the
diag
nosi
s of
alle
rgic
rhin
itis
(AR
) and
as
a di
seas
e m
odel
in
clin
ical
tria
ls e
valu
atin
g th
e ef
ficac
y of
tre
atm
ent.
How
ever
, bo
th t
he t
echn
ique
s us
ed f
or p
rovo
catio
n an
d th
e re
adou
ts o
f th
e ev
oked
upp
erai
rway
resp
onse
vary
, req
uirin
g st
anda
rdiz
atio
n to
ena
ble
the
use
of T
NPT
as
a to
ol in
mul
ticen
tre tr
ials
. We
perfo
rmed
a s
tand
ardi
zed
TNPT
an
d do
cum
ente
d th
e up
per a
irway
resp
onse
by
sym
ptom
sco
res
and
peak
nas
al in
spira
tory
flow
(PN
IF) m
easu
rem
ents
. In
addi
tion,
we
com
pare
d th
e up
per a
irway
resp
onse
s ac
ross
the
diffe
rent
alle
rgen
con
cent
ratio
ns a
nd in
vest
igat
ed a
pos
sibl
e co
rrela
tion
betw
een
both
read
outs
.
Con
clus
ion:
Whe
n ap
plie
d in
a m
ultic
entre
set
ting
in p
atie
nts
with
sus
pect
ed H
DM
-indu
ced
AR, a
sta
ndar
dize
d TN
PT w
ith a
HD
M e
xtra
ct in
duce
sa
cons
iste
nt g
radu
al in
crea
se in
alle
rgic
sym
ptom
s an
d si
gns
that
can
be
relia
bly
quan
tifie
d by
a w
ell-d
efin
ed c
ompo
site
sym
ptom
sc
ore
and
PNIF
mea
sure
men
ts.
Met
hods
:Pa
tient
s: M
/F a
ged
18-6
0 ye
ars
with
HD
M-re
late
d AR
, w
ith o
r w
ithou
t co
ncom
itant
, cl
inic
ally
sta
ble
asth
ma
(FEV
1 >7
0% p
redi
cted
) w
ere
scre
ened
prio
r to
enro
llmen
t int
o a
mul
ticen
tre e
ffica
cy s
tudy
with
HD
M im
mun
othe
rapy
. Pat
ient
s w
ith a
pos
itive
AR
-his
tory
and
a p
ositi
ve s
kin
pric
k te
st to
HD
M, u
nder
went
a s
tand
ardi
zed
TNPT
. M
etho
ds:
The
TNPT
sta
rted
with
the
alle
rgen
’s d
iluen
t fo
llow
ed b
y m
axim
al3
seria
l co
ncen
tratio
ns o
f a
stan
dard
ized
alle
rgen
ext
ract
(D
. Pt
eron
yssi
nus
100,
1,0
00 a
nd 1
0,00
0 AU
/ml,
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s), a
dmin
iste
red
by a
nas
al s
pray
ing
devi
ce (
1 pu
ff pe
r no
stril
) at
20
min
utes
int
erva
ls.
The
uppe
r ai
rway
res
pons
e w
as q
uant
ified
by
a co
mpo
site
sym
ptom
sco
re a
ccor
ding
to
Lebe
l an
d PN
IF
mea
sure
men
ts (h
ighe
st v
alue
of 3
mea
sure
d by
In-c
heck
nas
al in
spira
tory
flow
met
er [C
lem
ent C
lark
e, H
arlo
w, U
K])a
t bas
elin
e (p
re-d
iluen
t), 1
5 m
inut
es a
fter e
ach
adm
inis
tratio
n an
d 60
min
utes
pos
t-cha
lleng
e. S
ympt
oms
wer
e re
cord
ed u
sing
the
follo
win
g sc
orin
g sy
stem
: sne
ezes
2
= 0,
sn
eeze
s 3-
4 =
1 po
int,
snee
zes
5 =
3 po
ints
, ant
erio
r rh
inor
rhoe
a =
1 po
int,
post
erio
r rh
inor
rhoe
a =
1 po
int,
diffi
cult
brea
thin
g =
1 po
int,
1 bl
ocke
d no
stril
= 2
poi
nts,
2 b
lock
ed n
ostri
ls =
3 p
oint
s, n
asal
prur
itus
= 1
poin
t, pr
uritu
s in
pal
ate
or e
ar =
1 p
oint
, con
junc
tiviti
s =
1 po
int (
tota
l sc
ore
rang
e: 0
-11
poin
ts).
The
pre-
dilu
ent w
as n
ot a
llow
ed to
be
3. T
he T
NPT
was
con
side
red
posi
tive
once
a to
tal s
core
6
was
reac
hed.
Res
ults
: 15
0 pa
tient
s w
ere
scre
ened
. 103
pat
ient
s (T
able
1)h
ad a
pos
itive
TN
PT. T
he T
NPT
was
wel
l tol
erat
ed a
nd n
one
of th
epa
tient
s ex
perie
nced
any
cl
inic
ally
sig
nific
ant a
dver
se e
vent
s. T
he m
ost p
rom
inen
t sym
ptom
s w
ere
nasa
l blo
ckag
e an
d rh
inor
rhoe
a, fo
llow
ed b
y pr
uritu
s an
dsn
eezi
ng. A
co
nsis
tent
con
cent
ratio
n-re
spon
se r
elat
ions
hip
was
foun
d be
twee
n se
rial a
llerg
en c
once
ntra
tions
and
incr
easi
ng s
ympt
om s
core
s, m
atch
ing
a gr
adua
l dec
reas
e in
PN
IF. O
ne h
our a
fter t
he p
rovo
cativ
e al
lerg
en c
once
ntra
tion,
bot
h pa
ram
eter
s ha
d no
t com
plet
ely
retu
rned
to b
asel
ine
valu
es
(Fig
ure
1). T
here
was
a m
oder
ate
thou
gh h
ighl
y si
gnifi
cant
cor
rela
tion
betw
een
sym
ptom
sco
res
and
PNIF
mea
sure
men
ts (r
=0.5
4; p
<0.0
1). T
he
maj
ority
of p
atie
nts
had
a po
sitiv
e TN
PT-re
spon
se fo
llow
ing
adm
inis
tratio
n of
the
10,0
00 A
U/m
l con
cent
ratio
n (T
able
2).
Patie
nts
with
a p
ositi
ve
TNPT
-resp
onse
at a
low
er a
llerg
en c
once
ntra
tion
tend
ed to
hav
e a
larg
er w
heal
siz
e al
thou
gh d
iffer
ence
s w
ere
not s
tatis
tical
ly s
igni
fican
t (Ta
ble
2).
Tabl
e 2:
Pos
itive
TNP
T-re
spon
se a
nd c
orre
spon
ding
SP
T-w
heal
siz
es
Con
cent
ratio
n10
0 A
U/m
l1,
000
AU/
ml
10,0
00
AU/
ml
Posi
tive
resp
onse
(n)
1034
59M
ean
SPT-
whea
lsi
ze (m
m)
76
5Fi
gure
1: C
once
ntra
tion-
resp
onse
cur
ves
with
mea
n (
95%
CI)
chan
ges
in c
ompo
site
sym
ptom
sc
ores
(red
circ
les)
and
PNI
F m
easu
rem
ents
(blu
e ci
rcle
s) fo
llow
ing
TNPT
with
HDM
alle
rgen
.
Tabl
e 1:
Pat
ient
s’de
mog
raph
ic c
hara
cter
istic
s
Patie
nts
n =
103
Gen
der (
M (%
) / F
(%))
54(5
2.4%
)/ 49
(47.
6%)
Mea
n ag
e ±
SD in
yea
rs (r
ange
)32
±11
(18-
57)
SPT
to H
DM e
xtra
ct:
Mea
n wh
eal s
ize
in m
m ±
SD
(rang
e)6
±2
(3-1
2)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
C. B
ache
rt1 , O. P
faar
2 , M.J
. Nel
l3 , J.D
. Boo
t3 , H.E
. Nie
nhui
s3 , Z. D
iam
ant4,
5
1 UZ
Gen
t, Ea
r-, N
ose
and
Thro
at D
epar
tmen
t, G
ent,
Belg
ium
; 2 Cen
ter f
or R
hino
logy
and
Alle
rgol
ogy
Wie
sbad
en, G
erm
any;
3 H
AL A
llerg
y BV
, Le
iden
, The
Net
herla
nds;
4 Ska
ne U
nive
rsity
Hos
pita
l, D
ept o
f Res
pir M
ed &
Alle
rgol
, Lun
d, S
wed
en
and
5 QPS
-NL,
Gro
ning
en, T
he N
ethe
rland
s
37 -
Appl
icat
ion
of a
sta
ndar
dize
d tit
rate
d na
sal
prov
ocat
ion
test
with
HD
M e
xtra
ct in
a
mul
ticen
tre
clin
ical
stu
dy in
alle
rgic
rhin
itis
Bac
kgro
und
& Ai
m:
Titra
ted
nasa
l pro
voca
tion
test
s (T
NPT
) with
a re
leva
nt a
llerg
enca
n be
use
d in
the
diag
nosi
s of
alle
rgic
rhin
itis
(AR
) and
as
a di
seas
e m
odel
in
clin
ical
tria
ls e
valu
atin
g th
e ef
ficac
y of
tre
atm
ent.
How
ever
, bo
th t
he t
echn
ique
s us
ed f
or p
rovo
catio
n an
d th
e re
adou
ts o
f th
e ev
oked
upp
erai
rway
resp
onse
vary
, req
uirin
g st
anda
rdiz
atio
n to
ena
ble
the
use
of T
NPT
as
a to
ol in
mul
ticen
tre tr
ials
. We
perfo
rmed
a s
tand
ardi
zed
TNPT
an
d do
cum
ente
d th
e up
per a
irway
resp
onse
by
sym
ptom
sco
res
and
peak
nas
al in
spira
tory
flow
(PN
IF) m
easu
rem
ents
. In
addi
tion,
we
com
pare
d th
e up
per a
irway
resp
onse
s ac
ross
the
diffe
rent
alle
rgen
con
cent
ratio
ns a
nd in
vest
igat
ed a
pos
sibl
e co
rrela
tion
betw
een
both
read
outs
.
Con
clus
ion:
Whe
n ap
plie
d in
a m
ultic
entre
set
ting
in p
atie
nts
with
sus
pect
ed H
DM
-indu
ced
AR, a
sta
ndar
dize
d TN
PT w
ith a
HD
M e
xtra
ct in
duce
sa
cons
iste
nt g
radu
al in
crea
se in
alle
rgic
sym
ptom
s an
d si
gns
that
can
be
relia
bly
quan
tifie
d by
a w
ell-d
efin
ed c
ompo
site
sym
ptom
sc
ore
and
PNIF
mea
sure
men
ts.
Met
hods
:Pa
tient
s: M
/F a
ged
18-6
0 ye
ars
with
HD
M-re
late
d AR
, w
ith o
r w
ithou
t co
ncom
itant
, cl
inic
ally
sta
ble
asth
ma
(FEV
1 >7
0% p
redi
cted
) w
ere
scre
ened
prio
r to
enro
llmen
t int
o a
mul
ticen
tre e
ffica
cy s
tudy
with
HD
M im
mun
othe
rapy
. Pat
ient
s w
ith a
pos
itive
AR
-his
tory
and
a p
ositi
ve s
kin
pric
k te
st to
HD
M, u
nder
went
a s
tand
ardi
zed
TNPT
. M
etho
ds:
The
TNPT
sta
rted
with
the
alle
rgen
’s d
iluen
t fo
llow
ed b
y m
axim
al3
seria
l co
ncen
tratio
ns o
f a
stan
dard
ized
alle
rgen
ext
ract
(D
. Pt
eron
yssi
nus
100,
1,0
00 a
nd 1
0,00
0 AU
/ml,
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s), a
dmin
iste
red
by a
nas
al s
pray
ing
devi
ce (
1 pu
ff pe
r no
stril
) at
20
min
utes
int
erva
ls.
The
uppe
r ai
rway
res
pons
e w
as q
uant
ified
by
a co
mpo
site
sym
ptom
sco
re a
ccor
ding
to
Lebe
l an
d PN
IF
mea
sure
men
ts (h
ighe
st v
alue
of 3
mea
sure
d by
In-c
heck
nas
al in
spira
tory
flow
met
er [C
lem
ent C
lark
e, H
arlo
w, U
K])a
t bas
elin
e (p
re-d
iluen
t), 1
5 m
inut
es a
fter e
ach
adm
inis
tratio
n an
d 60
min
utes
pos
t-cha
lleng
e. S
ympt
oms
wer
e re
cord
ed u
sing
the
follo
win
g sc
orin
g sy
stem
: sne
ezes
2
= 0,
sn
eeze
s 3-
4 =
1 po
int,
snee
zes
5 =
3 po
ints
, ant
erio
r rh
inor
rhoe
a =
1 po
int,
post
erio
r rh
inor
rhoe
a =
1 po
int,
diffi
cult
brea
thin
g =
1 po
int,
1 bl
ocke
d no
stril
= 2
poi
nts,
2 b
lock
ed n
ostri
ls =
3 p
oint
s, n
asal
prur
itus
= 1
poin
t, pr
uritu
s in
pal
ate
or e
ar =
1 p
oint
, con
junc
tiviti
s =
1 po
int (
tota
l sc
ore
rang
e: 0
-11
poin
ts).
The
pre-
dilu
ent w
as n
ot a
llow
ed to
be
3. T
he T
NPT
was
con
side
red
posi
tive
once
a to
tal s
core
6
was
reac
hed.
Res
ults
: 15
0 pa
tient
s w
ere
scre
ened
. 103
pat
ient
s (T
able
1)h
ad a
pos
itive
TN
PT. T
he T
NPT
was
wel
l tol
erat
ed a
nd n
one
of th
epa
tient
s ex
perie
nced
any
cl
inic
ally
sig
nific
ant a
dver
se e
vent
s. T
he m
ost p
rom
inen
t sym
ptom
s w
ere
nasa
l blo
ckag
e an
d rh
inor
rhoe
a, fo
llow
ed b
y pr
uritu
s an
dsn
eezi
ng. A
co
nsis
tent
con
cent
ratio
n-re
spon
se r
elat
ions
hip
was
foun
d be
twee
n se
rial a
llerg
en c
once
ntra
tions
and
incr
easi
ng s
ympt
om s
core
s, m
atch
ing
a gr
adua
l dec
reas
e in
PN
IF. O
ne h
our a
fter t
he p
rovo
cativ
e al
lerg
en c
once
ntra
tion,
bot
h pa
ram
eter
s ha
d no
t com
plet
ely
retu
rned
to b
asel
ine
valu
es
(Fig
ure
1). T
here
was
a m
oder
ate
thou
gh h
ighl
y si
gnifi
cant
cor
rela
tion
betw
een
sym
ptom
sco
res
and
PNIF
mea
sure
men
ts (r
=0.5
4; p
<0.0
1). T
he
maj
ority
of p
atie
nts
had
a po
sitiv
e TN
PT-re
spon
se fo
llow
ing
adm
inis
tratio
n of
the
10,0
00 A
U/m
l con
cent
ratio
n (T
able
2).
Patie
nts
with
a p
ositi
ve
TNPT
-resp
onse
at a
low
er a
llerg
en c
once
ntra
tion
tend
ed to
hav
e a
larg
er w
heal
siz
e al
thou
gh d
iffer
ence
s w
ere
not s
tatis
tical
ly s
igni
fican
t (Ta
ble
2).
Tabl
e 2:
Pos
itive
TNP
T-re
spon
se a
nd c
orre
spon
ding
SP
T-w
heal
siz
es
Con
cent
ratio
n10
0 A
U/m
l1,
000
AU/
ml
10,0
00
AU/
ml
Posi
tive
resp
onse
(n)
1034
59M
ean
SPT-
whea
lsi
ze (m
m)
76
5Fi
gure
1: C
once
ntra
tion-
resp
onse
cur
ves
with
mea
n (
95%
CI)
chan
ges
in c
ompo
site
sym
ptom
sc
ores
(red
circ
les)
and
PNI
F m
easu
rem
ents
(blu
e ci
rcle
s) fo
llow
ing
TNPT
with
HDM
alle
rgen
.
Tabl
e 1:
Pat
ient
s’de
mog
raph
ic c
hara
cter
istic
s
Patie
nts
n =
103
Gen
der (
M (%
) / F
(%))
54(5
2.4%
)/ 49
(47.
6%)
Mea
n ag
e ±
SD in
yea
rs (r
ange
)32
±11
(18-
57)
SPT
to H
DM e
xtra
ct:
Mea
n wh
eal s
ize
in m
m ±
SD
(rang
e)6
±2
(3-1
2)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
C. B
ache
rt1 , O. P
faar
2 , M.J
. Nel
l3 , J.D
. Boo
t3 , H.E
. Nie
nhui
s3 , Z. D
iam
ant4,
5
EAAC
I Con
gres
s 20
12In
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er is
an
empl
oyee
of H
AL A
llerg
y.
Pa t i e nt Kn o w l e d ge, Pe rce p t i o n s , E xp e c t a t i o n s , a n d S a t isf a c t i o n , o n Su b cu t a n e o u s a n d Su b l ing u a l Al l e rge n Sp e c i f ic Im mu n o t h e rap y: a r ea l l i f e su r v e y
I. Baiardini (1), F. Puggioni (2), S. Menoni (3), J.D. Boot (4), Z. Diamant (5), F. Braido (6), G.W. Canonica (6).
(1) University of Genoa, Allergy and Respiratory Diseases, Department of Internal Medicine, Genoa, Italy, (2) IRCCS Istituto Clinico Humanitas,
Allergy and respiratory Disease Department, Milano, Italy, (3) University of Genoa, Biostatistic Unit, Department of Health Science, Genoa,
Italy, (4) HAL Allergy BV, Leiden, The Netherlands, (5) Skane University Hospital, Dept of Respir Med & Allergol, Lund, Sweden and (6) QPS-NL,
Groningen, The Netherlands.
BackgroundAssessing patient’s viewpoint on treatment provides useful information enabling customized therapeutic approach. Presently, only a few small studies addressing the patient’s perspective on allergen specific immunotherapy (SIT) are available. The aim of this cross-sectional multicentre survey was to evaluate subjective viewpoints on SIT in a large cohort of patients treated with subcutaneous (SCIT) or sublingual (SLIT) immunotherapy.
MethodsA novel survey of 28 questions assessing patient’s knowledge (6), perception (12), expectations (5) and satisfaction (5) was developed by an expert panel. Sixteen physicians from 15 allergology centres in North Italy asked 30 consecutive patients with allergic rhinitis with or without asthma treated with SIT to fill out the survey. The physicians were also asked to report for each patient their satisfaction level regarding SIT.
ResultsFully completed surveys from 434 patients (55.3% male; 66.7% polysensitized, 74% SLIT) were analyzed. SIT consisted of grass pollen (37.1%), mites (26.6%), tree pollen (17%), flower pollen (9.2%), moulds (5.6%), epithelia (2%) and other (2.5%); mean duration of treatment was 2.5 years. Most patients acquired their SIT knowledge through their physician (95%). Conversely, the physicians opinion in their choice to start with SIT was important. The majority of patients (70.6%) believed that SIT should be continued for more than two years. Most patients perceived SIT to be safe and easy to integrate in daily routine. The main motivation when starting SIT were its potential effect to alter the course of the disease (45.7%), less need of anti-allergy drugs (28.2%), and dissatisfaction with the current pharmacotherapy (19.3%). Both patient and physician satisfaction was high (VAS score 74/100 and 78/100, respectively) and a significant correlation between patients’ and physicians’ satisfaction scores was found in both groups (SCIT: r = 0.612, p <0.001; SLIT: r = 0.608, p <0.001). No major difference was found in patients answers based on the level of education. Compared to SCIT, SLIT was frequently considered easier to take (p=0.024) and without side effects (p=0.026)
ConclusionThis real life, cross-sectional survey evaluated in a large cohort different aspects of patient’s perspective on SIT. Despite some gaps and misconceptions, the majority of patients had an adequate level of knowledge, perception, expectations and satisfaction about SIT, which corresponded well with the physician’s satisfaction level.
10
EAACI, 16-20 June 2012, GenevaAbstract number: 874, Session date and time: Monday 18 June; 12:15 - 13:30 Session title: Poster 32 - Update in the treatment of allergic rhinitis
11
Poster Session 32 - Update in the treatment of allergic rhinitis
Pa t i e nt Kn o w l e d ge, Pe rce p t i o n s , E xp e c t a t i o n s , a n d S a t isf a c t i o n , o n Su b cu t a n e o u s a n d Su b l ing u a l Al l e rge n Sp e c i f ic Im mu n o t h e rap y: a r ea l l i f e su r v e y
1 Uni
vers
ity o
f Gen
oa, A
llerg
y an
d Re
spira
tory
Dis
ease
s, D
epar
tmen
t of I
nter
nal M
edic
ine,
Gen
oa, I
taly
, 2 IRCC
S Is
titut
oCl
inic
oHu
man
itas,
Alle
rgy
and
resp
irato
ry D
isea
se
Depa
rtmen
t, M
ilano
, Ita
ly,3 U
nive
rsity
of G
enoa
, Bio
stat
istic
Unit,
Dep
artm
ent o
f Hea
lth S
cien
ce, G
enoa
, Ita
ly,4 H
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds,
5 S
kane
Univ
ersi
ty H
ospi
tal,
Dep
t of R
espi
rMed
& A
llerg
ol, L
und,
Sw
eden
and
QPS
-NL,
Gro
ning
en, T
he N
ethe
rland
s
874
-Pat
ient
Kno
wle
dge,
Per
cept
ions
, Exp
ecta
tions
and
Sa
tisfa
ctio
n on
Sub
cuta
neou
s an
d Su
blin
gual
Al
lerg
en S
peci
fic Im
mun
othe
rapy
:a re
al li
fe s
urve
y
Tabl
e 1:
Pat
ient
dem
ogra
phic
s an
d cl
inic
al d
ata
Figu
re 1
: Cor
rela
tion
betw
een
patie
nts’
and
phys
icia
ns’s
atis
fact
ion
leve
l (V
AS
scor
e) o
n su
bcut
aneo
us (l
eft p
anel
; r =
0.6
12, p
<0.
001)
and
su
blin
gual
(rig
ht p
anel
r =
0.60
8, p
<0.
001)
SIT
Su
rvey
Patie
nts
n =
434
Gen
der
238
Mal
es/ 1
92 F
emal
es
Mea
n ag
e ±
SD in
yea
rs
31.5
12
Mea
n du
ratio
n of
trea
tmen
t ±SD
in y
ears
2.5
±1.
3
Rhi
nitis
–AR
IA c
lass
ificat
ion
Inte
rmitt
ent
40.6
%
Pers
isten
t59
.4%
Mild
11.3
%
Mod
erat
e88
.7%
Asth
ma
–G
INA
Cla
ssific
atio
n
129
.7%
254
.5%
314
.3%
41.
5%
Sens
itiza
tion
Mon
osen
sitiz
ed33
.3%
Poly
sens
itize
d66
.7%
SCIT
26.0
%
SLIT
74.0
%
Adm
inis
tere
d al
lerg
en
Gra
ss p
olle
n37
.1%
Mite
s26
.6%
Tree
pol
len
17.0
%
Flow
er p
olle
n9.
2%
Oth
er10
.1%
Tabl
e 2:
Pat
ient
s pe
rcep
tion
abou
t SIT
Que
stio
nsPa
tient
answ
ers
Com
plet
ely
True
Pa
rtly
True
Partl
yFa
lseC
ompl
etel
y Fa
lseD
on’t
know
SIT
is sa
fe54
.5%
37.4
%0.
2%0.
2%7.
6%SI
T is
easy
to ta
ke68
.6%
27.5
%0.
5%-
3.5%
SIT
is ha
ndy
to u
se in
da
ily ro
utin
e47
.1%
42.7
%1.
8%0.
2%8.
1%
It is
eas
y to
rem
embe
r to
take
SIT
50.8
%37
.0%
2.8%
0.2%
9.2%
SIT
allo
ws to
bet
ter
cont
rol y
our a
llerg
y56
.0%
35.2
%0.
7%5.
1%3.
0%
1.0%
Did
not
spe
cifiy
Poss
ible
ans
wer
st
Patie
nan
swer
sR
ecov
ery
from
alle
rgy
38.2
%Im
prov
emen
t in
heal
th re
late
d qu
ality
of l
ife21
.4%
Sym
ptom
redu
ctio
n22
.6%
Less
nee
d of
ant
i-alle
rgy
med
icatio
n10
.5%
Avoi
d wo
rsen
ing
of th
e di
seas
e6.
4%
Tabl
e 3:
Pat
ient
s ex
pect
atio
n ab
out S
IT
In re
latio
n to
this
pre
sent
atio
n, I
decl
are
the
follo
win
g, re
al o
r per
ceiv
ed c
onfli
cts
of in
tere
st:
the
pres
ente
r has
rece
ived
a tr
avel
gra
nt fr
om H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
:
®®
know
ledg
e
perc
eive
d
expe
cted
satis
fact
ion
I. B
aiar
dini
1 , F.
Pug
gion
i2 , S.
Men
oni3 ,
J.D
. Boo
t4 , Z.
Dia
man
t5 , F.
Bra
ido1 ,
G.W
. Can
onic
a1
1 Uni
vers
ity o
f Gen
oa, A
llerg
y an
d Re
spira
tory
Dis
ease
s, D
epar
tmen
t of I
nter
nal M
edic
ine,
Gen
oa, I
taly
, 2 IRCC
S Is
titut
oCl
inic
oHu
man
itas,
Alle
rgy
and
resp
irato
ry D
isea
se
Depa
rtmen
t, M
ilano
, Ita
ly,3 U
nive
rsity
of G
enoa
, Bio
stat
istic
Unit,
Dep
artm
ent o
f Hea
lth S
cien
ce, G
enoa
, Ita
ly,4 H
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds,
5 S
kane
Univ
ersi
ty H
ospi
tal,
Dep
t of R
espi
rMed
& A
llerg
ol, L
und,
Sw
eden
and
QPS
-NL,
Gro
ning
en, T
he N
ethe
rland
s
874
-Pat
ient
Kno
wle
dge,
Per
cept
ions
, Exp
ecta
tions
and
Sa
tisfa
ctio
n on
Sub
cuta
neou
s an
d Su
blin
gual
Al
lerg
en S
peci
fic Im
mun
othe
rapy
:a re
al li
fe s
urve
y
Tabl
e 1:
Pat
ient
dem
ogra
phic
s an
d cl
inic
al d
ata
Figu
re 1
: Cor
rela
tion
betw
een
patie
nts’
and
phys
icia
ns’s
atis
fact
ion
leve
l (V
AS
scor
e) o
n su
bcut
aneo
us (l
eft p
anel
; r =
0.6
12, p
<0.
001)
and
su
blin
gual
(rig
ht p
anel
r =
0.60
8, p
<0.
001)
SIT
Su
rvey
Patie
nts
n =
434
Gen
der
238
Mal
es/ 1
92 F
emal
es
Mea
n ag
e ±
SD in
yea
rs
31.5
12
Mea
n du
ratio
n of
trea
tmen
t ±SD
in y
ears
2.5
±1.
3
Rhi
nitis
–AR
IA c
lass
ificat
ion
Inte
rmitt
ent
40.6
%
Pers
isten
t59
.4%
Mild
11.3
%
Mod
erat
e88
.7%
Asth
ma
–G
INA
Cla
ssific
atio
n
129
.7%
254
.5%
314
.3%
41.
5%
Sens
itiza
tion
Mon
osen
sitiz
ed33
.3%
Poly
sens
itize
d66
.7%
SCIT
26.0
%
SLIT
74.0
%
Adm
inis
tere
d al
lerg
en
Gra
ss p
olle
n37
.1%
Mite
s26
.6%
Tree
pol
len
17.0
%
Flow
er p
olle
n9.
2%
Oth
er10
.1%
Tabl
e 2:
Pat
ient
s pe
rcep
tion
abou
t SIT
Que
stio
nsPa
tient
answ
ers
Com
plet
ely
True
Pa
rtly
True
Partl
yFa
lseC
ompl
etel
y Fa
lseD
on’t
know
SIT
is sa
fe54
.5%
37.4
%0.
2%0.
2%7.
6%SI
T is
easy
to ta
ke68
.6%
27.5
%0.
5%-
3.5%
SIT
is ha
ndy
to u
se in
da
ily ro
utin
e47
.1%
42.7
%1.
8%0.
2%8.
1%
It is
eas
y to
rem
embe
r to
take
SIT
50.8
%37
.0%
2.8%
0.2%
9.2%
SIT
allo
ws to
bet
ter
cont
rol y
our a
llerg
y56
.0%
35.2
%0.
7%5.
1%3.
0%
1.0%
Did
not
spe
cifiy
Poss
ible
ans
wer
st
Patie
nan
swer
sR
ecov
ery
from
alle
rgy
38.2
%Im
prov
emen
t in
heal
th re
late
d qu
ality
of l
ife21
.4%
Sym
ptom
redu
ctio
n22
.6%
Less
nee
d of
ant
i-alle
rgy
med
icatio
n10
.5%
Avoi
d wo
rsen
ing
of th
e di
seas
e6.
4%
Tabl
e 3:
Pat
ient
s ex
pect
atio
n ab
out S
IT
In re
latio
n to
this
pre
sent
atio
n, I
decl
are
the
follo
win
g, re
al o
r per
ceiv
ed c
onfli
cts
of in
tere
st:
the
pres
ente
r has
rece
ived
a tr
avel
gra
nt fr
om H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
:
®®
know
ledg
e
perc
eive
d
expe
cted
satis
fact
ion
I. B
aiar
dini
1 , F.
Pug
gion
i2 , S.
Men
oni3 ,
J.D
. Boo
t4 , Z.
Dia
man
t5 , F.
Bra
ido1 ,
G.W
. Can
onic
a1
1 Uni
vers
ity o
f Gen
oa, A
llerg
y an
d Re
spira
tory
Dis
ease
s, D
epar
tmen
t of I
nter
nal M
edic
ine,
Gen
oa, I
taly
, 2 IRCC
S Is
titut
oCl
inic
oHu
man
itas,
Alle
rgy
and
resp
irato
ry D
isea
se
Depa
rtmen
t, M
ilano
, Ita
ly,3 U
nive
rsity
of G
enoa
, Bio
stat
istic
Unit,
Dep
artm
ent o
f Hea
lth S
cien
ce, G
enoa
, Ita
ly,4 H
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds,
5 S
kane
Univ
ersi
ty H
ospi
tal,
Dep
t of R
espi
rMed
& A
llerg
ol, L
und,
Sw
eden
and
QPS
-NL,
Gro
ning
en, T
he N
ethe
rland
s
874
-Pat
ient
Kno
wle
dge,
Per
cept
ions
, Exp
ecta
tions
and
Sa
tisfa
ctio
n on
Sub
cuta
neou
s an
d Su
blin
gual
Al
lerg
en S
peci
fic Im
mun
othe
rapy
:a re
al li
fe s
urve
y
Tabl
e 1:
Pat
ient
dem
ogra
phic
s an
d cl
inic
al d
ata
Figu
re 1
: Cor
rela
tion
betw
een
patie
nts’
and
phys
icia
ns’s
atis
fact
ion
leve
l (V
AS
scor
e) o
n su
bcut
aneo
us (l
eft p
anel
; r =
0.6
12, p
<0.
001)
and
su
blin
gual
(rig
ht p
anel
r =
0.60
8, p
<0.
001)
SIT
Su
rvey
Patie
nts
n =
434
Gen
der
238
Mal
es/ 1
92 F
emal
es
Mea
n ag
e ±
SD in
yea
rs
31.5
12
Mea
n du
ratio
n of
trea
tmen
t ±SD
in y
ears
2.5
±1.
3
Rhi
nitis
–AR
IA c
lass
ificat
ion
Inte
rmitt
ent
40.6
%
Pers
isten
t59
.4%
Mild
11.3
%
Mod
erat
e88
.7%
Asth
ma
–G
INA
Cla
ssific
atio
n
129
.7%
254
.5%
314
.3%
41.
5%
Sens
itiza
tion
Mon
osen
sitiz
ed33
.3%
Poly
sens
itize
d66
.7%
SCIT
26.0
%
SLIT
74.0
%
Adm
inis
tere
d al
lerg
en
Gra
ss p
olle
n37
.1%
Mite
s26
.6%
Tree
pol
len
17.0
%
Flow
er p
olle
n9.
2%
Oth
er10
.1%
Tabl
e 2:
Pat
ient
s pe
rcep
tion
abou
t SIT
Que
stio
nsPa
tient
answ
ers
Com
plet
ely
True
Pa
rtly
True
Partl
yFa
lseC
ompl
etel
y Fa
lseD
on’t
know
SIT
is sa
fe54
.5%
37.4
%0.
2%0.
2%7.
6%SI
T is
easy
to ta
ke68
.6%
27.5
%0.
5%-
3.5%
SIT
is ha
ndy
to u
se in
da
ily ro
utin
e47
.1%
42.7
%1.
8%0.
2%8.
1%
It is
eas
y to
rem
embe
r to
take
SIT
50.8
%37
.0%
2.8%
0.2%
9.2%
SIT
allo
ws to
bet
ter
cont
rol y
our a
llerg
y56
.0%
35.2
%0.
7%5.
1%3.
0%
1.0%
Did
not
spe
cifiy
Poss
ible
ans
wer
st
Patie
nan
swer
sR
ecov
ery
from
alle
rgy
38.2
%Im
prov
emen
t in
heal
th re
late
d qu
ality
of l
ife21
.4%
Sym
ptom
redu
ctio
n22
.6%
Less
nee
d of
ant
i-alle
rgy
med
icatio
n10
.5%
Avoi
d wo
rsen
ing
of th
e di
seas
e6.
4%
Tabl
e 3:
Pat
ient
s ex
pect
atio
n ab
out S
IT
In re
latio
n to
this
pre
sent
atio
n, I
decl
are
the
follo
win
g, re
al o
r per
ceiv
ed c
onfli
cts
of in
tere
st:
the
pres
ente
r has
rece
ived
a tr
avel
gra
nt fr
om H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
:
®®
know
ledg
e
perc
eive
d
expe
cted
satis
fact
ion
I. B
aiar
dini
1 , F.
Pug
gion
i2 , S.
Men
oni3 ,
J.D
. Boo
t4 , Z.
Dia
man
t5 , F.
Bra
ido1 ,
G.W
. Can
onic
a1
In re
latio
n to
this
pre
sent
atio
n, I
decl
are
the
follo
win
g, re
al o
r per
ceiv
ed c
onfli
cts
of in
tere
st: t
he p
rese
nter
has
rece
ived
a tr
avel
gra
nt fr
om H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
In f l u e n ce o f c o - m o r b i d i t i e s a n d c o - m e d ic a t i o n s o n s a f e t y a n d t o l e ra b il i t y in t h e b u il d u p p h a s e o f v e n o m im mu n o t h e rap y
12
A. Bauer, K. Bernkopf, P. Spornraft-Ragaller.
Department of Dermatology, University Hospital Carl Gustav Carus, TU Dresden, Germany.
BackgroundSafety and tolerability of specific venom immunotherapy (VIT) may be influenced by co-morbidities and co-medications. In the current study safety and tolerability of a 3 day ultra-rush protocol in patients with co-morbidities and co-medications was assessed during the build-up phase.
MethodsSafety and tolerability of VIT (wasp venom n=114; bee venom n=11) was analysed in patients with co-morbidities and co-medications. Starting with 0.02 µg venom, the maintenance dose of 100µg was reached in 15 dose increments within 3 days. Local and systemic reactions were documented during the build-up phase.
Results124 patients (67 female/57 male, 51 years range 18-81 years) with an established history of venom allergy were included. 55 (44%) patients suffered from cardiovascular co-morbidities, 22 (17.7%) from metabolic disorders, 24 (19.3%) from skin or respiratory atopy and 18 (14.5%) showed increased baseline tryptase levels >11.4 µg/l. During the build-up phase of VIT local reactions were seen in 46 (37.1%) patients and large local reactions in 69 (55.6%) patients. No significant differences were seen between the groups. Systemic reactions (SR) grade I-III were seen in 10 (8,1%) patients (SR I: 5 (4%) patients, SR II: 4 (3.2%), SR III: 1 (0.8%)). No SR grade IV were seen. Multivariate logistic regression analysis revealed ACE-inhibitor medication (OR 16.14; 95%CI 2.5-104.27), female sex (OR 9.17; 95%CI 1.34-62.97), increased baseline serum tryptase levels (OR 6.68; 95%CI 1.04-43.01) and therapy with bee venom (OR 30.99; 95%CI 3.18-302.34) to be relevant risk factor for SR during the buildup phase of VIT.
ConclusionSafety and tolerability of ultra rush VIT was not influenced by cardiovascular, metabolic or atopic co-morbidities. However, female sex, bee venom sensitization, increased baseline serum tryptase levels and medication with ACE-inhibitors were significantly associated with an increased risk to develop SR during the build-up phase of VIT.
EAACI, 16-20 June 2012, GenevaAbstract number: 1087, Session date and time: Monday 18 June; 12:15 - 13:30 Session title: Poster 46 - Advances in immunotherapy: latest studies
13
Poster Session 46 - Advances in immunotherapy: latest studies
In f l u e n ce o f c o - m o r b i d i t i e s a n d c o - m e d ic a t i o n s o n s a f e t y a n d t o l e ra b il i t y in t h e b u il d u p p h a s e o f v e n o m im mu n o t h e rap y
D e sign o f a d o s e ra nge f in d ing s tu d y w i t h a l l e rge n s p e c i f ic im mu n o t h e rap y in p a t i e nt s w i t h h o u s e d u s t mi t e in d u ce d a l l e rgic r h ini t is / r h in o c o n j u n c t iv i t is
14
C. Bachert (1), O. Pfaar (2), A. Roger (3), H. Riechelmann (4), J.N.G. Oude Elberink (5), M.J. Nell (6), J.D. Boot (6).
(1) UZ Gent, Ear-, Nose and Throat Department, Gent, Belgium, (2) Center for Rhinology and Allergology Wiesbaden, Germany, (3) Unitat
d’Allèrgia, Hospital Universitari Germans Trias i Pujol, Badalona, Spain, (4) Universitätsklinik für Hals-, Nasen- und Ohrenheilkunde, Innsbruck,
Austria, (5) Dept of Allergology, University Medical Center Groningen, University of Groningen, The Netherlands, (6) HAL Allergy BV, Leiden,
The Netherlands.
BackgroundIn order to comply with the 2008 EMA guidelines on the development of specific immunotherapy (SIT) products, a clinical development program was started to obtain full marketing authorization for a subcutaneous SIT allergoid for the treatment of house dust mite (HDM) allergy. Safety and tolerability of increasing doses have previously been determined in an open-label clinical study. The next step is a dose range finding (DRF) study to identify the optimal, i.e. safe and effective dose in patients with HDM induced allergic rhinitis/rhinoconjunctivitis (AR). As there is a wide variety of study designs in terms of inclusion criteria, doses, study duration, end-points, analysis of data, and control of environmental variables in the evalu-ation of SIT products, this abstract highlights the design and rationale of the DRF study.
MethodThe current study is a 1 year, multicenter, randomized, double-blind, placebo-controlled, 5 arm parallel-group, DRF study (ClinicalTrials.gov NCT01438463). Approximately 250 patients (50 per arm) with persistent, symptomatic AR with or wit-hout concomitant asthma related to HDM will be randomized. Due to the need of at least 4 active study arms in a DRF study, the use of a classical endpoint for SIT (i.e. the symptom & medication score) as primary parameter was not feasible because of the necessity of a large sample size. We therefore selected the titrated nasal provocation test (TNPT) with HDM allergen as the primary parameter. The TNPT is a reproducible exacerbation model of allergic rhinitis often applied to evaluate the efficacy of anti-allergy medications. The allergen-induced upper airway response will be quantified by recording symptom scores (Lebel-Score) and nasal flow (Peak Nasal Inspiratory Flow) after each HDM challenge. To minimize seasonal influ-ences the absolute difference in mean symptom score in the TNPT after one year of treatment and baseline will be used as primary endpoint. Patients will only be subjected to a TNPT in the absence of baseline symptoms and patients with seasonal allergies will only be challenged outside the corresponding pollen season. The study is performed in 40 clinical study centers in 5 European countries.
ResultsThe first patient was recruited in October 2011 and results will be available in 2013.
ConclusionAccording to the EMA guidelines we designed a Phase II DRF study to identify the optimal dose of a subcutaneous SIT allergoid for patients with HDM induced AR.
EAACI, 16-20 June 2012, GenevaAbstract number: 1393, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 68 - New clinical studies in immunotherapy
15
Poster Session 68 - New clinical studies in immunotherapy
D e sign o f a d o s e ra nge f in d ing s tu d y w i t h a l l e rge n s p e c i f ic im mu n o t h e rap y in p a t i e nt s w i t h h o u s e d u s t mi t e in d u ce d a l l e rgic r h ini t is / r h in o c o n j u n c t iv i t is
1 UZ
Gen
t, Ea
r-, N
ose
and
Thro
at D
epar
tmen
t, G
ent,
Belg
ium
, 2Ce
nter
for R
hino
logy
and
Alle
rgol
ogy
Wie
sbad
en, G
erm
any,
3Un
itat d
’Allè
rgia
, Hos
pita
l Un
iver
sita
ri G
erm
ans
Tria
s i P
ujol
, Bad
alon
a, S
pain
, 4Un
iver
sitä
tskl
inik
für H
als-
, Nas
en-u
nd O
hren
heilk
unde
, Inn
sbru
ck, A
ustr
ia, 5
Dept
of A
llerg
olog
y,
Univ
ersi
ty M
edic
al C
ente
r Gro
ning
en, U
nive
rsity
of G
roni
ngen
, The
Net
herla
nds,
6 HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
1393
-D
esig
n of
a D
ose
Ran
ge F
indi
ng s
tudy
with
alle
rgen
sp
ecifi
c im
mun
othe
rapy
in p
atie
nts
with
H
DM
indu
ced
alle
rgic
rhin
itis/
rhin
ocon
junc
tiviti
s
Bac
kgro
und
& A
im:
In o
rder
to
com
ply
with
the
200
8 EM
A gu
idel
ines
on
the
deve
lopm
ent
of s
peci
fic i
mm
unot
hera
py (
SIT)
pro
duct
s, a
cl
inic
al d
evel
opm
ent
prog
ram
was
sta
rted
to o
btai
n fu
ll m
arke
ting
auth
oriz
atio
n fo
r a s
ubcu
tane
ous
SIT
alle
rgoi
d fo
r th
e tre
atm
ent o
f hou
se d
ust m
ite (H
DM
) alle
rgy
1 . Sa
fety
and
to
lera
bility
of
in
crea
sing
do
ses
have
pr
evio
usly
be
en
dete
rmin
ed in
an
open
-labe
l clin
ical
stu
dy. T
he n
ext s
tep
is a
do
se r
ange
find
ing
(DR
F) s
tudy
to id
entif
y th
e op
timal
, i.e
. sa
fe a
nd e
ffect
ive
dose
in
patie
nts
with
HD
M i
nduc
ed
alle
rgic
rhi
nitis
/rhin
ocon
junc
tivitis
(AR
). As
the
re i
s a
wide
va
riety
of s
tudy
des
igns
in te
rms
of in
clus
ion
crite
ria, d
oses
, st
udy
dura
tion,
end
-poi
nts,
ana
lysis
of
data
, an
d co
ntro
l of
envi
ronm
enta
l var
iabl
es in
the
eva
luat
ion
of S
IT p
rodu
cts,
th
is a
bstra
ct h
ighl
ight
s th
e de
sign
and
ratio
nale
of t
he D
RF
stud
y.
Figu
re 2
: Leb
el a
nd P
NIF
scor
e sh
eet f
or th
e TN
PT o
utco
me.
Figu
re 1
: Des
ign
of th
e st
udy
Con
clus
ion:
Acco
rdin
g to
the
EM
A gu
idel
ines
we
desi
gned
a P
hase
II
DR
F st
udy
to id
entif
y th
e op
timal
dos
e of
a s
ubcu
tane
ous
SIT
alle
rgoi
d fo
r pat
ient
s wi
th H
DM
indu
ced
AR.
Met
hods
:Th
e cu
rrent
stu
dy
is a
1 y
ear,
mul
ticen
ter,
rand
omiz
ed,
doub
le-b
lind,
pla
cebo
-con
trolle
d, 5
arm
par
alle
l-gro
up, D
RF
stud
y (C
linic
alTr
ials
.gov
NC
T014
3846
3). A
ppro
ximat
ely
250
patie
nts
(50
per a
rm)
with
per
sist
ent,
sym
ptom
atic
AR
with
or
with
out
conc
omita
nt a
sthm
a re
late
d to
HD
M w
ill be
ra
ndom
ized
. In
clus
ion
crite
ria a
re p
rese
nted
in
Tabl
e 1.
Pa
tient
s we
re tr
eate
d fo
r one
yea
r with
mite
s al
lerg
oid
(HAL
Al
lerg
y, L
eide
n, T
he N
ethe
rland
s) s
ee F
igur
e 1.
Due
to th
e ne
ed o
f at l
east
4 a
ctiv
e st
udy
arm
s in
a D
RF
stud
y, th
e us
e of
a
clas
sica
l en
dpoi
nt
for
SIT
(i.e.
th
e sy
mpt
om
& m
edic
atio
n sc
ore)
as
prim
ary
para
met
er w
as n
ot f
easi
ble
beca
use
of th
e ne
cess
ity o
f a la
rge
sam
ple
size
. The
refo
re,
the
titra
ted
nasa
l pr
ovoc
atio
n te
st
(TN
PT)
with
H
DM
al
lerg
en a
s th
e pr
imar
y pa
ram
eter
was
sel
ecte
d. T
he T
NPT
is
a re
prod
ucib
le e
xace
rbat
ion
mod
el o
f alle
rgic
rhin
itis
ofte
n ap
plie
d to
eva
luat
e th
e ef
ficac
y of
ant
i-alle
rgy
med
icat
ions
. Th
e al
lerg
en-in
duce
d up
per
airw
ay
resp
onse
w
ill be
qu
antif
ied
by r
ecor
ding
sym
ptom
sco
res
(Leb
el s
core
) an
d na
sal f
low
(Pe
ak N
asal
Insp
irato
ry F
low
, PN
IF)
afte
r ea
ch
HD
M
chal
leng
e (s
ee
Figu
res
2 an
d 3)
.To
m
inim
ize
seas
onal
in
fluen
ces
the
abso
lute
di
ffere
nce
in
mea
n sy
mpt
om s
core
in th
e TN
PT a
fter o
ne y
ear o
f tre
atm
ent a
nd
base
line
will
be u
sed
as p
rimar
y en
dpoi
nt. P
atie
nts
will
only
be
su
bjec
ted
to
a TN
PT
in
the
abse
nce
of
base
line
sym
ptom
s an
d pa
tient
s wi
th s
easo
nal a
llerg
ies
will
only
be
chal
leng
ed o
utsi
de t
he c
orre
spon
ding
pol
len
seas
on.
The
stud
y is
pe
rform
ed
in
40
clin
ical
st
udy
cent
ers
in
5 Eu
rope
an c
ount
ries.
Res
ults
: Th
e fir
st p
atie
nt w
as re
crui
ted
in O
ctob
er 2
011
and
the
last
pa
tient
was
enr
olle
d in
Mar
ch 2
012.
Res
ults
will
be a
vaila
ble
in 2
013.
Posi
tive
TNPT
for H
DM D
. pte
rext
ract
at s
cree
ning
(Leb
el s
core
6
at o
r bel
ow
10,0
00 A
U/m
l)
Seru
m s
peci
fic Ig
E-te
st (s
sIgE
) lev
el fo
r HD
M D
. pte
ror D
. far
at s
cree
ning
(>
0.7
U/m
l)
Posi
tive
SPT
to H
DM D
. pte
rand
/or D
. far
(mea
n wh
eal d
iam
eter
3m
m,
com
pare
d to
neg
ativ
e co
ntro
l and
neg
ativ
e co
ntro
l sho
uld
be n
egat
ive)
Patie
nts
with
a h
isto
ry o
f con
com
itant
ast
hma
shou
ld h
ave
a FE
V1>
70%
at
incl
usio
n. P
atie
nts
with
out a
his
tory
of a
sthm
a sh
ould
hav
e a
FEV1
> 7
0% o
r a
PEF
> 80
%
Patie
nts
with
alle
rgic
rhin
itis
or rh
inoc
onju
nctiv
itis
for a
t lea
st 1
yea
r; al
lerg
ic
sym
ptom
s re
late
d to
HDM
, with
or w
ithou
t con
com
itant
clin
ical
ly s
tabl
e co
ntro
lled
mild
to m
oder
ate
asth
ma
(acc
ordi
ng to
GIN
A cl
assi
ficat
ion)
Patie
nts
(mal
e or
fem
ale)
mus
t be
18 a
nd
60 y
ears
at s
cree
ning
Sign
ed in
form
ed c
onse
ntIn
clus
ion
crite
ria
Tabl
e 1:
Incl
usio
n cr
iteria
of t
he D
RF
stud
y.
Ref
eren
ces:
1EM
A G
uide
line
on th
e de
velo
pmen
t of p
rodu
cts
for
spec
ific im
mun
othe
rapy
for
the
treat
men
t of
al
lerg
ic
dise
ases
. C
HM
P/EW
P/18
504/
2006
, Lo
ndon
.20
N
ovem
ber 2
008.
PNIF
sco
re(h
ighe
stof
3
mea
sure
men
ts)
Tota
l LEB
EL s
core
Prur
itus
(Itch
yno
se=1
,Itc
hypa
late
and
/ore
ar=1
, Irr
itate
dey
es=1
)
Nas
alob
stru
ctio
n(D
ifficu
ltna
salb
reat
hing
=1,
One
nost
rilbl
ocke
d=2,
Bot
h no
stril
sbl
ocke
d=3)
Run
nyN
ose
(Pos
terio
r=1,
An
terio
r=1)
Snee
zing
(1-2
=0, 3
-4=1
,5
=3)
Tim
e of
mea
sure
men
t
-Ac
tual
Tim
e of
app
licat
ion
1h a
fter
last
ch.
20m
in.
afte
rpr
evio
us
20m
in.
afte
rpr
evio
us
20m
in.
afte
rdi
luen
t
--
Sche
dule
dtim
e fo
rnex
tch
alle
nge
Post
ch.
10,0
00
AU/m
l1,
000
AU/m
l10
0 AU
/ml
Post
D
iluen
tPr
e D
iluen
t(B
asel
ine)
Alle
rgen
Figu
re 3
: Per
form
ance
of a
nas
al c
halle
nge
(left)
and
PNI
F (r
ight
).
TNPT
& R
ando
mis
atio
nTN
PT12
mon
ths
10,0
00 A
Ueq/
dose
25,0
00 A
Ueq/
dose
50,0
00 A
Ueq/
dose
Plac
ebo
Scre
enin
g
3,33
3 AU
eq/d
ose
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
C. B
ache
rt1 , O
. Pfa
ar2 ,
A. R
oger
3 , H.
Rie
chel
man
n4 , J.
N.G
. Oud
e El
berin
k5 , M
.J. N
ell6 ,
J.D.
Boo
t6
1 UZ
Gen
t, Ea
r-, N
ose
and
Thro
at D
epar
tmen
t, G
ent,
Belg
ium
, 2Ce
nter
for R
hino
logy
and
Alle
rgol
ogy
Wie
sbad
en, G
erm
any,
3Un
itat d
’Allè
rgia
, Hos
pita
l Un
iver
sita
ri G
erm
ans
Tria
s i P
ujol
, Bad
alon
a, S
pain
, 4Un
iver
sitä
tskl
inik
für H
als-
, Nas
en-u
nd O
hren
heilk
unde
, Inn
sbru
ck, A
ustr
ia, 5
Dept
of A
llerg
olog
y,
Univ
ersi
ty M
edic
al C
ente
r Gro
ning
en, U
nive
rsity
of G
roni
ngen
, The
Net
herla
nds,
6 HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
1393
-D
esig
n of
a D
ose
Ran
ge F
indi
ng s
tudy
with
alle
rgen
sp
ecifi
c im
mun
othe
rapy
in p
atie
nts
with
H
DM
indu
ced
alle
rgic
rhin
itis/
rhin
ocon
junc
tiviti
s
Bac
kgro
und
& A
im:
In o
rder
to
com
ply
with
the
200
8 EM
A gu
idel
ines
on
the
deve
lopm
ent
of s
peci
fic i
mm
unot
hera
py (
SIT)
pro
duct
s, a
cl
inic
al d
evel
opm
ent
prog
ram
was
sta
rted
to o
btai
n fu
ll m
arke
ting
auth
oriz
atio
n fo
r a s
ubcu
tane
ous
SIT
alle
rgoi
d fo
r th
e tre
atm
ent o
f hou
se d
ust m
ite (H
DM
) alle
rgy
1 . Sa
fety
and
to
lera
bility
of
in
crea
sing
do
ses
have
pr
evio
usly
be
en
dete
rmin
ed in
an
open
-labe
l clin
ical
stu
dy. T
he n
ext s
tep
is a
do
se r
ange
find
ing
(DR
F) s
tudy
to id
entif
y th
e op
timal
, i.e
. sa
fe a
nd e
ffect
ive
dose
in
patie
nts
with
HD
M i
nduc
ed
alle
rgic
rhi
nitis
/rhin
ocon
junc
tivitis
(AR
). As
the
re i
s a
wide
va
riety
of s
tudy
des
igns
in te
rms
of in
clus
ion
crite
ria, d
oses
, st
udy
dura
tion,
end
-poi
nts,
ana
lysis
of
data
, an
d co
ntro
l of
envi
ronm
enta
l var
iabl
es in
the
eva
luat
ion
of S
IT p
rodu
cts,
th
is a
bstra
ct h
ighl
ight
s th
e de
sign
and
ratio
nale
of t
he D
RF
stud
y.
Figu
re 2
: Leb
el a
nd P
NIF
scor
e sh
eet f
or th
e TN
PT o
utco
me.
Figu
re 1
: Des
ign
of th
e st
udy
Con
clus
ion:
Acco
rdin
g to
the
EM
A gu
idel
ines
we
desi
gned
a P
hase
II
DR
F st
udy
to id
entif
y th
e op
timal
dos
e of
a s
ubcu
tane
ous
SIT
alle
rgoi
d fo
r pat
ient
s wi
th H
DM
indu
ced
AR.
Met
hods
:Th
e cu
rrent
stu
dy
is a
1 y
ear,
mul
ticen
ter,
rand
omiz
ed,
doub
le-b
lind,
pla
cebo
-con
trolle
d, 5
arm
par
alle
l-gro
up, D
RF
stud
y (C
linic
alTr
ials
.gov
NC
T014
3846
3). A
ppro
ximat
ely
250
patie
nts
(50
per a
rm)
with
per
sist
ent,
sym
ptom
atic
AR
with
or
with
out
conc
omita
nt a
sthm
a re
late
d to
HD
M w
ill be
ra
ndom
ized
. In
clus
ion
crite
ria a
re p
rese
nted
in
Tabl
e 1.
Pa
tient
s we
re tr
eate
d fo
r one
yea
r with
mite
s al
lerg
oid
(HAL
Al
lerg
y, L
eide
n, T
he N
ethe
rland
s) s
ee F
igur
e 1.
Due
to th
e ne
ed o
f at l
east
4 a
ctiv
e st
udy
arm
s in
a D
RF
stud
y, th
e us
e of
a
clas
sica
l en
dpoi
nt
for
SIT
(i.e.
th
e sy
mpt
om
& m
edic
atio
n sc
ore)
as
prim
ary
para
met
er w
as n
ot f
easi
ble
beca
use
of th
e ne
cess
ity o
f a la
rge
sam
ple
size
. The
refo
re,
the
titra
ted
nasa
l pr
ovoc
atio
n te
st
(TN
PT)
with
H
DM
al
lerg
en a
s th
e pr
imar
y pa
ram
eter
was
sel
ecte
d. T
he T
NPT
is
a re
prod
ucib
le e
xace
rbat
ion
mod
el o
f alle
rgic
rhin
itis
ofte
n ap
plie
d to
eva
luat
e th
e ef
ficac
y of
ant
i-alle
rgy
med
icat
ions
. Th
e al
lerg
en-in
duce
d up
per
airw
ay
resp
onse
w
ill be
qu
antif
ied
by r
ecor
ding
sym
ptom
sco
res
(Leb
el s
core
) an
d na
sal f
low
(Pe
ak N
asal
Insp
irato
ry F
low
, PN
IF)
afte
r ea
ch
HD
M
chal
leng
e (s
ee
Figu
res
2 an
d 3)
.To
m
inim
ize
seas
onal
in
fluen
ces
the
abso
lute
di
ffere
nce
in
mea
n sy
mpt
om s
core
in th
e TN
PT a
fter o
ne y
ear o
f tre
atm
ent a
nd
base
line
will
be u
sed
as p
rimar
y en
dpoi
nt. P
atie
nts
will
only
be
su
bjec
ted
to
a TN
PT
in
the
abse
nce
of
base
line
sym
ptom
s an
d pa
tient
s wi
th s
easo
nal a
llerg
ies
will
only
be
chal
leng
ed o
utsi
de t
he c
orre
spon
ding
pol
len
seas
on.
The
stud
y is
pe
rform
ed
in
40
clin
ical
st
udy
cent
ers
in
5 Eu
rope
an c
ount
ries.
Res
ults
: Th
e fir
st p
atie
nt w
as re
crui
ted
in O
ctob
er 2
011
and
the
last
pa
tient
was
enr
olle
d in
Mar
ch 2
012.
Res
ults
will
be a
vaila
ble
in 2
013.
Posi
tive
TNPT
for H
DM D
. pte
rext
ract
at s
cree
ning
(Leb
el s
core
6
at o
r bel
ow
10,0
00 A
U/m
l)
Seru
m s
peci
fic Ig
E-te
st (s
sIgE
) lev
el fo
r HD
M D
. pte
ror D
. far
at s
cree
ning
(>
0.7
U/m
l)
Posi
tive
SPT
to H
DM D
. pte
rand
/or D
. far
(mea
n wh
eal d
iam
eter
3m
m,
com
pare
d to
neg
ativ
e co
ntro
l and
neg
ativ
e co
ntro
l sho
uld
be n
egat
ive)
Patie
nts
with
a h
isto
ry o
f con
com
itant
ast
hma
shou
ld h
ave
a FE
V1>
70%
at
incl
usio
n. P
atie
nts
with
out a
his
tory
of a
sthm
a sh
ould
hav
e a
FEV1
> 7
0% o
r a
PEF
> 80
%
Patie
nts
with
alle
rgic
rhin
itis
or rh
inoc
onju
nctiv
itis
for a
t lea
st 1
yea
r; al
lerg
ic
sym
ptom
s re
late
d to
HDM
, with
or w
ithou
t con
com
itant
clin
ical
ly s
tabl
e co
ntro
lled
mild
to m
oder
ate
asth
ma
(acc
ordi
ng to
GIN
A cl
assi
ficat
ion)
Patie
nts
(mal
e or
fem
ale)
mus
t be
18 a
nd
60 y
ears
at s
cree
ning
Sign
ed in
form
ed c
onse
ntIn
clus
ion
crite
ria
Tabl
e 1:
Incl
usio
n cr
iteria
of t
he D
RF
stud
y.
Ref
eren
ces:
1EM
A G
uide
line
on th
e de
velo
pmen
t of p
rodu
cts
for
spec
ific im
mun
othe
rapy
for
the
treat
men
t of
al
lerg
ic
dise
ases
. C
HM
P/EW
P/18
504/
2006
, Lo
ndon
.20
N
ovem
ber 2
008.
PNIF
sco
re(h
ighe
stof
3
mea
sure
men
ts)
Tota
l LEB
EL s
core
Prur
itus
(Itch
yno
se=1
,Itc
hypa
late
and
/ore
ar=1
, Irr
itate
dey
es=1
)
Nas
alob
stru
ctio
n(D
ifficu
ltna
salb
reat
hing
=1,
One
nost
rilbl
ocke
d=2,
Bot
h no
stril
sbl
ocke
d=3)
Run
nyN
ose
(Pos
terio
r=1,
An
terio
r=1)
Snee
zing
(1-2
=0, 3
-4=1
,5
=3)
Tim
e of
mea
sure
men
t
-Ac
tual
Tim
e of
app
licat
ion
1h a
fter
last
ch.
20m
in.
afte
rpr
evio
us
20m
in.
afte
rpr
evio
us
20m
in.
afte
rdi
luen
t
--
Sche
dule
dtim
e fo
rnex
tch
alle
nge
Post
ch.
10,0
00
AU/m
l1,
000
AU/m
l10
0 AU
/ml
Post
D
iluen
tPr
e D
iluen
t(B
asel
ine)
Alle
rgen
Figu
re 3
: Per
form
ance
of a
nas
al c
halle
nge
(left)
and
PNI
F (r
ight
).
TNPT
& R
ando
mis
atio
nTN
PT12
mon
ths
10,0
00 A
Ueq/
dose
25,0
00 A
Ueq/
dose
50,0
00 A
Ueq/
dose
Plac
ebo
Scre
enin
g
3,33
3 AU
eq/d
ose
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
C. B
ache
rt1 , O
. Pfa
ar2 ,
A. R
oger
3 , H.
Rie
chel
man
n4 , J.
N.G
. Oud
e El
berin
k5 , M
.J. N
ell6 ,
J.D.
Boo
t6
1 UZ
Gen
t, Ea
r-, N
ose
and
Thro
at D
epar
tmen
t, G
ent,
Belg
ium
, 2Ce
nter
for R
hino
logy
and
Alle
rgol
ogy
Wie
sbad
en, G
erm
any,
3Un
itat d
’Allè
rgia
, Hos
pita
l Un
iver
sita
ri G
erm
ans
Tria
s i P
ujol
, Bad
alon
a, S
pain
, 4Un
iver
sitä
tskl
inik
für H
als-
, Nas
en-u
nd O
hren
heilk
unde
, Inn
sbru
ck, A
ustr
ia, 5
Dept
of A
llerg
olog
y,
Univ
ersi
ty M
edic
al C
ente
r Gro
ning
en, U
nive
rsity
of G
roni
ngen
, The
Net
herla
nds,
6 HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
1393
-D
esig
n of
a D
ose
Ran
ge F
indi
ng s
tudy
with
alle
rgen
sp
ecifi
c im
mun
othe
rapy
in p
atie
nts
with
H
DM
indu
ced
alle
rgic
rhin
itis/
rhin
ocon
junc
tiviti
s
Bac
kgro
und
& A
im:
In o
rder
to
com
ply
with
the
200
8 EM
A gu
idel
ines
on
the
deve
lopm
ent
of s
peci
fic i
mm
unot
hera
py (
SIT)
pro
duct
s, a
cl
inic
al d
evel
opm
ent
prog
ram
was
sta
rted
to o
btai
n fu
ll m
arke
ting
auth
oriz
atio
n fo
r a s
ubcu
tane
ous
SIT
alle
rgoi
d fo
r th
e tre
atm
ent o
f hou
se d
ust m
ite (H
DM
) alle
rgy
1 . Sa
fety
and
to
lera
bility
of
in
crea
sing
do
ses
have
pr
evio
usly
be
en
dete
rmin
ed in
an
open
-labe
l clin
ical
stu
dy. T
he n
ext s
tep
is a
do
se r
ange
find
ing
(DR
F) s
tudy
to id
entif
y th
e op
timal
, i.e
. sa
fe a
nd e
ffect
ive
dose
in
patie
nts
with
HD
M i
nduc
ed
alle
rgic
rhi
nitis
/rhin
ocon
junc
tivitis
(AR
). As
the
re i
s a
wide
va
riety
of s
tudy
des
igns
in te
rms
of in
clus
ion
crite
ria, d
oses
, st
udy
dura
tion,
end
-poi
nts,
ana
lysis
of
data
, an
d co
ntro
l of
envi
ronm
enta
l var
iabl
es in
the
eva
luat
ion
of S
IT p
rodu
cts,
th
is a
bstra
ct h
ighl
ight
s th
e de
sign
and
ratio
nale
of t
he D
RF
stud
y.
Figu
re 2
: Leb
el a
nd P
NIF
scor
e sh
eet f
or th
e TN
PT o
utco
me.
Figu
re 1
: Des
ign
of th
e st
udy
Con
clus
ion:
Acco
rdin
g to
the
EM
A gu
idel
ines
we
desi
gned
a P
hase
II
DR
F st
udy
to id
entif
y th
e op
timal
dos
e of
a s
ubcu
tane
ous
SIT
alle
rgoi
d fo
r pat
ient
s wi
th H
DM
indu
ced
AR.
Met
hods
:Th
e cu
rrent
stu
dy
is a
1 y
ear,
mul
ticen
ter,
rand
omiz
ed,
doub
le-b
lind,
pla
cebo
-con
trolle
d, 5
arm
par
alle
l-gro
up, D
RF
stud
y (C
linic
alTr
ials
.gov
NC
T014
3846
3). A
ppro
ximat
ely
250
patie
nts
(50
per a
rm)
with
per
sist
ent,
sym
ptom
atic
AR
with
or
with
out
conc
omita
nt a
sthm
a re
late
d to
HD
M w
ill be
ra
ndom
ized
. In
clus
ion
crite
ria a
re p
rese
nted
in
Tabl
e 1.
Pa
tient
s we
re tr
eate
d fo
r one
yea
r with
mite
s al
lerg
oid
(HAL
Al
lerg
y, L
eide
n, T
he N
ethe
rland
s) s
ee F
igur
e 1.
Due
to th
e ne
ed o
f at l
east
4 a
ctiv
e st
udy
arm
s in
a D
RF
stud
y, th
e us
e of
a
clas
sica
l en
dpoi
nt
for
SIT
(i.e.
th
e sy
mpt
om
& m
edic
atio
n sc
ore)
as
prim
ary
para
met
er w
as n
ot f
easi
ble
beca
use
of th
e ne
cess
ity o
f a la
rge
sam
ple
size
. The
refo
re,
the
titra
ted
nasa
l pr
ovoc
atio
n te
st
(TN
PT)
with
H
DM
al
lerg
en a
s th
e pr
imar
y pa
ram
eter
was
sel
ecte
d. T
he T
NPT
is
a re
prod
ucib
le e
xace
rbat
ion
mod
el o
f alle
rgic
rhin
itis
ofte
n ap
plie
d to
eva
luat
e th
e ef
ficac
y of
ant
i-alle
rgy
med
icat
ions
. Th
e al
lerg
en-in
duce
d up
per
airw
ay
resp
onse
w
ill be
qu
antif
ied
by r
ecor
ding
sym
ptom
sco
res
(Leb
el s
core
) an
d na
sal f
low
(Pe
ak N
asal
Insp
irato
ry F
low
, PN
IF)
afte
r ea
ch
HD
M
chal
leng
e (s
ee
Figu
res
2 an
d 3)
.To
m
inim
ize
seas
onal
in
fluen
ces
the
abso
lute
di
ffere
nce
in
mea
n sy
mpt
om s
core
in th
e TN
PT a
fter o
ne y
ear o
f tre
atm
ent a
nd
base
line
will
be u
sed
as p
rimar
y en
dpoi
nt. P
atie
nts
will
only
be
su
bjec
ted
to
a TN
PT
in
the
abse
nce
of
base
line
sym
ptom
s an
d pa
tient
s wi
th s
easo
nal a
llerg
ies
will
only
be
chal
leng
ed o
utsi
de t
he c
orre
spon
ding
pol
len
seas
on.
The
stud
y is
pe
rform
ed
in
40
clin
ical
st
udy
cent
ers
in
5 Eu
rope
an c
ount
ries.
Res
ults
: Th
e fir
st p
atie
nt w
as re
crui
ted
in O
ctob
er 2
011
and
the
last
pa
tient
was
enr
olle
d in
Mar
ch 2
012.
Res
ults
will
be a
vaila
ble
in 2
013.
Posi
tive
TNPT
for H
DM D
. pte
rext
ract
at s
cree
ning
(Leb
el s
core
6
at o
r bel
ow
10,0
00 A
U/m
l)
Seru
m s
peci
fic Ig
E-te
st (s
sIgE
) lev
el fo
r HD
M D
. pte
ror D
. far
at s
cree
ning
(>
0.7
U/m
l)
Posi
tive
SPT
to H
DM D
. pte
rand
/or D
. far
(mea
n wh
eal d
iam
eter
3m
m,
com
pare
d to
neg
ativ
e co
ntro
l and
neg
ativ
e co
ntro
l sho
uld
be n
egat
ive)
Patie
nts
with
a h
isto
ry o
f con
com
itant
ast
hma
shou
ld h
ave
a FE
V1>
70%
at
incl
usio
n. P
atie
nts
with
out a
his
tory
of a
sthm
a sh
ould
hav
e a
FEV1
> 7
0% o
r a
PEF
> 80
%
Patie
nts
with
alle
rgic
rhin
itis
or rh
inoc
onju
nctiv
itis
for a
t lea
st 1
yea
r; al
lerg
ic
sym
ptom
s re
late
d to
HDM
, with
or w
ithou
t con
com
itant
clin
ical
ly s
tabl
e co
ntro
lled
mild
to m
oder
ate
asth
ma
(acc
ordi
ng to
GIN
A cl
assi
ficat
ion)
Patie
nts
(mal
e or
fem
ale)
mus
t be
18 a
nd
60 y
ears
at s
cree
ning
Sign
ed in
form
ed c
onse
ntIn
clus
ion
crite
ria
Tabl
e 1:
Incl
usio
n cr
iteria
of t
he D
RF
stud
y.
Ref
eren
ces:
1EM
A G
uide
line
on th
e de
velo
pmen
t of p
rodu
cts
for
spec
ific im
mun
othe
rapy
for
the
treat
men
t of
al
lerg
ic
dise
ases
. C
HM
P/EW
P/18
504/
2006
, Lo
ndon
.20
N
ovem
ber 2
008.
PNIF
sco
re(h
ighe
stof
3
mea
sure
men
ts)
Tota
l LEB
EL s
core
Prur
itus
(Itch
yno
se=1
,Itc
hypa
late
and
/ore
ar=1
, Irr
itate
dey
es=1
)
Nas
alob
stru
ctio
n(D
ifficu
ltna
salb
reat
hing
=1,
One
nost
rilbl
ocke
d=2,
Bot
h no
stril
sbl
ocke
d=3)
Run
nyN
ose
(Pos
terio
r=1,
An
terio
r=1)
Snee
zing
(1-2
=0, 3
-4=1
,5
=3)
Tim
e of
mea
sure
men
t
-Ac
tual
Tim
e of
app
licat
ion
1h a
fter
last
ch.
20m
in.
afte
rpr
evio
us
20m
in.
afte
rpr
evio
us
20m
in.
afte
rdi
luen
t
--
Sche
dule
dtim
e fo
rnex
tch
alle
nge
Post
ch.
10,0
00
AU/m
l1,
000
AU/m
l10
0 AU
/ml
Post
D
iluen
tPr
e D
iluen
t(B
asel
ine)
Alle
rgen
Figu
re 3
: Per
form
ance
of a
nas
al c
halle
nge
(left)
and
PNI
F (r
ight
).
TNPT
& R
ando
mis
atio
nTN
PT12
mon
ths
10,0
00 A
Ueq/
dose
25,0
00 A
Ueq/
dose
50,0
00 A
Ueq/
dose
Plac
ebo
Scre
enin
g
3,33
3 AU
eq/d
ose
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
C. B
ache
rt1 , O
. Pfa
ar2 ,
A. R
oger
3 , H.
Rie
chel
man
n4 , J.
N.G
. Oud
e El
berin
k5 , M
.J. N
ell6 ,
J.D.
Boo
t6
EAAC
I Con
gres
s 20
12In
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er is
an
empl
oyee
of H
AL A
llerg
y.
S a f e t y o f p r e - a n d c o s ea s o n a l s t ar t o f su b l ing u a l im mu n o t h e rap y tr ea t m e nt in p a t i e nt s w i t h p o l l e n a l l e rg y
16
A. Distler (1), H. Kietzmann (2), H. Brüning (2), A. Roger (3), N. Angelova (4), J.D. Boot (4).
(1) HAL Allergie GmbH, Medical Department, Düsseldorf, Germany, (2) CutiCon, Kiel, Germany, (3) Allergy Unit University Hospital Germans
Trias Pujol. Badalona, Spain, (4) HAL Allergy BV, Leiden, Netherlands.
BackgroundStart of treatment with sublingual allergen immunotherapy (SLIT) during the relevant pollen season (co-seasonal) may result in more and/or worse adverse events (AEs) compared to the start outside the pollen season (pre-seasonal). A post marketing surveillance study in adults and children treated with SLIT pollen was performed to evaluate safety and clinical efficacy during routine application at home.
MethodsSubjects with allergic rhinoconjunctivitis with or without mild asthma due to pollen were treated with SLIT Pollen for approximately 1 year, according to onsite routine. Patients started with one sublingual drop and add one drop each consecutive day until on day 5 the maintenance dose is reached. At each visit, the number of daily drops and AEs were recorded. The patients filled in a dairy during the first 30 treatment days to monitor AEs. The treatment’s efficacy was evaluated after 1 year. The start of the respective pollen season was based on local pollen counts in Germany and Spain.
Results129 pts (88 female, mean age 38 years) were included in the study: 43.4% received grasses, 8.5% birch, and 47.3% received a combination or other pollen antigens. In total 40.3% of the patients started SLIT co- and 59.7% pre-seasonally. 89.1% of patients reached maintenance dose within 5 days (88.5% co- vs 89.6% pre-seasonal). During up-dosing 37.9% of the patients (38.4% co- vs 37.6% pre-seasonal) experienced a total of 82 adverse drug reactions. 60 local reactions (most common were allergic reactions in the mouth and abdominal pain) and 22 systemic reactions (most common were pruritus of eyes and ears). No difference in the number and severity of adverse drug reactions was observed between the two groups. Patients’ assessment of their own situation improved in 74.4%, remained equal in 11.6% and was unknown in 13.9% of the patients. No patient reported a worsening of their situation. The satisfaction of patients about the use of SLIT pollen was (very) satisfied in 75.2%, (very) unsatisfied in 11.6% and unknown in 12.4% of the patients.
ConclusionCo-seasonal start of treatment was found to be as safe as the pre-seasonal start of treatment with SLIT pollen. This is practical since it is difficult to up-dose before the season due to lengthening of the pollen season, new pollen allergens and a high number of patients with multiple pollen allergies. Subjects were generally satisfied with their treatment.
EAACI, 16-20 June 2012, GenevaAbstract number: 1394, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 68 - New clinical studies in immunotherapy
17
Poster Session 68 - New clinical studies in immunotherapy
S a f e t y o f p r e - a n d c o s ea s o n a l s t ar t o f su b l ing u a l im mu n o t h e rap y tr ea t m e nt in p a t i e nt s w i t h p o l l e n a l l e rg y
1394
-Sa
fety
of p
re-a
nd c
o-se
ason
al s
tart
of s
ublin
gual
im
mun
othe
rapy
trea
tmen
t in
patie
nts
with
pol
len
alle
rgy
, B
adal
ona,
Spa
in; 4 H
AL A
llerg
y B
VLe
iden
, Net
herla
nds
Bac
kgro
und
& A
im:
Diff
eren
t up
dosi
ngsc
hedu
les
for
subl
ingu
al
alle
rgen
im
mun
othe
rapy
(SL
IT)
exis
t. St
art
of t
reat
men
t du
ring
the
rele
vant
pol
len
seas
on (
co-s
easo
nal)
may
res
ult
in m
ore
and/
or w
orse
adv
erse
reac
tions
com
pare
d to
a s
tart
outs
ide
the
polle
n se
ason
(pre
-sea
sona
l). T
o in
vest
igat
e th
is, a
non
in
terv
entio
nal s
tudy
in a
dults
and
chi
ldre
n tre
ated
with
SLI
T po
llen
was
per
form
ed to
eva
luat
e sa
fety
and
clin
ical
effi
cacy
du
ring
rout
ine
appl
icat
ion.
Met
hods
:Th
is s
tudy
was
per
form
ed in
18
clin
ical
cen
tres
in G
erm
any
and
Spai
n.
Subj
ects
w
ith
a hi
stor
y of
al
lerg
ic
rhin
ocon
junc
tiviti
sw
ith o
r w
ithou
t mild
con
com
itant
ast
hma
due
to p
olle
n w
ere
treat
ed w
ith S
LIT
polle
n (S
UBL
IVAC
®
HAL
Al
lerg
y BV
, Le
iden
, Th
e N
ethe
rland
s)fo
r ap
prox
imat
ely
1 ye
ar, a
ccor
ding
to o
nsite
rou
tine.
Pat
ient
s (p
ts)
star
ted
with
one
sub
lingu
al d
rop
and
adde
d on
e dr
op
each
con
secu
tive
day
until
on
day
5 th
e m
aint
enan
ce d
ose
was
rea
ched
. Tr
eatm
ent
was
con
tinue
d du
ring
the
polle
n se
ason
. At
eac
h vi
sit,
the
num
ber
of d
aily
dro
ps a
nd A
Esw
ere
reco
rded
. The
pat
ient
s fil
led
in a
dia
ry d
urin
g th
e fir
st
30 tr
eatm
ent d
ays
to m
onito
r AEs
. Afte
r 1 y
ear o
f tre
atm
ent
patie
nts
scor
ed th
eir a
llerg
y co
mpl
aint
s co
mpa
red
to b
efor
e tre
atm
ent.
The
star
t of
the
res
pect
ive
polle
n se
ason
was
ba
sed
on lo
cal p
olle
n co
unts
in G
erm
any
and
Spai
n. B
ased
on
this
dat
a pa
tient
s w
ere
clas
sifie
d as
co-
or p
re-s
easo
nal.
Con
clus
ion:
Co-
seas
onal
sta
rt of
trea
tmen
t was
foun
d to
be
as s
afe
as
the
pre-
seas
onal
sta
rt of
trea
tmen
t with
SLI
T po
llen.
Thi
s is
pr
actic
al s
ince
it is
diff
icul
t to
up-
dose
bef
ore
the
seas
on
due
to
leng
then
ing
of
the
polle
n se
ason
, ne
w
polle
n al
lerg
ens
and
a hi
gh n
umbe
r of p
atie
nts
with
mul
tiple
pol
len
alle
rgie
s.
Subj
ects
w
ere
gene
rally
sa
tisfie
d w
ith
thei
r tre
atm
ent.
Res
ults
: 12
9 pt
s w
ere
incl
uded
in th
e st
udy
(Tab
le 1
). At
the
time
of
the
stud
y al
l pa
tient
s w
ere
only
pre
scrib
ed o
ne t
ype
of
polle
n SL
IT. T
he a
llerg
ens
are
liste
d in
Tab
le 1
.In
tot
al 4
0.3%
of
the
patie
nts
star
ted
SLIT
in t
he s
easo
n (c
o-se
ason
al)
and
59.7
%
outs
ide
(pre
-sea
sona
l) th
e se
ason
. 89
.1%
of
pa
tient
s re
ache
d m
aint
enan
ce d
ose
with
in 5
day
s (8
8.5%
co-
vs. 8
9.6%
pre
-sea
sona
l).
Dur
ing
up-d
osin
g a
tota
l of 8
2 ad
vers
e re
actio
ns in
49
pts
wer
e re
porte
d; 6
0 lo
cal
reac
tions
(m
ost
com
mon
wer
e al
lerg
ic re
actio
ns in
the
mou
th a
nd a
bdom
inal
pai
n) a
nd 2
2 sy
stem
ic r
eact
ions
(m
ost
com
mon
wer
e pr
uritu
sof
eye
s an
d ea
rs).
No
diffe
renc
es i
n th
e nu
mbe
r an
d se
verit
y of
ad
vers
e dr
ug r
eact
ions
wer
e ob
serv
ed b
etw
een
the
two
grou
ps (T
able
2 a
nd 3
). Al
mos
t all
patie
nts
repo
rted
an im
prov
emen
t and
no
patie
nt
repo
rted
a w
orse
ning
of t
heir
alle
rgic
com
plai
nts
(Fig
ure
1).
No
diffe
renc
e w
as s
een
betw
een
the
co-a
nd p
re-s
easo
nal
grou
ps. P
atie
nts
wer
e ge
nera
lly s
atis
fied
abou
t the
use
of
SLIT
po
llen:
34
.1%
w
ere
very
sa
tisfie
d,
41.1
%
wer
e sa
tisfie
d,
9.3%
w
ere
unsa
tisfie
d,
2.3%
w
ere
very
un
satis
fied
and
13.2
% w
ere
unkn
own.
Co-
seas
onal
Pre-
seas
onal
Tota
l(n
= 1
29)
Gen
der (
M /
F)20
/ 33
20 /
5640
/ 89
Mea
n ag
e ±
SD in
yea
rs36
.3 ±
15.3
38.3
±15
.337
.8 ±
15.2
Adol
esce
nts
75
12
Pres
crib
ed a
llerg
ens
-Gra
sses
-Tre
es-P
arie
taria
sp.
-Ole
aeu
rope
a
53 20 21 11 1
76 37 33 5 1
129 57
54 16
2
Tabl
e 1:
Pat
ient
dem
ogra
phic
s an
d ad
min
iste
red
alle
rgen
s
Tabl
e 3:
Ove
rvie
w o
f mos
t fre
quen
t adv
erse
reac
tions
Figu
re 1
: Pat
ient
s’as
sess
men
t of i
mpr
ovem
ent i
n th
e co
-(le
ft) a
nd p
re-
seas
onal
(rig
ht) g
roup
.
Tabl
e 2:
Occ
urre
nce
of a
dver
se re
actio
ns
Co-
seas
onal
Pre-
seas
onal
Tota
l
Patie
nts
with
adv
erse
reac
tions
38.4
%37
.6%
37.9
%
Patie
nts
with
loca
l rea
ctio
ns34
%34
.2%
34.1
%
Patie
nts
with
sys
tem
ic re
actio
ns9.
4%13
.2%
11.6
%
33%
53%14
%0%
35%
53%12
%0%
Muc
h im
prov
edIm
prov
edEq
ual
Wor
sene
d
Med
DR
APr
efer
red
Term
Num
ber o
f adv
erse
re
actio
nsN
umbe
r of
patie
nts
[%]
Swol
len
tong
ue
Ora
lpru
ritus
Abdo
min
alpa
in
Burn
ing
sens
atio
n
Para
esth
esia
oral
Lip
swel
ling
Ear p
rurit
us
Glo
ssod
ynia
Ora
ldisc
omfo
rt
Thro
atirr
itatio
n
Dys
peps
ia
Gin
giva
lpru
ritus
Lip
prur
itus
Prur
itus
Swel
ling
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
alor
perc
eive
dco
nflic
tsof
inte
rest
: th
e pr
esen
ter i
s an
empl
oyee
of H
AL A
llerg
yEA
ACI C
ongr
ess
2012
A. D
istle
r1 , H.
Kie
tzm
ann2 ,
H. B
rüni
ng2 ,
A. R
oger
3 , N.
Ang
elov
a4 , J.
D. B
oot4
Düs
seld
orf,
Ger
man
y; 2 C
utiC
on, K
iel,
Ger
man
y; 3 A
llerg
y Un
it Un
iver
sity
Hos
pita
l Ger
man
s Tr
ias
Pujo
l. 1 H
AL A
llerg
ieG
mbH
,
1394
-Sa
fety
of p
re-a
nd c
o-se
ason
al s
tart
of s
ublin
gual
im
mun
othe
rapy
trea
tmen
t in
patie
nts
with
pol
len
alle
rgy
, Ba
dalo
na, S
pain
; 4 HAL
Alle
rgy
BVLe
iden
, Net
herla
nds
Bac
kgro
und
& A
im:
Diff
eren
t up
dosi
ngsc
hedu
les
for
subl
ingu
al
alle
rgen
im
mun
othe
rapy
(SL
IT)
exis
t. St
art
of t
reat
men
t du
ring
the
rele
vant
pol
len
seas
on (
co-s
easo
nal)
may
res
ult
in m
ore
and/
or w
orse
adv
erse
reac
tions
com
pare
d to
a s
tart
outs
ide
the
polle
n se
ason
(pre
-sea
sona
l). T
o in
vest
igat
e th
is, a
non
in
terv
entio
nal s
tudy
in a
dults
and
chi
ldre
n tre
ated
with
SLI
T po
llen
was
perfo
rmed
to e
valu
ate
safe
ty a
nd c
linic
al e
ffica
cy
durin
g ro
utin
e ap
plic
atio
n.
Met
hods
:Th
is s
tudy
was
per
form
ed in
18
clin
ical
cen
tres
in G
erm
any
and
Spai
n.
Subj
ects
w
ith
a hi
stor
y of
al
lerg
ic
rhin
ocon
junc
tivitis
with
or
with
out m
ild c
onco
mita
nt a
sthm
a du
e to
pol
len
were
tre
ated
with
SLI
T po
llen
(SU
BLIV
AC®
HAL
Al
lerg
y BV
, Le
iden
, Th
e N
ethe
rland
s)fo
r ap
prox
imat
ely
1 ye
ar, a
ccor
ding
to o
nsite
rou
tine.
Pat
ient
s (p
ts) s
tarte
d w
ith o
ne s
ublin
gual
dro
p an
d ad
ded
one
drop
ea
ch c
onse
cutiv
e da
y un
til o
n da
y 5
the
mai
nten
ance
dos
e wa
s re
ache
d. T
reat
men
t wa
s co
ntin
ued
durin
g th
e po
llen
seas
on.
At e
ach
visi
t, th
e nu
mbe
r of
dai
ly d
rops
and
AEs
were
reco
rded
. The
pat
ient
s fil
led
in a
dia
ry d
urin
g th
e fir
st
30 tr
eatm
ent d
ays
to m
onito
r AEs
. Afte
r 1 y
ear o
f tre
atm
ent
patie
nts
scor
ed th
eir a
llerg
y co
mpl
aint
s co
mpa
red
to b
efor
e tre
atm
ent.
The
star
t of
the
res
pect
ive
polle
n se
ason
was
ba
sed
on lo
cal p
olle
n co
unts
in G
erm
any
and
Spai
n. B
ased
on
this
dat
a pa
tient
s w
ere
clas
sifie
d as
co-
or p
re-s
easo
nal.
Con
clus
ion:
Co-
seas
onal
sta
rt of
trea
tmen
t was
foun
d to
be
as s
afe
as
the
pre-
seas
onal
sta
rt of
trea
tmen
t with
SLI
T po
llen.
Thi
s is
pr
actic
al s
ince
it is
diff
icul
t to
up-
dose
bef
ore
the
seas
on
due
to
leng
then
ing
of
the
polle
n se
ason
, ne
w po
llen
alle
rgen
s an
d a
high
num
ber o
f pat
ient
s wi
th m
ultip
le p
olle
n al
lerg
ies.
Su
bjec
ts
were
ge
nera
lly
satis
fied
with
th
eir
treat
men
t.
Res
ults
: 12
9 pt
s we
re in
clud
ed in
the
stud
y (T
able
1).
At th
e tim
e of
th
e st
udy
all
patie
nts
wer
e on
ly p
resc
ribed
one
typ
e of
po
llen
SLIT
. The
alle
rgen
s ar
e lis
ted
in T
able
1.
In t
otal
40.
3% o
f th
e pa
tient
s st
arte
d SL
IT in
the
sea
son
(co-
seas
onal
) an
d 59
.7%
ou
tsid
e (p
re-s
easo
nal)
the
seas
on.
89.1
% o
f pa
tient
s re
ache
d m
aint
enan
ce d
ose
with
in 5
day
s (8
8.5%
co-
vs. 8
9.6%
pre
-sea
sona
l).
Dur
ing
up-d
osin
g a
tota
l of 8
2 ad
vers
e re
actio
ns in
49
pts
were
rep
orte
d; 6
0 lo
cal
reac
tions
(m
ost
com
mon
wer
e al
lerg
ic re
actio
ns in
the
mou
th a
nd a
bdom
inal
pai
n) a
nd 2
2 sy
stem
ic r
eact
ions
(m
ost
com
mon
wer
e pr
uritu
sof
eye
s an
d ea
rs).
No
diffe
renc
es i
n th
e nu
mbe
r an
d se
verit
y of
ad
vers
e dr
ug r
eact
ions
wer
e ob
serv
ed b
etw
een
the
two
grou
ps (T
able
2 a
nd 3
). Al
mos
t all
patie
nts
repo
rted
an im
prov
emen
t and
no
patie
nt
repo
rted
a w
orse
ning
of t
heir
alle
rgic
com
plai
nts
(Fig
ure
1).
No
diffe
renc
e w
as s
een
betw
een
the
co-a
nd p
re-s
easo
nal
grou
ps. P
atie
nts
were
gen
eral
ly s
atis
fied
abou
t the
use
of
SLIT
po
llen:
34
.1%
we
re
very
sa
tisfie
d,
41.1
%
were
sa
tisfie
d,
9.3%
we
re
unsa
tisfie
d,
2.3%
we
re
very
un
satis
fied
and
13.2
% w
ere
unkn
own.
Co-
seas
onal
Pre-
seas
onal
Tota
l(n
= 1
29)
Gen
der (
M /
F)20
/ 33
20 /
5640
/ 89
Mea
n ag
e ±
SD in
yea
rs36
.3 ±
15.3
38.3
±15
.337
.8 ±
15.2
Adol
esce
nts
75
12
Pres
crib
ed a
llerg
ens
-Gra
sses
-Tre
es-P
arie
taria
sp.
-Ole
aeu
rope
a
53 20 21 11 1
76 37 33 5 1
129 57
54 16
2
Tabl
e 1:
Pat
ient
dem
ogra
phic
s an
d ad
min
iste
red
alle
rgen
s
Tabl
e 3:
Ove
rvie
w o
f mos
t fre
quen
t adv
erse
reac
tions
Figu
re 1
: Pat
ient
s’as
sess
men
t of i
mpr
ovem
ent i
n th
e co
-(le
ft) a
nd p
re-
seas
onal
(rig
ht) g
roup
.
Tabl
e 2:
Occ
urre
nce
of a
dver
se re
actio
ns
Co-
seas
onal
Pre-
seas
onal
Tota
l
Patie
nts
with
adv
erse
reac
tions
38.4
%37
.6%
37.9
%
Patie
nts
with
loca
l rea
ctio
ns34
%34
.2%
34.1
%
Patie
nts
with
sys
tem
ic re
actio
ns9.
4%13
.2%
11.6
%
33%
53%14
%0%
35%
53%12
%0%
Muc
h im
prov
edIm
prov
edEq
ual
Wor
sene
d
Med
DR
APr
efer
red
Term
Num
ber o
f adv
erse
re
actio
nsN
umbe
r of
patie
nts
[%]
Swol
len
tong
ue
Ora
lpru
ritus
Abdo
min
alpa
in
Burn
ing
sens
atio
n
Para
esth
esia
oral
Lip
swel
ling
Ear p
rurit
us
Glo
ssod
ynia
Ora
ldisc
omfo
rt
Thro
atirr
itatio
n
Dys
peps
ia
Gin
giva
lpru
ritus
Lip
prur
itus
Prur
itus
Swel
ling
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
wing
, rea
lorp
erce
ived
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy
EAAC
I Con
gres
s 20
12
A. D
istle
r1 , H.
Kie
tzm
ann2 ,
H. B
rüni
ng2 ,
A. R
oger
3 , N.
Ang
elov
a4 , J.
D. B
oot4
Düs
seld
orf,
Ger
man
y; 2 C
utiC
on, K
iel,
Ger
man
y; 3 A
llerg
y Un
it Un
iver
sity
Hos
pita
l Ger
man
s Tr
ias
Pujo
l. 1 H
AL A
llerg
ieG
mbH
,
1394
-Sa
fety
of p
re-a
nd c
o-se
ason
al s
tart
of s
ublin
gual
im
mun
othe
rapy
trea
tmen
t in
patie
nts
with
pol
len
alle
rgy
, B
adal
ona,
Spa
in; 4 H
AL A
llerg
y B
VLe
iden
, Net
herla
nds
Bac
kgro
und
& A
im:
Diff
eren
t up
dosi
ngsc
hedu
les
for
subl
ingu
al
alle
rgen
im
mun
othe
rapy
(SL
IT)
exis
t. St
art
of t
reat
men
t du
ring
the
rele
vant
pol
len
seas
on (
co-s
easo
nal)
may
res
ult
in m
ore
and/
or w
orse
adv
erse
reac
tions
com
pare
d to
a s
tart
outs
ide
the
polle
n se
ason
(pre
-sea
sona
l). T
o in
vest
igat
e th
is, a
non
in
terv
entio
nal s
tudy
in a
dults
and
chi
ldre
n tre
ated
with
SLI
T po
llen
was
per
form
ed to
eva
luat
e sa
fety
and
clin
ical
effi
cacy
du
ring
rout
ine
appl
icat
ion.
Met
hods
:Th
is s
tudy
was
per
form
ed in
18
clin
ical
cen
tres
in G
erm
any
and
Spai
n.
Subj
ects
w
ith
a hi
stor
y of
al
lerg
ic
rhin
ocon
junc
tiviti
sw
ith o
r w
ithou
t mild
con
com
itant
ast
hma
due
to p
olle
n w
ere
treat
ed w
ith S
LIT
polle
n (S
UBL
IVAC
®
HAL
Al
lerg
y BV
, Le
iden
, Th
e N
ethe
rland
s)fo
r ap
prox
imat
ely
1 ye
ar, a
ccor
ding
to o
nsite
rou
tine.
Pat
ient
s (p
ts)
star
ted
with
one
sub
lingu
al d
rop
and
adde
d on
e dr
op
each
con
secu
tive
day
until
on
day
5 th
e m
aint
enan
ce d
ose
was
rea
ched
. Tr
eatm
ent
was
con
tinue
d du
ring
the
polle
n se
ason
. At
eac
h vi
sit,
the
num
ber
of d
aily
dro
ps a
nd A
Esw
ere
reco
rded
. The
pat
ient
s fil
led
in a
dia
ry d
urin
g th
e fir
st
30 tr
eatm
ent d
ays
to m
onito
r AEs
. Afte
r 1 y
ear o
f tre
atm
ent
patie
nts
scor
ed th
eir a
llerg
y co
mpl
aint
s co
mpa
red
to b
efor
e tre
atm
ent.
The
star
t of
the
res
pect
ive
polle
n se
ason
was
ba
sed
on lo
cal p
olle
n co
unts
in G
erm
any
and
Spai
n. B
ased
on
this
dat
a pa
tient
s w
ere
clas
sifie
d as
co-
or p
re-s
easo
nal.
Con
clus
ion:
Co-
seas
onal
sta
rt of
trea
tmen
t was
foun
d to
be
as s
afe
as
the
pre-
seas
onal
sta
rt of
trea
tmen
t with
SLI
T po
llen.
Thi
s is
pr
actic
al s
ince
it is
diff
icul
t to
up-
dose
bef
ore
the
seas
on
due
to
leng
then
ing
of
the
polle
n se
ason
, ne
w
polle
n al
lerg
ens
and
a hi
gh n
umbe
r of p
atie
nts
with
mul
tiple
pol
len
alle
rgie
s.
Subj
ects
w
ere
gene
rally
sa
tisfie
d w
ith
thei
r tre
atm
ent.
Res
ults
: 12
9 pt
s w
ere
incl
uded
in th
e st
udy
(Tab
le 1
). At
the
time
of
the
stud
y al
l pa
tient
s w
ere
only
pre
scrib
ed o
ne t
ype
of
polle
n SL
IT. T
he a
llerg
ens
are
liste
d in
Tab
le 1
.In
tot
al 4
0.3%
of
the
patie
nts
star
ted
SLIT
in t
he s
easo
n (c
o-se
ason
al)
and
59.7
%
outs
ide
(pre
-sea
sona
l) th
e se
ason
. 89
.1%
of
pa
tient
s re
ache
d m
aint
enan
ce d
ose
with
in 5
day
s (8
8.5%
co-
vs. 8
9.6%
pre
-sea
sona
l).
Dur
ing
up-d
osin
g a
tota
l of 8
2 ad
vers
e re
actio
ns in
49
pts
wer
e re
porte
d; 6
0 lo
cal
reac
tions
(m
ost
com
mon
wer
e al
lerg
ic re
actio
ns in
the
mou
th a
nd a
bdom
inal
pai
n) a
nd 2
2 sy
stem
ic r
eact
ions
(m
ost
com
mon
wer
e pr
uritu
sof
eye
s an
d ea
rs).
No
diffe
renc
es i
n th
e nu
mbe
r an
d se
verit
y of
ad
vers
e dr
ug r
eact
ions
wer
e ob
serv
ed b
etw
een
the
two
grou
ps (T
able
2 a
nd 3
). Al
mos
t all
patie
nts
repo
rted
an im
prov
emen
t and
no
patie
nt
repo
rted
a w
orse
ning
of t
heir
alle
rgic
com
plai
nts
(Fig
ure
1).
No
diffe
renc
e w
as s
een
betw
een
the
co-a
nd p
re-s
easo
nal
grou
ps. P
atie
nts
wer
e ge
nera
lly s
atis
fied
abou
t the
use
of
SLIT
po
llen:
34
.1%
w
ere
very
sa
tisfie
d,
41.1
%
wer
e sa
tisfie
d,
9.3%
w
ere
unsa
tisfie
d,
2.3%
w
ere
very
un
satis
fied
and
13.2
% w
ere
unkn
own.
Co-
seas
onal
Pre-
seas
onal
Tota
l(n
= 1
29)
Gen
der (
M /
F)20
/ 33
20 /
5640
/ 89
Mea
n ag
e ±
SD in
yea
rs36
.3 ±
15.3
38.3
±15
.337
.8 ±
15.2
Adol
esce
nts
75
12
Pres
crib
ed a
llerg
ens
-Gra
sses
-Tre
es-P
arie
taria
sp.
-Ole
aeu
rope
a
53 20 21 11 1
76 37 33 5 1
129 57
54 16
2
Tabl
e 1:
Pat
ient
dem
ogra
phic
s an
d ad
min
iste
red
alle
rgen
s
Tabl
e 3:
Ove
rvie
w o
f mos
t fre
quen
t adv
erse
reac
tions
Figu
re 1
: Pat
ient
s’as
sess
men
t of i
mpr
ovem
ent i
n th
e co
-(le
ft) a
nd p
re-
seas
onal
(rig
ht) g
roup
.
Tabl
e 2:
Occ
urre
nce
of a
dver
se re
actio
ns
Co-
seas
onal
Pre-
seas
onal
Tota
l
Patie
nts
with
adv
erse
reac
tions
38.4
%37
.6%
37.9
%
Patie
nts
with
loca
l rea
ctio
ns34
%34
.2%
34.1
%
Patie
nts
with
sys
tem
ic re
actio
ns9.
4%13
.2%
11.6
%
33%
53%14
%0%
35%
53%12
%0%
Muc
h im
prov
edIm
prov
edEq
ual
Wor
sene
d
Med
DR
APr
efer
red
Term
Num
ber o
f adv
erse
re
actio
nsN
umbe
r of
patie
nts
[%]
Swol
len
tong
ue
Ora
lpru
ritus
Abdo
min
alpa
in
Burn
ing
sens
atio
n
Para
esth
esia
oral
Lip
swel
ling
Ear p
rurit
us
Glo
ssod
ynia
Ora
ldisc
omfo
rt
Thro
atirr
itatio
n
Dys
peps
ia
Gin
giva
lpru
ritus
Lip
prur
itus
Prur
itus
Swel
ling
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
alor
perc
eive
dco
nflic
tsof
inte
rest
: th
e pr
esen
ter i
s an
empl
oyee
of H
AL A
llerg
yEA
ACI C
ongr
ess
2012
A. D
istle
r1 , H.
Kie
tzm
ann2 ,
H. B
rüni
ng2 ,
A. R
oger
3 , N.
Ang
elov
a4 , J.
D. B
oot4
Düs
seld
orf,
Ger
man
y; 2 C
utiC
on, K
iel,
Ger
man
y; 3 A
llerg
y Un
it Un
iver
sity
Hos
pita
l Ger
man
s Tr
ias
Pujo
l. 1 H
AL A
llerg
ieG
mbH
,
EAAC
I Con
gres
s 20
12In
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er is
an
empl
oyee
of H
AL A
llerg
y.
C h e mic a l m o d i f ic a t i o n o f a p ea n u t ex tra c t d e cr ea s e s Ig E b in d ing w h il e t h e im mu n o ge nic i t y is m a int a in e d
18
H. van der Kleij (1), J. Smit (2), S.C. Dreskin (3), H. Warmenhoven (1), D. Verbart (1), E. Knol (4), A. Knulst (4), E. Kerkvliet (1),
R. van den Hout (1), R. van Ree (5), R. Pieters (2), E. van Hoffen (4), S.J. Koppelman (1).
(1) HAL Allergy BV, Leiden, The Netherlands, (2) Institute for Risk Assessment Sciences, Immunotoxicology, Utrecht University, Utrecht,
The Netherlands, (3) University of Colorado Denver School of Medicine, Aurora, CO, USA, (4) Dermatology/Allergology, University Medical
Center Utrecht, Utrecht, The Netherlands, (5) Experimental Immunology, Academic Medical Centre, Amsterdam, The Netherlands.
RationalePeanuts are one of the most common foods responsible for food-induced anaphylaxis in adults. Until now, a curative treatment is not available for peanut-allergic patients. Peanut sIT could be an option but earlier attempts with aqueous peanut extract were hampered by safety issues. Therefore, a chemically modified peanut extract with improved safety characteristics is being investigated for its potential use in immunotherapy.
MethodsA peanut extract (PE) was prepared and modified by reduction of the disulfide bonds and subsequent alkylation of the free Cys residues. The modified extract (mPE) was analysed for biochemical parameters using electrophoreses and far UV CD spectroscopy. Using a set of sera obtained from 20 US and 9 Dutch peanut allergic patients, solid-state IgE-binding assays and mediator release assays (MRA) using RBL SX38 cells and human basophils were performed. The potency ofPE and mPE to activate T-cells was evaluated using PE-specific T-cell lines from 9 peanut-allergic patients. The potency of PE and mPE to induce PE-specific IgG was evaluated in mice too. Furthermore, the in vivo safety of PE and mPE was tested by measuring body temperature upon subcutaneous challenges in mice allergic to PE.
ResultsThe IgE-binding of mPE was for all patients tested lower than for PE (mean remaining potency: 7.2% ± 5%), which was confirmed by IgE-immunoblotting. The potency of mPE in MRA was for all patients tested lower than for PE (mean reduction: 10 to100 fold, range 3 to >10,000 fold). PE-specific T-cell lines were activated equally well with mPE and PE. Mice sensitized with PE experienced a severe anaphylactic reaction upon subcutaneous challenge starting at 0.01 mg PE, while 0.3 mg mPE was tolerated without signs of an allergic reaction. PE and mPE were equally potent in inducing PE-specific IgG antibodies in mice.
ConclusionsUsing in vitro and in vivo models, we have shown that an allergoid preparation of peanut extract has a reduced allergenicity compared to its native counterpart, while the immunogenicity is maintained.
EAACI, 16-20 June 2012, GenevaAbstract number: 4, Session date and time: Sunday 17 June; 10:30 - 12:00 Session title: OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment
19
OAS 1 - Allergen immunotherapy: new aspects in diagnostics and treatment
C h e mic a l m o d i f ic a t i o n o f a p ea n u t ex tra c t d e cr ea s e s Ig E b in d ing w h il e t h e im mu n o ge nic i t y is m a int a in e d
1 HAL
Alle
rgy
BV,
Lei
den,
The
Net
herla
nds,
2 Ins
titut
e fo
r Ris
k A
sses
smen
t Sci
ence
s an
d U
trec
ht C
entr
e fo
r Foo
d Al
lerg
y, U
trec
ht,
The
Net
herla
nds,
3 Uni
vers
ity o
f Col
orad
o D
enve
r Sch
ool o
f Med
icin
e, A
uror
a, C
O, U
SA,
4 Dep
. Der
mat
olog
y/Al
lerg
olog
y, U
MC
Utr
echt
, Utr
echt
, Th
e N
ethe
rland
s, 5 E
xper
imen
tal I
mm
unol
ogy,
Aca
dem
ic M
edic
al C
entr
e, A
mst
erda
m, T
heN
ethe
rland
s
4 -C
hem
ical
mod
ifica
tion
of a
pea
nut e
xtra
ct d
ecre
ases
Ig
Ebi
ndin
g w
hile
the
imm
unog
enic
ity is
mai
ntai
ned
Bac
kgro
und
& A
im:
•Pea
nuts
are
one
of t
he m
ost c
omm
onfo
ods
resp
onsi
ble
for f
ood-
indu
ced
anap
hyla
xis
in a
dults
.•A
cur
ativ
e tre
atm
ent i
s no
t ava
ilabl
e.
•A m
odifi
ed p
eanu
t ext
ract
(alle
rgoi
d) is
bein
g in
vest
igat
ed fo
r its
pot
entia
l use
in im
mun
othe
rapy
.
Con
clus
ion:
An
alle
rgoi
dpr
epar
atio
n of
pe
anut
ex
tract
ha
s a
redu
ced
alle
rgen
icity
com
pare
d to
its
nativ
e co
unte
rpar
t, w
hile
th
e im
mun
ogen
icity
is m
aint
aine
d.
Met
hods
:Th
e Pe
anut
ext
ract
(PE)
pre
para
tion
isch
emic
ally
mod
ified
by
redu
ctio
n of
the
intra
mol
ecul
ardi
sulfi
de b
onds
of P
E,an
d al
kyla
tion
of
the
resu
lting
fre
e su
lfhyd
rylg
roup
s (R
A).
For i
mm
unog
enic
ity, t
he a
llerg
oid
was
adso
rbed
to a
lum
iniu
m h
ydro
xide
.
Res
ults
:Ef
ficac
y:•P
E-sp
ecifi
c hu
man
T-c
ell l
ines
wer
eac
tivat
ed e
qual
ly w
ell w
ith P
E an
d R
A-PE
.•I
n m
ice,
PE
and
RA-
PE w
ere
equa
llypo
tent
in in
duci
ng P
E-sp
ecifi
c Ig
Gan
tibod
ies
in m
ice.
Safe
ty:
•Mod
ifica
tion
low
ered
the
IgE-
bind
ing
inal
l pat
ient
s to
a m
ean
rem
aini
ng p
oten
cyof
7.2
%5%
. •T
he m
edia
tor r
elea
se a
ssay
(MR
A)sh
owed
a re
duce
d po
tenc
y of
10-
100
fold
(ran
ge 3
to >
10,0
00 fo
ld).
•Mic
e se
nsiti
zed
with
PE
expe
rienc
ed a
seve
re a
naph
ylac
tic re
actio
n up
onsu
bcut
aneo
us c
halle
nge
with
0.1
mg
PE, w
hile
3 m
g R
A-PE
was
stil
lto
lera
ted
with
out p
robl
ems.
Figu
re 1
: Res
pons
e of
PE-
spec
ific
TCLs
(T c
ell l
ines
) to
nativ
e PE
and
RA
-PE.
Prol
ifera
tion
was
expr
esse
d as
the
stim
ulat
ion
inde
x (S
I, i.e
., pr
olife
ratio
n to
alle
rgen
stim
ulat
ion
divi
ded
by b
lanc
).A.
Rep
rese
ntat
ive
exam
ples
of a
TC
L of
an
alle
rgic
pat
ient
. B. I
mm
unog
enic
ity o
f PE
and
RA-
PE in
9 T
CLs
.
A. Fi
gure
3: I
nhib
ition
ELI
SA
. A
. Inh
ibiti
on c
urve
s of
the
nativ
e an
d m
odifie
d PE
of a
US
seru
m
pool
of 2
0 pa
tient
s. T
est s
ampl
es c
ompe
te w
ith P
E co
ated
to E
LISA
pl
ates
for b
indi
ng to
ser
um Ig
E. In
crea
sing
con
cent
ratio
ns o
f tes
t sa
mpl
e re
sult
in in
hibi
tion
of b
indi
ng. T
he a
mou
nt o
f the
IgE
bind
ing
in p
rese
nce
of P
E co
mpa
red
to th
at in
abs
ence
of P
E is
cal
cula
ted
as
perc
enta
ge in
hibi
tion.
B. Ig
Epo
tenc
y of
RA-
CPE
rela
tive
to n
ativ
e C
PE d
eter
min
ed in
an
IgE
inhi
bitio
n EL
ISA
usin
g se
ra o
f 20
pean
ut a
llerg
ic p
atie
nts
from
the
US.
An
in-h
ouse
refe
renc
e (IH
R) a
llerg
en p
repa
ratio
n is
test
ed to
com
pare
the
inhi
bitio
n cu
rves
by
para
llel l
ine
anal
yses
. The
IgE
bind
ing
effic
ienc
y is
exp
ress
ed a
s re
lativ
e po
tenc
y (R
P) c
ompa
red
to th
e IH
R.
1,3
pool
5,9
D11
48,
8D
107
6,2
D10
512
,2D
103
8,2
D98
8,2
D81
2,8
D80
11,8
D77
4,5
D74
9,1
D70
11,3
D69
4,3
D68
0,3
D65
17,8
D64
4,1
D62
5,3
D60
1,4
D50
16,9
D48
0,4
D44
1,3
D19
Rea
lativ
epo
tenc
y (%
) Sa
mpl
eB
.
Figu
re4:
The
pot
ency
of n
ativ
ePE
and
RA
-PE
in th
e in
dire
ct M
RA
. A. R
epre
sent
ativ
eex
ampl
e.
B.R
elat
ive
pote
ncy
of n
ativ
ePE
and
RA-
PE to
act
ivat
eba
soph
ilsFigu
re 2
: Spe
cific
IgG
resp
onse
s to
nat
ive
vers
us m
odifi
ed P
E.Sp
ecific
IgG
titre
s at
day
63
afte
r 5 im
mun
izat
ions
(i.p
.) wi
th 1
0 or
100
µg
prod
uct.
Con
trol m
ice
were
inje
cted
with
mat
rix o
nly.
Dot
s re
pres
ent t
he
IgG
titre
of i
ndiv
idua
l mic
e in
arb
itrar
y un
its (A
U) a
nd b
ars
repr
esen
t the
m
edia
n tit
re o
f eac
h gr
oup.
010
2030
4050
6070
8090
100
28303234363840
0.6
mg
PE0.
1 m
g PE
3 m
g PE
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celcius)
010
2030
4050
6070
8090
100
283032343638400.
1 m
g PE
-RA
0.6
mg
PE-R
A
3 m
g PE
-RA
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celsius)
Cha
lleng
eby
subc
utan
eous
inje
ctio
n
6 m
g PE
+ 1
5 g
Cho
lera
Tox
inby
oral
gava
ge
Day
: 0
1
2
7
14
21
2
8
42
Figu
re 5
: Ani
mal
mod
el.M
ice
were
sen
sitiz
ed w
ith 6
mg
PE
and
15 µ
g C
hole
ra T
oxin
via
ora
l gav
age
and
chal
leng
ed (i
.p.)
with
PE,
PE
adso
rbed
to a
lum
, RA-
PE p
repa
ratio
n an
d R
A-PE
ad
sorb
ed to
alu
m (n
=6 p
er g
roup
).
Figu
re 6
: Bod
y te
mpe
ratu
re fo
llow
ing
i.p. c
halle
nge;
Ana
phyl
axis
to th
e na
tive
prep
arat
ion.
An i.
p. c
halle
nge
with
0.6
mg
PE p
er
mou
se re
sulte
d in
an
anap
hyla
ctic
sho
ck re
spon
se. T
he m
ice
in th
e gr
oups
chal
leng
edwi
thm
odifie
dPE
sho
wed
smal
ldec
reas
esin
te
mpe
ratu
reat
the
high
estd
ose
(3 m
g R
A-PE
). C
ontro
lmic
edi
dno
tres
pond
to th
e ch
alle
nge
(with
3 m
g of
PE
orPE
-RA)
.
blan
c50
2512
.56.
253.
131.
560255075100
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)Proliferation (SI)
blan
c50
2512
.56.
253.
131.
56050100
150
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)
Proliferation (SI)
0.010.
1110100
p<0.
01
PE
R
A-PE
relative basophil activation (%)
02550PE PE
-RA
con
0.0
3 0
.01
0.3
1
3
10
100
10
00
conc
entr
atio
n (n
g/m
l)
% CD63+ basophils
0.01
0.1
110
100
0255075100
PE PE-R
A
inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)110100
p=0.
25
PE
PE-
RA
stimulation index
con
0
100
200
300
10μ μμμ
g
100
μ μμμg
10μ μμμg
100μ μμμg
PEPE
-RA
IgG (AU)
A.
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
H. v
an d
er K
leij1 ,
J. S
mit2 ,
S.C
. Dre
skin
3 , H
. War
men
hove
n1 , D
. Ver
bart
1 , E.
Kno
l4 , A
. Knu
lst4 ,
E. K
erkv
liet1 ,
R. v
an d
en H
out1 ,
R. v
an R
ee5 ,
R. P
iete
rs2 ,
E. v
an H
offe
n4 , S.
J. K
oppe
lman
1
1 HAL
Alle
rgy
BV,
Lei
den,
The
Net
herla
nds,
2 Ins
titut
e fo
r Ris
k As
sess
men
t Sci
ence
s an
d U
trec
ht C
entr
e fo
r Foo
d Al
lerg
y, U
trec
ht,
The
Net
herla
nds,
3 Uni
vers
ity o
f Col
orad
o D
enve
r Sch
ool o
f Med
icin
e, A
uror
a, C
O, U
SA,
4 Dep
. Der
mat
olog
y/Al
lerg
olog
y, U
MC
Utr
echt
, Utr
echt
, Th
e N
ethe
rland
s, 5 E
xper
imen
tal I
mm
unol
ogy,
Aca
dem
ic M
edic
al C
entr
e, A
mst
erda
m, T
heN
ethe
rland
s
4 -C
hem
ical
mod
ifica
tion
of a
pea
nut e
xtra
ct d
ecre
ases
Ig
Ebi
ndin
g w
hile
the
imm
unog
enic
ity is
mai
ntai
ned
Bac
kgro
und
& A
im:
•Pea
nuts
are
one
of t
he m
ost c
omm
onfo
ods
resp
onsi
ble
for f
ood-
indu
ced
anap
hyla
xis
in a
dults
.•A
cur
ativ
e tre
atm
ent i
s no
t ava
ilabl
e.
•A m
odifi
ed p
eanu
t ext
ract
(alle
rgoi
d) is
bein
g in
vest
igat
ed fo
r its
pot
entia
l use
in im
mun
othe
rapy
.
Con
clus
ion:
An
alle
rgoi
dpr
epar
atio
n of
pe
anut
ex
tract
ha
s a
redu
ced
alle
rgen
icity
com
pare
d to
its
nativ
e co
unte
rpar
t, w
hile
th
e im
mun
ogen
icity
is m
aint
aine
d.
Met
hods
:Th
e Pe
anut
ext
ract
(PE)
pre
para
tion
isch
emic
ally
mod
ified
by
redu
ctio
n of
the
intra
mol
ecul
ardi
sulfi
de b
onds
of P
E,an
d al
kyla
tion
of
the
resu
lting
fre
e su
lfhyd
rylg
roup
s (R
A).
For i
mm
unog
enic
ity, t
he a
llerg
oid
was
adso
rbed
to a
lum
iniu
m h
ydro
xide
.
Res
ults
:Ef
ficac
y:•P
E-sp
ecifi
c hu
man
T-c
ell l
ines
wer
eac
tivat
ed e
qual
ly w
ell w
ith P
E an
d R
A-PE
.•I
n m
ice,
PE
and
RA-
PE w
ere
equa
llypo
tent
in in
duci
ng P
E-sp
ecifi
c Ig
Gan
tibod
ies
in m
ice.
Safe
ty:
•Mod
ifica
tion
low
ered
the
IgE-
bind
ing
inal
l pat
ient
s to
a m
ean
rem
aini
ng p
oten
cyof
7.2
%5%
. •T
he m
edia
tor r
elea
se a
ssay
(MR
A)sh
owed
a re
duce
d po
tenc
y of
10-
100
fold
(ran
ge 3
to >
10,0
00 fo
ld).
•Mic
e se
nsiti
zed
with
PE
expe
rienc
ed a
seve
re a
naph
ylac
tic re
actio
n up
onsu
bcut
aneo
us c
halle
nge
with
0.1
mg
PE, w
hile
3 m
g R
A-PE
was
stil
lto
lera
ted
with
out p
robl
ems.
Figu
re 1
: Res
pons
e of
PE-
spec
ific
TCLs
(T c
ell l
ines
) to
nativ
e PE
and
RA
-PE.
Prol
ifera
tion
was
expr
esse
d as
the
stim
ulat
ion
inde
x (S
I, i.e
., pr
olife
ratio
n to
alle
rgen
stim
ulat
ion
divi
ded
by b
lanc
).A.
Rep
rese
ntat
ive
exam
ples
of a
TC
L of
an
alle
rgic
pat
ient
. B. I
mm
unog
enic
ity o
f PE
and
RA-
PE in
9 T
CLs
.
A. Fi
gure
3: I
nhib
ition
ELI
SA
. A
. Inh
ibiti
on c
urve
s of
the
nativ
e an
d m
odifie
d PE
of a
US
seru
m
pool
of 2
0 pa
tient
s. T
est s
ampl
es c
ompe
te w
ith P
E co
ated
to E
LISA
pl
ates
for b
indi
ng to
ser
um Ig
E. In
crea
sing
con
cent
ratio
ns o
f tes
t sa
mpl
e re
sult
in in
hibi
tion
of b
indi
ng. T
he a
mou
nt o
f the
IgE
bind
ing
in p
rese
nce
of P
E co
mpa
red
to th
at in
abs
ence
of P
E is
cal
cula
ted
as
perc
enta
ge in
hibi
tion.
B. Ig
Epo
tenc
y of
RA-
CPE
rela
tive
to n
ativ
e C
PE d
eter
min
ed in
an
IgE
inhi
bitio
n EL
ISA
usin
g se
ra o
f 20
pean
ut a
llerg
ic p
atie
nts
from
the
US.
An
in-h
ouse
refe
renc
e (IH
R) a
llerg
en p
repa
ratio
n is
test
ed to
com
pare
the
inhi
bitio
n cu
rves
by
para
llel l
ine
anal
yses
. The
IgE
bind
ing
effic
ienc
y is
exp
ress
ed a
s re
lativ
e po
tenc
y (R
P) c
ompa
red
to th
e IH
R.
1,3
pool
5,9
D11
48,
8D
107
6,2
D10
512
,2D
103
8,2
D98
8,2
D81
2,8
D80
11,8
D77
4,5
D74
9,1
D70
11,3
D69
4,3
D68
0,3
D65
17,8
D64
4,1
D62
5,3
D60
1,4
D50
16,9
D48
0,4
D44
1,3
D19
Rea
lativ
epo
tenc
y (%
) Sa
mpl
eB
.
Figu
re4:
The
pot
ency
of n
ativ
ePE
and
RA
-PE
in th
e in
dire
ct M
RA
. A. R
epre
sent
ativ
eex
ampl
e.
B.R
elat
ive
pote
ncy
of n
ativ
ePE
and
RA-
PE to
act
ivat
eba
soph
ilsFigu
re 2
: Spe
cific
IgG
resp
onse
s to
nat
ive
vers
us m
odifi
ed P
E.Sp
ecific
IgG
titre
s at
day
63
afte
r 5 im
mun
izat
ions
(i.p
.) wi
th 1
0 or
100
µg
prod
uct.
Con
trol m
ice
were
inje
cted
with
mat
rix o
nly.
Dot
s re
pres
ent t
he
IgG
titre
of i
ndiv
idua
l mic
e in
arb
itrar
y un
its (A
U) a
nd b
ars
repr
esen
t the
m
edia
n tit
re o
f eac
h gr
oup.
010
2030
4050
6070
8090
100
28303234363840
0.6
mg
PE0.
1 m
g PE
3 m
g PE
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celcius)
010
2030
4050
6070
8090
100
283032343638400.
1 m
g PE
-RA
0.6
mg
PE-R
A
3 m
g PE
-RA
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celsius)
Cha
lleng
eby
subc
utan
eous
inje
ctio
n
6 m
g PE
+ 1
5 g
Cho
lera
Tox
inby
oral
gava
ge
Day
: 0
1
2
7
14
21
2
8
42
Figu
re 5
: Ani
mal
mod
el.M
ice
were
sen
sitiz
ed w
ith 6
mg
PE
and
15 µ
g C
hole
ra T
oxin
via
ora
l gav
age
and
chal
leng
ed (i
.p.)
with
PE,
PE
adso
rbed
to a
lum
, RA-
PE p
repa
ratio
n an
d R
A-PE
ad
sorb
ed to
alu
m (n
=6 p
er g
roup
).
Figu
re 6
: Bod
y te
mpe
ratu
re fo
llow
ing
i.p. c
halle
nge;
Ana
phyl
axis
to th
e na
tive
prep
arat
ion.
An i.
p. c
halle
nge
with
0.6
mg
PE p
er
mou
se re
sulte
d in
an
anap
hyla
ctic
sho
ck re
spon
se. T
he m
ice
in th
e gr
oups
chal
leng
edwi
thm
odifie
dPE
sho
wed
smal
ldec
reas
esin
te
mpe
ratu
reat
the
high
estd
ose
(3 m
g R
A-PE
). C
ontro
lmic
edi
dno
tres
pond
to th
e ch
alle
nge
(with
3 m
g of
PE
orPE
-RA)
.
blan
c50
2512
.56.
253.
131.
560255075100
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)Proliferation (SI)
blan
c50
2512
.56.
253.
131.
56050100
150
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)
Proliferation (SI)
0.010.
1110100
p<0.
01
PE
R
A-PE
relative basophil activation (%)
02550PE PE
-RA
con
0.0
3 0
.01
0.3
1
3
10
100
10
00
conc
entr
atio
n (n
g/m
l)
% CD63+ basophils
0.01
0.1
110
100
0255075100
PE PE-R
A
inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)110100
p=0.
25
PE
PE-
RA
stimulation index
con
0
100
200
300
10μ μμμ
g
100
μ μμμg
10μ μμμg
100μ μμμg
PEPE
-RA
IgG (AU)
A.
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
H. v
an d
er K
leij1 ,
J. S
mit2 ,
S.C
. Dre
skin
3 , H
. War
men
hove
n1 , D
. Ver
bart
1 , E.
Kno
l4 , A
. Knu
lst4 ,
E. K
erkv
liet1 ,
R. v
an d
en H
out1 ,
R. v
an R
ee5 ,
R. P
iete
rs2 ,
E. v
an H
offe
n4 , S.
J. K
oppe
lman
1
1 HAL
Alle
rgy
BV,
Lei
den,
The
Net
herla
nds,
2 Inst
itute
for R
isk
Asse
ssm
ent S
cien
ces
and
Utr
echt
Cen
tre
for F
ood
Alle
rgy,
Utr
echt
, Th
e N
ethe
rland
s, 3 U
nive
rsity
of C
olor
ado
Den
ver S
choo
l of M
edic
ine,
Aur
ora,
CO
, USA
,4 D
ep. D
erm
atol
ogy/
Alle
rgol
ogy,
UM
C U
trec
ht, U
trec
ht,
The
Net
herla
nds,
5 Exp
erim
enta
l Im
mun
olog
y, A
cade
mic
Med
ical
Cen
tre,
Am
ster
dam
, The
Net
herla
nds
4 -C
hem
ical
mod
ifica
tion
of a
pea
nut e
xtra
ct d
ecre
ases
Ig
Ebi
ndin
g w
hile
the
imm
unog
enic
ity is
mai
ntai
ned
Bac
kgro
und
& A
im:
•Pea
nuts
are
one
of t
he m
ost c
omm
onfo
ods
resp
onsi
ble
for f
ood-
indu
ced
anap
hyla
xis
in a
dults
.•A
cur
ativ
e tre
atm
ent i
s no
t ava
ilabl
e.
•A m
odifi
ed p
eanu
t ext
ract
(alle
rgoi
d) is
bein
g in
vest
igat
ed fo
r its
pot
entia
l use
in im
mun
othe
rapy
.
Con
clus
ion:
An
alle
rgoi
dpr
epar
atio
n of
pe
anut
ex
tract
ha
s a
redu
ced
alle
rgen
icity
com
pare
d to
its
nativ
e co
unte
rpar
t, w
hile
th
e im
mun
ogen
icity
is m
aint
aine
d.
Met
hods
:Th
e Pe
anut
ext
ract
(PE)
pre
para
tion
isch
emic
ally
mod
ified
by
redu
ctio
n of
the
intra
mol
ecul
ardi
sulfi
de b
onds
of P
E,an
d al
kyla
tion
of
the
resu
lting
fre
e su
lfhyd
rylg
roup
s (R
A).
For i
mm
unog
enic
ity, t
he a
llerg
oid
was
adso
rbed
to a
lum
iniu
m h
ydro
xide
.
Res
ults
:Ef
ficac
y:•P
E-sp
ecifi
c hu
man
T-c
ell l
ines
wer
eac
tivat
ed e
qual
ly w
ell w
ith P
E an
d R
A-PE
.•I
n m
ice, P
E an
d R
A-PE
wer
e eq
ually
pote
nt in
indu
cing
PE-
spec
ific
IgG
antib
odie
s in
mic
e.Sa
fety
:•M
odifi
catio
n lo
wer
ed th
e Ig
E-bi
ndin
g in
all p
atie
nts
to a
mea
n re
mai
ning
pot
ency
of 7
.2%
5%.
•The
med
iato
r rel
ease
ass
ay (M
RA)
show
ed a
redu
ced
pote
ncy
of 1
0-10
0fo
ld (r
ange
3 to
>10
,000
fold
). •M
ice
sens
itize
d w
ith P
E ex
perie
nced
ase
vere
ana
phyl
actic
reac
tion
upon
subc
utan
eous
cha
lleng
e w
ith 0
.1 m
gPE
, whi
le 3
mg
RA-
PE w
as s
till
tole
rate
d w
ithou
t pro
blem
s.
Figu
re 1
: Res
pons
e of
PE-
spec
ific
TCLs
(T c
ell l
ines
) to
nativ
e PE
and
RA-
PE.
Prol
ifera
tion
was
expr
esse
d as
the
stim
ulat
ion
inde
x (S
I, i.e
., pr
olife
ratio
n to
alle
rgen
stim
ulat
ion
divi
ded
by b
lanc
).A.
Rep
rese
ntat
ive
exam
ples
of a
TC
L of
an
alle
rgic
pat
ient
. B. I
mm
unog
enic
ity o
f PE
and
RA-
PE in
9 T
CLs
.
A.
Figu
re 3
: Inh
ibiti
on E
LISA
. A.
Inhi
bitio
n cu
rves
of t
he n
ativ
e an
d m
odifie
d PE
of a
US
seru
m
pool
of 2
0 pa
tient
s. T
est s
ampl
es c
ompe
te w
ith P
E co
ated
to E
LISA
pl
ates
for b
indi
ng to
ser
um Ig
E. In
crea
sing
conc
entra
tions
of t
est
sam
ple
resu
lt in
inhi
bitio
n of
bin
ding
. The
am
ount
of t
he Ig
Ebi
ndin
g in
pre
senc
e of
PE
com
pare
d to
that
in a
bsen
ce o
f PE
is c
alcu
late
d as
pe
rcen
tage
inhi
bitio
n.
B. Ig
Epo
tenc
y of
RA-
CPE
rela
tive
to n
ativ
e C
PE d
eter
min
ed in
an
IgE
inhi
bitio
n EL
ISA
usin
g se
ra o
f 20
pean
ut a
llerg
ic p
atie
nts
from
the
US.
An
in-h
ouse
refe
renc
e (IH
R) a
llerg
en p
repa
ratio
n is
test
ed to
com
pare
the
inhi
bitio
n cu
rves
by
para
llel l
ine
anal
yses
. The
IgE
bind
ing
effic
ienc
y is
exp
ress
ed a
s re
lativ
e po
tenc
y (R
P) c
ompa
red
to th
e IH
R.
1,3
pool
5,9
D11
48,
8D
107
6,2
D10
512
,2D
103
8,2
D98
8,2
D81
2,8
D80
11,8
D77
4,5
D74
9,1
D70
11,3
D69
4,3
D68
0,3
D65
17,8
D64
4,1
D62
5,3
D60
1,4
D50
16,9
D48
0,4
D44
1,3
D19
Rea
lativ
epo
tenc
y (%
) Sa
mpl
eB
.
Figu
re4:
The
pot
ency
of n
ativ
ePE
and
RA-
PE
in th
e in
dire
ct M
RA.
A. R
epre
sent
ativ
eex
ampl
e.
B.R
elat
ive
pote
ncy
of n
ativ
ePE
and
RA-
PE to
act
ivat
eba
soph
ilsFigu
re 2
: Spe
cific
IgG
resp
onse
s to
nat
ive
vers
us m
odifi
ed P
E.Sp
ecific
IgG
titre
s at
day
63
afte
r 5 im
mun
izatio
ns (i
.p.)
with
10
or 1
00 µ
g pr
oduc
t. C
ontro
l mic
e we
re in
ject
ed w
ith m
atrix
onl
y. D
ots
repr
esen
t the
Ig
Gtit
re o
f ind
ivid
ual m
ice
in a
rbitr
ary
units
(AU
) and
bar
s re
pres
ent t
he
med
ian
titre
of e
ach
grou
p.
010
2030
4050
6070
8090
100
28303234363840
0.6
mg
PE0.
1 m
g PE
3 m
g PE
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celcius)
010
2030
4050
6070
8090
100
283032343638400.
1 m
g PE
-RA
0.6
mg
PE-R
A
3 m
g PE
-RA
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celsius)
Cha
lleng
eby
subc
utan
eous
inje
ctio
n
6 m
g PE
+ 1
5 g
Cho
lera
Tox
inby
oral
gava
ge
Day
: 0
1
2
7
14
21
2
8
42
Figu
re 5
: Ani
mal
mod
el.M
ice
were
sen
sitiz
ed w
ith 6
mg
PE
and
15 µ
g C
hole
ra T
oxin
via
ora
l gav
age
and
chal
leng
ed (i
.p.)
with
PE,
PE
adso
rbed
to a
lum
, RA-
PE p
repa
ratio
n an
d R
A-PE
ad
sorb
ed to
alu
m (n
=6 p
er g
roup
).
Figu
re 6
: Bod
y te
mpe
ratu
re fo
llow
ing
i.p. c
halle
nge;
Ana
phyl
axis
to th
e na
tive
prep
arat
ion.
An i.
p. c
halle
nge
with
0.6
mg
PE p
er
mou
se re
sulte
d in
an
anap
hyla
ctic
sho
ck re
spon
se. T
he m
ice
in th
e gr
oups
chal
leng
edwi
thm
odifie
dPE
sho
wed
smal
ldec
reas
esin
te
mpe
ratu
reat
the
high
estd
ose
(3 m
g R
A-PE
). C
ontro
lmic
edi
dno
tres
pond
to th
e ch
alle
nge
(with
3 m
g of
PE
orPE
-RA)
.
blan
c50
2512
.56.
253.
131.
560255075100
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)
Proliferation (SI)bl
anc
5025
12.5
6.25
3.13
1.56
050100
150
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)
Proliferation (SI)
0.010.
1110100
p<0.
01
PE
R
A-PE
relative basophil activation (%)
02550PE PE
-RA
con
0.0
3 0
.01
0.3
1
3
10
100
10
00
conc
entr
atio
n (n
g/m
l)
% CD63+ basophils
0.01
0.1
110
100
0255075100
PE PE-R
A
inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
110100
p=0.
25
PE
PE-
RA
stimulation index
con
0
100
200
300
10μ μμμ
g
100
μ μμμg
10μ μμμg
100μ μμμg
PEPE
-RA
IgG (AU)
A.
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
H. v
an d
er K
leij1 ,
J. S
mit2 ,
S.C
. Dre
skin
3 , H
. War
men
hove
n1 , D
. Ver
bart
1 , E.
Kno
l4 , A
. Knu
lst4 ,
E. K
erkv
liet1 ,
R. v
an d
en H
out1 ,
R. v
an R
ee5 ,
R. P
iete
rs2 ,
E. v
an H
offe
n4 , S.
J. K
oppe
lman
1
1 HAL
Alle
rgy
BV,
Lei
den,
The
Net
herla
nds,
2 Ins
titut
e fo
r Ris
k A
sses
smen
t Sci
ence
s an
d U
trec
ht C
entr
e fo
r Foo
d Al
lerg
y, U
trec
ht,
The
Net
herla
nds,
3 Uni
vers
ity o
f Col
orad
o D
enve
r Sch
ool o
f Med
icin
e, A
uror
a, C
O, U
SA,
4 Dep
. Der
mat
olog
y/Al
lerg
olog
y, U
MC
Utr
echt
, Utr
echt
, Th
e N
ethe
rland
s, 5 E
xper
imen
tal I
mm
unol
ogy,
Aca
dem
ic M
edic
al C
entr
e, A
mst
erda
m, T
heN
ethe
rland
s
4 -C
hem
ical
mod
ifica
tion
of a
pea
nut e
xtra
ct d
ecre
ases
Ig
Ebi
ndin
g w
hile
the
imm
unog
enic
ity is
mai
ntai
ned
Bac
kgro
und
& A
im:
•Pea
nuts
are
one
of t
he m
ost c
omm
onfo
ods
resp
onsi
ble
for f
ood-
indu
ced
anap
hyla
xis
in a
dults
.•A
cur
ativ
e tre
atm
ent i
s no
t ava
ilabl
e.
•A m
odifi
ed p
eanu
t ext
ract
(alle
rgoi
d) is
bein
g in
vest
igat
ed fo
r its
pot
entia
l use
in im
mun
othe
rapy
.
Con
clus
ion:
An
alle
rgoi
dpr
epar
atio
n of
pe
anut
ex
tract
ha
s a
redu
ced
alle
rgen
icity
com
pare
d to
its
nativ
e co
unte
rpar
t, w
hile
th
e im
mun
ogen
icity
is m
aint
aine
d.
Met
hods
:Th
e Pe
anut
ext
ract
(PE)
pre
para
tion
isch
emic
ally
mod
ified
by
redu
ctio
n of
the
intra
mol
ecul
ardi
sulfi
de b
onds
of P
E,an
d al
kyla
tion
of
the
resu
lting
fre
e su
lfhyd
rylg
roup
s (R
A).
For i
mm
unog
enic
ity, t
he a
llerg
oid
was
adso
rbed
to a
lum
iniu
m h
ydro
xide
.
Res
ults
:Ef
ficac
y:•P
E-sp
ecifi
c hu
man
T-c
ell l
ines
wer
eac
tivat
ed e
qual
ly w
ell w
ith P
E an
d R
A-PE
.•I
n m
ice,
PE
and
RA-
PE w
ere
equa
llypo
tent
in in
duci
ng P
E-sp
ecifi
c Ig
Gan
tibod
ies
in m
ice.
Safe
ty:
•Mod
ifica
tion
low
ered
the
IgE-
bind
ing
inal
l pat
ient
s to
a m
ean
rem
aini
ng p
oten
cyof
7.2
%5%
. •T
he m
edia
tor r
elea
se a
ssay
(MR
A)sh
owed
a re
duce
d po
tenc
y of
10-
100
fold
(ran
ge 3
to >
10,0
00 fo
ld).
•Mic
e se
nsiti
zed
with
PE
expe
rienc
ed a
seve
re a
naph
ylac
tic re
actio
n up
onsu
bcut
aneo
us c
halle
nge
with
0.1
mg
PE, w
hile
3 m
g R
A-PE
was
stil
lto
lera
ted
with
out p
robl
ems.
Figu
re 1
: Res
pons
e of
PE-
spec
ific
TCLs
(T c
ell l
ines
) to
nativ
e PE
and
RA
-PE.
Prol
ifera
tion
was
expr
esse
d as
the
stim
ulat
ion
inde
x (S
I, i.e
., pr
olife
ratio
n to
alle
rgen
stim
ulat
ion
divi
ded
by b
lanc
).A.
Rep
rese
ntat
ive
exam
ples
of a
TC
L of
an
alle
rgic
pat
ient
. B. I
mm
unog
enic
ity o
f PE
and
RA-
PE in
9 T
CLs
.
A. Fi
gure
3: I
nhib
ition
ELI
SA
. A
. Inh
ibiti
on c
urve
s of
the
nativ
e an
d m
odifie
d PE
of a
US
seru
m
pool
of 2
0 pa
tient
s. T
est s
ampl
es c
ompe
te w
ith P
E co
ated
to E
LISA
pl
ates
for b
indi
ng to
ser
um Ig
E. In
crea
sing
con
cent
ratio
ns o
f tes
t sa
mpl
e re
sult
in in
hibi
tion
of b
indi
ng. T
he a
mou
nt o
f the
IgE
bind
ing
in p
rese
nce
of P
E co
mpa
red
to th
at in
abs
ence
of P
E is
cal
cula
ted
as
perc
enta
ge in
hibi
tion.
B. Ig
Epo
tenc
y of
RA-
CPE
rela
tive
to n
ativ
e C
PE d
eter
min
ed in
an
IgE
inhi
bitio
n EL
ISA
usin
g se
ra o
f 20
pean
ut a
llerg
ic p
atie
nts
from
the
US.
An
in-h
ouse
refe
renc
e (IH
R) a
llerg
en p
repa
ratio
n is
test
ed to
com
pare
the
inhi
bitio
n cu
rves
by
para
llel l
ine
anal
yses
. The
IgE
bind
ing
effic
ienc
y is
exp
ress
ed a
s re
lativ
e po
tenc
y (R
P) c
ompa
red
to th
e IH
R.
1,3
pool
5,9
D11
48,
8D
107
6,2
D10
512
,2D
103
8,2
D98
8,2
D81
2,8
D80
11,8
D77
4,5
D74
9,1
D70
11,3
D69
4,3
D68
0,3
D65
17,8
D64
4,1
D62
5,3
D60
1,4
D50
16,9
D48
0,4
D44
1,3
D19
Rea
lativ
epo
tenc
y (%
) Sa
mpl
eB
.
Figu
re4:
The
pot
ency
of n
ativ
ePE
and
RA
-PE
in th
e in
dire
ct M
RA
. A. R
epre
sent
ativ
eex
ampl
e.
B.R
elat
ive
pote
ncy
of n
ativ
ePE
and
RA-
PE to
act
ivat
eba
soph
ilsFigu
re 2
: Spe
cific
IgG
resp
onse
s to
nat
ive
vers
us m
odifi
ed P
E.Sp
ecific
IgG
titre
s at
day
63
afte
r 5 im
mun
izat
ions
(i.p
.) wi
th 1
0 or
100
µg
prod
uct.
Con
trol m
ice
were
inje
cted
with
mat
rix o
nly.
Dot
s re
pres
ent t
he
IgG
titre
of i
ndiv
idua
l mic
e in
arb
itrar
y un
its (A
U) a
nd b
ars
repr
esen
t the
m
edia
n tit
re o
f eac
h gr
oup.
010
2030
4050
6070
8090
100
28303234363840
0.6
mg
PE0.
1 m
g PE
3 m
g PE
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celcius)
010
2030
4050
6070
8090
100
283032343638400.
1 m
g PE
-RA
0.6
mg
PE-R
A
3 m
g PE
-RA
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celsius)
Cha
lleng
eby
subc
utan
eous
inje
ctio
n
6 m
g PE
+ 1
5 g
Cho
lera
Tox
inby
oral
gava
ge
Day
: 0
1
2
7
14
21
2
8
42
Figu
re 5
: Ani
mal
mod
el.M
ice
were
sen
sitiz
ed w
ith 6
mg
PE
and
15 µ
g C
hole
ra T
oxin
via
ora
l gav
age
and
chal
leng
ed (i
.p.)
with
PE,
PE
adso
rbed
to a
lum
, RA-
PE p
repa
ratio
n an
d R
A-PE
ad
sorb
ed to
alu
m (n
=6 p
er g
roup
).
Figu
re 6
: Bod
y te
mpe
ratu
re fo
llow
ing
i.p. c
halle
nge;
Ana
phyl
axis
to th
e na
tive
prep
arat
ion.
An i.
p. c
halle
nge
with
0.6
mg
PE p
er
mou
se re
sulte
d in
an
anap
hyla
ctic
sho
ck re
spon
se. T
he m
ice
in th
e gr
oups
chal
leng
edwi
thm
odifie
dPE
sho
wed
smal
ldec
reas
esin
te
mpe
ratu
reat
the
high
estd
ose
(3 m
g R
A-PE
). C
ontro
lmic
edi
dno
tres
pond
to th
e ch
alle
nge
(with
3 m
g of
PE
orPE
-RA)
.
blan
c50
2512
.56.
253.
131.
560255075100
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)
Proliferation (SI)
blan
c50
2512
.56.
253.
131.
56050100
150
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)
Proliferation (SI)
0.010.
1110100
p<0.
01
PE
R
A-PE
relative basophil activation (%)
02550PE PE
-RA
con
0.0
3 0
.01
0.3
1
3
10
100
10
00
conc
entr
atio
n (n
g/m
l)
% CD63+ basophils
0.01
0.1
110
100
0255075100
PE PE-R
A
inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
110100
p=0.
25
PE
PE-
RA
stimulation index
con
0
100
200
300
10μ μμμ
g
100
μ μμμg
10μ μμμg
100μ μμμg
PEPE
-RA
IgG (AU)
A.
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
H. v
an d
er K
leij1 ,
J. S
mit2 ,
S.C
. Dre
skin
3 , H
. War
men
hove
n1 , D
. Ver
bart
1 , E.
Kno
l4 , A
. Knu
lst4 ,
E. K
erkv
liet1 ,
R. v
an d
en H
out1 ,
R. v
an R
ee5 ,
R. P
iete
rs2 ,
E. v
an H
offe
n4 , S.
J. K
oppe
lman
1
1 HAL
Alle
rgy
BV,
Lei
den,
The
Net
herla
nds,
2 Ins
titut
e fo
r Ris
k As
sess
men
t Sci
ence
s an
d U
trec
ht C
entr
e fo
r Foo
d Al
lerg
y, U
trec
ht,
The
Net
herla
nds,
3 Uni
vers
ity o
f Col
orad
o D
enve
r Sch
ool o
f Med
icin
e, A
uror
a, C
O, U
SA,
4 Dep
. Der
mat
olog
y/Al
lerg
olog
y, U
MC
Utr
echt
, Utr
echt
, Th
e N
ethe
rland
s, 5 E
xper
imen
tal I
mm
unol
ogy,
Aca
dem
ic M
edic
al C
entr
e, A
mst
erda
m, T
heN
ethe
rland
s
4 -C
hem
ical
mod
ifica
tion
of a
pea
nut e
xtra
ct d
ecre
ases
Ig
Ebi
ndin
g w
hile
the
imm
unog
enic
ity is
mai
ntai
ned
Bac
kgro
und
& A
im:
•Pea
nuts
are
one
of t
he m
ost c
omm
onfo
ods
resp
onsi
ble
for f
ood-
indu
ced
anap
hyla
xis
in a
dults
.•A
cur
ativ
e tre
atm
ent i
s no
t ava
ilabl
e.
•A m
odifi
ed p
eanu
t ext
ract
(alle
rgoi
d) is
bein
g in
vest
igat
ed fo
r its
pot
entia
l use
in im
mun
othe
rapy
.
Con
clus
ion:
An
alle
rgoi
dpr
epar
atio
n of
pe
anut
ex
tract
ha
s a
redu
ced
alle
rgen
icity
com
pare
d to
its
nativ
e co
unte
rpar
t, w
hile
th
e im
mun
ogen
icity
is m
aint
aine
d.
Met
hods
:Th
e Pe
anut
ext
ract
(PE)
pre
para
tion
isch
emic
ally
mod
ified
by
redu
ctio
n of
the
intra
mol
ecul
ardi
sulfi
de b
onds
of P
E,an
d al
kyla
tion
of
the
resu
lting
fre
e su
lfhyd
rylg
roup
s (R
A).
For i
mm
unog
enic
ity, t
he a
llerg
oid
was
adso
rbed
to a
lum
iniu
m h
ydro
xide
.
Res
ults
:Ef
ficac
y:•P
E-sp
ecifi
c hu
man
T-c
ell l
ines
wer
eac
tivat
ed e
qual
ly w
ell w
ith P
E an
d R
A-PE
.•I
n m
ice,
PE
and
RA-
PE w
ere
equa
llypo
tent
in in
duci
ng P
E-sp
ecifi
c Ig
Gan
tibod
ies
in m
ice.
Safe
ty:
•Mod
ifica
tion
low
ered
the
IgE-
bind
ing
inal
l pat
ient
s to
a m
ean
rem
aini
ng p
oten
cyof
7.2
%5%
. •T
he m
edia
tor r
elea
se a
ssay
(MR
A)sh
owed
a re
duce
d po
tenc
y of
10-
100
fold
(ran
ge 3
to >
10,0
00 fo
ld).
•Mic
e se
nsiti
zed
with
PE
expe
rienc
ed a
seve
re a
naph
ylac
tic re
actio
n up
onsu
bcut
aneo
us c
halle
nge
with
0.1
mg
PE, w
hile
3 m
g R
A-PE
was
stil
lto
lera
ted
with
out p
robl
ems.
Figu
re 1
: Res
pons
e of
PE-
spec
ific
TCLs
(T c
ell l
ines
) to
nativ
e PE
and
RA
-PE.
Prol
ifera
tion
was
expr
esse
d as
the
stim
ulat
ion
inde
x (S
I, i.e
., pr
olife
ratio
n to
alle
rgen
stim
ulat
ion
divi
ded
by b
lanc
).A.
Rep
rese
ntat
ive
exam
ples
of a
TC
L of
an
alle
rgic
pat
ient
. B. I
mm
unog
enic
ity o
f PE
and
RA-
PE in
9 T
CLs
.
A. Fi
gure
3: I
nhib
ition
ELI
SA
. A
. Inh
ibiti
on c
urve
s of
the
nativ
e an
d m
odifie
d PE
of a
US
seru
m
pool
of 2
0 pa
tient
s. T
est s
ampl
es c
ompe
te w
ith P
E co
ated
to E
LISA
pl
ates
for b
indi
ng to
ser
um Ig
E. In
crea
sing
con
cent
ratio
ns o
f tes
t sa
mpl
e re
sult
in in
hibi
tion
of b
indi
ng. T
he a
mou
nt o
f the
IgE
bind
ing
in p
rese
nce
of P
E co
mpa
red
to th
at in
abs
ence
of P
E is
cal
cula
ted
as
perc
enta
ge in
hibi
tion.
B. Ig
Epo
tenc
y of
RA-
CPE
rela
tive
to n
ativ
e C
PE d
eter
min
ed in
an
IgE
inhi
bitio
n EL
ISA
usin
g se
ra o
f 20
pean
ut a
llerg
ic p
atie
nts
from
the
US.
An
in-h
ouse
refe
renc
e (IH
R) a
llerg
en p
repa
ratio
n is
test
ed to
com
pare
the
inhi
bitio
n cu
rves
by
para
llel l
ine
anal
yses
. The
IgE
bind
ing
effic
ienc
y is
exp
ress
ed a
s re
lativ
e po
tenc
y (R
P) c
ompa
red
to th
e IH
R.
1,3
pool
5,9
D11
48,
8D
107
6,2
D10
512
,2D
103
8,2
D98
8,2
D81
2,8
D80
11,8
D77
4,5
D74
9,1
D70
11,3
D69
4,3
D68
0,3
D65
17,8
D64
4,1
D62
5,3
D60
1,4
D50
16,9
D48
0,4
D44
1,3
D19
Rea
lativ
epo
tenc
y (%
) Sa
mpl
eB
.
Figu
re4:
The
pot
ency
of n
ativ
ePE
and
RA
-PE
in th
e in
dire
ct M
RA
. A. R
epre
sent
ativ
eex
ampl
e.
B.R
elat
ive
pote
ncy
of n
ativ
ePE
and
RA-
PE to
act
ivat
eba
soph
ilsFigu
re 2
: Spe
cific
IgG
resp
onse
s to
nat
ive
vers
us m
odifi
ed P
E.Sp
ecific
IgG
titre
s at
day
63
afte
r 5 im
mun
izat
ions
(i.p
.) wi
th 1
0 or
100
µg
prod
uct.
Con
trol m
ice
were
inje
cted
with
mat
rix o
nly.
Dot
s re
pres
ent t
he
IgG
titre
of i
ndiv
idua
l mic
e in
arb
itrar
y un
its (A
U) a
nd b
ars
repr
esen
t the
m
edia
n tit
re o
f eac
h gr
oup.
010
2030
4050
6070
8090
100
28303234363840
0.6
mg
PE0.
1 m
g PE
3 m
g PE
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celcius)
010
2030
4050
6070
8090
100
283032343638400.
1 m
g PE
-RA
0.6
mg
PE-R
A
3 m
g PE
-RA
Tim
e af
ter c
halle
nge
(min
utes
)
Temperature (Celsius)
Cha
lleng
eby
subc
utan
eous
inje
ctio
n
6 m
g PE
+ 1
5 g
Cho
lera
Tox
inby
oral
gava
ge
Day
: 0
1
2
7
14
21
2
8
42
Figu
re 5
: Ani
mal
mod
el.M
ice
were
sen
sitiz
ed w
ith 6
mg
PE
and
15 µ
g C
hole
ra T
oxin
via
ora
l gav
age
and
chal
leng
ed (i
.p.)
with
PE,
PE
adso
rbed
to a
lum
, RA-
PE p
repa
ratio
n an
d R
A-PE
ad
sorb
ed to
alu
m (n
=6 p
er g
roup
).
Figu
re 6
: Bod
y te
mpe
ratu
re fo
llow
ing
i.p. c
halle
nge;
Ana
phyl
axis
to th
e na
tive
prep
arat
ion.
An i.
p. c
halle
nge
with
0.6
mg
PE p
er
mou
se re
sulte
d in
an
anap
hyla
ctic
sho
ck re
spon
se. T
he m
ice
in th
e gr
oups
chal
leng
edwi
thm
odifie
dPE
sho
wed
smal
ldec
reas
esin
te
mpe
ratu
reat
the
high
estd
ose
(3 m
g R
A-PE
). C
ontro
lmic
edi
dno
tres
pond
to th
e ch
alle
nge
(with
3 m
g of
PE
orPE
-RA)
.
blan
c50
2512
.56.
253.
131.
560255075100
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)
Proliferation (SI)
blan
c50
2512
.56.
253.
131.
56050100
150
PE RA-
PE
conc
entr
atio
n (μ μμμ
g/m
l)
Proliferation (SI)
0.010.
1110100
p<0.
01
PE
R
A-PE
relative basophil activation (%)
02550PE PE
-RA
con
0.0
3 0
.01
0.3
1
3
10
100
10
00
conc
entr
atio
n (n
g/m
l)
% CD63+ basophils
0.01
0.1
110
100
0255075100
PE PE-R
A
inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
110100
p=0.
25
PE
PE-
RA
stimulation index
con
0
100
200
300
10μ μμμ
g
100
μ μμμg
10μ μμμg
100μ μμμg
PEPE
-RA
IgG (AU)
A.
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
H. v
an d
er K
leij1 ,
J. S
mit2 ,
S.C
. Dre
skin
3 , H
. War
men
hove
n1 , D
. Ver
bart
1 , E.
Kno
l4 , A
. Knu
lst4 ,
E. K
erkv
liet1 ,
R. v
an d
en H
out1 ,
R. v
an R
ee5 ,
R. P
iete
rs2 ,
E. v
an H
offe
n4 , S.
J. K
oppe
lman
1
EAAC
I Con
gres
s 20
12In
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er is
an
empl
oyee
of H
AL A
llerg
y.
Al l e rge nic p o t e n c y o f c h e mic a l l y m o d i f i e d Ara h 2 / h 6 eva l u a t e d w i t h b a s o p h il a c t iva t i o n , R B L- S X 3 8 a c t iva t i o n , a n d s o l i d p h a s e Ig E - im mu n o a ss ay
20
H. van der Kleij (1), H. Warmenhoven (1), D. Verbart (1), Q. Wang (2), R. van Ree (3), S.C. Dreskin (2), S.J. Koppelman (1).
(1) HAL Allergy BV, Leiden, The Netherlands, (2) University of Colorado Denver School of Medicine, Aurora, CO, USA, (3) Experimental
Immunology, Academic Medical Centre, Amsterdam, The Netherlands.
RationalePeanuts are one of the most common foods responsible for food-induced anaphylaxis and there is no curative treatment for peanut allergy. Peanut sIT could be an option but earlier attempts with aqueous peanut extract were hampered by safety issues. Major peanut allergens Ara h2 and Ara h6 have been modified to make them suitable for immunotherapy. This study evaluates the potency of modified Ara h2 and Ara h6 in mediator release assays in comparison with native, not modified Ara h2 and Ara h6.
MethodsAra h2 and Ara h6 were purified and chemically modified by means of reduction of disulfide bonds and subsequent alkylation of free Cys. Twenty patients from the greater Denver area were recruited and serum was collected. Total- and peanut-specific IgE was determined as well as IgE to individual peanut allergens. RBL SX38 cell expressing the human IgE receptor and basophils from healthy donors were loaded with the serum samples and were subsequently exposed to different concentrations of either native or modified Ara h2 / Ara h6. Mediator release was determine and expressed as percentage of maximum release. Potency of native and modified Ara h2 / Ara h6 was also evaluated applying a solid phase IgE- binding assay performed with individual patient sera.
ResultsFor all tested patient sera it was observed that the potency of modified Ara h2 /Ara h 6 was reduced compared to native Ara h2 / Ara h6. Using the RBL-SX38 assay a mean reduction of 100- fold was found (range: 3 to <10,000 fold). Similar data were found with the basophil assay and the fold reduction observed per patient correlated between the two mediator release tests. For modified Ara h2 / Ara h6, IgE-binding as determined using the solid phase IgE-binding assay was for all patient sera reduced compared to native Ara h2 / Ara h6. Sera for which the modification had the largest effect on the activity in mediator release assays also showed the largest reduction in potency using the solid-phase IgE binding test.
ConclusionsThe allergenic potency of modified Ara h2/Ara h6 is about 100-fold lower that that of native Ara h2/Ara h6, making them more suitable candidates for subcutaneous immunotherapy.
EAACI, 16-20 June 2012, GenevaAbstract number: 62, Session date and time: Sunday 17 June; 15:30 - 17:00 Session title: OAS 11 - Molecules and multiplexing in allergy diagnosis
21
OAS 11 - Molecules and multiplexing in allergy diagnosis
Al l e rge nic p o t e n c y o f c h e mic a l l y m o d i f i e d Ara h 2 / h 6 eva l u a t e d w i t h b a s o p h il a c t iva t i o n , R B L- S X 3 8 a c t iva t i o n , a n d s o l i d p h a s e Ig E - im mu n o a ss ay
1H
AL A
llerg
y B
V, L
eide
n, T
he N
ethe
rland
s, 2
Uni
vers
ity o
f Col
orad
o D
enve
r Sch
ool o
f Med
icin
e, A
uror
a, C
O, U
SA3 Ex
perim
enta
l Im
mun
olog
y, A
cade
mic
Med
ical
Cen
tre,
Am
ster
dam
, The
Net
herla
nds
62 -
Alle
rgen
ic p
oten
cy o
f che
mic
ally
mod
ified
Ara
h2/h
6 ev
alua
ted
with
bas
ophi
lact
ivat
ion,
RB
L-SX
38 a
ctiv
atio
n, a
nd
solid
pha
se Ig
E-im
mun
oass
ay
Bac
kgro
und
& A
im:
•Pea
nuts
are
one
of t
he m
ost c
omm
onfo
ods
resp
onsi
ble
for f
ood-
indu
ced
anap
hyla
xis
in a
dults
.•P
eanu
t sIT
with
aqu
eous
pea
nut e
xtra
ctha
s fa
iled
beca
use
of s
afet
y is
sues
.•A
mod
ified
pre
para
tion
of th
e 2
maj
orpe
anut
alle
rgen
s Ar
ah2
and
Ara
h6 is
bein
g in
vest
igat
ed fo
r its
pot
entia
l use
in im
mun
othe
rapy
IgE
inhi
bitio
n cu
rves
of t
he n
ativ
e an
d m
odifie
d Ar
ah2
/6 o
f ind
ivid
ual
patie
nts.
Te
st s
ampl
es c
ompe
te w
ith n
ativ
e Ar
ah2
/6 c
oate
d to
ELI
SA p
late
s fo
r bin
ding
to
ser
um Ig
E. In
crea
sing
con
cent
ratio
ns o
f tes
t sam
ple
resu
lt in
inhi
bitio
n of
bi
ndin
g. T
he a
mou
nt o
f the
IgE
bind
ing
in p
rese
nce
of A
rah2
/6 c
ompa
red
to
that
in a
bsen
ce o
f Ara
h2/6
is c
alcu
late
d as
per
cent
age
inhi
bitio
n.
Tabl
e1:
Ser
olog
yof
recr
uite
dpa
tient
s
Con
clus
ion:
The
alle
rgen
ic p
oten
cy o
f mod
ified
Ara
h2/6
is a
bout
100
-fold
low
er th
an th
at o
fna
tive
Ara
h2/A
ra h
6, m
akin
g th
em m
ore
suita
ble
cand
idat
es fo
r sub
cuta
neou
sim
mun
othe
rapy
.
Met
hods
:•A
rah2
and
Ara
h6 w
ere
purif
ied
and
chem
ical
ly m
odifi
ed b
y m
eans
of
redu
ctio
n (R
) of d
isul
fide
bond
s an
dsu
bseq
uent
alk
ylat
ion
(A) o
f fre
e C
ys.
Prep
arat
ions
wer
e ch
arac
teriz
ed b
ySD
S-PA
GE
and
by fa
r-UV
CD
spec
tosc
ropy
.•T
wen
ty p
atie
nts
from
the
grea
ter
Den
ver a
rea
wer
e re
crui
ted
and
seru
mw
as c
olle
cted
. •R
BL S
X38
cells
and
bas
ophi
lsfro
mhe
alth
y do
nors
wer
e lo
aded
with
the
seru
m s
ampl
es a
nd s
ubse
quen
tlyex
pose
d to
eith
er n
ativ
e Ar
ah2
/6 o
rm
odifi
ed A
rah2
/6 (R
A-Ar
ah2
/6).
•Pot
ency
was
als
o ev
alua
ted
appl
ying
aso
lid p
hase
IgE-
bind
ing
assa
ype
rform
ed w
ith in
divi
dual
pat
ient
ser
a.
Res
ults
: Al
l tes
ted
patie
nt s
era
disp
laye
d a
redu
ced
resp
onse
to R
A-Ar
ah2
/6:
•A m
ean
redu
ctio
n of
100
-fol
d w
asfo
und
(rang
e: 3
to <
10,0
00 fo
ld) u
sing
both
the
RBL
SX3
8 an
d th
e in
dire
ctba
soph
ilas
say.
•T
he fo
ld re
duct
ion
obse
rved
per
pat
ient
corre
late
d be
twee
n th
e tw
o m
edia
tor
rele
ase
test
s.
•Ser
a fo
r whi
ch th
e m
odifi
catio
n ha
d th
ela
rges
t effe
ct o
n th
e ac
tivity
in m
edia
tor
rele
ase
assa
ys a
lso
show
ed th
e la
rges
t re
duct
ion
in p
oten
cy u
sing
the
solid
-ph
ase
IgE
bind
ing
test
.
A.
Mw
1
2
B.N
ativ
e co
nglu
tinsh
ows
a ty
pica
l sp
ectru
m fo
r pro
tein
s co
nsis
ting
mai
nly
of a
lpha
-hel
ices
(min
imal
ellip
ticity
valu
es a
t 208
and
222
nm
) but
als
o
beta
-stru
ctur
e. In
con
trast
, RA
show
s a
typi
cal s
pect
rum
for u
nfol
ded
prot
ein.
St
rong
dec
reas
e of
ellip
ticity
at 2
22 n
m
indi
cate
s lo
ss o
f alp
ha-h
elic
es a
nd
incr
ease
of r
ando
m c
oil s
truct
ure.
Thi
s ca
n be
exp
lain
ed b
y th
e fa
ct th
at th
e di
sulp
hide
brid
ges
have
bee
n cl
eave
d.
200
210
220
230
240
250
260
-15
-10-5051015
Nat
ive
RA
Wav
elen
gth
(nm
)
CD (mDeg)
The
far-U
V C
D s
pect
ra o
f nat
ive
and
mod
ified
con
glut
in.
117
199
88179780219
545
505
194
1112
290
293
2421
280337306
533
138
101573
Tota
l IgE
226514787
1681285
4929500
1352180
591
135638716539
Pean
ut-
Spec
ific
IgE
2,0
D11
40,
7D
107
0D
105
6,4
D10
30,
4D
98
9,4
D81
9,7
D80
1,5
D77
0,5
D74
0D
70
3,4
D69
0D
68
0D
650,
8D
640,
3D
620,
5D
60
0,3
D50
0,4
D48
0,2
D44
0D
19
Rel
ativ
e po
tenc
y (%
) Sa
mpl
e
0.01
0.1
110
100
1000
0255075100
D10
3
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.1
110
100
1000
0102030Se
rum
poo
l
nativ
e
RA
ng/m
l
net % degranulationFigu
re 3
: Deg
ranu
latio
nof
RB
L SX
-38
cells
to n
ativ
e or
mod
ified
Ara
h2/6
1010
010
000102030
D65
nativ
e
RA
ng/m
l
net degranulation
110
100
1000
0102030D8
0
nativ
e
RA
ng/m
l
net degranulation
A.T
he n
ativ
e (1
) and
RA
(2)
Ara
h2/h
6 pr
epar
atio
ns
were
ana
lyzed
by
SDS-
PAG
E. T
he p
rote
ins
were
vi
sual
ized
usi
ng C
oom
assi
e-st
aini
ng.
0.01
110
010
000
0102030405060D
65
nativ
e
RA
ng/m
l
release (%)
0.01
110
010
000
0102030405060D
68
nativ
e
RA
ng/m
l
release (%)
0.01
110
010
000
0102030405060D
64 nativ
e
RA
ng/m
l
release (%)
Figu
re 4
: Deg
ranu
latio
nof
hum
an b
asop
hils
to n
ativ
e or
mod
ified
Ara
h2/6
RBL
SX-
38 c
ells
wer
e se
nsitiz
ed e
ither
with
a s
erum
poo
l (A)
or i
ndiv
idua
l ser
a (B
and
C) a
nd tr
igge
red
with
nat
ive
Ara
h 2/
6 or
RA-
Ara
h 2/
6.
Dat
a ar
e sh
own
as “n
et”d
egra
nula
tion
(tota
l deg
ranu
latio
nm
inus
bac
kgro
und)
.
In th
e in
dire
ct B
AT, h
uman
bas
ophi
lswe
re s
ensit
ized
with
indi
vidu
al s
era
and
trig
gere
d wi
th n
ativ
e Ar
ah
2/6
or R
A-Ar
ah
2/6.
Dat
a ar
e sh
own
as %
rele
ase
(in re
latio
n to
tota
l deg
ranu
latio
nm
inus
bac
kgro
und)
.
B.
Figu
re 1
. Cha
ract
eris
tics
of p
eanu
t pre
para
tions
Figu
re 2
: Sol
id p
hase
IgE-
bind
ing
test
0.01
0.1
110
100
1000
0255075100
D64
nativ
eR
A
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.01
0.1
110
100
1000
0255075100
D10
5
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.01
0.1
110
100
1000
0255075100
D11
4
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
IgE
bind
ing
pote
ncy
of th
e na
tive
and
mod
ified
prep
arat
ions
wer
e de
term
ined
in 2
0 U
S pa
tient
s wi
th
esta
blis
hed
clin
ical
alle
rgy
for p
eanu
t.Th
e Ig
Ebi
ndin
g is
exp
ress
ed a
s re
lativ
e po
tenc
y (R
P) c
ompa
red
to a
n in
-hou
se re
fere
nce
alle
rgen
pre
para
tion.
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
H. v
an d
er K
leij1 ,
H. W
arm
enho
ven1 ,
D. V
erba
rt1 ,
Q. W
ang2 ,
R. v
an R
ee3 ,
S.C
. Dre
skin
2 , S.
J. K
oppe
lman
11
HAL
Alle
rgy
BV,
Lei
den,
The
Net
herla
nds,
2U
nive
rsity
of C
olor
ado
Den
ver S
choo
l of M
edic
ine,
Aur
ora,
CO
, USA
3 Ex
perim
enta
l Im
mun
olog
y, A
cade
mic
Med
ical
Cen
tre,
Am
ster
dam
, The
Net
herla
nds
62 -
Alle
rgen
ic p
oten
cy o
f che
mic
ally
mod
ified
Ara
h2/h
6 ev
alua
ted
with
bas
ophi
lact
ivat
ion,
RB
L-SX
38 a
ctiv
atio
n, a
nd
solid
pha
se Ig
E-im
mun
oass
ay
Bac
kgro
und
& A
im:
•Pea
nuts
are
one
of t
he m
ost c
omm
onfo
ods
resp
onsi
ble
for f
ood-
indu
ced
anap
hyla
xis
in a
dults
.•P
eanu
t sIT
with
aqu
eous
pea
nut e
xtra
ctha
s fa
iled
beca
use
of s
afet
y is
sues
.•A
mod
ified
pre
para
tion
of th
e 2
maj
orpe
anut
alle
rgen
s Ar
ah2
and
Ara
h6 is
bein
g in
vest
igat
ed fo
r its
pot
entia
l use
in im
mun
othe
rapy
IgE
inhi
bitio
n cu
rves
of t
he n
ativ
e an
d m
odifie
d Ar
ah2
/6 o
f ind
ivid
ual
patie
nts.
Te
st s
ampl
es c
ompe
te w
ith n
ativ
e Ar
ah2
/6 c
oate
d to
ELI
SA p
late
s fo
r bin
ding
to
ser
um Ig
E. In
crea
sing
con
cent
ratio
ns o
f tes
t sam
ple
resu
lt in
inhi
bitio
n of
bi
ndin
g. T
he a
mou
nt o
f the
IgE
bind
ing
in p
rese
nce
of A
rah2
/6 c
ompa
red
to
that
in a
bsen
ce o
f Ara
h2/6
is c
alcu
late
d as
per
cent
age
inhi
bitio
n.
Tabl
e1:
Ser
olog
yof
recr
uite
dpa
tient
s
Con
clus
ion:
The
alle
rgen
ic p
oten
cy o
f mod
ified
Ara
h2/6
is a
bout
100
-fold
low
er th
an th
at o
fna
tive
Ara
h2/A
ra h
6, m
akin
g th
em m
ore
suita
ble
cand
idat
es fo
r sub
cuta
neou
sim
mun
othe
rapy
.
Met
hods
:•A
rah2
and
Ara
h6 w
ere
purif
ied
and
chem
ical
ly m
odifi
ed b
y m
eans
of
redu
ctio
n (R
) of d
isul
fide
bond
s an
dsu
bseq
uent
alk
ylat
ion
(A) o
f fre
e C
ys.
Prep
arat
ions
wer
e ch
arac
teriz
ed b
ySD
S-PA
GE
and
by fa
r-UV
CD
spec
tosc
ropy
.•T
wen
ty p
atie
nts
from
the
grea
ter
Den
ver a
rea
wer
e re
crui
ted
and
seru
mw
as c
olle
cted
. •R
BL S
X38
cells
and
bas
ophi
lsfro
mhe
alth
y do
nors
wer
e lo
aded
with
the
seru
m s
ampl
es a
nd s
ubse
quen
tlyex
pose
d to
eith
er n
ativ
e Ar
ah2
/6 o
rm
odifi
ed A
rah2
/6 (R
A-Ar
ah2
/6).
•Pot
ency
was
als
o ev
alua
ted
appl
ying
aso
lid p
hase
IgE-
bind
ing
assa
ype
rform
ed w
ith in
divi
dual
pat
ient
ser
a.
Res
ults
: Al
l tes
ted
patie
nt s
era
disp
laye
d a
redu
ced
resp
onse
to R
A-Ar
ah2
/6:
•A m
ean
redu
ctio
n of
100
-fol
d w
asfo
und
(rang
e: 3
to <
10,0
00 fo
ld) u
sing
both
the
RBL
SX3
8 an
d th
e in
dire
ctba
soph
ilas
say.
•T
he fo
ld re
duct
ion
obse
rved
per
pat
ient
corre
late
d be
twee
n th
e tw
o m
edia
tor
rele
ase
test
s.
•Ser
a fo
r whi
ch th
e m
odifi
catio
n ha
d th
ela
rges
t effe
ct o
n th
e ac
tivity
in m
edia
tor
rele
ase
assa
ys a
lso
show
ed th
e la
rges
t re
duct
ion
in p
oten
cy u
sing
the
solid
-ph
ase
IgE
bind
ing
test
.
A.
Mw
1
2
B.N
ativ
e co
nglu
tinsh
ows
a ty
pica
l sp
ectru
m fo
r pro
tein
s co
nsis
ting
mai
nly
of a
lpha
-hel
ices
(min
imal
ellip
ticity
valu
es a
t 208
and
222
nm
) but
als
o
beta
-stru
ctur
e. In
con
trast
, RA
show
s a
typi
cal s
pect
rum
for u
nfol
ded
prot
ein.
St
rong
dec
reas
e of
ellip
ticity
at 2
22 n
m
indi
cate
s lo
ss o
f alp
ha-h
elic
es a
nd
incr
ease
of r
ando
m c
oil s
truct
ure.
Thi
s ca
n be
exp
lain
ed b
y th
e fa
ct th
at th
e di
sulp
hide
brid
ges
have
bee
n cl
eave
d.
200
210
220
230
240
250
260
-15
-10-5051015
Nat
ive
RA
Wav
elen
gth
(nm
)
CD (mDeg)
The
far-U
V C
D s
pect
ra o
f nat
ive
and
mod
ified
con
glut
in.
117
199
88179780219
545
505
194
1112
290
293
2421
280337306
533
138
101573
Tota
l IgE
226514787
1681285
4929500
1352180
591
135638716539
Pean
ut-
Spec
ific
IgE
2,0
D11
40,
7D
107
0D
105
6,4
D10
30,
4D
98
9,4
D81
9,7
D80
1,5
D77
0,5
D74
0D
70
3,4
D69
0D
68
0D
650,
8D
640,
3D
620,
5D
60
0,3
D50
0,4
D48
0,2
D44
0D
19
Rel
ativ
e po
tenc
y (%
) Sa
mpl
e
0.01
0.1
110
100
1000
0255075100
D10
3
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.1
110
100
1000
0102030Se
rum
poo
l
nativ
e
RA
ng/m
l
net % degranulationFigu
re 3
: Deg
ranu
latio
nof
RB
L SX
-38
cells
to n
ativ
e or
mod
ified
Ara
h2/6
1010
010
000102030
D65
nativ
e
RA
ng/m
l
net degranulation
110
100
1000
0102030D8
0
nativ
e
RA
ng/m
l
net degranulation
A.T
he n
ativ
e (1
) and
RA
(2)
Ara
h2/h
6 pr
epar
atio
ns
were
ana
lyzed
by
SDS-
PAG
E. T
he p
rote
ins
were
vi
sual
ized
usi
ng C
oom
assi
e-st
aini
ng.
0.01
110
010
000
0102030405060D
65
nativ
e
RA
ng/m
l
release (%)
0.01
110
010
000
0102030405060D
68
nativ
e
RA
ng/m
l
release (%)
0.01
110
010
000
0102030405060D
64 nativ
e
RA
ng/m
l
release (%)
Figu
re 4
: Deg
ranu
latio
nof
hum
an b
asop
hils
to n
ativ
e or
mod
ified
Ara
h2/6
RBL
SX-
38 c
ells
wer
e se
nsitiz
ed e
ither
with
a s
erum
poo
l (A)
or i
ndiv
idua
l ser
a (B
and
C) a
nd tr
igge
red
with
nat
ive
Ara
h 2/
6 or
RA-
Ara
h 2/
6.
Dat
a ar
e sh
own
as “n
et”d
egra
nula
tion
(tota
l deg
ranu
latio
nm
inus
bac
kgro
und)
.
In th
e in
dire
ct B
AT, h
uman
bas
ophi
lswe
re s
ensit
ized
with
indi
vidu
al s
era
and
trig
gere
d wi
th n
ativ
e Ar
ah
2/6
or R
A-Ar
ah
2/6.
Dat
a ar
e sh
own
as %
rele
ase
(in re
latio
n to
tota
l deg
ranu
latio
nm
inus
bac
kgro
und)
.
B.
Figu
re 1
. Cha
ract
eris
tics
of p
eanu
t pre
para
tions
Figu
re 2
: Sol
id p
hase
IgE-
bind
ing
test
0.01
0.1
110
100
1000
0255075100
D64
nativ
eR
A
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.01
0.1
110
100
1000
0255075100
D10
5
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.01
0.1
110
100
1000
0255075100
D11
4
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
IgE
bind
ing
pote
ncy
of th
e na
tive
and
mod
ified
prep
arat
ions
wer
e de
term
ined
in 2
0 U
S pa
tient
s wi
th
esta
blis
hed
clin
ical
alle
rgy
for p
eanu
t.Th
e Ig
Ebi
ndin
g is
exp
ress
ed a
s re
lativ
e po
tenc
y (R
P) c
ompa
red
to a
n in
-hou
se re
fere
nce
alle
rgen
pre
para
tion.
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
H. v
an d
er K
leij1 ,
H. W
arm
enho
ven1 ,
D. V
erba
rt1 ,
Q. W
ang2 ,
R. v
an R
ee3 ,
S.C
. Dre
skin
2 , S.
J. K
oppe
lman
1
1H
AL A
llerg
y B
V, L
eide
n, T
he N
ethe
rland
s, 2
Uni
vers
ity o
f Col
orad
o D
enve
r Sch
ool o
f Med
icin
e, A
uror
a, C
O, U
SA3 Ex
perim
enta
l Im
mun
olog
y, A
cade
mic
Med
ical
Cen
tre,
Am
ster
dam
, The
Net
herla
nds
62 -
Alle
rgen
ic p
oten
cy o
f che
mic
ally
mod
ified
Ara
h2/h
6 ev
alua
ted
with
bas
ophi
lact
ivat
ion,
RB
L-SX
38 a
ctiv
atio
n, a
nd
solid
pha
se Ig
E-im
mun
oass
ay
Bac
kgro
und
& A
im:
•Pea
nuts
are
one
of t
he m
ost c
omm
onfo
ods
resp
onsi
ble
for f
ood-
indu
ced
anap
hyla
xis
in a
dults
.•P
eanu
t sIT
with
aqu
eous
pea
nut e
xtra
ctha
s fa
iled
beca
use
of s
afet
y is
sues
.•A
mod
ified
pre
para
tion
of th
e 2
maj
orpe
anut
alle
rgen
s Ar
ah2
and
Ara
h6 is
bein
g in
vest
igat
ed fo
r its
pot
entia
l use
in im
mun
othe
rapy
IgE
inhi
bitio
n cu
rves
of t
he n
ativ
e an
d m
odifie
d Ar
ah2
/6 o
f ind
ivid
ual
patie
nts.
Te
st s
ampl
es c
ompe
te w
ith n
ativ
e Ar
ah2
/6 c
oate
d to
ELI
SA p
late
s fo
r bin
ding
to
ser
um Ig
E. In
crea
sing
con
cent
ratio
ns o
f tes
t sam
ple
resu
lt in
inhi
bitio
n of
bi
ndin
g. T
he a
mou
nt o
f the
IgE
bind
ing
in p
rese
nce
of A
rah2
/6 c
ompa
red
to
that
in a
bsen
ce o
f Ara
h2/6
is c
alcu
late
d as
per
cent
age
inhi
bitio
n.
Tabl
e1:
Ser
olog
yof
recr
uite
dpa
tient
s
Con
clus
ion:
The
alle
rgen
ic p
oten
cy o
f mod
ified
Ara
h2/6
is a
bout
100
-fold
low
er th
an th
at o
fna
tive
Ara
h2/A
ra h
6, m
akin
g th
em m
ore
suita
ble
cand
idat
es fo
r sub
cuta
neou
sim
mun
othe
rapy
.
Met
hods
:•A
rah2
and
Ara
h6 w
ere
purif
ied
and
chem
ical
ly m
odifi
ed b
y m
eans
of
redu
ctio
n (R
) of d
isul
fide
bond
s an
dsu
bseq
uent
alk
ylat
ion
(A) o
f fre
e C
ys.
Prep
arat
ions
wer
e ch
arac
teriz
ed b
ySD
S-PA
GE
and
by fa
r-UV
CD
spec
tosc
ropy
.•T
wen
ty p
atie
nts
from
the
grea
ter
Den
ver a
rea
wer
e re
crui
ted
and
seru
mw
as c
olle
cted
. •R
BL S
X38
cells
and
bas
ophi
lsfro
mhe
alth
y do
nors
wer
e lo
aded
with
the
seru
m s
ampl
es a
nd s
ubse
quen
tlyex
pose
d to
eith
er n
ativ
e Ar
ah2
/6 o
rm
odifi
ed A
rah2
/6 (R
A-Ar
ah2
/6).
•Pot
ency
was
als
o ev
alua
ted
appl
ying
aso
lid p
hase
IgE-
bind
ing
assa
ype
rform
ed w
ith in
divi
dual
pat
ient
ser
a.
Res
ults
: Al
l tes
ted
patie
nt s
era
disp
laye
d a
redu
ced
resp
onse
to R
A-Ar
ah2
/6:
•A m
ean
redu
ctio
n of
100
-fol
d w
asfo
und
(rang
e: 3
to <
10,0
00 fo
ld) u
sing
both
the
RBL
SX3
8 an
d th
e in
dire
ctba
soph
ilas
say.
•T
he fo
ld re
duct
ion
obse
rved
per
pat
ient
corre
late
d be
twee
n th
e tw
o m
edia
tor
rele
ase
test
s.
•Ser
a fo
r whi
ch th
e m
odifi
catio
n ha
d th
ela
rges
t effe
ct o
n th
e ac
tivity
in m
edia
tor
rele
ase
assa
ys a
lso
show
ed th
e la
rges
t re
duct
ion
in p
oten
cy u
sing
the
solid
-ph
ase
IgE
bind
ing
test
.
A.
Mw
1
2
B.N
ativ
e co
nglu
tinsh
ows
a ty
pica
l sp
ectru
m fo
r pro
tein
s co
nsis
ting
mai
nly
of a
lpha
-hel
ices
(min
imal
ellip
ticity
valu
es a
t 208
and
222
nm
) but
als
o
beta
-stru
ctur
e. In
con
trast
, RA
show
s a
typi
cal s
pect
rum
for u
nfol
ded
prot
ein.
St
rong
dec
reas
e of
ellip
ticity
at 2
22 n
m
indi
cate
s lo
ss o
f alp
ha-h
elic
es a
nd
incr
ease
of r
ando
m c
oil s
truct
ure.
Thi
s ca
n be
exp
lain
ed b
y th
e fa
ct th
at th
e di
sulp
hide
brid
ges
have
bee
n cl
eave
d.
200
210
220
230
240
250
260
-15
-10-5051015
Nat
ive
RA
Wav
elen
gth
(nm
)
CD (mDeg)
The
far-U
V C
D s
pect
ra o
f nat
ive
and
mod
ified
con
glut
in.
117
199
88179780219
545
505
194
1112
290
293
2421
280337306
533
138
101573
Tota
l IgE
226514787
1681285
4929500
1352180
591
135638716539
Pean
ut-
Spec
ific
IgE
2,0
D11
40,
7D
107
0D
105
6,4
D10
30,
4D
98
9,4
D81
9,7
D80
1,5
D77
0,5
D74
0D
70
3,4
D69
0D
68
0D
650,
8D
640,
3D
620,
5D
60
0,3
D50
0,4
D48
0,2
D44
0D
19
Rel
ativ
e po
tenc
y (%
) Sa
mpl
e
0.01
0.1
110
100
1000
0255075100
D10
3
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.1
110
100
1000
0102030Se
rum
poo
l
nativ
e
RA
ng/m
l
net % degranulationFigu
re 3
: Deg
ranu
latio
nof
RB
L SX
-38
cells
to n
ativ
e or
mod
ified
Ara
h2/6
1010
010
000102030
D65
nativ
e
RA
ng/m
l
net degranulation
110
100
1000
0102030D8
0
nativ
e
RA
ng/m
l
net degranulation
A.T
he n
ativ
e (1
) and
RA
(2)
Ara
h2/h
6 pr
epar
atio
ns
were
ana
lyzed
by
SDS-
PAG
E. T
he p
rote
ins
were
vi
sual
ized
usi
ng C
oom
assi
e-st
aini
ng.
0.01
110
010
000
0102030405060D
65
nativ
e
RA
ng/m
l
release (%)
0.01
110
010
000
0102030405060D
68
nativ
e
RA
ng/m
l
release (%)
0.01
110
010
000
0102030405060D
64 nativ
e
RA
ng/m
l
release (%)
Figu
re 4
: Deg
ranu
latio
nof
hum
an b
asop
hils
to n
ativ
e or
mod
ified
Ara
h2/6
RBL
SX-
38 c
ells
wer
e se
nsitiz
ed e
ither
with
a s
erum
poo
l (A)
or i
ndiv
idua
l ser
a (B
and
C) a
nd tr
igge
red
with
nat
ive
Ara
h 2/
6 or
RA-
Ara
h 2/
6.
Dat
a ar
e sh
own
as “n
et”d
egra
nula
tion
(tota
l deg
ranu
latio
nm
inus
bac
kgro
und)
.
In th
e in
dire
ct B
AT, h
uman
bas
ophi
lswe
re s
ensit
ized
with
indi
vidu
al s
era
and
trig
gere
d wi
th n
ativ
e Ar
ah
2/6
or R
A-Ar
ah
2/6.
Dat
a ar
e sh
own
as %
rele
ase
(in re
latio
n to
tota
l deg
ranu
latio
nm
inus
bac
kgro
und)
.
B.
Figu
re 1
. Cha
ract
eris
tics
of p
eanu
t pre
para
tions
Figu
re 2
: Sol
id p
hase
IgE-
bind
ing
test
0.01
0.1
110
100
1000
0255075100
D64
nativ
eR
A
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.01
0.1
110
100
1000
0255075100
D10
5
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
0.01
0.1
110
100
1000
0255075100
D11
4
nativ
e
RA
Inhi
bito
r con
cent
ratio
n (μ μμμ
g/m
l)
Inhibition (%)
IgE
bind
ing
pote
ncy
of th
e na
tive
and
mod
ified
prep
arat
ions
wer
e de
term
ined
in 2
0 U
S pa
tient
s wi
th
esta
blis
hed
clin
ical
alle
rgy
for p
eanu
t.Th
e Ig
Ebi
ndin
g is
exp
ress
ed a
s re
lativ
e po
tenc
y (R
P) c
ompa
red
to a
n in
-hou
se re
fere
nce
alle
rgen
pre
para
tion.
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
H. v
an d
er K
leij1 ,
H. W
arm
enho
ven1 ,
D. V
erba
rt1 ,
Q. W
ang2 ,
R. v
an R
ee3 ,
S.C
. Dre
skin
2 , S.
J. K
oppe
lman
1
EAAC
I Con
gres
s 20
12In
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er is
an
empl
oyee
of H
AL A
llerg
y.
T h e c o nt e nt o f a l l e rge n s Ara h 1, Ara h 2, Ara h 3 a n d Ara h 6 in d i f f e r e nt p ea n u t cu l t ivars c o m m o n l y c o n su m e d in Eu ro p e a n d t h e U SA
22
S.J. Koppelman(1,2), D. Apostolovic (1), H. Warmenhoven (1), D. Verbart (1), S. L. Taylor (2), T. Isleib (3), S. Maleki (3).
(1) HAL Allergy BV, Leiden, The Netherlands, (2) Food Allergy Research and Resources Programme, University of Nebraska, Lincoln NE, USA,
(3) US Dept of Agriculture, New Orleans LA, USA.
RationalePeanut allergens Ara h2 and Ara h6 are considered to be the most relevant peanut allergens, while Ara h1 and to a lesser extent Ara h3, may play an important role as well. There are four main peanut cultivars commonly consumed in the Western world and their major allergen content is only partially known. Also, the effect of roasting on the extractability of major allergens is not well known.
MethodsTwelve peanuts samples comprising the four main cultivars Runner, Spanish, Valencia, and Virginia, were obtained and part of these sample were roasted (140 °C for 15 minutes in a preheated circulating hot air oven). Extracts were made and total protein content was measured using Bradford analysis. SDS-PAGE was used to visualize the protein bands patterns and the content of Ara h1, Ara h2, Ara h3, and Ara h6 was quantified using rp-HPLC. A solid phase IgE binding test was applied for evaluation of allergenic potency.
ResultsProtein concentration in varietal extracts varied from 13.9 to 21.2 mg/ml, and roasting reduced the extractability by 22 to 50%. Band patterns indicated that the four allergens of interest were present in samples of all varieties. The ranges of individual allergens in the tested samples were as follows: Ara h1: 11-32%; Ara h2: 7-16%; Ara h3: 38-76%; and Ara h6: 4-14%. In extracts of roasted peanuts, the relative content of Ara h1 was lower while that of Ara h3 was higher compared to non-roasted. The relative content of Ara h2 and Ara h6 did not vary between raw and roasted. The allergenic potencies compared to in-house reference extracts varied from 99 to 139% for the raw samples, and roasting increased the potency only marginally with an increase of 14 to 30% relative to non-roasted counterparts.
ConclusionsThe main peanut cultivars consumed in the Western world all contain the main peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 with a slight variation of the relative content of these allergens between the different cultivars. Roasting had minimal effects on allergenic potency with the sera used in this study.
EAACI, 16-20 June 2012, GenevaAbstract number: 1463, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 73 - Molecular components in allergy diagnosis
23
Poster Session 73 - Molecular components in allergy diagnosis
T h e c o nt e nt o f a l l e rge n s Ara h 1, Ara h 2, Ara h 3 a n d Ara h 6 in d i f f e r e nt p ea n u t cu l t ivars c o m m o n l y c o n su m e d in Eu ro p e a n d t h e U SA
1 HAL
Alle
rgy
BV,
Lei
den,
The
Net
herla
nds,
2 Foo
d Al
lerg
y R
esea
rch
and
Reso
urce
s Pr
ogra
m, U
nive
rsity
of
Nebr
aska
, Lin
coln
, NE,
USA
, 3 US
Dep
t of A
gric
ultu
re, N
ew O
rlean
s LA
, USA
Figu
re 3
: Pot
ency
det
erm
inat
ion
of th
e va
rious
ext
ract
s us
ing
hum
an Ig
EEL
ISA.
Extra
cts
of th
e pe
anut
ext
ract
s we
re d
ilute
d in
a
rang
e of
0.0
1 to
10
g/m
l. Ex
ampl
es o
f of V
irgin
ia
pean
ut (G
reen
: Bra
ntle
y ra
w; Y
ello
w: G
rego
ry
raw;
Red
: NC
-v11
roas
ted;
Blu
e: N
C-v
11 ra
w) a
re
show
n in
com
paris
on to
the
IHR
.Th
e pl
asm
a po
ol u
sed
cons
iste
d of
14
plas
mas
fro
m p
atie
nts,
all c
linic
ally
alle
rgic
to p
eanu
t, m
ean
IgE
to p
eanu
36,9
kU
/L.
1463
-Th
e co
nten
t of a
llerg
ens
Ara
h1, A
rah2
, Ar
ah3
and
Ara
h6 in
diff
eren
t pea
nut c
ultiv
ars
com
mon
ly c
onsu
med
in E
urop
e an
d th
e U
SA
Bac
kgro
und
& A
im:
Pean
utal
lerg
ens
Ara
h2 a
nd A
ra h
6 ar
e co
nsid
ered
to b
eth
e m
ost
rele
vant
pea
nut
alle
rgen
s, w
hile
Ara
h1 a
nd t
o a
less
erex
tent
Ara
h3, m
aypl
ayan
impo
rtant
role
as
wel
l. Th
ere
are
four
mai
npe
anut
culti
vars
co
mm
only
cons
umed
in t
he W
este
rn w
orld
and
thei
rm
ajor
al
lerg
enco
nten
t is
on
lypa
rtial
lykn
own.
Als
o, t
he e
ffect
of
roas
ting
onth
e ex
tract
abilit
yof
maj
or a
llerg
ens
is n
otw
ellk
now
n.
Tabl
e 1:
Ext
ract
able
pro
tein
, con
tent
of i
ndiv
idua
l alle
rgen
s an
d Ig
E-bi
ndin
g po
tenc
y of
the
vario
us p
eanu
t sam
ples
Figu
re 2
: Det
erm
inat
ion
of p
eanu
t alle
rgen
s by
rp-H
PLC
. A.
Exa
mpl
e of
chr
omat
ogra
m o
f sam
ples
of V
irgin
ia (G
reen
: Bra
ntle
y ra
w; Y
ello
w: G
rego
ry ra
w; R
ed: N
C-v
11 ro
aste
d; B
lue:
NC
-v11
raw)
. In
divi
dual
cur
ves
were
shi
fted
verti
cally
, ran
ge fo
r eac
h sa
mpl
eis
0-8
00 m
AU.
B. C
hrom
atog
ram
of p
urifie
d pe
anut
alle
rgen
refe
renc
es A
rah1
: blu
e; A
rah2
: red
; Ara
h3: y
ello
w; A
rah6
: gre
en.
Col
umn:
X-B
ridge
BEH
Phe
nyl;
3.5
m; 1
35Å;
[2.1
x 1
50 m
m].
Tem
pera
ture
: 30
1 C
. Sam
ple
volu
me:
0.0
2 m
l, flo
w ra
te: 0
.3 m
l/min
. El
uent
A: 0
.1 %
TFA
in M
Q W
ater
; elu
ent B
: 0.0
85 %
TFA
in M
eOH
. Abs
orba
nces
were
mea
sure
d by
dio
de a
rray
and
reco
rded
for 2
15
and
280
nm. S
ampl
e si
ze fo
r ext
ract
s: 2
0 lo
f 1 m
g/m
l sol
utio
n.
Con
clus
ion:
The
mai
n pe
anut
cul
tivar
s co
nsum
ed in
the
Wes
tern
wor
ld a
ll co
ntai
n th
e m
ain
pean
ut
alle
rgen
s Ar
ah1
, Ara
h2, A
rah3
, and
Ara
h6
with
a s
light
var
iatio
n of
the
rela
tive
cont
ent
of
thes
e al
lerg
ens
betw
een
the
diffe
rent
cu
ltiva
rs.
Roa
stin
g ha
d m
inim
al e
ffect
s on
al
lerg
enic
pot
ency
with
the
sera
use
d in
this
st
udy.
Met
hods
:Tw
elve
pea
nuts
sam
ples
com
pris
ing
the
four
m
ain
culti
vars
Run
ner,
Span
ish,
Val
enci
a,
and
Virg
inia
, w
ere
obta
ined
an
d pa
rt of
th
ese
sam
ple
wer
e ro
aste
d (1
40
Cfo
r 15
m
inut
es i
n a
preh
eate
d ci
rcul
atin
g ho
t ai
r ov
en).
Extra
cts
wer
e m
ade
and
tota
l pro
tein
co
nten
t w
as
mea
sure
d us
ing
Brad
ford
an
alys
is.
SDS-
PAG
E w
as u
sed
to v
isua
lize
the
prot
ein
band
s pa
ttern
s an
d th
e co
nten
t of
Ara
h1, A
rah2
, Ara
h3, a
nd A
rah6
was
qu
antif
ied
usin
g rp
-HPL
C. A
sol
id p
hase
IgE
bind
ing
test
was
app
lied
for
eval
uatio
n of
al
lerg
enic
pot
ency
.An
extra
ct f
rom
Virg
inia
pe
anut
s(ra
w, G
reer
, Len
oir
NC
, USA
) w
as
used
as in
-hou
sere
fere
nce
(IHR
).
Res
ults
: Pr
otei
n co
ncen
tratio
n in
va
rieta
l ex
tract
s va
ried
from
13.
9 to
21.
2 m
g/m
l, an
d ro
astin
g re
duce
d th
e ex
tract
abilit
y by
22
to 5
0%.
Band
pa
ttern
s in
dica
ted
that
th
e fo
ur
alle
rgen
s of
in
tere
st
wer
e pr
esen
t in
sa
mpl
es
of
all
varie
ties.
Th
e ra
nges
of
in
divi
dual
alle
rgen
s in
the
tes
ted
sam
ples
w
ere
as fo
llow
s: A
rah1
: 11-
32%
; Ara
h2: 7
-16
%; A
rah3
: 38-
76%
; and
Ara
h6: 4
-14%
. In
extra
cts
of
roas
ted
pean
uts,
th
e re
lativ
e co
nten
t of A
rah1
was
low
er w
hile
that
of A
rah3
was
hig
her
com
pare
d to
non
-roas
ted.
Th
e re
lativ
e co
nten
t of
Ara
h2 a
nd A
rah6
di
d no
t var
y be
twee
n ra
w a
nd r
oast
ed. T
he
alle
rgen
ic
pote
ncie
s co
mpa
red
to in
-hou
se
refe
renc
e ex
tract
s va
ried
from
99
to 1
39%
fo
r the
raw
sam
ples
, and
roas
ting
incr
ease
d th
e po
tenc
y on
ly m
argi
nally
with
an
incr
ease
of
14
to
30
%
rela
tive
to
non-
roas
ted
coun
terp
arts
.
Figu
re 1
: SD
S-PA
GE
of th
e va
rious
pea
nut s
ampl
es
and
purif
ied
pean
ut a
llerg
en re
fere
nces
A. P
eanu
t sam
ple
anal
ysed
und
er re
duci
ng c
ondi
tions
. B
. Pea
nut s
ampl
e an
alys
ed u
nder
non
-redu
cing
con
ditio
ns.
1. B
io-R
ad M
olec
ular
wei
ght m
arke
r (kD
a)
2. L
aem
mlis
ampl
e bu
ffer
3. R
unne
r; G
AO6G
Run
ner (
Raw
) 4.
Run
ner;
Tiftg
uard
Run
ner (
Raw
)5.
Run
ner;
Flor
unne
r4/1
4 (R
aw)
6. R
unne
r; Fl
orun
ner4
/14
(Roa
sted
) 7.
Spa
nish
; Olin
Spa
nish
(Raw
) 8.
Spa
nish
; Olin
Spa
nish
(Roa
sted
) 9.
Val
encia
; Val
enci
a A
(Raw
) 10
. Val
enci
a; V
alen
cia
A (R
oast
ed)
11. V
irgin
ia; N
c-v1
1 Vi
rgin
ia (R
aw)
12. V
irgin
ia; N
c-v1
1 Vi
rgin
ia (R
oast
ed)
13. V
irgin
ia; G
rego
ry V
irgin
ia (R
aw)
14. V
irgin
ia; B
rant
ley
Virg
inia
(Raw
)
Purif
ied
alle
rgen
s Ar
ah1
, Ara
h2, A
rah3
, and
Ara
h6 w
ere
used
for b
and
assi
gnm
ent (
right
mar
gin)
.
A. B.
5 10
15
A215 (mAU)
Tim
e (m
in)
0
300
600
900
1200
5 10
15
A215 (mAU)
Tim
e (m
in)
B.
A. 800 0
0,0
25,0
50,0
75,0
100,
0 0,01
0,
10
1,00
10
,00
Inhibition (%)
Inhi
bito
r con
cent
ratio
n (µ
g/m
l)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
or p
erce
ived
conf
licts
of in
tere
st:
the
pres
ente
r was
an
empl
oyee
of H
AL A
llerg
ydu
ring
whe
nth
isre
sear
ch w
as c
ondu
cted
EAAC
I Con
gres
s 20
12
S.J.
Kop
pelm
an1,
2 , D
. Apo
stol
ovic
1 , H
. War
men
hove
n1, D
. Ver
bart
1 , S.
L. T
aylo
r2, T
. Isl
eib3
and
S. M
alek
i3
1 HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s, 2 F
ood
Alle
rgy
Rese
arch
and
Res
ourc
es P
rogr
am, U
nive
rsity
of
Nebr
aska
, Lin
coln
, NE,
USA
, 3 US
Dept
of A
gric
ultu
re, N
ew O
rlean
s LA
, USA
Figu
re 3
: Pot
ency
det
erm
inat
ion
of th
e va
rious
ext
ract
s us
ing
hum
an Ig
EEL
ISA.
Extra
cts
of th
e pe
anut
ext
ract
s we
re d
ilute
d in
a
rang
e of
0.0
1 to
10
g/m
l. Ex
ampl
es o
f of V
irgin
ia
pean
ut (G
reen
: Bra
ntle
y ra
w; Y
ello
w: G
rego
ry
raw;
Red
: NC
-v11
roas
ted;
Blu
e: N
C-v
11 ra
w) a
re
show
n in
com
paris
on to
the
IHR
.Th
e pl
asm
a po
ol u
sed
cons
isted
of 1
4 pl
asm
as
from
pat
ient
s, a
ll clin
ically
alle
rgic
to p
eanu
t, m
ean
IgE
to p
eanu
36,9
kU
/L.
1463
-Th
e co
nten
t of a
llerg
ens
Ara
h1, A
rah2
, Ar
ah3
and
Ara
h6 in
diff
eren
t pea
nut c
ultiv
ars
com
mon
ly c
onsu
med
in E
urop
e an
d th
e U
SA
Bac
kgro
und
& A
im:
Pean
utal
lerg
ens
Ara
h2 a
nd A
ra h
6 ar
e co
nsid
ered
to b
eth
e m
ost
rele
vant
pea
nut
alle
rgen
s, w
hile
Ara
h1 a
nd t
o a
less
erex
tent
Ara
h3, m
aypl
ayan
impo
rtant
role
as
wel
l. Th
ere
are
four
mai
npe
anut
culti
vars
co
mm
only
cons
umed
in t
he W
este
rn w
orld
and
thei
rm
ajor
al
lerg
enco
nten
t is
on
lypa
rtial
lykn
own.
Als
o, t
he e
ffect
of
roas
ting
onth
e ex
tract
abilit
yof
maj
or a
llerg
ens
is n
otw
ellk
now
n.
Tabl
e 1:
Ext
ract
able
pro
tein
, con
tent
of i
ndiv
idua
l alle
rgen
s an
d Ig
E-bi
ndin
g po
tenc
y of
the
vario
us p
eanu
t sam
ples
Figu
re 2
: Det
erm
inat
ion
of p
eanu
t alle
rgen
s by
rp-H
PLC
. A.
Exa
mpl
e of
chr
omat
ogra
m o
f sam
ples
of V
irgin
ia (G
reen
: Bra
ntle
y ra
w; Y
ello
w: G
rego
ry ra
w; R
ed: N
C-v
11 ro
aste
d; B
lue:
NC
-v11
raw)
. In
divi
dual
cur
ves
were
shi
fted
verti
cally
, ran
ge fo
r eac
h sa
mpl
eis
0-80
0 m
AU.
B. C
hrom
atog
ram
of p
urifie
d pe
anut
alle
rgen
refe
renc
es A
rah1
: blu
e; A
rah2
: red
; Ara
h3: y
ello
w; A
rah6
: gre
en.
Col
umn:
X-B
ridge
BEH
Phe
nyl;
3.5
m; 1
35Å;
[2.1
x 1
50 m
m].
Tem
pera
ture
: 30
1 C
. Sam
ple
volu
me:
0.0
2 m
l, flo
w ra
te: 0
.3 m
l/min
. El
uent
A: 0
.1 %
TFA
in M
Q W
ater
; elu
ent B
: 0.0
85 %
TFA
in M
eOH
. Abs
orba
nces
were
mea
sure
d by
dio
de a
rray
and
reco
rded
for 2
15
and
280
nm. S
ampl
e si
ze fo
r ext
ract
s: 2
0 lo
f 1 m
g/m
l sol
utio
n.
Con
clus
ion:
The
mai
n pe
anut
cul
tivar
s co
nsum
ed in
the
Wes
tern
wor
ld a
ll co
ntai
n th
e m
ain
pean
ut
alle
rgen
s Ar
ah1
, Ara
h2, A
rah3
, and
Ara
h6
with
a s
light
var
iatio
n of
the
rela
tive
cont
ent
of
thes
e al
lerg
ens
betw
een
the
diffe
rent
cu
ltiva
rs.
Roa
stin
g ha
d m
inim
al e
ffect
s on
al
lerg
enic
pot
ency
with
the
sera
use
d in
this
st
udy.
Met
hods
:Tw
elve
pea
nuts
sam
ples
com
pris
ing
the
four
m
ain
culti
vars
Run
ner,
Span
ish,
Val
enci
a,
and
Virg
inia
, w
ere
obta
ined
an
d pa
rt of
th
ese
sam
ple
wer
e ro
aste
d (1
40
Cfo
r 15
m
inut
es i
n a
preh
eate
d ci
rcul
atin
g ho
t ai
r ov
en).
Extra
cts
wer
e m
ade
and
tota
l pro
tein
co
nten
t w
as
mea
sure
d us
ing
Brad
ford
an
alys
is.
SDS-
PAG
E w
as u
sed
to v
isua
lize
the
prot
ein
band
s pa
ttern
s an
d th
e co
nten
t of
Ara
h1, A
rah2
, Ara
h3, a
nd A
rah6
was
qu
antif
ied
usin
g rp
-HPL
C. A
sol
id p
hase
IgE
bind
ing
test
was
app
lied
for
eval
uatio
n of
al
lerg
enic
pot
ency
.An
extra
ct f
rom
Virg
inia
pe
anut
s(ra
w, G
reer
, Len
oirN
C, U
SA )
was
us
edas
in-h
ouse
refe
renc
e(IH
R).
Res
ults
: Pr
otei
n co
ncen
tratio
n in
va
rieta
l ex
tract
s va
ried
from
13.
9 to
21.
2 m
g/m
l, an
d ro
astin
g re
duce
d th
e ex
tract
abilit
y by
22
to 5
0%.
Band
pa
ttern
s in
dica
ted
that
th
e fo
ur
alle
rgen
s of
in
tere
st
wer
e pr
esen
t in
sa
mpl
es
of
all
varie
ties.
Th
e ra
nges
of
in
divi
dual
alle
rgen
s in
the
tes
ted
sam
ples
w
ere
as fo
llow
s: A
rah1
: 11-
32%
; Ara
h2: 7
-16
%; A
rah3
: 38-
76%
; and
Ara
h6: 4
-14%
. In
extra
cts
of
roas
ted
pean
uts,
th
e re
lativ
e co
nten
t of A
rah1
was
low
er w
hile
that
of A
rah3
was
hig
her
com
pare
d to
non
-roas
ted.
Th
e re
lativ
e co
nten
t of
Ara
h2 a
nd A
rah6
di
d no
t var
y be
twee
n ra
w a
nd r
oast
ed. T
he
alle
rgen
ic
pote
ncie
s co
mpa
red
to in
-hou
se
refe
renc
e ex
tract
s va
ried
from
99
to 1
39%
fo
r the
raw
sam
ples
, and
roas
ting
incr
ease
d th
e po
tenc
y on
ly m
argi
nally
with
an
incr
ease
of
14
to
30
%
rela
tive
to
non-
roas
ted
coun
terp
arts
.
Figu
re 1
: SD
S-PA
GE
of th
e va
rious
pea
nut s
ampl
es
and
purif
ied
pean
ut a
llerg
en re
fere
nces
A. P
eanu
t sam
ple
anal
ysed
und
er re
ducin
g co
nditio
ns.
B. P
eanu
t sam
ple
anal
ysed
und
er n
on-re
ducin
g co
nditio
ns.
1. B
io-R
ad M
olec
ular
wei
ght m
arke
r (kD
a)
2. L
aem
mlis
ampl
e bu
ffer
3. R
unne
r; G
AO6G
Run
ner (
Raw)
4.
Run
ner;
Tiftg
uard
Runn
er (R
aw)
5. R
unne
r; Fl
orun
ner4
/14
(Raw
) 6.
Run
ner;
Flor
unne
r4/1
4 (R
oast
ed)
7. S
pani
sh; O
lin S
pani
sh (R
aw)
8. S
pani
sh; O
lin S
pani
sh (R
oast
ed)
9. V
alen
cia; V
alen
cia A
(Raw
) 10
. Val
encia
; Val
encia
A (R
oast
ed)
11. V
irgin
ia; N
c-v1
1 Vi
rgin
ia (R
aw)
12. V
irgin
ia; N
c-v1
1 Vi
rgin
ia (R
oast
ed)
13. V
irgin
ia; G
rego
ry V
irgin
ia (R
aw)
14. V
irgin
ia; B
rant
ley
Virg
inia
(Raw
)
Purif
ied
alle
rgen
s Ar
ah1
, Ara
h2, A
rah3
, and
Ara
h6 w
ere
used
for b
and
assig
nmen
t (rig
ht m
argi
n).
A. B.
5 10
15
A215 (mAU)
Tim
e (m
in)
0
300
600
900
1200
5 10
15
A215 (mAU)
Tim
e (m
in)
B.A. 80
0 0
0,0
25,0
50,0
75,0
100,
0 0,01
0,
10
1,00
10
,00
Inhibition (%)
Inhi
bito
r con
cent
ratio
n (µ
g/m
l)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
or p
erce
ived
conf
licts
of in
tere
st:
the
pres
ente
r was
an
empl
oyee
of H
AL A
llerg
ydu
ring
whe
nth
isre
sear
ch w
as c
ondu
cted
EAAC
I Con
gres
s 20
12
S.J.
Kop
pelm
an1,
2 , D
. Apo
stol
ovic
1 , H
. War
men
hove
n1, D
. Ver
bart
1 , S.
L. T
aylo
r2, T
. Isl
eib3
and
S. M
alek
i3
1 HAL
Alle
rgy
BV,
Lei
den,
The
Net
herla
nds,
2 Foo
d Al
lerg
y R
esea
rch
and
Reso
urce
s Pr
ogra
m, U
nive
rsity
of
Nebr
aska
, Lin
coln
, NE,
USA
, 3 US
Dep
t of A
gric
ultu
re, N
ew O
rlean
s LA
, USA
Figu
re 3
: Pot
ency
det
erm
inat
ion
of th
e va
rious
ext
ract
s us
ing
hum
an Ig
EEL
ISA.
Extra
cts
of th
e pe
anut
ext
ract
s we
re d
ilute
d in
a
rang
e of
0.0
1 to
10
g/m
l. Ex
ampl
es o
f of V
irgin
ia
pean
ut (G
reen
: Bra
ntle
y ra
w; Y
ello
w: G
rego
ry
raw;
Red
: NC
-v11
roas
ted;
Blu
e: N
C-v
11 ra
w) a
re
show
n in
com
paris
on to
the
IHR
.Th
e pl
asm
a po
ol u
sed
cons
iste
d of
14
plas
mas
fro
m p
atie
nts,
all c
linic
ally
alle
rgic
to p
eanu
t, m
ean
IgE
to p
eanu
36,9
kU
/L.
1463
-Th
e co
nten
t of a
llerg
ens
Ara
h1, A
rah2
, Ar
ah3
and
Ara
h6 in
diff
eren
t pea
nut c
ultiv
ars
com
mon
ly c
onsu
med
in E
urop
e an
d th
e U
SA
Bac
kgro
und
& A
im:
Pean
utal
lerg
ens
Ara
h2 a
nd A
ra h
6 ar
e co
nsid
ered
to b
eth
e m
ost
rele
vant
pea
nut
alle
rgen
s, w
hile
Ara
h1 a
nd t
o a
less
erex
tent
Ara
h3, m
aypl
ayan
impo
rtant
role
as
wel
l. Th
ere
are
four
mai
npe
anut
culti
vars
co
mm
only
cons
umed
in t
he W
este
rn w
orld
and
thei
rm
ajor
al
lerg
enco
nten
t is
on
lypa
rtial
lykn
own.
Als
o, t
he e
ffect
of
roas
ting
onth
e ex
tract
abilit
yof
maj
or a
llerg
ens
is n
otw
ellk
now
n.
Tabl
e 1:
Ext
ract
able
pro
tein
, con
tent
of i
ndiv
idua
l alle
rgen
s an
d Ig
E-bi
ndin
g po
tenc
y of
the
vario
us p
eanu
t sam
ples
Figu
re 2
: Det
erm
inat
ion
of p
eanu
t alle
rgen
s by
rp-H
PLC
. A.
Exa
mpl
e of
chr
omat
ogra
m o
f sam
ples
of V
irgin
ia (G
reen
: Bra
ntle
y ra
w; Y
ello
w: G
rego
ry ra
w; R
ed: N
C-v
11 ro
aste
d; B
lue:
NC
-v11
raw)
. In
divi
dual
cur
ves
were
shi
fted
verti
cally
, ran
ge fo
r eac
h sa
mpl
eis
0-8
00 m
AU.
B. C
hrom
atog
ram
of p
urifie
d pe
anut
alle
rgen
refe
renc
es A
rah1
: blu
e; A
rah2
: red
; Ara
h3: y
ello
w; A
rah6
: gre
en.
Col
umn:
X-B
ridge
BEH
Phe
nyl;
3.5
m; 1
35Å;
[2.1
x 1
50 m
m].
Tem
pera
ture
: 30
1 C
. Sam
ple
volu
me:
0.0
2 m
l, flo
w ra
te: 0
.3 m
l/min
. El
uent
A: 0
.1 %
TFA
in M
Q W
ater
; elu
ent B
: 0.0
85 %
TFA
in M
eOH
. Abs
orba
nces
were
mea
sure
d by
dio
de a
rray
and
reco
rded
for 2
15
and
280
nm. S
ampl
e si
ze fo
r ext
ract
s: 2
0 lo
f 1 m
g/m
l sol
utio
n.
Con
clus
ion:
The
mai
n pe
anut
cul
tivar
s co
nsum
ed in
the
Wes
tern
wor
ld a
ll co
ntai
n th
e m
ain
pean
ut
alle
rgen
s Ar
ah1
, Ara
h2, A
rah3
, and
Ara
h6
with
a s
light
var
iatio
n of
the
rela
tive
cont
ent
of
thes
e al
lerg
ens
betw
een
the
diffe
rent
cu
ltiva
rs.
Roa
stin
g ha
d m
inim
al e
ffect
s on
al
lerg
enic
pot
ency
with
the
sera
use
d in
this
st
udy.
Met
hods
:Tw
elve
pea
nuts
sam
ples
com
pris
ing
the
four
m
ain
culti
vars
Run
ner,
Span
ish,
Val
enci
a,
and
Virg
inia
, w
ere
obta
ined
an
d pa
rt of
th
ese
sam
ple
wer
e ro
aste
d (1
40
Cfo
r 15
m
inut
es i
n a
preh
eate
d ci
rcul
atin
g ho
t ai
r ov
en).
Extra
cts
wer
e m
ade
and
tota
l pro
tein
co
nten
t w
as
mea
sure
d us
ing
Brad
ford
an
alys
is.
SDS-
PAG
E w
as u
sed
to v
isua
lize
the
prot
ein
band
s pa
ttern
s an
d th
e co
nten
t of
Ara
h1, A
rah2
, Ara
h3, a
nd A
rah6
was
qu
antif
ied
usin
g rp
-HPL
C. A
sol
id p
hase
IgE
bind
ing
test
was
app
lied
for
eval
uatio
n of
al
lerg
enic
pot
ency
.An
extra
ct f
rom
Virg
inia
pe
anut
s(ra
w, G
reer
, Len
oir
NC
, USA
) w
as
used
as in
-hou
sere
fere
nce
(IHR
).
Res
ults
: Pr
otei
n co
ncen
tratio
n in
va
rieta
l ex
tract
s va
ried
from
13.
9 to
21.
2 m
g/m
l, an
d ro
astin
g re
duce
d th
e ex
tract
abilit
y by
22
to 5
0%.
Band
pa
ttern
s in
dica
ted
that
th
e fo
ur
alle
rgen
s of
in
tere
st
wer
e pr
esen
t in
sa
mpl
es
of
all
varie
ties.
Th
e ra
nges
of
in
divi
dual
alle
rgen
s in
the
tes
ted
sam
ples
w
ere
as fo
llow
s: A
rah1
: 11-
32%
; Ara
h2: 7
-16
%; A
rah3
: 38-
76%
; and
Ara
h6: 4
-14%
. In
extra
cts
of
roas
ted
pean
uts,
th
e re
lativ
e co
nten
t of A
rah1
was
low
er w
hile
that
of A
rah3
was
hig
her
com
pare
d to
non
-roas
ted.
Th
e re
lativ
e co
nten
t of
Ara
h2 a
nd A
rah6
di
d no
t var
y be
twee
n ra
w a
nd r
oast
ed. T
he
alle
rgen
ic
pote
ncie
s co
mpa
red
to in
-hou
se
refe
renc
e ex
tract
s va
ried
from
99
to 1
39%
fo
r the
raw
sam
ples
, and
roas
ting
incr
ease
d th
e po
tenc
y on
ly m
argi
nally
with
an
incr
ease
of
14
to
30
%
rela
tive
to
non-
roas
ted
coun
terp
arts
.
Figu
re 1
: SD
S-PA
GE
of th
e va
rious
pea
nut s
ampl
es
and
purif
ied
pean
ut a
llerg
en re
fere
nces
A. P
eanu
t sam
ple
anal
ysed
und
er re
duci
ng c
ondi
tions
. B
. Pea
nut s
ampl
e an
alys
ed u
nder
non
-redu
cing
con
ditio
ns.
1. B
io-R
ad M
olec
ular
wei
ght m
arke
r (kD
a)
2. L
aem
mlis
ampl
e bu
ffer
3. R
unne
r; G
AO6G
Run
ner (
Raw
) 4.
Run
ner;
Tiftg
uard
Run
ner (
Raw
)5.
Run
ner;
Flor
unne
r4/1
4 (R
aw)
6. R
unne
r; Fl
orun
ner4
/14
(Roa
sted
) 7.
Spa
nish
; Olin
Spa
nish
(Raw
) 8.
Spa
nish
; Olin
Spa
nish
(Roa
sted
) 9.
Val
encia
; Val
enci
a A
(Raw
) 10
. Val
enci
a; V
alen
cia
A (R
oast
ed)
11. V
irgin
ia; N
c-v1
1 Vi
rgin
ia (R
aw)
12. V
irgin
ia; N
c-v1
1 Vi
rgin
ia (R
oast
ed)
13. V
irgin
ia; G
rego
ry V
irgin
ia (R
aw)
14. V
irgin
ia; B
rant
ley
Virg
inia
(Raw
)
Purif
ied
alle
rgen
s Ar
ah1
, Ara
h2, A
rah3
, and
Ara
h6 w
ere
used
for b
and
assi
gnm
ent (
right
mar
gin)
.
A. B.
5 10
15
A215 (mAU)
Tim
e (m
in)
0
300
600
900
1200
5 10
15
A215 (mAU)
Tim
e (m
in)
B.
A. 800 0
0,0
25,0
50,0
75,0
100,
0 0,01
0,
10
1,00
10
,00
Inhibition (%)
Inhi
bito
r con
cent
ratio
n (µ
g/m
l)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
or p
erce
ived
conf
licts
of in
tere
st:
the
pres
ente
r was
an
empl
oyee
of H
AL A
llerg
ydu
ring
whe
nth
isre
sear
ch w
as c
ondu
cted
EAAC
I Con
gres
s 20
12
S.J.
Kop
pelm
an1,
2 , D
. Apo
stol
ovic
1 , H
. War
men
hove
n1, D
. Ver
bart
1 , S.
L. T
aylo
r2, T
. Isl
eib3
and
S. M
alek
i3
EAAC
I Con
gres
s 20
12In
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er w
as a
n em
ploy
ee o
f HAL
Alle
rgy
whe
n th
is re
sear
ch w
as c
ondu
cted
.
Co m p aris o n o f a nt i b o d y - b a s e d a ss ay w i t h p h y sic o c h e mic a l a ss ay s f o r m o ni t o ring t h e s t a b il i t y o f a l u m - a ds o r b e d mi t e a l l e rg o i d
24
D. Luykx, E. Kerkvliet, J. de Bruijn, N. Sinnige, R. van den Hout.
HAL Allergy BV, Leiden, The Netherlands.
BackgroundAn IgG inhibition assay has been developed previously to monitor the stability of alum-adsorbed mite allergoids in a mite allergy vaccine (ALU-M). In addition, two physicochemical methods have been developed to monitor the structural stability of the mite vaccine. These methods concern circular dichroism (CD) and front-face fluorescence spectroscopy (FF-fluorescence). In order to find out whether there is a relation between the antibody-based assay and physicochemical methods, temperature-stressed samples were analysed with both types of methods and the results were compared.
MethodsALU-M: D. pteronyssinus and D. farinae mite extracts were modified with glutaraldehyde and adsorbed onto aluminium hydroxide. After adsorption the species were mixed. IgG inhibition assay: Mite allergoid specific IgG antibodies were used to determine the 50% IgG inhibition value. CD: Far-UV CD spectra were recorded from 195 to 260 nm while stirring the sample suspension. FF-fluorescence: Fluorescence emission spectra were recorded from 290 to 400 nm (excitation at 280 nm).
ResultsStorage of the ALU-M vaccine at various elevated temperatures resulted in increased IgG inhibition values and spectral alterations with CD and FF-fluorescence, meaning structural alterations of the adsorbed protein. The increase of the 50% IgG inhibition value indicates a reduction of IgG binding to the epitopes. The spectral alterations observed with CD indicate partial loss of α-helices and increase of β-structure at higher temperatures. The spectral alterations observed with FF-fluorescence indicate formation of a more rigid protein structure at higher temperatures. When the temperature stress stability data obtained with the three methods were compared, a relation was observed between the IgG inhibition assay and CD assay by means of the CD-ratio 207/222 nm. Samples stored at elevated temperatures showed a similar trend with respect to IgG inhibition and CD ratio; both values increased in time.
ConclusionBy performing stress stability studies on ALU-M it was shown that the IgG inhibition assay and physicochemical assays were stability indicating. The results obtained with the IgG inhibition assay and CD assay relate well suggesting a relation between the IgG binding and secondary protein structure of the adsorbed mite allergoid.
EAACI, 16-20 June 2012, GenevaAbstract number: 278, Session date and time: Monday 18 June; 10:45 - 12:15 Session title: PDS 5 - Immunotherapy: vaccines and mechanisms
25
PDS 5 - Immunotherapy: vaccines and mechanisms
Co m p aris o n o f a nt i b o d y - b a s e d a ss ay w i t h p h y sic o c h e mic a l a ss ay s f o r m o ni t o ring t h e s t a b il i t y o f a l u m - a ds o r b e d mi t e a l l e rg o i d
278
-Com
paris
on o
f ant
ibod
y-ba
sed
assa
y w
ith p
hysi
coch
emic
al a
ssay
s fo
r mon
itorin
g th
e st
abili
ty o
f alu
m-a
dsor
bed
mite
alle
rgoi
dD.
Luy
kx, E
. Ker
kvlie
t, J.
de
Brui
jn, N
. Sin
nige
and
R. v
an d
en H
out
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Back
grou
nd &
Aim
:An
IgG
inhi
bitio
n as
say
has
been
dev
elop
ed p
revi
ousl
y to
mon
itor
the
stab
ility
of a
lum
-ads
orbe
d m
ite a
llerg
oids
in a
mite
alle
rgy
vacc
ine
(ALU
-M
). In
add
ition,
two
phys
icoc
hem
ical
met
hods
hav
e be
en d
evel
oped
to
mon
itor
the
stru
ctur
al s
tabi
lity o
f th
e m
ite v
acci
ne:
circ
ular
dich
roism
(CD)
and
fro
nt-fa
ce f
luor
esce
nce
spec
trosc
opy
(FF-
fluor
esce
nce)
. In
or
der t
o fin
d ou
t whe
ther
ther
e is
a re
latio
n be
twee
n th
e an
tibod
y-ba
sed
assa
y an
d ph
ysic
oche
mica
l m
etho
ds,
tem
pera
ture
-stre
ssed
sam
ples
we
re a
nalys
ed
with
bot
h ty
pes
of m
etho
ds a
nd t
he r
esul
ts w
ere
com
pare
d.
Met
hods
:AL
U-M
: D. p
tero
nyss
inus
and
D. fa
rinae
mite
ext
ract
s we
re s
epar
atel
y m
odifi
ed w
ith g
luta
rald
ehyd
ean
d ad
sorb
ed o
nto
alum
iniu
m h
ydro
xide
. Af
terw
ards
the
two
spec
ies
were
mix
ed.
Stre
ss c
ondi
tions
: ALU
-M w
as s
tore
d at
5 o
r 37º
C fo
r sev
eral
mon
ths,
at
50ºC
for s
ever
al d
ays,
or a
t 90º
C fo
r 6 h
ours
.
IgG
inhi
bitio
n as
say :
Mite
alle
rgoi
dsp
ecifi
c Ig
Gan
tibod
ies
were
use
d to
de
term
ine
the
50%
IgG
inhi
bitio
n va
lue.
CD: F
ar-U
V CD
spe
ctra
wer
e re
cord
ed fr
om 1
95 to
260
nm
and
und
er
stirr
ing
cond
itions
of
the
sam
ple.
The
CD
ratio
207
/222
nm
wa
s m
onito
red.
FF-fl
uore
scen
ce:
Fluo
resc
ence
em
issio
n sp
ectra
wer
e re
cord
ed f
rom
29
0 to
400
nm
with
exc
itatio
n at
280
nm
. The
fluo
resc
ence
inte
nsity
at
326
nm w
as m
onito
red.
Resu
lts:
The
stab
ility
of A
LU-M
was
stu
died
by
mon
itorin
g th
e 50
% Ig
Gin
hibi
tion
beha
viou
r, CD
-ratio
207
/222
nm
and
FF-
fluor
esce
nce
inte
nsity
at
326
nm a
t var
ious
tem
pera
ture
s fo
r diff
eren
t per
iods
of t
ime
(Fig
. 1an
d 2)
. Th
e an
tibod
y-ba
sed
assa
y an
d ph
ysic
oche
mica
l ass
ays
did
not
show
an
y ch
ange
s wh
en A
LU-M
was
sto
red
at 5
ºC fo
r a p
erio
d of
1 y
ear (
Fig.
1)
. St
orag
e of
the
ALU
-M v
acci
ne a
t 37
ºC r
esul
ted
in in
crea
sed
IgG
inhi
bitio
n va
lues
and
spe
ctra
l alte
ratio
ns w
ith C
D an
d FF
-fluo
resc
ence
(F
ig.
1),
mea
ning
stru
ctur
al a
ltera
tions
of
the
adso
rbed
pro
tein
. Th
e in
crea
se o
f th
e 50
% I
gGin
hibi
tion
valu
e in
dica
tes
a re
duct
ion
of I
gGbi
ndin
g to
the
epi
tope
s. T
he s
pect
ral
alte
ratio
ns o
bser
ved
with
CD
in
dica
te p
artia
l los
s of
-h
elic
es a
nd in
crea
se o
f -s
truct
ure
at h
ighe
r te
mpe
ratu
res
(incr
ease
of
CD
ra
tio
207/
222
nm).
The
spec
tral
alte
ratio
ns o
bser
ved
with
FF-
fluor
esce
nce
indi
cate
form
atio
n of
a m
ore
rigid
pro
tein
stru
ctur
e at
hig
her t
empe
ratu
res
(incr
ease
of f
luor
esce
nce
at 3
26 n
m).
Com
paris
on o
f th
e re
sults
of
the
thre
e di
ffere
nt a
ssay
s ob
tain
ed a
t 37
ºC in
dica
te t
hat
the
antib
ody-
base
d an
d CD
ass
ay a
re
mor
e se
nsitiv
e to
de
tect
st
ruct
ural
ch
ange
s fo
r AL
U-M
th
an
FF-
fluor
esce
nce
(i.e.
, afte
r 1 m
onth
). Th
e sa
me
hold
s wh
en th
e va
ccin
e is
stor
ed a
t 50
ºC f
or a
few
day
s (F
ig.
2).
In t
his
case
the
flu
ores
cenc
e in
tens
ity f
irst
decr
ease
s in
tim
e be
fore
it in
crea
ses
agai
n. A
ppar
ently
, th
e co
nditi
ons
at 5
0ºC
quen
ch th
e flu
ores
cenc
e si
gnal
.
Whe
n th
e te
mpe
ratu
re s
tress
sta
bility
dat
a ob
tain
ed w
ith t
he t
hree
m
etho
ds w
ere
com
pare
d, a
rel
atio
n is
obs
erve
d be
twee
n th
e Ig
Gin
hibi
tion
assa
y an
d CD
ass
ay b
y m
eans
of t
he C
D-ra
tio 2
07/2
22 n
m
(Fig
. 3).
Sam
ples
sto
red
at e
leva
ted
tem
pera
ture
s (i.
e., 3
7, 5
0 an
d 90
ºC)
show
ed a
sim
ilar t
rend
with
resp
ect t
o Ig
Gin
hibi
tion
and
CD ra
tio. T
he
data
of t
hese
ass
ays
supp
ort e
ach
othe
r ind
icat
ing
that
bot
h as
says
can
be
use
d as
sta
bilit
y in
dica
ting
assa
y fo
r ALU
-M.
Conc
lusi
on:
By p
erfo
rmin
g st
ress
sta
bility
stu
dies
on
ALU
-M it
was
sho
wn th
at th
e Ig
Gin
hibi
tion
assa
y an
d ph
ysico
chem
ical
assa
ys
were
st
abilit
y in
dica
ting.
The
res
ults
obt
aine
d wi
th t
he I
gGin
hibi
tion
assa
y an
d CD
as
say
rela
te w
ell s
ugge
stin
g a
rela
tion
betw
een
the
IgG
bind
ing
and
seco
ndar
y pr
otei
n st
ruct
ure
of th
e ad
sorb
ed m
ite a
llerg
oid.
Bot
h as
says
we
re s
hown
to b
e m
ore
sens
itive
than
the
FF-fl
uore
scen
ce a
ssay
.
Figu
re
1:
(A)
50%
Ig
Gin
hibi
tion
valu
es,
(B)
CD
ra
tio 2
07/2
22 n
m a
nd (C
) FF-
fluor
esce
nce
inte
nsity
of
AL
U-M
sto
red
at 5
ºC (
blue
) or
37º
C(g
reen
) fo
r se
vera
l m
onth
s.
Figu
re 2
: (A
) 50
% I
gGin
hibi
tion
valu
es,
(B)
CD
rat
io 2
07/2
22 n
m a
nd (
C)
FF-
fluor
esce
nce
inte
nsity
of A
LU-M
sto
red
at 5
0ºC
(red
) for
sev
eral
day
s.
Figu
re 3
: 50%
IgG
inhi
bitio
n da
ta v
sC
D r
atio
207
/222
nm
da
ta o
btai
ned
afte
r st
orag
e of
ALU
-M a
t 5º
C(b
lue)
37º
C
(gre
en),
50ºC
(red
) and
90º
C
(yel
low
).
0
500
1000
1500
2000
2500
3000
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
50% IgG inhibition (AUeq/ml)
5°C
37°C
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
CD ratio
5°C
37°C
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
FF fluorescence (x1E7)
5°C
37°C
50°C
0
500
1000
1500
2000
2500
3000
02
46
810
Stor
age
time
in d
ays
50 % IgG inhibition(AUeq/ml)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
02
46
810
Stor
age
time
in d
ays
CD ratio
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
02
46
810
Stor
age
time
in d
ays
FF fluoresence (x1E7)
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
050
010
0015
0020
0025
0030
0035
0040
00
50%
IgG
Inhi
bitio
n (A
Ueq/
ml)
CD ratio
5°C
37°C
50°C
1000
0
1.50
90° °°°C
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
050
010
0015
0020
0025
0030
0035
0040
00
50%
IgG
Inhi
bitio
n (A
Ueq/
ml)
CD ratio
5°C
37°C
50°C
1000
0
1.50
90° °°°C
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12
278
-Com
paris
on o
f ant
ibod
y-ba
sed
assa
y w
ith p
hysi
coch
emic
al a
ssay
s fo
r mon
itorin
g th
e st
abili
ty o
f alu
m-a
dsor
bed
mite
alle
rgoi
dD.
Luy
kx, E
. Ker
kvlie
t, J.
de
Brui
jn, N
. Sin
nige
and
R. v
an d
en H
out
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Back
grou
nd &
Aim
:An
IgG
inhi
bitio
n as
say
has
been
dev
elop
ed p
revi
ousl
y to
mon
itor
the
stab
ility
of a
lum
-ads
orbe
d m
ite a
llerg
oids
in a
mite
alle
rgy
vacc
ine
(ALU
-M
). In
add
ition,
two
phys
icoc
hem
ical
met
hods
hav
e be
en d
evel
oped
to
mon
itor
the
stru
ctur
al s
tabi
lity o
f th
e m
ite v
acci
ne:
circ
ular
dich
roism
(CD)
and
fro
nt-fa
ce f
luor
esce
nce
spec
trosc
opy
(FF-
fluor
esce
nce)
. In
or
der t
o fin
d ou
t whe
ther
ther
e is
a re
latio
n be
twee
n th
e an
tibod
y-ba
sed
assa
y an
d ph
ysic
oche
mica
l m
etho
ds,
tem
pera
ture
-stre
ssed
sam
ples
we
re a
naly
sed
with
bot
h ty
pes
of m
etho
ds a
nd t
he r
esul
ts w
ere
com
pare
d.
Met
hods
:AL
U-M
: D. p
tero
nyss
inus
and
D. fa
rinae
mite
ext
ract
s we
re s
epar
atel
y m
odifi
ed w
ith g
luta
rald
ehyd
ean
d ad
sorb
ed o
nto
alum
iniu
m h
ydro
xide
. Af
terw
ards
the
two
spec
ies
were
mix
ed.
Stre
ss c
ondi
tions
: ALU
-M w
as s
tore
d at
5 o
r 37º
C fo
r sev
eral
mon
ths,
at
50ºC
for s
ever
al d
ays,
or a
t 90º
C fo
r 6 h
ours
.
IgG
inhi
bitio
n as
say :
Mite
alle
rgoi
dsp
ecifi
c Ig
Gan
tibod
ies
were
use
d to
de
term
ine
the
50%
IgG
inhi
bitio
n va
lue.
CD: F
ar-U
V CD
spe
ctra
wer
e re
cord
ed fr
om 1
95 to
260
nm
and
und
er
stirr
ing
cond
itions
of
the
sam
ple.
The
CD
ratio
207
/222
nm
wa
s m
onito
red.
FF-fl
uore
scen
ce:
Fluo
resc
ence
em
issio
n sp
ectra
wer
e re
cord
ed f
rom
29
0 to
400
nm
with
exc
itatio
n at
280
nm
. The
fluo
resc
ence
inte
nsity
at
326
nm w
as m
onito
red.
Resu
lts:
The
stab
ility
of A
LU-M
was
stu
died
by
mon
itorin
g th
e 50
% Ig
Gin
hibi
tion
beha
viou
r, C
D-ra
tio 2
07/2
22 n
m a
nd F
F-flu
ores
cenc
e in
tens
ity a
t 32
6 nm
at v
ario
us te
mpe
ratu
res
for d
iffer
ent p
erio
ds o
f tim
e (F
ig. 1
and
2).
The
antib
ody-
base
d as
say
and
phys
icoc
hem
ical a
ssay
s di
d no
t sh
ow
any
chan
ges
when
ALU
-M w
as s
tore
d at
5ºC
for a
per
iod
of 1
yea
r (Fi
g.
1).
Stor
age
of t
he A
LU-M
vac
cine
at
37ºC
res
ulte
d in
incr
ease
d Ig
Gin
hibi
tion
valu
es a
nd s
pect
ral a
ltera
tions
with
CD
and
FF-fl
uore
scen
ce
(Fig
. 1)
, m
eani
ng s
truct
ural
alte
ratio
ns o
f th
e ad
sorb
ed p
rote
in.
The
incr
ease
of
the
50%
IgG
inhi
bitio
n va
lue
indi
cate
s a
redu
ctio
n of
IgG
bind
ing
to t
he e
pito
pes.
The
spe
ctra
l al
tera
tions
obs
erve
d wi
th C
D
indi
cate
par
tial l
oss
of
-hel
ices
and
incr
ease
of
-stru
ctur
e at
hig
her
tem
pera
ture
s (in
crea
se
of
CD
ratio
20
7/22
2 nm
). Th
e sp
ectra
l al
tera
tions
obs
erve
d wi
th F
F-flu
ores
cenc
e in
dica
te fo
rmat
ion
of a
mor
e rig
id p
rote
in s
truct
ure
at h
ighe
r tem
pera
ture
s (in
crea
se o
f flu
ores
cenc
e at
326
nm
). Co
mpa
rison
of
the
resu
lts o
f th
e th
ree
diffe
rent
ass
ays
obta
ined
at
37ºC
indi
cate
tha
t th
e an
tibod
y-ba
sed
and
CD a
ssay
are
m
ore
sens
itive
to
dete
ct
stru
ctur
al
chan
ges
for
ALU
-M
than
FF
-flu
ores
cenc
e (i.
e., a
fter 1
mon
th).
The
sam
e ho
lds
when
the
vacc
ine
is st
ored
at
50ºC
for
a f
ew d
ays
(Fig
. 2)
. In
thi
s ca
se t
he f
luor
esce
nce
inte
nsity
firs
t de
crea
ses
in t
ime
befo
re it
incr
ease
s ag
ain.
App
aren
tly,
the
cond
ition
s at
50º
C qu
ench
the
fluor
esce
nce
sign
al.
Whe
n th
e te
mpe
ratu
re s
tress
sta
bility
dat
a ob
tain
ed w
ith t
he t
hree
m
etho
ds w
ere
com
pare
d, a
rel
atio
n is
obs
erve
d be
twee
n th
e Ig
Gin
hibi
tion
assa
y an
d CD
ass
ay b
y m
eans
of t
he C
D-ra
tio 2
07/2
22 n
m
(Fig
. 3).
Sam
ples
sto
red
at e
leva
ted
tem
pera
ture
s (i.
e., 3
7, 5
0 an
d 90
ºC)
show
ed a
sim
ilar t
rend
with
resp
ect t
o Ig
Gin
hibi
tion
and
CD ra
tio. T
he
data
of t
hese
ass
ays
supp
ort e
ach
othe
r ind
icat
ing
that
bot
h as
says
can
be
use
d as
sta
bilit
y in
dica
ting
assa
y fo
r ALU
-M.
Conc
lusi
on:
By p
erfo
rmin
g st
ress
sta
bility
stu
dies
on
ALU
-M it
was
sho
wn th
at th
e Ig
Gin
hibi
tion
assa
y an
d ph
ysico
chem
ical
assa
ys
were
st
abilit
y in
dica
ting.
The
res
ults
obt
aine
d wi
th t
he I
gGin
hibi
tion
assa
y an
d CD
as
say
rela
te w
ell s
ugge
stin
g a
rela
tion
betw
een
the
IgG
bind
ing
and
seco
ndar
y pr
otei
n st
ruct
ure
of th
e ad
sorb
ed m
ite a
llerg
oid.
Bot
h as
says
we
re s
hown
to b
e m
ore
sens
itive
than
the
FF-fl
uore
scen
ce a
ssay
.
Figu
re
1:
(A)
50%
Ig
Gin
hibi
tion
valu
es,
(B)
CD
ra
tio 2
07/2
22 n
m a
nd (C
) FF-
fluor
esce
nce
inte
nsity
of
AL
U-M
sto
red
at 5
ºC (
blue
) or
37º
C(g
reen
) fo
r se
vera
l m
onth
s.
Figu
re 2
: (A
) 50
% I
gGin
hibi
tion
valu
es,
(B)
CD
rat
io 2
07/2
22 n
m a
nd (
C)
FF-
fluor
esce
nce
inte
nsity
of A
LU-M
sto
red
at 5
0ºC
(red
) for
sev
eral
day
s.
Figu
re 3
: 50%
IgG
inhi
bitio
n da
ta v
sC
D r
atio
207
/222
nm
da
ta o
btai
ned
afte
r st
orag
e of
ALU
-M a
t 5º
C(b
lue)
37º
C
(gre
en),
50ºC
(red
) and
90º
C
(yel
low
).
0
500
1000
1500
2000
2500
3000
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
50% IgG inhibition (AUeq/ml)
5°C
37°C
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
CD ratio
5°C
37°C
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
FF fluorescence (x1E7)
5°C
37°C
50°C
0
500
1000
1500
2000
2500
3000
02
46
810
Stor
age
time
in d
ays
50 % IgG inhibition(AUeq/ml)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
02
46
810
Stor
age
time
in d
ays
CD ratio
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
02
46
810
Stor
age
time
in d
ays
FF fluoresence (x1E7)
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
050
010
0015
0020
0025
0030
0035
0040
00
50%
IgG
Inhi
bitio
n (A
Ueq/
ml)
CD ratio
5°C
37°C
50°C
1000
0
1.50
90° °°°C
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
050
010
0015
0020
0025
0030
0035
0040
00
50%
IgG
Inhi
bitio
n (A
Ueq/
ml)
CD ratio
5°C
37°C
50°C
1000
0
1.50
90° °°°C
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12
278
-Com
paris
on o
f ant
ibod
y-ba
sed
assa
y w
ith p
hysi
coch
emic
al a
ssay
s fo
r mon
itorin
g th
e st
abili
ty o
f alu
m-a
dsor
bed
mite
alle
rgoi
dD.
Luy
kx, E
. Ker
kvlie
t, J.
de
Brui
jn, N
. Sin
nige
and
R. v
an d
en H
out
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Back
grou
nd &
Aim
:An
IgG
inhi
bitio
n as
say
has
been
dev
elop
ed p
revi
ousl
y to
mon
itor
the
stab
ility
of a
lum
-ads
orbe
d m
ite a
llerg
oids
in a
mite
alle
rgy
vacc
ine
(ALU
-M
). In
add
ition,
two
phys
icoc
hem
ical
met
hods
hav
e be
en d
evel
oped
to
mon
itor
the
stru
ctur
al s
tabi
lity o
f th
e m
ite v
acci
ne:
circ
ular
dich
roism
(CD)
and
fro
nt-fa
ce f
luor
esce
nce
spec
trosc
opy
(FF-
fluor
esce
nce)
. In
or
der t
o fin
d ou
t whe
ther
ther
e is
a re
latio
n be
twee
n th
e an
tibod
y-ba
sed
assa
y an
d ph
ysic
oche
mica
l m
etho
ds,
tem
pera
ture
-stre
ssed
sam
ples
we
re a
nalys
ed
with
bot
h ty
pes
of m
etho
ds a
nd t
he r
esul
ts w
ere
com
pare
d.
Met
hods
:AL
U-M
: D. p
tero
nyss
inus
and
D. fa
rinae
mite
ext
ract
s we
re s
epar
atel
y m
odifi
ed w
ith g
luta
rald
ehyd
ean
d ad
sorb
ed o
nto
alum
iniu
m h
ydro
xide
. Af
terw
ards
the
two
spec
ies
were
mix
ed.
Stre
ss c
ondi
tions
: ALU
-M w
as s
tore
d at
5 o
r 37º
C fo
r sev
eral
mon
ths,
at
50ºC
for s
ever
al d
ays,
or a
t 90º
C fo
r 6 h
ours
.
IgG
inhi
bitio
n as
say :
Mite
alle
rgoi
dsp
ecifi
c Ig
Gan
tibod
ies
were
use
d to
de
term
ine
the
50%
IgG
inhi
bitio
n va
lue.
CD: F
ar-U
V CD
spe
ctra
wer
e re
cord
ed fr
om 1
95 to
260
nm
and
und
er
stirr
ing
cond
itions
of
the
sam
ple.
The
CD
ratio
207
/222
nm
wa
s m
onito
red.
FF-fl
uore
scen
ce:
Fluo
resc
ence
em
issio
n sp
ectra
wer
e re
cord
ed f
rom
29
0 to
400
nm
with
exc
itatio
n at
280
nm
. The
fluo
resc
ence
inte
nsity
at
326
nm w
as m
onito
red.
Resu
lts:
The
stab
ility
of A
LU-M
was
stu
died
by
mon
itorin
g th
e 50
% Ig
Gin
hibi
tion
beha
viou
r, CD
-ratio
207
/222
nm
and
FF-
fluor
esce
nce
inte
nsity
at
326
nm a
t var
ious
tem
pera
ture
s fo
r diff
eren
t per
iods
of t
ime
(Fig
. 1an
d 2)
. Th
e an
tibod
y-ba
sed
assa
y an
d ph
ysic
oche
mica
l ass
ays
did
not
show
an
y ch
ange
s wh
en A
LU-M
was
sto
red
at 5
ºC fo
r a p
erio
d of
1 y
ear (
Fig.
1)
. St
orag
e of
the
ALU
-M v
acci
ne a
t 37
ºC r
esul
ted
in in
crea
sed
IgG
inhi
bitio
n va
lues
and
spe
ctra
l alte
ratio
ns w
ith C
D an
d FF
-fluo
resc
ence
(F
ig.
1),
mea
ning
stru
ctur
al a
ltera
tions
of
the
adso
rbed
pro
tein
. Th
e in
crea
se o
f th
e 50
% I
gGin
hibi
tion
valu
e in
dica
tes
a re
duct
ion
of I
gGbi
ndin
g to
the
epi
tope
s. T
he s
pect
ral
alte
ratio
ns o
bser
ved
with
CD
in
dica
te p
artia
l los
s of
-h
elic
es a
nd in
crea
se o
f -s
truct
ure
at h
ighe
r te
mpe
ratu
res
(incr
ease
of
CD
ra
tio
207/
222
nm).
The
spec
tral
alte
ratio
ns o
bser
ved
with
FF-
fluor
esce
nce
indi
cate
form
atio
n of
a m
ore
rigid
pro
tein
stru
ctur
e at
hig
her t
empe
ratu
res
(incr
ease
of f
luor
esce
nce
at 3
26 n
m).
Com
paris
on o
f th
e re
sults
of
the
thre
e di
ffere
nt a
ssay
s ob
tain
ed a
t 37
ºC in
dica
te t
hat
the
antib
ody-
base
d an
d CD
ass
ay a
re
mor
e se
nsitiv
e to
de
tect
st
ruct
ural
ch
ange
s fo
r AL
U-M
th
an
FF-
fluor
esce
nce
(i.e.
, afte
r 1 m
onth
). Th
e sa
me
hold
s wh
en th
e va
ccin
e is
stor
ed a
t 50
ºC f
or a
few
day
s (F
ig.
2).
In t
his
case
the
flu
ores
cenc
e in
tens
ity f
irst
decr
ease
s in
tim
e be
fore
it in
crea
ses
agai
n. A
ppar
ently
, th
e co
nditi
ons
at 5
0ºC
quen
ch th
e flu
ores
cenc
e si
gnal
.
Whe
n th
e te
mpe
ratu
re s
tress
sta
bility
dat
a ob
tain
ed w
ith t
he t
hree
m
etho
ds w
ere
com
pare
d, a
rel
atio
n is
obs
erve
d be
twee
n th
e Ig
Gin
hibi
tion
assa
y an
d CD
ass
ay b
y m
eans
of t
he C
D-ra
tio 2
07/2
22 n
m
(Fig
. 3).
Sam
ples
sto
red
at e
leva
ted
tem
pera
ture
s (i.
e., 3
7, 5
0 an
d 90
ºC)
show
ed a
sim
ilar t
rend
with
resp
ect t
o Ig
Gin
hibi
tion
and
CD ra
tio. T
he
data
of t
hese
ass
ays
supp
ort e
ach
othe
r ind
icat
ing
that
bot
h as
says
can
be
use
d as
sta
bilit
y in
dica
ting
assa
y fo
r ALU
-M.
Conc
lusi
on:
By p
erfo
rmin
g st
ress
sta
bility
stu
dies
on
ALU
-M it
was
sho
wn th
at th
e Ig
Gin
hibi
tion
assa
y an
d ph
ysico
chem
ical
assa
ys
were
st
abilit
y in
dica
ting.
The
res
ults
obt
aine
d wi
th t
he I
gGin
hibi
tion
assa
y an
d CD
as
say
rela
te w
ell s
ugge
stin
g a
rela
tion
betw
een
the
IgG
bind
ing
and
seco
ndar
y pr
otei
n st
ruct
ure
of th
e ad
sorb
ed m
ite a
llerg
oid.
Bot
h as
says
we
re s
hown
to b
e m
ore
sens
itive
than
the
FF-fl
uore
scen
ce a
ssay
.
Figu
re
1:
(A)
50%
Ig
Gin
hibi
tion
valu
es,
(B)
CD
ra
tio 2
07/2
22 n
m a
nd (C
) FF-
fluor
esce
nce
inte
nsity
of
AL
U-M
sto
red
at 5
ºC (
blue
) or
37º
C(g
reen
) fo
r se
vera
l m
onth
s.
Figu
re 2
: (A
) 50
% I
gGin
hibi
tion
valu
es,
(B)
CD
rat
io 2
07/2
22 n
m a
nd (
C)
FF-
fluor
esce
nce
inte
nsity
of A
LU-M
sto
red
at 5
0ºC
(red
) for
sev
eral
day
s.
Figu
re 3
: 50%
IgG
inhi
bitio
n da
ta v
sC
D r
atio
207
/222
nm
da
ta o
btai
ned
afte
r st
orag
e of
ALU
-M a
t 5º
C(b
lue)
37º
C
(gre
en),
50ºC
(red
) and
90º
C
(yel
low
).
0
500
1000
1500
2000
2500
3000
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
50% IgG inhibition (AUeq/ml)
5°C
37°C
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
CD ratio
5°C
37°C
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
01
23
45
67
89
1011
12St
orag
e tim
e in
mon
ths
FF fluorescence (x1E7)
5°C
37°C
50°C
0
500
1000
1500
2000
2500
3000
02
46
810
Stor
age
time
in d
ays
50 % IgG inhibition(AUeq/ml)
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
02
46
810
Stor
age
time
in d
ays
CD ratio
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
02
46
810
Stor
age
time
in d
ays
FF fluoresence (x1E7)
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
050
010
0015
0020
0025
0030
0035
0040
00
50%
IgG
Inhi
bitio
n (A
Ueq/
ml)
CD ratio
5°C
37°C
50°C
1000
0
1.50
90° °°°C
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
050
010
0015
0020
0025
0030
0035
0040
00
50%
IgG
Inhi
bitio
n (A
Ueq/
ml)
CD ratio
5°C
37°C
50°C
1000
0
1.50
90° °°°C
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12In
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er is
an
empl
oyee
of H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
C h ara c t e ris a t i o n o f p o l l e n a l l e rg o i ds w i t h p h y sic o c h e mic a l t e c h ni q u e s
26
D. Luykx (1), J. de Bruijn (1), J. Cordewener (2), T. America (2), R. van den Hout (1).
(1) HAL Allergy BV, Leiden, The Netherlands, (2) Plant Research International, Wageningen, The Netherlands.
BackgroundBirch and grass allergoids are drug substances for birch and grass allergy vaccines, respectively. Until now these allergoids have been poorly characterised due to their complexity. Nevertheless, a better characterisation is needed. This includes identification of the relevant allergens, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures. Several physicochemical techniques were tested for their suitability to analyse these characteristics in the allergoid and corresponding allergen extract: Mass spectrometry (MS), SDS-PAGE, HPLC-SEC, lysine determination and fluorescence spectroscopy.
MethodsAllergoids: Allergen extracts from birch and grasses were treated with glutaraldehyde to obtain birch and grass allergoids, respectively. MS: Preparation of tryptic digests and peptide separation via nano-LC before electrospray ionisation. Ionised peptides were fragmented revealing sequences. SDS-PAGE: 10-20% gels with Coomassie Blue staining and reduced samples. HPLC: GF250 and GF450 SEC columns were used combined with UV-detection. Lysine determination: Samples were hydrolysed into amino acids followed by free lysine determination with HPLC. Fluorescence: Emission spectra were recorded from 290-400 nm, with excitation at 280 nm.
ResultsSeveral isoforms of relevant birch allergen Bet v1 were identified in the birch allergoid with MS. In the grass allergoid relevant grass allergens group 1 and 5 were identified. SDS-PAGE showed for both allergoids the formation of various high molecular weight molecules including masses ≥250kDa. HPLC-SEC showed for both allergoids the formation of molecules ≥670 kDa. For both pollen extracts a minority of HPLC peaks (6 and 9%) was corresponding to proteins with a molecular mass >44 kDa. For the allergoids the majority of peaks (76 and 75%) was corresponding to proteins with a molecular mass (far) >44 kDa. Determination of free lysines showed that the majority of the lysines were modified in both pollen allergoids (73 and 74%). Fluorescence indicated hydrophobic tertiary protein structures after modification of the pollen extracts.
ConclusionApplying a combination of physicochemical techniques was shown to be a suitable approach to characterise the pollen allergoids well: Identification of the relevant allergens, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures were accomplished.
EAACI, 16-20 June 2012, GenevaAbstract number: 287, Session date and time: Monday 18 June; 10:45 - 12:15 Session title: PDS 5 - Immunotherapy: vaccines and mechanisms
27
PDS 5 - Immunotherapy: vaccines and mechanisms
C h ara c t e ris a t i o n o f p o l l e n a l l e rg o i ds w i t h p h y sic o c h e mic a l t e c h ni q u e s
287
-Cha
ract
eris
atio
n of
pol
len
alle
rgoi
dsw
ith p
hysi
coch
emic
al te
chni
ques
D. L
uykx
1 , J.
de
Brui
jn1 1, J
. Cor
dew
ener
2
2
, T. A
mer
ica2
and
R. v
an d
en H
out1
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Plan
t Res
earc
h In
tern
atio
nal,
Wag
enin
gen,
The
Net
herla
nds
Back
grou
nd &
Aim
:Bi
rch
and
gras
s al
lerg
oids
are
used
as
drug
sub
stan
ces
for b
irch
and
gras
s al
lerg
y va
ccin
es, r
espe
ctive
ly. U
ntil
now
thes
e al
lerg
oids
have
bee
n po
orly
ch
arac
teris
ed d
ue to
thei
r co
mpl
exity
. A b
ette
r ch
arac
teris
atio
nis
need
ed.
This
inc
lude
s id
entif
icat
ion
of t
he m
ajor
alle
rgen
s, d
eter
min
atio
n of
the
de
gree
of p
olym
eris
atio
n an
d cr
oss-
linki
ng, a
nd in
vest
igat
ion
of th
e pr
otei
n st
ruct
ures
. Se
vera
l ph
ysic
oche
mica
l te
chni
ques
wer
e ap
plie
d to
ana
lyse
th
ese
char
acte
ristic
s in
thr
ee s
ucce
ssiv
e, s
epar
atel
y pr
oduc
ed b
irch
and
gras
s al
lerg
oids
and
corre
spon
ding
alle
rgen
ext
ract
s: M
ass
spec
trom
etry
(M
S),
SDS-
PAG
E,
HPL
C-S
EC,
lysin
e de
term
inat
ion
and
fluor
esce
nce
spec
trosc
opy.
Met
hods
:Al
lerg
oids
: Alle
rgen
ext
ract
s fro
m b
irch
and
mixe
d gr
ass
polle
n we
re tr
eate
d wi
th g
luta
rald
ehyd
eto
obt
ain
birc
h an
d gr
ass
alle
rgoi
ds, r
espe
ctiv
ely.
MS:
Firs
t try
ptic
dige
sts
were
pre
pare
d fro
m th
e sa
mpl
es. S
ubse
quen
tly, t
he
pept
ides
wer
e se
para
ted
with
nan
o-liq
uid
chro
mat
ogra
phy
befo
re e
nter
ing
the
MS
inst
rum
ent
via
elec
trosp
ray
ioni
satio
n.
Ioni
sed
pept
ides
we
re
fragm
ente
d re
veal
ing
amin
o ac
id s
eque
nces
(i.e
., id
entif
icatio
n).
SDS-
PAG
E: R
educ
ed s
ampl
es w
ere
appl
ied
onto
10-
20%
pol
yacr
ylam
ide
gels
and
sta
ined
with
Coo
mas
sie
Blue
.
HPL
C-S
EC:
A G
F250
or
GF4
50 s
ize e
xclu
sion
chr
omat
ogra
phy
(SEC
) co
lum
n wa
s us
ed c
ombi
ned
with
UV-
dete
ctio
n (2
15 n
m).
Lysi
ne d
eter
min
atio
n : S
ampl
es w
ere
hydr
olys
ed in
to a
min
o ac
ids
follo
wed
by l
ysin
e de
term
inat
ion
with
HPL
C. B
y de
term
inin
g th
e am
ount
of
free
lysi
nes
in t
he e
xtra
ct a
nd a
llerg
oid,
the
am
ount
of m
odifi
ed ly
sine
sin
the
al
lerg
oid
can
be c
alcu
late
d.
Fluo
resc
ence
spe
ctro
scop
y : E
mis
sion
spec
tra w
ere
reco
rded
from
290
-400
nm
with
exc
itatio
n at
280
nm
.
Resu
lts:
MS:
Sev
eral
isof
orm
sof
the
maj
or b
irch
alle
rgen
Bet
v1
were
iden
tifie
d in
th
e th
ree
birc
h al
lerg
oids
(a, b
, c, d
/h, f
/i). N
ext t
o th
e m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5, a
lso
grou
p 2,
3, 4
and
6 a
llerg
ens
were
iden
tifie
d in
the
thre
e gr
ass
alle
rgoi
ds(d
ata
not s
hown
).
SDS-
PAG
E: F
or t
he t
hree
birc
h ex
tract
s, s
imila
r pr
otei
n pr
ofile
s we
re
obse
rved
(Fi
g. 1
). Th
e ba
nd c
orre
spon
ding
to
15-1
8 kD
ain
dica
tes
the
pres
ence
of m
ajor
alle
rgen
Bet
v1.
For
the
thre
e gr
ass
extra
cts
also
sim
ilar
prot
ein
prof
iles
were
obs
erve
d. H
ere,
the
band
s co
rresp
ondi
ng to
27-3
4 kD
ain
dica
te th
e pr
esen
ce o
f maj
or a
llerg
ens
grou
p 1
and
5. F
or a
ll al
lerg
oids
the
form
atio
n of
var
ious
hig
h m
olec
ular
wei
ght m
olec
ules
is s
hown
via
a s
mea
r an
d a
band
aro
und
250
kDa
(mol
ecul
es
250
kDa)
on
gel.
HPL
C-S
EC:
For
the
thre
e bi
rch
and
gras
s ex
tract
s a
min
ority
of
HPL
C
peak
s is
cor
resp
ondi
ng t
o pr
otei
ns w
ith a
mol
ecul
ar m
ass
abov
e 44
kDa
(Fig
. 2)
. Th
e ch
rom
atog
ram
s of
the
birc
h an
d gr
ass
alle
rgoi
dba
tche
s sh
owed
the
pre
senc
e of
a v
arie
ty o
f hi
gh m
olec
ular
wei
ght
mol
ecul
es
incl
udin
g m
olec
ules
with
mol
ecul
ar m
asse
s 67
0 kD
a. H
ere,
a c
onsi
sten
t m
ajor
ity
of
peak
s (7
4-77
%
for
birc
h an
d 68
-79%
fo
r gr
asse
s)
is co
rresp
ondi
ng t
o pr
otei
ns w
ith a
mol
ecul
ar m
ass
(far)
abov
e 44
kDa
show
ing
a co
nsis
tent
deg
ree
of p
rote
in p
olym
eris
atio
n (T
able
1-2
).
Lysi
ne d
eter
min
atio
n: F
or t
he b
irch
alle
rgoi
ds74
-79%
of t
he ly
sine
swe
re
mod
ified
and
for
the
gras
s al
lerg
oids
73-7
6% o
f the
lysi
nes
were
mod
ified
(Tab
le 1
-2).
This
sho
ws a
con
sist
ent d
egre
e of
pro
tein
cro
ss-li
nkin
g.
Fluo
resc
ence
spe
ctro
scop
y : M
odific
atio
n of
the
birc
h an
d gr
ass
extra
cts
resu
lted
in a
shi
ft of
the
emis
sion
max
imum
to a
lowe
r wa
vele
ngth
and
a
decr
ease
of f
luor
esce
nce
inte
nsity
(dat
a no
t sho
wn).
This
indi
cate
s th
at th
e su
rroun
ding
s of
the
arom
atic
am
ino
acid
s in
the
prot
eins
cha
nge
to a
mor
e hy
drop
hobi
c en
viro
nmen
t whi
ch is
indi
cativ
e fo
r pro
tein
pol
ymer
isat
ion.
The
em
issi
on
max
imum
va
lues
ob
tain
ed
for
the
polle
n al
lerg
oids
show
co
nsis
tenc
y as
wel
l (Ta
ble
1-2)
.
Conc
lusi
on:
Appl
ying
a c
ombi
natio
n of
phy
sicoc
hem
ical
tech
niqu
es w
as s
hown
to b
e a
suita
ble
appr
oach
to c
hara
cter
ise th
e po
llen
alle
rgoi
dswe
ll: Id
entif
icat
ion
of
the
maj
or a
llerg
ens,
det
erm
inat
ion
of t
he d
egre
e of
pol
ymer
isat
ion
and
cros
s-lin
king
, and
inve
stig
atio
n of
the
prot
ein
stru
ctur
es w
ere
acco
mpl
ishe
d.
This
stu
dy s
hows
con
sist
ency
for t
he m
odifi
catio
n of
the
polle
n ex
tract
s.
Figu
re 2
: H
PLC
-SEC
pat
tern
s (A
215
nm)
of c
alib
ratio
n st
anda
rd (
A1 a
nd B
1), t
hree
birc
h ex
trac
ts (
blue
lin
e) a
nd c
orre
spon
ding
alle
rgoi
ds(r
ed l
ine)
(A2
-A4)
, an
d th
ree
gras
s ex
trac
ts (b
lue
line)
and
cor
resp
ondi
ng a
llerg
oids
(red
line)
(B2-
B4)
. The
bla
ck v
ertic
al li
ne
in th
e ch
rom
atog
ram
spl
its p
eaks
cor
resp
ondi
ng to
mol
ecul
ar m
asse
s ab
ove
and
belo
w
44 k
Da.
A G
F250
SEC
col
umn
was
app
lied
for b
irch,
a G
F450
SEC
col
umn
for g
rass
es.
Tabl
e 1:
Phy
sico
chem
ical
cha
ract
eris
tics
of th
ree
batc
hes
of b
irch
alle
rgoi
ds
670
kDa
158
kDa44
kDa 17
kDa 1.35
kDa
Absorbance 215 nm
Rete
ntio
ntim
e (m
in)
670
kDa 15
8 kD
a44 k
Da 17 k
Da 1.35
kDa
Stan
dard
Stan
dard
Birc
h1
Birc
h2
Birc
h3
Gra
ss1
Gra
ss2
Gra
ss3
A1 A2 A3 A4
B1 B2 B3 B4
Tabl
e 2:
Phy
sico
chem
ical
cha
ract
eris
tics
of th
ree
batc
hes
of g
rass
alle
rgoi
ds
Ch
ara
cte
ris
tic
Bir
ch
1
Bir
ch
2
B
irc
h 3
HP
LC
: P
eak
are
a a
bo
ve 4
4 k
Da
(%
)
75
7
4
77
Mo
difi
ed
lysi
ne
s (%
)
74
7
9
78
Flu
ore
scen
ce e
mis
sion m
axi
mum
(nm
) 3
11
3
11
3
11
C
ha
rac
teri
sti
c
G
ras
s 1
G
ras
s 2
Gra
ss 3
HP
LC
: P
eak
are
a a
bo
ve 4
4 k
Da
(%
)
76
6
8
79
Mo
difi
ed
lysi
ne
s (%
)
73
7
3
76
Flu
ore
sce
nce
em
issi
on m
axi
mu
m (
nm
) 3
29
3
29
3
30
Figu
re 1
: SD
S-PA
GE
patte
rns
of m
olec
ular
wei
ght
mar
ker
(M),
thre
e bi
rch
(1-3
) an
d th
ree
gras
s (4
-6)
extr
acts
and
cor
resp
ondi
ng a
llerg
oids
(1-6
). M
ajor
birc
h al
lerg
en B
et v
1 an
d m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5 ar
e bo
xed.
M1
23
54
61
24
35
6
Extr
acts
Alle
rgoi
dsG
rass
Birc
hB
irch
Birc
hG
rass
Birc
hB
irch
Gra
ssG
rass
Birc
hG
rass
Birc
hG
rass
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12
287
-Cha
ract
eris
atio
n of
pol
len
alle
rgoi
dsw
ith p
hysi
coch
emic
al te
chni
ques
D. L
uykx
1 , J.
de
Brui
jn1 1, J
. Cor
dew
ener
2
2
, T. A
mer
ica2
and
R. v
an d
en H
out1
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Plan
t Res
earc
h In
tern
atio
nal,
Wag
enin
gen,
The
Net
herla
nds
Back
grou
nd &
Aim
:Bi
rch
and
gras
s al
lerg
oids
are
used
as
drug
sub
stan
ces
for b
irch
and
gras
s al
lerg
y va
ccin
es, r
espe
ctive
ly. U
ntil
now
thes
e al
lerg
oids
have
bee
n po
orly
ch
arac
teris
ed d
ue to
thei
r co
mpl
exity
. A b
ette
r ch
arac
teris
atio
nis
need
ed.
This
inc
lude
s id
entif
icat
ion
of t
he m
ajor
alle
rgen
s, d
eter
min
atio
n of
the
de
gree
of p
olym
eris
atio
n an
d cr
oss-
linki
ng, a
nd in
vest
igat
ion
of th
e pr
otei
n st
ruct
ures
. Se
vera
l ph
ysic
oche
mica
l te
chni
ques
wer
e ap
plie
d to
ana
lyse
th
ese
char
acte
ristic
s in
thr
ee s
ucce
ssiv
e, s
epar
atel
y pr
oduc
ed b
irch
and
gras
s al
lerg
oids
and
corre
spon
ding
alle
rgen
ext
ract
s: M
ass
spec
trom
etry
(M
S),
SDS-
PAG
E,
HPL
C-S
EC,
lysin
e de
term
inat
ion
and
fluor
esce
nce
spec
trosc
opy.
Met
hods
:Al
lerg
oids
: Alle
rgen
ext
ract
s fro
m b
irch
and
mixe
d gr
ass
polle
n we
re tr
eate
d wi
th g
luta
rald
ehyd
eto
obt
ain
birc
h an
d gr
ass
alle
rgoi
ds, r
espe
ctiv
ely.
MS:
Firs
t try
ptic
dige
sts
were
pre
pare
d fro
m th
e sa
mpl
es. S
ubse
quen
tly, t
he
pept
ides
wer
e se
para
ted
with
nan
o-liq
uid
chro
mat
ogra
phy
befo
re e
nter
ing
the
MS
inst
rum
ent
via
elec
trosp
ray
ioni
satio
n.
Ioni
sed
pept
ides
we
re
fragm
ente
d re
veal
ing
amin
o ac
id s
eque
nces
(i.e
., id
entif
icatio
n).
SDS-
PAG
E: R
educ
ed s
ampl
es w
ere
appl
ied
onto
10-
20%
pol
yacr
ylam
ide
gels
and
sta
ined
with
Coo
mas
sie
Blue
.
HPL
C-S
EC:
A G
F250
or
GF4
50 s
ize e
xclu
sion
chr
omat
ogra
phy
(SEC
) co
lum
n wa
s us
ed c
ombi
ned
with
UV-
dete
ctio
n (2
15 n
m).
Lysi
ne d
eter
min
atio
n : S
ampl
es w
ere
hydr
olys
ed in
to a
min
o ac
ids
follo
wed
by l
ysin
e de
term
inat
ion
with
HPL
C. B
y de
term
inin
g th
e am
ount
of
free
lysi
nes
in t
he e
xtra
ct a
nd a
llerg
oid,
the
am
ount
of m
odifi
ed ly
sine
sin
the
al
lerg
oid
can
be c
alcu
late
d.
Fluo
resc
ence
spe
ctro
scop
y : E
mis
sion
spec
tra w
ere
reco
rded
from
290
-400
nm
with
exc
itatio
n at
280
nm
.
Resu
lts:
MS:
Sev
eral
isof
orm
sof
the
maj
or b
irch
alle
rgen
Bet
v1
were
iden
tifie
d in
th
e th
ree
birc
h al
lerg
oids
(a, b
, c, d
/h, f
/i). N
ext t
o th
e m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5, a
lso
grou
p 2,
3, 4
and
6 a
llerg
ens
were
iden
tifie
d in
the
thre
e gr
ass
alle
rgoi
ds(d
ata
not s
hown
).
SDS-
PAG
E: F
or t
he t
hree
birc
h ex
tract
s, s
imila
r pr
otei
n pr
ofile
s we
re
obse
rved
(Fi
g. 1
). Th
e ba
nd c
orre
spon
ding
to
15-1
8 kD
ain
dica
tes
the
pres
ence
of m
ajor
alle
rgen
Bet
v1.
For
the
thre
e gr
ass
extra
cts
also
sim
ilar
prot
ein
prof
iles
were
obs
erve
d. H
ere,
the
band
s co
rresp
ondi
ng to
27-3
4 kD
ain
dica
te th
e pr
esen
ce o
f maj
or a
llerg
ens
grou
p 1
and
5. F
or a
ll al
lerg
oids
the
form
atio
n of
var
ious
hig
h m
olec
ular
wei
ght m
olec
ules
is s
hown
via
a s
mea
r an
d a
band
aro
und
250
kDa
(mol
ecul
es
250
kDa)
on
gel.
HPL
C-S
EC:
For
the
thre
e bi
rch
and
gras
s ex
tract
s a
min
ority
of
HPL
C
peak
s is
cor
resp
ondi
ng t
o pr
otei
ns w
ith a
mol
ecul
ar m
ass
abov
e 44
kDa
(Fig
. 2)
. Th
e ch
rom
atog
ram
s of
the
birc
h an
d gr
ass
alle
rgoi
dba
tche
s sh
owed
the
pre
senc
e of
a v
arie
ty o
f hi
gh m
olec
ular
wei
ght
mol
ecul
es
incl
udin
g m
olec
ules
with
mol
ecul
ar m
asse
s 67
0 kD
a. H
ere,
a c
onsi
sten
t m
ajor
ity
of
peak
s (7
4-77
%
for
birc
h an
d 68
-79%
fo
r gr
asse
s)
is co
rresp
ondi
ng t
o pr
otei
ns w
ith a
mol
ecul
ar m
ass
(far)
abov
e 44
kDa
show
ing
a co
nsis
tent
deg
ree
of p
rote
in p
olym
eris
atio
n (T
able
1-2
).
Lysi
ne d
eter
min
atio
n: F
or t
he b
irch
alle
rgoi
ds74
-79%
of t
he ly
sine
swe
re
mod
ified
and
for
the
gras
s al
lerg
oids
73-7
6% o
f the
lysi
nes
were
mod
ified
(Tab
le 1
-2).
This
sho
ws a
con
sist
ent d
egre
e of
pro
tein
cro
ss-li
nkin
g.
Fluo
resc
ence
spe
ctro
scop
y : M
odific
atio
n of
the
birc
h an
d gr
ass
extra
cts
resu
lted
in a
shi
ft of
the
emis
sion
max
imum
to a
lowe
r wa
vele
ngth
and
a
decr
ease
of f
luor
esce
nce
inte
nsity
(dat
a no
t sho
wn).
This
indi
cate
s th
at th
e su
rroun
ding
s of
the
arom
atic
am
ino
acid
s in
the
prot
eins
cha
nge
to a
mor
e hy
drop
hobi
c en
viro
nmen
t whi
ch is
indi
cativ
e fo
r pro
tein
pol
ymer
isat
ion.
The
em
issi
on
max
imum
va
lues
ob
tain
ed
for
the
polle
n al
lerg
oids
show
co
nsis
tenc
y as
wel
l (Ta
ble
1-2)
.
Conc
lusi
on:
Appl
ying
a c
ombi
natio
n of
phy
sicoc
hem
ical
tech
niqu
es w
as s
hown
to b
e a
suita
ble
appr
oach
to c
hara
cter
ise th
e po
llen
alle
rgoi
dswe
ll: Id
entif
icat
ion
of
the
maj
or a
llerg
ens,
det
erm
inat
ion
of t
he d
egre
e of
pol
ymer
isat
ion
and
cros
s-lin
king
, and
inve
stig
atio
n of
the
prot
ein
stru
ctur
es w
ere
acco
mpl
ishe
d.
This
stu
dy s
hows
con
sist
ency
for t
he m
odifi
catio
n of
the
polle
n ex
tract
s.
Figu
re 2
: H
PLC
-SEC
pat
tern
s (A
215
nm)
of c
alib
ratio
n st
anda
rd (
A1 a
nd B
1), t
hree
birc
h ex
trac
ts (
blue
lin
e) a
nd c
orre
spon
ding
alle
rgoi
ds(r
ed l
ine)
(A2
-A4)
, an
d th
ree
gras
s ex
trac
ts (b
lue
line)
and
cor
resp
ondi
ng a
llerg
oids
(red
line)
(B2-
B4)
. The
bla
ck v
ertic
al li
ne
in th
e ch
rom
atog
ram
spl
its p
eaks
cor
resp
ondi
ng to
mol
ecul
ar m
asse
s ab
ove
and
belo
w
44 k
Da.
A G
F250
SEC
col
umn
was
app
lied
for b
irch,
a G
F450
SEC
col
umn
for g
rass
es.
Tabl
e 1:
Phy
sico
chem
ical
cha
ract
eris
tics
of th
ree
batc
hes
of b
irch
alle
rgoi
ds
670
kDa
158
kDa44
kDa 17
kDa 1.35
kDa
Absorbance 215 nm
Rete
ntio
ntim
e (m
in)
670
kDa 15
8 kD
a44 k
Da 17 k
Da 1.35
kDa
Stan
dard
Stan
dard
Birc
h1
Birc
h2
Birc
h3
Gra
ss1
Gra
ss2
Gra
ss3
A1 A2 A3 A4
B1 B2 B3 B4
Tabl
e 2:
Phy
sico
chem
ical
cha
ract
eris
tics
of th
ree
batc
hes
of g
rass
alle
rgoi
ds
Ch
ara
cte
ris
tic
Bir
ch
1
Bir
ch
2
B
irc
h 3
HP
LC
: P
eak
are
a a
bo
ve 4
4 k
Da
(%
)
75
7
4
77
Mo
difi
ed
lysi
ne
s (%
)
74
7
9
78
Flu
ore
scen
ce e
mis
sion m
axi
mum
(nm
) 3
11
3
11
3
11
C
ha
rac
teri
sti
c
G
ras
s 1
G
ras
s 2
Gra
ss 3
HP
LC
: P
eak
are
a a
bo
ve 4
4 k
Da
(%
)
76
6
8
79
Mo
difi
ed
lysi
ne
s (%
)
73
7
3
76
Flu
ore
sce
nce
em
issi
on m
axi
mu
m (
nm
) 3
29
3
29
3
30
Figu
re 1
: SD
S-PA
GE
patte
rns
of m
olec
ular
wei
ght
mar
ker
(M),
thre
e bi
rch
(1-3
) an
d th
ree
gras
s (4
-6)
extr
acts
and
cor
resp
ondi
ng a
llerg
oids
(1-6
). M
ajor
birc
h al
lerg
en B
et v
1 an
d m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5 ar
e bo
xed.
M1
23
54
61
24
35
6
Extr
acts
Alle
rgoi
dsG
rass
Birc
hB
irch
Birc
hG
rass
Birc
hB
irch
Gra
ssG
rass
Birc
hG
rass
Birc
hG
rass
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12
287
-Cha
ract
eris
atio
n of
pol
len
alle
rgoi
dsw
ith p
hysi
coch
emic
al te
chni
ques
D. L
uykx
1 , J.
de
Brui
jn1 1, J
. Cor
dew
ener
2
2
, T. A
mer
ica2
and
R. v
an d
en H
out1
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Plan
t Res
earc
h In
tern
atio
nal,
Wag
enin
gen,
The
Net
herla
nds
Back
grou
nd &
Aim
:Bi
rch
and
gras
s al
lerg
oids
are
used
as
drug
sub
stan
ces
for b
irch
and
gras
s al
lerg
y va
ccin
es, r
espe
ctive
ly. U
ntil
now
thes
e al
lerg
oids
have
bee
n po
orly
ch
arac
teris
ed d
ue to
thei
r co
mpl
exity
. A b
ette
r ch
arac
teris
atio
nis
need
ed.
This
inc
lude
s id
entif
icat
ion
of t
he m
ajor
alle
rgen
s, d
eter
min
atio
n of
the
de
gree
of p
olym
eris
atio
n an
d cr
oss-
linki
ng, a
nd in
vest
igat
ion
of th
e pr
otei
n st
ruct
ures
. Se
vera
l ph
ysic
oche
mica
l te
chni
ques
wer
e ap
plie
d to
ana
lyse
th
ese
char
acte
ristic
s in
thr
ee s
ucce
ssiv
e, s
epar
atel
y pr
oduc
ed b
irch
and
gras
s al
lerg
oids
and
corre
spon
ding
alle
rgen
ext
ract
s: M
ass
spec
trom
etry
(M
S),
SDS-
PAG
E,
HPL
C-S
EC,
lysin
e de
term
inat
ion
and
fluor
esce
nce
spec
trosc
opy.
Met
hods
:Al
lerg
oids
: Alle
rgen
ext
ract
s fro
m b
irch
and
mixe
d gr
ass
polle
n we
re tr
eate
d wi
th g
luta
rald
ehyd
eto
obt
ain
birc
h an
d gr
ass
alle
rgoi
ds, r
espe
ctiv
ely.
MS:
Firs
t try
ptic
dige
sts
were
pre
pare
d fro
m th
e sa
mpl
es. S
ubse
quen
tly, t
he
pept
ides
wer
e se
para
ted
with
nan
o-liq
uid
chro
mat
ogra
phy
befo
re e
nter
ing
the
MS
inst
rum
ent
via
elec
trosp
ray
ioni
satio
n.
Ioni
sed
pept
ides
we
re
fragm
ente
d re
veal
ing
amin
o ac
id s
eque
nces
(i.e
., id
entif
icatio
n).
SDS-
PAG
E: R
educ
ed s
ampl
es w
ere
appl
ied
onto
10-
20%
pol
yacr
ylam
ide
gels
and
sta
ined
with
Coo
mas
sie
Blue
.
HPL
C-S
EC:
A G
F250
or
GF4
50 s
ize e
xclu
sion
chr
omat
ogra
phy
(SEC
) co
lum
n wa
s us
ed c
ombi
ned
with
UV-
dete
ctio
n (2
15 n
m).
Lysi
ne d
eter
min
atio
n : S
ampl
es w
ere
hydr
olys
ed in
to a
min
o ac
ids
follo
wed
by l
ysin
e de
term
inat
ion
with
HPL
C. B
y de
term
inin
g th
e am
ount
of
free
lysi
nes
in t
he e
xtra
ct a
nd a
llerg
oid,
the
am
ount
of m
odifi
ed ly
sine
sin
the
al
lerg
oid
can
be c
alcu
late
d.
Fluo
resc
ence
spe
ctro
scop
y : E
mis
sion
spec
tra w
ere
reco
rded
from
290
-400
nm
with
exc
itatio
n at
280
nm
.
Resu
lts:
MS:
Sev
eral
isof
orm
sof
the
maj
or b
irch
alle
rgen
Bet
v1
were
iden
tifie
d in
th
e th
ree
birc
h al
lerg
oids
(a, b
, c, d
/h, f
/i). N
ext t
o th
e m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5, a
lso
grou
p 2,
3, 4
and
6 a
llerg
ens
were
iden
tifie
d in
the
thre
e gr
ass
alle
rgoi
ds(d
ata
not s
hown
).
SDS-
PAG
E: F
or t
he t
hree
birc
h ex
tract
s, s
imila
r pr
otei
n pr
ofile
s we
re
obse
rved
(Fi
g. 1
). Th
e ba
nd c
orre
spon
ding
to
15-1
8 kD
ain
dica
tes
the
pres
ence
of m
ajor
alle
rgen
Bet
v1.
For
the
thre
e gr
ass
extra
cts
also
sim
ilar
prot
ein
prof
iles
were
obs
erve
d. H
ere,
the
band
s co
rresp
ondi
ng to
27-3
4 kD
ain
dica
te th
e pr
esen
ce o
f maj
or a
llerg
ens
grou
p 1
and
5. F
or a
ll al
lerg
oids
the
form
atio
n of
var
ious
hig
h m
olec
ular
wei
ght m
olec
ules
is s
hown
via
a s
mea
r an
d a
band
aro
und
250
kDa
(mol
ecul
es
250
kDa)
on
gel.
HPL
C-S
EC:
For
the
thre
e bi
rch
and
gras
s ex
tract
s a
min
ority
of
HPL
C
peak
s is
cor
resp
ondi
ng t
o pr
otei
ns w
ith a
mol
ecul
ar m
ass
abov
e 44
kDa
(Fig
. 2)
. Th
e ch
rom
atog
ram
s of
the
birc
h an
d gr
ass
alle
rgoi
dba
tche
s sh
owed
the
pre
senc
e of
a v
arie
ty o
f hi
gh m
olec
ular
wei
ght
mol
ecul
es
incl
udin
g m
olec
ules
with
mol
ecul
ar m
asse
s 67
0 kD
a. H
ere,
a c
onsi
sten
t m
ajor
ity
of
peak
s (7
4-77
%
for
birc
h an
d 68
-79%
fo
r gr
asse
s)
is co
rresp
ondi
ng t
o pr
otei
ns w
ith a
mol
ecul
ar m
ass
(far)
abov
e 44
kDa
show
ing
a co
nsis
tent
deg
ree
of p
rote
in p
olym
eris
atio
n (T
able
1-2
).
Lysi
ne d
eter
min
atio
n: F
or t
he b
irch
alle
rgoi
ds74
-79%
of t
he ly
sine
swe
re
mod
ified
and
for
the
gras
s al
lerg
oids
73-7
6% o
f the
lysi
nes
were
mod
ified
(Tab
le 1
-2).
This
sho
ws a
con
sist
ent d
egre
e of
pro
tein
cro
ss-li
nkin
g.
Fluo
resc
ence
spe
ctro
scop
y : M
odific
atio
n of
the
birc
h an
d gr
ass
extra
cts
resu
lted
in a
shi
ft of
the
emis
sion
max
imum
to a
lowe
r wa
vele
ngth
and
a
decr
ease
of f
luor
esce
nce
inte
nsity
(dat
a no
t sho
wn).
This
indi
cate
s th
at th
e su
rroun
ding
s of
the
arom
atic
am
ino
acid
s in
the
prot
eins
cha
nge
to a
mor
e hy
drop
hobi
c en
viro
nmen
t whi
ch is
indi
cativ
e fo
r pro
tein
pol
ymer
isat
ion.
The
em
issi
on
max
imum
va
lues
ob
tain
ed
for
the
polle
n al
lerg
oids
show
co
nsis
tenc
y as
wel
l (Ta
ble
1-2)
.
Conc
lusi
on:
Appl
ying
a c
ombi
natio
n of
phy
sicoc
hem
ical
tech
niqu
es w
as s
hown
to b
e a
suita
ble
appr
oach
to c
hara
cter
ise th
e po
llen
alle
rgoi
dswe
ll: Id
entif
icat
ion
of
the
maj
or a
llerg
ens,
det
erm
inat
ion
of t
he d
egre
e of
pol
ymer
isat
ion
and
cros
s-lin
king
, and
inve
stig
atio
n of
the
prot
ein
stru
ctur
es w
ere
acco
mpl
ishe
d.
This
stu
dy s
hows
con
sist
ency
for t
he m
odifi
catio
n of
the
polle
n ex
tract
s.
Figu
re 2
: H
PLC
-SEC
pat
tern
s (A
215
nm)
of c
alib
ratio
n st
anda
rd (
A1 a
nd B
1), t
hree
birc
h ex
trac
ts (
blue
lin
e) a
nd c
orre
spon
ding
alle
rgoi
ds(r
ed l
ine)
(A2
-A4)
, an
d th
ree
gras
s ex
trac
ts (b
lue
line)
and
cor
resp
ondi
ng a
llerg
oids
(red
line)
(B2-
B4)
. The
bla
ck v
ertic
al li
ne
in th
e ch
rom
atog
ram
spl
its p
eaks
cor
resp
ondi
ng to
mol
ecul
ar m
asse
s ab
ove
and
belo
w
44 k
Da.
A G
F250
SEC
col
umn
was
app
lied
for b
irch,
a G
F450
SEC
col
umn
for g
rass
es.
Tabl
e 1:
Phy
sico
chem
ical
cha
ract
eris
tics
of th
ree
batc
hes
of b
irch
alle
rgoi
ds
670
kDa
158
kDa44
kDa 17
kDa 1.35
kDa
Absorbance 215 nm
Rete
ntio
ntim
e (m
in)
670
kDa 15
8 kD
a44 k
Da 17 k
Da 1.35
kDa
Stan
dard
Stan
dard
Birc
h1
Birc
h2
Birc
h3
Gra
ss1
Gra
ss2
Gra
ss3
A1 A2 A3 A4
B1 B2 B3 B4
Tabl
e 2:
Phy
sico
chem
ical
cha
ract
eris
tics
of th
ree
batc
hes
of g
rass
alle
rgoi
ds
Ch
ara
cte
ris
tic
Bir
ch
1
Bir
ch
2
B
irc
h 3
HP
LC
: P
eak
are
a a
bo
ve 4
4 k
Da
(%
)
75
7
4
77
Mo
difi
ed
lysi
ne
s (%
)
74
7
9
78
Flu
ore
scen
ce e
mis
sion m
axi
mum
(nm
) 3
11
3
11
3
11
C
ha
rac
teri
sti
c
G
ras
s 1
G
ras
s 2
Gra
ss 3
HP
LC
: P
eak
are
a a
bo
ve 4
4 k
Da
(%
)
76
6
8
79
Mo
difi
ed
lysi
ne
s (%
)
73
7
3
76
Flu
ore
sce
nce
em
issi
on m
axi
mu
m (
nm
) 3
29
3
29
3
30
Figu
re 1
: SD
S-PA
GE
patte
rns
of m
olec
ular
wei
ght
mar
ker
(M),
thre
e bi
rch
(1-3
) an
d th
ree
gras
s (4
-6)
extr
acts
and
cor
resp
ondi
ng a
llerg
oids
(1-6
). M
ajor
birc
h al
lerg
en B
et v
1 an
d m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5 ar
e bo
xed.
M1
23
54
61
24
35
6
Extr
acts
Alle
rgoi
dsG
rass
Birc
hB
irch
Birc
hG
rass
Birc
hB
irch
Gra
ssG
rass
Birc
hG
rass
Birc
hG
rass
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12
In re
latio
n to
this
pre
sent
atio
n, I
decl
are
the
follo
win
g, re
al o
r per
ceiv
ed c
onfli
cts
of in
tere
st: t
he p
rese
nter
is a
n em
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
C h ara c t e ris a t i o n o f a l u m - a ds o r b e d p o l l e n a l l e rg o i ds w i t h p h y sic o c h e mic a l t e c h ni q u e s
28
D. Luykx (1), J. de Bruijn (1), J. Cordewener (2), T. America (2), R. van den Hout (1).
(1) HAL Allergy BV, Leiden, The Netherlands, (2) Plant Research International, Wageningen, The Netherlands.
BackgroundSeveral physicochemical techniques have been selected to investigate their suitability to characterise a birch allergy vaccine (ALU-B) and grass allergy vaccine (ALU-G). These techniques are mass spectrometry (MS), circular dichroism (CD), and front-face fluorescence (FF-fluorescence). They should be able to investigate the alum-adsorbed and modified allergens on all structural levels, i.e. primary structure (for protein identification), secondary and tertiary structure (for structural stability). Samples were temperature stressed to find out if these techniques are stability indicating.
MethodAlum-adsorbed pollen allergoids: Allergen extracts from birch and grasses were modified with glutaraldehyde and adsorbed onto aluminium hydroxide to obtain ALU-B and ALU-G, respectively. MS: Preparation of tryptic digests and peptide separation via nano-LC before electrospray ionisation. Ionised peptides were fragmented revealing amino acid sequences. CD: Far-UV CD spectra were recorded from 195 to 260 nm while stirring the sample suspension. FF-fluorescence: Fluorescence emission spectra were recorded from 290 to 400 nm, with excitation at 280 nm.
ResultsVia MS several isoforms of relevant birch allergen Bet v1 were identified in ALU-B. In ALU-G relevant grass allergens group 1 and 5 were identified. The CD-spectrum of ALU-B and ALU-G represented protein structures consisting of mainly α-helices and ß-structure. The FF-fluorescence spectrum of both products showed an emission maximum at 326 nm suggesting hydrophobic surroundings for the aromatic amino acids present in both protein samples. Storage of ALU-B and ALU-G at several elevated temperatures resulted in spectral alterations meaning structural alterations of protein. CD spectra indicated partially loss of α-helices (increase of ratio 207/222 nm) while fluorescence spectra indicated formation of a less rigid structure (decrease of fluorescence at 326 nm).
ConclusionMS, CD and FF-fluorescence were able to characterise the complex birch and grass allergy vaccines on all structural levels, i.e. primary, secondary and tertiary structure. Via MS the relevant birch and grass allergens could be identified while via CD and FF-fluorescence the protein structures could be investigated. Moreover, CD and FF-fluorescence were shown to be promising stability indicating assays.
EAACI, 16-20 June 2012, GenevaAbstract number: 560, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations
29
Poster Session 13 - Immunotherapy: allergens and allergen-preparations
C h ara c t e ris a t i o n o f a l u m - a ds o r b e d p o l l e n a l l e rg o i ds w i t h p h y sic o c h e mic a l t e c h ni q u e s
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
Time (
mont
hs)
CD ratio 207/222 nm
5°C
37°C
0510152025
01
23
Time (
month
s)
Fluorescence intensity (106 x LU)
5°C
37°C
0510152025
01
23
Time (
mont
hs)
Fluorescence intensity (106 x LU)
5°C
37°C
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
Time (
mont
hs)
CD ratio 207/222 nm
5°C
37°C
0510152025
290
300
310
320
330
340
350
360
370
380
390
400
Wave
length
(nm)
Fluorescence intensity (106 x LU)
0510152025
290
300
310
320
330
340
350
360
370
380
390
400
Wave
length
(nm)
Fluorescence intensity (106 x LU)
560
-Cha
ract
eris
atio
nof
alu
m-a
dsor
bed
polle
n al
lerg
oids
with
ph
ysic
oche
mic
al te
chni
ques
D. L
uykx
1
1, J
. de
Brui
jn1
2
, J. C
orde
wen
er2 ,
T. A
mer
ica2
and
R. v
an d
en H
out1
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Plan
t Res
earc
h In
tern
atio
nal,
Wag
enin
gen,
The
Net
herla
nds
Back
grou
nd &
Aim
:Se
vera
l phy
sico
chem
ical t
echn
ique
s ha
ve b
een
sele
cted
to in
vest
igat
e th
eir s
uita
bilit
y to
cha
ract
eris
e a
birc
h al
lerg
y va
ccin
e (A
LU-B
) and
gra
ss
alle
rgy
vacc
ine
(ALU
-G).
Thes
e te
chni
ques
are
mas
s sp
ectro
met
ry
(MS)
, ci
rcul
ar
dich
roism
(CD)
, an
d fro
nt-fa
ce
fluor
esce
nce
(FF-
fluor
esce
nce)
. Th
ey s
houl
d be
abl
e to
inv
estig
ate
the
alum
-ads
orbe
d m
odifi
ed a
llerg
ens
on a
ll st
ruct
ural
lev
els,
i.e
. pr
imar
y st
ruct
ure
(for
prot
ein
iden
tific
atio
n),
seco
ndar
y an
d te
rtiar
y st
ruct
ure
(for
stru
ctur
al
stab
ility)
. Sa
mpl
es w
ere
tem
pera
ture
stre
ssed
to
find
out
if th
ese
tech
niqu
es a
re s
tabi
lity
indi
catin
g.
Met
hods
:Al
um-a
dsor
bed
polle
n al
lerg
oids
: Alle
rgen
ext
ract
s fro
m b
irch
and
mixe
d gr
ass
polle
n we
re m
odifi
ed w
ith g
luta
rald
ehyd
e, a
nd a
dsor
bed
onto
al
umin
ium
hyd
roxi
de to
obt
ain
ALU
-B a
nd A
LU-G
, res
pect
ivel
y.
Stre
ss c
ondi
tions
: ALU
-B a
nd A
LU-G
wer
e st
ored
at 5
and
37º
C fo
r 1-3
m
onth
s, a
t 50º
Cfo
r 12
hour
s, a
nd a
t 90º
C fo
r 1 h
our.
MS:
The
app
lied
proc
edur
e fir
stly
invo
lved
prep
arat
ion
of tr
yptic
dige
sts
from
the
sam
ple.
Nex
t, th
e pe
ptid
es w
ere
sepa
rate
d vi
a 2D
nan
o-liq
uid
chro
mat
ogra
phy
befo
re e
lect
rosp
ray
ioni
satio
n an
d en
terin
g th
e M
S in
stru
men
t. Fo
r pr
otei
n id
entif
icatio
n,
the
ioni
sed
pept
ides
we
re
fragm
ente
d re
veal
ing
the
amin
o ac
id s
eque
nce.
CD: F
ar-U
V CD
spe
ctra
wer
e re
cord
ed fr
om 1
95 to
260
nm
and
und
er
stirr
ing
cond
itions
of
the
sam
ple.
The
CD
ratio
207
/222
nm
wa
s m
onito
red.
FF-fl
uore
scen
ce:
Fluo
resc
ence
em
issi
on s
pect
ra w
ere
reco
rded
fro
m
290
to 4
00 n
m w
ith e
xcita
tion
at 2
80 n
m. T
he fl
uore
scen
ce in
tens
ity a
t 32
6 nm
was
mon
itore
d.
Resu
lts:
Via
MS
seve
ral
isofo
rms
of t
he m
ajor
birc
h al
lerg
en B
et v
1 we
re
iden
tifie
d in
ALU
-B (a
, b, c
, d/h
, f/i)
. In
ALU
-G th
e m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5 we
re id
entif
ied
(dat
a no
t sho
wn).
The
CD-
spec
trum
of A
LU-B
and
ALU
-G r
epre
sent
ed p
rote
in s
truct
ures
co
nsis
ting
of m
ainl
y -h
elic
es (i
.e.,
ellip
ticity
at 2
08 a
nd 2
22 n
m) a
nd
-st
ruct
ure
(Fig
. 1)
. Th
e FF
-fluo
resc
ence
spe
ctru
m o
f bo
th v
acci
nes
show
ed a
n em
issi
on m
axim
um a
t 32
6 nm
sug
gest
ing
hydr
opho
bic
surro
undi
ngs
for
the
arom
atic
am
ino
acid
s pr
esen
t in
bot
h pr
otei
n sa
mpl
es (F
ig. 2
).
Stor
age
of A
LU-B
and
ALU
-G a
t sev
eral
ele
vate
d te
mpe
ratu
res
resu
lted
in s
pect
ral
alte
ratio
ns m
eani
ng s
truct
ural
alte
ratio
ns o
f pr
otei
n. C
D sp
ectra
indi
cate
d pa
rtial
ly lo
ss o
f -h
elic
es (
incr
ease
of
ratio
207
/222
nm
) wh
ile f
luor
esce
nce
spec
tra i
ndic
ated
for
mat
ion
of a
les
s rig
id
stru
ctur
e (d
ecre
ase
of fl
uore
scen
ce a
t 326
nm
).By
mon
itorin
g th
e C
D-ra
tio 2
07/2
22 n
m (
i.e.,
loss
of
-hel
ices
) an
d flu
ores
cenc
e in
tens
ity a
t 326
nm
(i.e
., rig
idity
), th
e st
ruct
ural
sta
bilit
y of
AL
U-B
and
ALU
-G w
as fo
llowe
d at
5 a
nd 3
7ºC
for 1
-3 m
onth
s (F
ig. 3
-4).
Stor
age
at
5ºC
di
d no
t af
fect
the
se
cond
ary
and
terti
ary
prot
ein
stru
ctur
es o
f bot
h va
ccin
es. H
owev
er, s
truct
ural
cha
nges
wer
e ob
serv
ed
for
the
polle
n va
ccin
es w
hen
stor
ed a
t 37
ºC.
Stor
age
at 5
0ºC
for
12
hour
s di
d re
sult
in s
imila
r st
ruct
ural
cha
nges
as
was
foun
d af
ter
1-2
mon
ths
at 3
7ºC
(dat
a no
t sho
wn).
The
prot
ein
stru
ctur
es o
f ALU
-B a
nd
ALU
-G w
ere
mos
tly e
ffect
ed w
hen
stor
ed a
t 90º
C fo
r 1
hour
(da
ta n
ot
show
n). I
n th
is ca
se th
e C
D-ra
tio in
crea
sed
to 1
.20
and
1.56
for A
LU-B
an
d AL
U-G
, re
spec
tivel
y.
With
re
spec
t to
th
e flu
ores
cenc
e,
the
inte
nsiti
es d
ecre
ased
for b
oth
vacc
ines
to 9
.5 x
106 .
Conc
lusi
on:
MS,
CD
and
FF-fl
uore
scen
ce w
ere
able
to
char
acte
rise
the
com
plex
bi
rch
and
gras
s al
lerg
y va
ccin
es o
n al
l stru
ctur
al l
evel
s, i
.e.
prim
ary,
se
cond
ary
and
terti
ary
stru
ctur
e. V
ia M
S th
e m
ajor
birc
h an
d gr
ass
alle
rgen
s we
re id
entif
ied
while
via
CD
and
FF-fl
uore
scen
ce th
e pr
otei
n st
ruct
ures
wer
e el
ucid
ated
. CD
and
FF-fl
uore
scen
ce w
ere
show
n to
be
prom
isin
g st
abilit
y in
dica
ting
assa
ys.
Figu
re 4
: Flu
ores
cenc
e in
tens
ity a
t 326
nm
of A
LU-B
(A) a
nd A
LU-G
(B) s
tore
d at
5 a
nd
37ºC
for 1
-3 m
onth
s.
Figu
re 1
: Fa
r-UV
CD
-spe
ctru
m o
f AL
U-B
(A)
and
ALU
-G (
B).
The
arro
ws
indi
cate
the
el
liptic
ityat
207
and
222
nm
.
Figu
re 3
: CD
-ratio
207
/222
nm
of
ALU
-B (
A) a
nd A
LU-G
(B) s
tore
d at
5 a
nd 3
7ºC
for
1-3
mon
ths.
Figu
re 2
: FF-
fluor
esce
nce
spec
trum
of A
LU-B
(A) a
nd A
LU-G
(B).
The
arro
w in
dica
tes
the
fluor
esce
nce
inte
nsity
at 3
26 n
m. T
he p
eak
at 3
10 n
m re
pres
ents
the
wat
er ra
man
peak
.
-20-15-10-50510
190
200
210
220
230
240
250
260
Wav
eleng
th (n
m)
Ellipticity (mDeg)
-10-50510
190
200
210
220
230
240
250
260
Wav
eleng
th (nm
)
Ellipticity (mDeg)
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
Time (
mont
hs)
CD ratio 207/222 nm
5°C
37°C
0510152025
01
23
Time (
month
s)
Fluorescence intensity (106 x LU)
5°C
37°C
0510152025
01
23
Time (
mont
hs)
Fluorescence intensity (106 x LU)
5°C
37°C
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
Time (
mont
hs)
CD ratio 207/222 nm
5°C
37°C
0510152025
290
300
310
320
330
340
350
360
370
380
390
400
Wave
length
(nm)
Fluorescence intensity (106 x LU)
0510152025
290
300
310
320
330
340
350
360
370
380
390
400
Wave
length
(nm)
Fluorescence intensity (106 x LU)
560
-Cha
ract
eris
atio
nof
alu
m-a
dsor
bed
polle
n al
lerg
oids
with
ph
ysic
oche
mic
al te
chni
ques
D. L
uykx
1
1, J
. de
Brui
jn1
2
, J. C
orde
wen
er2 ,
T. A
mer
ica2
and
R. v
an d
en H
out1
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Plan
t Res
earc
h In
tern
atio
nal,
Wag
enin
gen,
The
Net
herla
nds
Back
grou
nd &
Aim
:Se
vera
l phy
sico
chem
ical t
echn
ique
s ha
ve b
een
sele
cted
to in
vest
igat
e th
eir s
uita
bilit
y to
cha
ract
eris
e a
birc
h al
lerg
y va
ccin
e (A
LU-B
) and
gra
ss
alle
rgy
vacc
ine
(ALU
-G).
Thes
e te
chni
ques
are
mas
s sp
ectro
met
ry
(MS)
, ci
rcul
ar
dich
roism
(CD)
, an
d fro
nt-fa
ce
fluor
esce
nce
(FF-
fluor
esce
nce)
. Th
ey s
houl
d be
abl
e to
inv
estig
ate
the
alum
-ads
orbe
d m
odifi
ed a
llerg
ens
on a
ll st
ruct
ural
lev
els,
i.e
. pr
imar
y st
ruct
ure
(for
prot
ein
iden
tific
atio
n),
seco
ndar
y an
d te
rtiar
y st
ruct
ure
(for
stru
ctur
al
stab
ility)
. Sa
mpl
es w
ere
tem
pera
ture
stre
ssed
to
find
out
if th
ese
tech
niqu
es a
re s
tabi
lity
indi
catin
g.
Met
hods
:Al
um-a
dsor
bed
polle
n al
lerg
oids
: Alle
rgen
ext
ract
s fro
m b
irch
and
mixe
d gr
ass
polle
n we
re m
odifi
ed w
ith g
luta
rald
ehyd
e, a
nd a
dsor
bed
onto
al
umin
ium
hyd
roxi
de to
obt
ain
ALU
-B a
nd A
LU-G
, res
pect
ivel
y.
Stre
ss c
ondi
tions
: ALU
-B a
nd A
LU-G
wer
e st
ored
at 5
and
37º
C fo
r 1-3
m
onth
s, a
t 50º
Cfo
r 12
hour
s, a
nd a
t 90º
C fo
r 1 h
our.
MS:
The
app
lied
proc
edur
e fir
stly
invo
lved
prep
arat
ion
of tr
yptic
dige
sts
from
the
sam
ple.
Nex
t, th
e pe
ptid
es w
ere
sepa
rate
d vi
a 2D
nan
o-liq
uid
chro
mat
ogra
phy
befo
re e
lect
rosp
ray
ioni
satio
n an
d en
terin
g th
e M
S in
stru
men
t. Fo
r pr
otei
n id
entif
icatio
n,
the
ioni
sed
pept
ides
we
re
fragm
ente
d re
veal
ing
the
amin
o ac
id s
eque
nce.
CD: F
ar-U
V CD
spe
ctra
wer
e re
cord
ed fr
om 1
95 to
260
nm
and
und
er
stirr
ing
cond
itions
of
the
sam
ple.
The
CD
ratio
207
/222
nm
wa
s m
onito
red.
FF-fl
uore
scen
ce:
Fluo
resc
ence
em
issi
on s
pect
ra w
ere
reco
rded
fro
m
290
to 4
00 n
m w
ith e
xcita
tion
at 2
80 n
m. T
he fl
uore
scen
ce in
tens
ity a
t 32
6 nm
was
mon
itore
d.
Resu
lts:
Via
MS
seve
ral
isofo
rms
of t
he m
ajor
birc
h al
lerg
en B
et v
1 we
re
iden
tifie
d in
ALU
-B (a
, b, c
, d/h
, f/i)
. In
ALU
-G th
e m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5 we
re id
entif
ied
(dat
a no
t sho
wn).
The
CD-
spec
trum
of A
LU-B
and
ALU
-G r
epre
sent
ed p
rote
in s
truct
ures
co
nsis
ting
of m
ainl
y -h
elic
es (i
.e.,
ellip
ticity
at 2
08 a
nd 2
22 n
m) a
nd
-st
ruct
ure
(Fig
. 1)
. Th
e FF
-fluo
resc
ence
spe
ctru
m o
f bo
th v
acci
nes
show
ed a
n em
issi
on m
axim
um a
t 32
6 nm
sug
gest
ing
hydr
opho
bic
surro
undi
ngs
for
the
arom
atic
am
ino
acid
s pr
esen
t in
bot
h pr
otei
n sa
mpl
es (F
ig. 2
).
Stor
age
of A
LU-B
and
ALU
-G a
t sev
eral
ele
vate
d te
mpe
ratu
res
resu
lted
in s
pect
ral
alte
ratio
ns m
eani
ng s
truct
ural
alte
ratio
ns o
f pr
otei
n. C
D sp
ectra
indi
cate
d pa
rtial
ly lo
ss o
f -h
elic
es (
incr
ease
of
ratio
207
/222
nm
) wh
ile f
luor
esce
nce
spec
tra i
ndic
ated
for
mat
ion
of a
les
s rig
id
stru
ctur
e (d
ecre
ase
of fl
uore
scen
ce a
t 326
nm
).By
mon
itorin
g th
e C
D-ra
tio 2
07/2
22 n
m (
i.e.,
loss
of
-hel
ices
) an
d flu
ores
cenc
e in
tens
ity a
t 326
nm
(i.e
., rig
idity
), th
e st
ruct
ural
sta
bilit
y of
AL
U-B
and
ALU
-G w
as fo
llowe
d at
5 a
nd 3
7ºC
for 1
-3 m
onth
s (F
ig. 3
-4).
Stor
age
at
5ºC
di
d no
t af
fect
the
se
cond
ary
and
terti
ary
prot
ein
stru
ctur
es o
f bot
h va
ccin
es. H
owev
er, s
truct
ural
cha
nges
wer
e ob
serv
ed
for
the
polle
n va
ccin
es w
hen
stor
ed a
t 37
ºC.
Stor
age
at 5
0ºC
for
12
hour
s di
d re
sult
in s
imila
r st
ruct
ural
cha
nges
as
was
foun
d af
ter
1-2
mon
ths
at 3
7ºC
(dat
a no
t sho
wn).
The
prot
ein
stru
ctur
es o
f ALU
-B a
nd
ALU
-G w
ere
mos
tly e
ffect
ed w
hen
stor
ed a
t 90º
C fo
r 1
hour
(da
ta n
ot
show
n). I
n th
is ca
se th
e C
D-ra
tio in
crea
sed
to 1
.20
and
1.56
for A
LU-B
an
d AL
U-G
, re
spec
tivel
y.
With
re
spec
t to
th
e flu
ores
cenc
e,
the
inte
nsiti
es d
ecre
ased
for b
oth
vacc
ines
to 9
.5 x
106 .
Conc
lusi
on:
MS,
CD
and
FF-fl
uore
scen
ce w
ere
able
to
char
acte
rise
the
com
plex
bi
rch
and
gras
s al
lerg
y va
ccin
es o
n al
l stru
ctur
al l
evel
s, i
.e.
prim
ary,
se
cond
ary
and
terti
ary
stru
ctur
e. V
ia M
S th
e m
ajor
birc
h an
d gr
ass
alle
rgen
s we
re id
entif
ied
while
via
CD
and
FF-fl
uore
scen
ce th
e pr
otei
n st
ruct
ures
wer
e el
ucid
ated
. CD
and
FF-fl
uore
scen
ce w
ere
show
n to
be
prom
isin
g st
abilit
y in
dica
ting
assa
ys.
Figu
re 4
: Flu
ores
cenc
e in
tens
ity a
t 326
nm
of A
LU-B
(A) a
nd A
LU-G
(B) s
tore
d at
5 a
nd
37ºC
for 1
-3 m
onth
s.
Figu
re 1
: Fa
r-UV
CD
-spe
ctru
m o
f AL
U-B
(A)
and
ALU
-G (
B).
The
arro
ws
indi
cate
the
el
liptic
ityat
207
and
222
nm
.
Figu
re 3
: CD
-ratio
207
/222
nm
of
ALU
-B (
A) a
nd A
LU-G
(B) s
tore
d at
5 a
nd 3
7ºC
for
1-3
mon
ths.
Figu
re 2
: FF-
fluor
esce
nce
spec
trum
of A
LU-B
(A) a
nd A
LU-G
(B).
The
arro
w in
dica
tes
the
fluor
esce
nce
inte
nsity
at 3
26 n
m. T
he p
eak
at 3
10 n
m re
pres
ents
the
wat
er ra
man
peak
.
-20-15-10-50510
190
200
210
220
230
240
250
260
Wav
eleng
th (n
m)
Ellipticity (mDeg)
-10-50510
190
200
210
220
230
240
250
260
Wav
eleng
th (nm
)
Ellipticity (mDeg)
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
Time (
mont
hs)
CD ratio 207/222 nm
5°C
37°C
0510152025
01
23
Time (
month
s)
Fluorescence intensity (106 x LU)
5°C
37°C
0510152025
01
23
Time (
mont
hs)
Fluorescence intensity (106 x LU)
5°C
37°C
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
01
23
Time (
mont
hs)
CD ratio 207/222 nm
5°C
37°C
0510152025
290
300
310
320
330
340
350
360
370
380
390
400
Wave
length
(nm)
Fluorescence intensity (106 x LU)
0510152025
290
300
310
320
330
340
350
360
370
380
390
400
Wave
length
(nm)
Fluorescence intensity (106 x LU)
560
-Cha
ract
eris
atio
nof
alu
m-a
dsor
bed
polle
n al
lerg
oids
with
ph
ysic
oche
mic
al te
chni
ques
D. L
uykx
1
1, J
. de
Brui
jn1
2
, J. C
orde
wen
er2 ,
T. A
mer
ica2
and
R. v
an d
en H
out1
HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s
Plan
t Res
earc
h In
tern
atio
nal,
Wag
enin
gen,
The
Net
herla
nds
Back
grou
nd &
Aim
:Se
vera
l phy
sico
chem
ical t
echn
ique
s ha
ve b
een
sele
cted
to in
vest
igat
e th
eir s
uita
bilit
y to
cha
ract
eris
e a
birc
h al
lerg
y va
ccin
e (A
LU-B
) and
gra
ss
alle
rgy
vacc
ine
(ALU
-G).
Thes
e te
chni
ques
are
mas
s sp
ectro
met
ry
(MS)
, ci
rcul
ar
dich
roism
(CD)
, an
d fro
nt-fa
ce
fluor
esce
nce
(FF-
fluor
esce
nce)
. Th
ey s
houl
d be
abl
e to
inv
estig
ate
the
alum
-ads
orbe
d m
odifi
ed a
llerg
ens
on a
ll st
ruct
ural
lev
els,
i.e
. pr
imar
y st
ruct
ure
(for
prot
ein
iden
tific
atio
n),
seco
ndar
y an
d te
rtiar
y st
ruct
ure
(for
stru
ctur
al
stab
ility)
. Sa
mpl
es w
ere
tem
pera
ture
stre
ssed
to
find
out
if th
ese
tech
niqu
es a
re s
tabi
lity
indi
catin
g.
Met
hods
:Al
um-a
dsor
bed
polle
n al
lerg
oids
: Alle
rgen
ext
ract
s fro
m b
irch
and
mixe
d gr
ass
polle
n we
re m
odifi
ed w
ith g
luta
rald
ehyd
e, a
nd a
dsor
bed
onto
al
umin
ium
hyd
roxi
de to
obt
ain
ALU
-B a
nd A
LU-G
, res
pect
ivel
y.
Stre
ss c
ondi
tions
: ALU
-B a
nd A
LU-G
wer
e st
ored
at 5
and
37º
C fo
r 1-3
m
onth
s, a
t 50º
Cfo
r 12
hour
s, a
nd a
t 90º
C fo
r 1 h
our.
MS:
The
app
lied
proc
edur
e fir
stly
invo
lved
prep
arat
ion
of tr
yptic
dige
sts
from
the
sam
ple.
Nex
t, th
e pe
ptid
es w
ere
sepa
rate
d vi
a 2D
nan
o-liq
uid
chro
mat
ogra
phy
befo
re e
lect
rosp
ray
ioni
satio
n an
d en
terin
g th
e M
S in
stru
men
t. Fo
r pr
otei
n id
entif
icatio
n,
the
ioni
sed
pept
ides
we
re
fragm
ente
d re
veal
ing
the
amin
o ac
id s
eque
nce.
CD: F
ar-U
V CD
spe
ctra
wer
e re
cord
ed fr
om 1
95 to
260
nm
and
und
er
stirr
ing
cond
itions
of
the
sam
ple.
The
CD
ratio
207
/222
nm
wa
s m
onito
red.
FF-fl
uore
scen
ce:
Fluo
resc
ence
em
issi
on s
pect
ra w
ere
reco
rded
fro
m
290
to 4
00 n
m w
ith e
xcita
tion
at 2
80 n
m. T
he fl
uore
scen
ce in
tens
ity a
t 32
6 nm
was
mon
itore
d.
Resu
lts:
Via
MS
seve
ral
isofo
rms
of t
he m
ajor
birc
h al
lerg
en B
et v
1 we
re
iden
tifie
d in
ALU
-B (a
, b, c
, d/h
, f/i)
. In
ALU
-G th
e m
ajor
gra
ss a
llerg
ens
grou
p 1
and
5 we
re id
entif
ied
(dat
a no
t sho
wn).
The
CD-
spec
trum
of A
LU-B
and
ALU
-G r
epre
sent
ed p
rote
in s
truct
ures
co
nsis
ting
of m
ainl
y -h
elic
es (i
.e.,
ellip
ticity
at 2
08 a
nd 2
22 n
m) a
nd
-st
ruct
ure
(Fig
. 1)
. Th
e FF
-fluo
resc
ence
spe
ctru
m o
f bo
th v
acci
nes
show
ed a
n em
issi
on m
axim
um a
t 32
6 nm
sug
gest
ing
hydr
opho
bic
surro
undi
ngs
for
the
arom
atic
am
ino
acid
s pr
esen
t in
bot
h pr
otei
n sa
mpl
es (F
ig. 2
).
Stor
age
of A
LU-B
and
ALU
-G a
t sev
eral
ele
vate
d te
mpe
ratu
res
resu
lted
in s
pect
ral
alte
ratio
ns m
eani
ng s
truct
ural
alte
ratio
ns o
f pr
otei
n. C
D sp
ectra
indi
cate
d pa
rtial
ly lo
ss o
f -h
elic
es (
incr
ease
of
ratio
207
/222
nm
) wh
ile f
luor
esce
nce
spec
tra i
ndic
ated
for
mat
ion
of a
les
s rig
id
stru
ctur
e (d
ecre
ase
of fl
uore
scen
ce a
t 326
nm
).By
mon
itorin
g th
e C
D-ra
tio 2
07/2
22 n
m (
i.e.,
loss
of
-hel
ices
) an
d flu
ores
cenc
e in
tens
ity a
t 326
nm
(i.e
., rig
idity
), th
e st
ruct
ural
sta
bilit
y of
AL
U-B
and
ALU
-G w
as fo
llowe
d at
5 a
nd 3
7ºC
for 1
-3 m
onth
s (F
ig. 3
-4).
Stor
age
at
5ºC
di
d no
t af
fect
the
se
cond
ary
and
terti
ary
prot
ein
stru
ctur
es o
f bot
h va
ccin
es. H
owev
er, s
truct
ural
cha
nges
wer
e ob
serv
ed
for
the
polle
n va
ccin
es w
hen
stor
ed a
t 37
ºC.
Stor
age
at 5
0ºC
for
12
hour
s di
d re
sult
in s
imila
r st
ruct
ural
cha
nges
as
was
foun
d af
ter
1-2
mon
ths
at 3
7ºC
(dat
a no
t sho
wn).
The
prot
ein
stru
ctur
es o
f ALU
-B a
nd
ALU
-G w
ere
mos
tly e
ffect
ed w
hen
stor
ed a
t 90º
C fo
r 1
hour
(da
ta n
ot
show
n). I
n th
is ca
se th
e C
D-ra
tio in
crea
sed
to 1
.20
and
1.56
for A
LU-B
an
d AL
U-G
, re
spec
tivel
y.
With
re
spec
t to
th
e flu
ores
cenc
e,
the
inte
nsiti
es d
ecre
ased
for b
oth
vacc
ines
to 9
.5 x
106 .
Conc
lusi
on:
MS,
CD
and
FF-fl
uore
scen
ce w
ere
able
to
char
acte
rise
the
com
plex
bi
rch
and
gras
s al
lerg
y va
ccin
es o
n al
l stru
ctur
al l
evel
s, i
.e.
prim
ary,
se
cond
ary
and
terti
ary
stru
ctur
e. V
ia M
S th
e m
ajor
birc
h an
d gr
ass
alle
rgen
s we
re id
entif
ied
while
via
CD
and
FF-fl
uore
scen
ce th
e pr
otei
n st
ruct
ures
wer
e el
ucid
ated
. CD
and
FF-fl
uore
scen
ce w
ere
show
n to
be
prom
isin
g st
abilit
y in
dica
ting
assa
ys.
Figu
re 4
: Flu
ores
cenc
e in
tens
ity a
t 326
nm
of A
LU-B
(A) a
nd A
LU-G
(B) s
tore
d at
5 a
nd
37ºC
for 1
-3 m
onth
s.
Figu
re 1
: Fa
r-UV
CD
-spe
ctru
m o
f AL
U-B
(A)
and
ALU
-G (
B).
The
arro
ws
indi
cate
the
el
liptic
ityat
207
and
222
nm
.
Figu
re 3
: CD
-ratio
207
/222
nm
of
ALU
-B (
A) a
nd A
LU-G
(B) s
tore
d at
5 a
nd 3
7ºC
for
1-3
mon
ths.
Figu
re 2
: FF-
fluor
esce
nce
spec
trum
of A
LU-B
(A) a
nd A
LU-G
(B).
The
arro
w in
dica
tes
the
fluor
esce
nce
inte
nsity
at 3
26 n
m. T
he p
eak
at 3
10 n
m re
pres
ents
the
wat
er ra
man
peak
.
-20-15-10-50510
190
200
210
220
230
240
250
260
Wav
eleng
th (n
m)
Ellipticity (mDeg)
-10-50510
190
200
210
220
230
240
250
260
Wav
eleng
th (nm
)
Ellipticity (mDeg)
In re
latio
n to
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
al o
r per
ceiv
ed c
onflic
ts o
f int
eres
t: th
e pr
esen
ter i
s an
em
ploy
ee o
f HAL
EAAC
I Con
gres
s 20
12
In re
latio
n to
this
pre
sent
atio
n, I
decl
are
the
follo
win
g, re
al o
r per
ceiv
ed c
onfli
cts
of in
tere
st: t
he p
rese
nter
is a
n em
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
An In h i b i t i o n E LI SA m e t h o d f o r t h e d e t e r min a t i o n o f R e l a t iv e Po t e n c y o f M i t e p r e p ara t i o n s
30
E. Kerkvliet, I. Peekel, R. van den Hout.
HAL Allergy BV, Leiden, The Netherlands.
BackgroundWe have developed and (pre-)validated a new method for the determination of IgE potency of mites. Advantages of the new method are i) all components are well characterised and verifiable, ii) the method is specific for different mite cultures and has a high precision, and iii) the costs of reagents are relatively low.
Method The assay starts with a pre-incubation of mite preparations with a sera pool obtained from patients with established clinical allergy for mite allergens. Thereafter, the mixture is transferred to 96-well plates coated with mite allergens from the mite species and mite source of interest. Unbound specific IgE binds to the coated wells and the total amount of IgE is quantitatively stained by using an anti-IgE streptavidine conjugate. The extent to which the IgE binds to the coat is compared with the maximum amount of IgE that was bound if the sera pool was not pre-incubated (E-max). The difference of the pre-incubated samples and E-max is indicated as percentage inhibition of the IgE preparation. As control, an in-house reference (IHR) is included with the assay to compare the inhibition curves by parallel line analyses. The IgE binding efficiency of an allergen preparation is expressed by relative potency (RP) compared to the IHR. The allergen preparations that were tested with the new method are different mite drug substances from Dermatophagoides pteronyssinus and Dermatophagoides farinae. The results were compared with results obtained with the ImmunoCap assay.
Results All mite preparations tested showed linear inhibition curves with high correlations (R2≥0,99) and reproducible RP data (intermediate precision (CV) of 10%). Furthermore, RP data measured with the new method were comparable with the RP data measured with the ImmunoCap.
Conclusion The newly developed IgE inhibition ELISA is well suited for use of potency determination of different mite species from bodies and cultures. In comparison with the ImmunoCap assay, the newly developed assay has an improved precision and all reagents are well characterised.
EAACI, 16-20 June 2012, GenevaAbstract number: 571, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations
31
Poster Session 13 - Immunotherapy: allergens and allergen-preparations
An In h i b i t i o n E LI SA m e t h o d f o r t h e d e t e r min a t i o n o f R e l a t iv e Po t e n c y o f M i t e p r e p ara t i o n s
571
-An
Inhi
bitio
n EL
ISA
met
hod
for t
he d
eter
min
atio
n of
R
elat
ive
Pote
ncy
of M
ite p
repa
ratio
nsE.
Ker
kvlie
t, I.
Peek
el a
nd R
. van
den
Hou
tH
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds
Back
grou
nd a
nd a
im:
IgE
pote
ncy
assa
ys w
ith s
peci
fic re
agen
ts s
uch
as b
iotin
ylat
edal
lerg
en e
xtra
cts
or a
llerg
en c
aps
used
in th
e Im
mun
oCap
syst
em a
re re
lativ
ely
expe
nsiv
e an
d co
ntai
n re
agen
ts th
at c
an n
ot b
e ch
arac
teriz
ed.I
n ad
ditio
n, th
e Im
mun
oCap
assa
y fo
r mite
s wa
s no
t dev
elop
ed fo
r pot
ency
test
ing
of d
iffer
ent m
ite c
ultu
res.
Ther
efor
e, a
new
met
hod
for t
he d
eter
min
atio
n of
IgE
pote
ncy
of m
ites
was
deve
lope
d an
d pr
e-va
lidat
ed
Conc
lusi
on:
The
newl
y de
velo
ped
IgE
inhi
bitio
n EL
ISA
is w
ell s
uite
d fo
r use
of p
oten
cy d
eter
min
atio
n of
diff
eren
t mite
spe
cies
from
bod
ies
and
cultu
res.
In c
ompa
rison
with
the
Imm
unoC
apas
say,
the
newl
y de
velo
ped
assa
y ha
s an
impr
oved
pre
cisi
onan
d al
l rea
gent
s ar
e we
ll ch
arac
teris
ed.
Tabl
e 1.
RP
data
and
var
iatio
n (C
V%) f
rom
4 d
iffer
ent
sam
ples
test
ed in
two
conc
entra
tions
. The
RP
valu
e is
an
ave
rage
of
3 di
ffere
nt r
uns.
Eac
h in
divi
dual
RP
valu
e is
an a
vera
ge o
f an
inde
pend
ent d
uplic
ate.
Met
hods
:iE
LISA
: The
ass
ay s
tarts
with
coa
ting
of m
icot
iterp
late
s us
ing
mite
ext
ract
s of
the
mite
sou
rce
of in
tere
st (F
ig 1
, A),
follo
wed
by a
n in
cuba
tion
in th
e co
ated
we
lls o
f mite
pre
para
tions
with
a s
era
pool
obt
aine
d fro
m p
atie
nts
with
est
ablis
hed
clin
ical
alle
rgy
for m
ite a
llerg
ens
(Fig
1, B
). U
nbou
nd s
peci
fic Ig
Ebi
nds
to th
e co
ated
wel
ls a
nd th
e to
tal a
mou
nt o
f IgE
is qu
antit
ative
ly st
aine
d by
usin
g an
HRP
ant
i-IgE
conj
ugat
e (F
ig 1
, C).
The
exte
nt to
whi
ch Ig
Ebi
nds
to th
e co
at is
co
mpa
red
with
the
max
imum
am
ount
of I
gEth
at w
as b
ound
if o
nly
seru
m w
as in
cuba
ted
(E-m
ax).
The
diffe
renc
e of
the
pre-
incu
bate
d sa
mpl
es a
nd E
-max
is
indi
cate
d as
per
cent
age
inhi
bitio
n of
the
IgE
prep
arat
ion.
As
cont
rol,
an in
-hou
se re
fere
nce
(IHR
) is
incl
uded
to c
ompa
re th
e in
hibi
tion
curv
es b
y pa
ralle
l lin
e an
alys
es (
PLA)
(Fi
g 1,
D).
The
IgE
bind
ing
effic
ienc
y of
an
alle
rgen
pre
para
tion
is e
xpre
ssed
by
rela
tive
pote
ncy
(RP)
com
pare
d to
the
IHR.
The
alle
rgen
pr
epar
atio
ns th
at w
ere
test
ed w
ith th
e ne
w m
etho
d ar
e di
ffere
nt m
ite e
xtra
cts
from
Der
mat
opha
goid
espt
eron
yssi
nus
and
Der
mat
opha
goid
esfa
rinae
.The
re
sults
of t
he iE
LISA
were
com
pare
d wi
th re
sults
obt
aine
d wi
th th
e Im
mun
oCap
assa
y
Res
ults
:Al
l mite
pre
para
tions
test
ed s
howe
d lin
ear i
nhib
ition
curv
es w
ith h
igh
corre
latio
ns (R
20,
99).
Diffe
rent
mite
spe
cies
from
bot
h bo
dies
and
cul
ture
s we
rete
sted
in
two
conc
entra
tions
in tr
iplic
ate
(Tab
le 1
). Fu
rther
mor
e, in
tra-a
ssay
(Tab
le 2
) and
inte
r-ass
ay (T
able
3) p
reci
sion
was
det
erm
ined
for 4
diff
eren
t mite
pro
duct
s an
d sh
ow a
max
imal
day
-to-d
ay v
aria
tion
of 1
0% (n
=6).
No s
igni
fican
t diff
eren
ces
were
foun
d wh
en R
P da
ta m
easu
red
with
the
new
met
hod
were
com
pare
d wi
th R
P da
ta m
easu
red
with
the
Imm
unoC
ap.
80.
550.
5
31.
051
Mite
mix
cultu
res
90.
680.
5
11.
191
D. f
arin
ae c
ultu
res
170.
400.
5
120.
741
D. p
terb
odie
s
70.
470.
5
30.
851
D. f
arin
aebo
dies
CV%
Mea
nR
PTe
sted
dose
Test
sam
ple
104106
CV%
1.20
1.60
0.60
0.61
Mea
nR
P
Imm
unoC
apiE
LISA
201.
02M
item
ix cu
lture
s
461.
15D
. far
inae
cul
ture
s
170.
75D
. pte
rbod
ies
230.
84D
. far
inae
bodi
es
CV%
Mea
nR
PTe
st s
ampl
e
Tabl
e 3.
RP
data
of 4
diff
eren
t sam
ples
test
ed w
ith th
e iE
LISA
and
the
Imm
unoC
ap.
The
resu
lts o
f th
e iE
LISA
are
from
the
int
er-a
ssay
pr
ecis
ion
dete
rmin
atio
n. iE
LISA
: ave
rage
and
CV%
of 6
inde
pend
ent
runs
; Im
mun
oCap
: ave
rage
and
CV%
of 3
inde
pend
ent r
uns.
0.93
1.19
0.69
0.91
Mea
nR
P
11M
item
ix cu
lture
s
12D
. far
inae
cu
lture
s
4D
. pte
rbo
dies
3D
. far
inae
bodi
es
CV%
Test
sam
ple
Tabl
e 2.
Int
ra-m
edia
te p
reci
sion
of
RP
data
and
var
iatio
n (C
V%)
of 4
diff
eren
t sa
mpl
es.
Intra
med
iate
prec
isio
n is
de
term
ined
in
th
ree
runs
; th
ese
data
re
pres
ent
the
data
fro
m t
he r
un w
ith t
he
high
est
varia
tion.
The
mea
n R
P va
lue
is
an a
vera
ge o
f 3 d
iffer
ent s
ampl
e di
lutio
ns
anal
ysed
in o
ne ru
n.
Imm
unoC
ap: T
he Im
mun
oCap
assa
y in
brie
f: th
e as
say
star
ts w
ith a
pre
-incu
batio
n of
mite
ext
ract
and
ser
um, a
fter
whic
h th
e m
ixtur
e is
trans
ferre
d to
the
Imm
unoC
APsy
stem
(Ph
adia
AB, S
wede
n) lo
aded
with
CAP
sco
ated
with
mite
alle
rgen
s. U
nbou
nd Ig
E, w
hich
was
una
ble
to b
ind
the
prod
uct d
urin
g pr
e-in
cuba
tion,
bin
ds to
the
CAP
and
is d
etec
ted
by a
n an
ti-Ig
Eco
njug
ate.
A fl
uoro
geni
csu
bstra
te is
add
ed a
nd th
e am
ount
of I
gEon
the
CAP
is m
easu
red
quan
titat
ively
by
fluor
esce
nce.
RP
is de
term
ined
by
usin
g PL
A.
020406080100
110
100
1000
mite
con
cent
ratio
n in
AU
/ml
IgE inhibition (IgE)
IHR
mite
ext
ract
Coa
ting
of m
ite e
xtra
ct
ME-
BM
E-C
Mixt
ure
of m
ite p
rote
ins
and
patie
nt s
erum
is
adde
d
Com
petit
ion
betw
een
co
at &
mite
sam
ple
for
bind
ing
of Ig
Ein
the
seru
m
Wash
Wash
Boun
d Ig
Eis
det
ecte
d wi
th H
RP la
bele
dα
-IgE
HRP
is s
tain
ed w
ith T
MB
Col
our i
s de
tect
ed a
t 45
0nm
Line
ar c
urve
s ar
e co
mpu
ted
by u
sing
ratio
(%) b
ound
IgE
vsE-
max
. Pot
ency
is
dete
rmin
ed b
y us
ing
PLA.
AB
CD
= m
iteex
tract
= Ig
E=
TMB
= H
RP-la
bele
d co
njug
ate
020406080100
110
100
1000
mite
con
cent
ratio
n in
AU
/ml
IgE inhibition (IgE)
IHR
mite
ext
ract
Coa
ting
of m
ite e
xtra
ct
ME-
BM
E-C
Mixt
ure
of m
ite p
rote
ins
and
patie
nt s
erum
is
adde
d
Com
petit
ion
betw
een
co
at &
mite
sam
ple
for
bind
ing
of Ig
Ein
the
seru
m
Wash
Wash
Boun
d Ig
Eis
det
ecte
d wi
th H
RP la
bele
dα
-IgE
HRP
is s
tain
ed w
ith T
MB
Col
our i
s de
tect
ed a
t 45
0nm
Line
ar c
urve
s ar
e co
mpu
ted
by u
sing
ratio
(%) b
ound
IgE
vsE-
max
. Pot
ency
is
dete
rmin
ed b
y us
ing
PLA.
AB
CD
= m
iteex
tract
= Ig
E=
TMB
= H
RP-la
bele
d co
njug
ate
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
alor
perc
eive
dco
nflic
tsof
inte
rest
: th
e pr
esen
ter i
s an
empl
oyee
of H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
571
-An
Inhi
bitio
n EL
ISA
met
hod
for t
he d
eter
min
atio
n of
R
elat
ive
Pote
ncy
of M
ite p
repa
ratio
nsE.
Ker
kvlie
t, I.
Peek
el a
nd R
. van
den
Hou
tH
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds
Back
grou
nd a
nd a
im:
IgE
pote
ncy
assa
ys w
ith s
peci
fic re
agen
ts s
uch
as b
iotin
ylat
edal
lerg
en e
xtra
cts
or a
llerg
en c
aps
used
in th
e Im
mun
oCap
syst
em a
re re
lativ
ely
expe
nsiv
e an
d co
ntai
n re
agen
ts th
at c
an n
ot b
e ch
arac
teriz
ed.I
n ad
ditio
n, th
e Im
mun
oCap
assa
y fo
r mite
s wa
s no
t dev
elop
ed fo
r pot
ency
test
ing
of d
iffer
ent m
ite c
ultu
res.
Ther
efor
e, a
new
met
hod
for t
he d
eter
min
atio
n of
IgE
pote
ncy
of m
ites
was
deve
lope
d an
d pr
e-va
lidat
ed
Conc
lusi
on:
The
newl
y de
velo
ped
IgE
inhi
bitio
n EL
ISA
is w
ell s
uite
d fo
r use
of p
oten
cy d
eter
min
atio
n of
diff
eren
t mite
spe
cies
from
bod
ies
and
cultu
res.
In c
ompa
rison
with
the
Imm
unoC
apas
say,
the
newl
y de
velo
ped
assa
y ha
s an
impr
oved
pre
cisi
onan
d al
l rea
gent
s ar
e we
ll ch
arac
teris
ed.
Tabl
e 1.
RP
data
and
var
iatio
n (C
V%) f
rom
4 d
iffer
ent
sam
ples
test
ed in
two
conc
entra
tions
. The
RP
valu
e is
an
ave
rage
of
3 di
ffere
nt r
uns.
Eac
h in
divi
dual
RP
valu
e is
an a
vera
ge o
f an
inde
pend
ent d
uplic
ate.
Met
hods
:iE
LISA
: The
ass
ay s
tarts
with
coa
ting
of m
icot
iterp
late
s us
ing
mite
ext
ract
s of
the
mite
sou
rce
of in
tere
st (F
ig 1
, A),
follo
wed
by a
n in
cuba
tion
in th
e co
ated
we
lls o
f mite
pre
para
tions
with
a s
era
pool
obt
aine
d fro
m p
atie
nts
with
est
ablis
hed
clin
ical
alle
rgy
for m
ite a
llerg
ens
(Fig
1, B
). U
nbou
nd s
peci
fic Ig
Ebi
nds
to th
e co
ated
wel
ls a
nd th
e to
tal a
mou
nt o
f IgE
is qu
antit
ative
ly st
aine
d by
usin
g an
HRP
ant
i-IgE
conj
ugat
e (F
ig 1
, C).
The
exte
nt to
whi
ch Ig
Ebi
nds
to th
e co
at is
co
mpa
red
with
the
max
imum
am
ount
of I
gEth
at w
as b
ound
if o
nly
seru
m w
as in
cuba
ted
(E-m
ax).
The
diffe
renc
e of
the
pre-
incu
bate
d sa
mpl
es a
nd E
-max
is
indi
cate
d as
per
cent
age
inhi
bitio
n of
the
IgE
prep
arat
ion.
As
cont
rol,
an in
-hou
se re
fere
nce
(IHR
) is
incl
uded
to c
ompa
re th
e in
hibi
tion
curv
es b
y pa
ralle
l lin
e an
alys
es (
PLA)
(Fi
g 1,
D).
The
IgE
bind
ing
effic
ienc
y of
an
alle
rgen
pre
para
tion
is e
xpre
ssed
by
rela
tive
pote
ncy
(RP)
com
pare
d to
the
IHR.
The
alle
rgen
pr
epar
atio
ns th
at w
ere
test
ed w
ith th
e ne
w m
etho
d ar
e di
ffere
nt m
ite e
xtra
cts
from
Der
mat
opha
goid
espt
eron
yssi
nus
and
Der
mat
opha
goid
esfa
rinae
.The
re
sults
of t
he iE
LISA
were
com
pare
d wi
th re
sults
obt
aine
d wi
th th
e Im
mun
oCap
assa
y
Res
ults
:Al
l mite
pre
para
tions
test
ed s
howe
d lin
ear i
nhib
ition
curv
es w
ith h
igh
corre
latio
ns (R
20,
99).
Diffe
rent
mite
spe
cies
from
bot
h bo
dies
and
cul
ture
s we
rete
sted
in
two
conc
entra
tions
in tr
iplic
ate
(Tab
le 1
). Fu
rther
mor
e, in
tra-a
ssay
(Tab
le 2
) and
inte
r-ass
ay (T
able
3) p
reci
sion
was
det
erm
ined
for 4
diff
eren
t mite
pro
duct
s an
d sh
ow a
max
imal
day
-to-d
ay v
aria
tion
of 1
0% (n
=6).
No s
igni
fican
t diff
eren
ces
were
foun
d wh
en R
P da
ta m
easu
red
with
the
new
met
hod
were
com
pare
d wi
th R
P da
ta m
easu
red
with
the
Imm
unoC
ap.
80.
550.
5
31.
051
Mite
mix
cultu
res
90.
680.
5
11.
191
D. f
arin
ae c
ultu
res
170.
400.
5
120.
741
D. p
terb
odie
s
70.
470.
5
30.
851
D. f
arin
aebo
dies
CV%
Mea
nR
PTe
sted
dose
Test
sam
ple
104106
CV%
1.20
1.60
0.60
0.61
Mea
nR
P
Imm
unoC
apiE
LISA
201.
02M
item
ix cu
lture
s
461.
15D
. far
inae
cul
ture
s
170.
75D
. pte
rbod
ies
230.
84D
. far
inae
bodi
es
CV%
Mea
nR
PTe
st s
ampl
e
Tabl
e 3.
RP
data
of 4
diff
eren
t sam
ples
test
ed w
ith th
e iE
LISA
and
the
Imm
unoC
ap.
The
resu
lts o
f th
e iE
LISA
are
from
the
int
er-a
ssay
pr
ecis
ion
dete
rmin
atio
n. iE
LISA
: ave
rage
and
CV%
of 6
inde
pend
ent
runs
; Im
mun
oCap
: ave
rage
and
CV%
of 3
inde
pend
ent r
uns.
0.93
1.19
0.69
0.91
Mea
nR
P
11M
item
ix cu
lture
s
12D
. far
inae
cu
lture
s
4D
. pte
rbo
dies
3D
. far
inae
bodi
es
CV%
Test
sam
ple
Tabl
e 2.
Int
ra-m
edia
te p
reci
sion
of
RP
data
and
var
iatio
n (C
V%)
of 4
diff
eren
t sa
mpl
es.
Intra
med
iate
prec
isio
n is
de
term
ined
in
th
ree
runs
; th
ese
data
re
pres
ent
the
data
fro
m t
he r
un w
ith t
he
high
est
varia
tion.
The
mea
n R
P va
lue
is
an a
vera
ge o
f 3 d
iffer
ent s
ampl
e di
lutio
ns
anal
ysed
in o
ne ru
n.
Imm
unoC
ap: T
he Im
mun
oCap
assa
y in
brie
f: th
e as
say
star
ts w
ith a
pre
-incu
batio
n of
mite
ext
ract
and
ser
um, a
fter
whic
h th
e m
ixtur
e is
trans
ferre
d to
the
Imm
unoC
APsy
stem
(Ph
adia
AB, S
wede
n) lo
aded
with
CAP
sco
ated
with
mite
alle
rgen
s. U
nbou
nd Ig
E, w
hich
was
una
ble
to b
ind
the
prod
uct d
urin
g pr
e-in
cuba
tion,
bin
ds to
the
CAP
and
is d
etec
ted
by a
n an
ti-Ig
Eco
njug
ate.
A fl
uoro
geni
csu
bstra
te is
add
ed a
nd th
e am
ount
of I
gEon
the
CAP
is m
easu
red
quan
titat
ively
by
fluor
esce
nce.
RP
is de
term
ined
by
usin
g PL
A.
020406080100
110
100
1000
mite
con
cent
ratio
n in
AU
/ml
IgE inhibition (IgE)
IHR
mite
ext
ract
Coa
ting
of m
ite e
xtra
ct
ME-
BM
E-C
Mixt
ure
of m
ite p
rote
ins
and
patie
nt s
erum
is
adde
d
Com
petit
ion
betw
een
co
at &
mite
sam
ple
for
bind
ing
of Ig
Ein
the
seru
m
Wash
Wash
Boun
d Ig
Eis
det
ecte
d wi
th H
RP la
bele
dα
-IgE
HRP
is s
tain
ed w
ith T
MB
Col
our i
s de
tect
ed a
t 45
0nm
Line
ar c
urve
s ar
e co
mpu
ted
by u
sing
ratio
(%) b
ound
IgE
vsE-
max
. Pot
ency
is
dete
rmin
ed b
y us
ing
PLA.
AB
CD
= m
iteex
tract
= Ig
E=
TMB
= H
RP-la
bele
d co
njug
ate
020406080100
110
100
1000
mite
con
cent
ratio
n in
AU
/ml
IgE inhibition (IgE)
IHR
mite
ext
ract
Coa
ting
of m
ite e
xtra
ct
ME-
BM
E-C
Mixt
ure
of m
ite p
rote
ins
and
patie
nt s
erum
is
adde
d
Com
petit
ion
betw
een
co
at &
mite
sam
ple
for
bind
ing
of Ig
Ein
the
seru
m
Wash
Wash
Boun
d Ig
Eis
det
ecte
d wi
th H
RP la
bele
dα
-IgE
HRP
is s
tain
ed w
ith T
MB
Col
our i
s de
tect
ed a
t 45
0nm
Line
ar c
urve
s ar
e co
mpu
ted
by u
sing
ratio
(%) b
ound
IgE
vsE-
max
. Pot
ency
is
dete
rmin
ed b
y us
ing
PLA.
AB
CD
= m
iteex
tract
= Ig
E=
TMB
= H
RP-la
bele
d co
njug
ate
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
alor
perc
eive
dco
nflic
tsof
inte
rest
: th
e pr
esen
ter i
s an
empl
oyee
of H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
571
-An
Inhi
bitio
n EL
ISA
met
hod
for t
he d
eter
min
atio
n of
R
elat
ive
Pote
ncy
of M
ite p
repa
ratio
nsE.
Ker
kvlie
t, I.
Peek
el a
nd R
. van
den
Hou
tH
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds
Back
grou
nd a
nd a
im:
IgE
pote
ncy
assa
ys w
ith s
peci
fic re
agen
ts s
uch
as b
iotin
ylat
edal
lerg
en e
xtra
cts
or a
llerg
en c
aps
used
in th
e Im
mun
oCap
syst
em a
re re
lativ
ely
expe
nsiv
e an
d co
ntai
n re
agen
ts th
at c
an n
ot b
e ch
arac
teriz
ed.I
n ad
ditio
n, th
e Im
mun
oCap
assa
y fo
r mite
s wa
s no
t dev
elop
ed fo
r pot
ency
test
ing
of d
iffer
ent m
ite c
ultu
res.
Ther
efor
e, a
new
met
hod
for t
he d
eter
min
atio
n of
IgE
pote
ncy
of m
ites
was
deve
lope
d an
d pr
e-va
lidat
ed
Conc
lusi
on:
The
newl
y de
velo
ped
IgE
inhi
bitio
n EL
ISA
is w
ell s
uite
d fo
r use
of p
oten
cy d
eter
min
atio
n of
diff
eren
t mite
spe
cies
from
bod
ies
and
cultu
res.
In c
ompa
rison
with
the
Imm
unoC
apas
say,
the
newl
y de
velo
ped
assa
y ha
s an
impr
oved
pre
cisi
onan
d al
l rea
gent
s ar
e we
ll ch
arac
teris
ed.
Tabl
e 1.
RP
data
and
var
iatio
n (C
V%) f
rom
4 d
iffer
ent
sam
ples
test
ed in
two
conc
entra
tions
. The
RP
valu
e is
an
ave
rage
of
3 di
ffere
nt r
uns.
Eac
h in
divi
dual
RP
valu
e is
an a
vera
ge o
f an
inde
pend
ent d
uplic
ate.
Met
hods
:iE
LISA
: The
ass
ay s
tarts
with
coa
ting
of m
icot
iterp
late
s us
ing
mite
ext
ract
s of
the
mite
sou
rce
of in
tere
st (F
ig 1
, A),
follo
wed
by a
n in
cuba
tion
in th
e co
ated
we
lls o
f mite
pre
para
tions
with
a s
era
pool
obt
aine
d fro
m p
atie
nts
with
est
ablis
hed
clin
ical
alle
rgy
for m
ite a
llerg
ens
(Fig
1, B
). U
nbou
nd s
peci
fic Ig
Ebi
nds
to th
e co
ated
wel
ls a
nd th
e to
tal a
mou
nt o
f IgE
is qu
antit
ative
ly st
aine
d by
usin
g an
HRP
ant
i-IgE
conj
ugat
e (F
ig 1
, C).
The
exte
nt to
whi
ch Ig
Ebi
nds
to th
e co
at is
co
mpa
red
with
the
max
imum
am
ount
of I
gEth
at w
as b
ound
if o
nly
seru
m w
as in
cuba
ted
(E-m
ax).
The
diffe
renc
e of
the
pre-
incu
bate
d sa
mpl
es a
nd E
-max
is
indi
cate
d as
per
cent
age
inhi
bitio
n of
the
IgE
prep
arat
ion.
As
cont
rol,
an in
-hou
se re
fere
nce
(IHR
) is
incl
uded
to c
ompa
re th
e in
hibi
tion
curv
es b
y pa
ralle
l lin
e an
alys
es (
PLA)
(Fi
g 1,
D).
The
IgE
bind
ing
effic
ienc
y of
an
alle
rgen
pre
para
tion
is e
xpre
ssed
by
rela
tive
pote
ncy
(RP)
com
pare
d to
the
IHR.
The
alle
rgen
pr
epar
atio
ns th
at w
ere
test
ed w
ith th
e ne
w m
etho
d ar
e di
ffere
nt m
ite e
xtra
cts
from
Der
mat
opha
goid
espt
eron
yssi
nus
and
Der
mat
opha
goid
esfa
rinae
.The
re
sults
of t
he iE
LISA
were
com
pare
d wi
th re
sults
obt
aine
d wi
th th
e Im
mun
oCap
assa
y
Res
ults
:Al
l mite
pre
para
tions
test
ed s
howe
d lin
ear i
nhib
ition
curv
es w
ith h
igh
corre
latio
ns (R
20,
99).
Diffe
rent
mite
spe
cies
from
bot
h bo
dies
and
cul
ture
s we
rete
sted
in
two
conc
entra
tions
in tr
iplic
ate
(Tab
le 1
). Fu
rther
mor
e, in
tra-a
ssay
(Tab
le 2
) and
inte
r-ass
ay (T
able
3) p
reci
sion
was
det
erm
ined
for 4
diff
eren
t mite
pro
duct
s an
d sh
ow a
max
imal
day
-to-d
ay v
aria
tion
of 1
0% (n
=6).
No s
igni
fican
t diff
eren
ces
were
foun
d wh
en R
P da
ta m
easu
red
with
the
new
met
hod
were
com
pare
d wi
th R
P da
ta m
easu
red
with
the
Imm
unoC
ap.
80.
550.
5
31.
051
Mite
mix
cultu
res
90.
680.
5
11.
191
D. f
arin
ae c
ultu
res
170.
400.
5
120.
741
D. p
terb
odie
s
70.
470.
5
30.
851
D. f
arin
aebo
dies
CV%
Mea
nR
PTe
sted
dose
Test
sam
ple
104106
CV%
1.20
1.60
0.60
0.61
Mea
nR
P
Imm
unoC
apiE
LISA
201.
02M
item
ix cu
lture
s
461.
15D
. far
inae
cul
ture
s
170.
75D
. pte
rbod
ies
230.
84D
. far
inae
bodi
es
CV%
Mea
nR
PTe
st s
ampl
e
Tabl
e 3.
RP
data
of 4
diff
eren
t sam
ples
test
ed w
ith th
e iE
LISA
and
the
Imm
unoC
ap.
The
resu
lts o
f th
e iE
LISA
are
from
the
int
er-a
ssay
pr
ecis
ion
dete
rmin
atio
n. iE
LISA
: ave
rage
and
CV%
of 6
inde
pend
ent
runs
; Im
mun
oCap
: ave
rage
and
CV%
of 3
inde
pend
ent r
uns.
0.93
1.19
0.69
0.91
Mea
nR
P
11M
item
ix cu
lture
s
12D
. far
inae
cu
lture
s
4D
. pte
rbo
dies
3D
. far
inae
bodi
es
CV%
Test
sam
ple
Tabl
e 2.
Int
ra-m
edia
te p
reci
sion
of
RP
data
and
var
iatio
n (C
V%)
of 4
diff
eren
t sa
mpl
es.
Intra
med
iate
prec
isio
n is
de
term
ined
in
th
ree
runs
; th
ese
data
re
pres
ent
the
data
fro
m t
he r
un w
ith t
he
high
est
varia
tion.
The
mea
n R
P va
lue
is
an a
vera
ge o
f 3 d
iffer
ent s
ampl
e di
lutio
ns
anal
ysed
in o
ne ru
n.
Imm
unoC
ap: T
he Im
mun
oCap
assa
y in
brie
f: th
e as
say
star
ts w
ith a
pre
-incu
batio
n of
mite
ext
ract
and
ser
um, a
fter
whic
h th
e m
ixtur
e is
trans
ferre
d to
the
Imm
unoC
APsy
stem
(Ph
adia
AB, S
wede
n) lo
aded
with
CAP
sco
ated
with
mite
alle
rgen
s. U
nbou
nd Ig
E, w
hich
was
una
ble
to b
ind
the
prod
uct d
urin
g pr
e-in
cuba
tion,
bin
ds to
the
CAP
and
is d
etec
ted
by a
n an
ti-Ig
Eco
njug
ate.
A fl
uoro
geni
csu
bstra
te is
add
ed a
nd th
e am
ount
of I
gEon
the
CAP
is m
easu
red
quan
titat
ively
by
fluor
esce
nce.
RP
is de
term
ined
by
usin
g PL
A.
020406080100
110
100
1000
mite
con
cent
ratio
n in
AU
/ml
IgE inhibition (IgE)
IHR
mite
ext
ract
Coa
ting
of m
ite e
xtra
ct
ME-
BM
E-C
Mixt
ure
of m
ite p
rote
ins
and
patie
nt s
erum
is
adde
d
Com
petit
ion
betw
een
co
at &
mite
sam
ple
for
bind
ing
of Ig
Ein
the
seru
m
Wash
Wash
Boun
d Ig
Eis
det
ecte
d wi
th H
RP la
bele
dα
-IgE
HRP
is s
tain
ed w
ith T
MB
Col
our i
s de
tect
ed a
t 45
0nm
Line
ar c
urve
s ar
e co
mpu
ted
by u
sing
ratio
(%) b
ound
IgE
vsE-
max
. Pot
ency
is
dete
rmin
ed b
y us
ing
PLA.
AB
CD
= m
iteex
tract
= Ig
E=
TMB
= H
RP-la
bele
d co
njug
ate
020406080100
110
100
1000
mite
con
cent
ratio
n in
AU
/ml
IgE inhibition (IgE)
IHR
mite
ext
ract
Coa
ting
of m
ite e
xtra
ct
ME-
BM
E-C
Mixt
ure
of m
ite p
rote
ins
and
patie
nt s
erum
is
adde
d
Com
petit
ion
betw
een
co
at &
mite
sam
ple
for
bind
ing
of Ig
Ein
the
seru
m
Wash
Wash
Boun
d Ig
Eis
det
ecte
d wi
th H
RP la
bele
dα
-IgE
HRP
is s
tain
ed w
ith T
MB
Col
our i
s de
tect
ed a
t 45
0nm
Line
ar c
urve
s ar
e co
mpu
ted
by u
sing
ratio
(%) b
ound
IgE
vsE-
max
. Pot
ency
is
dete
rmin
ed b
y us
ing
PLA.
AB
CD
= m
iteex
tract
= Ig
E=
TMB
= H
RP-la
bele
d co
njug
ate
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
alor
perc
eive
dco
nflic
tsof
inte
rest
: th
e pr
esen
ter i
s an
empl
oyee
of H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
In re
latio
n to
this
pre
sent
atio
n, I
decl
are
the
follo
win
g, re
al o
r per
ceiv
ed c
onfli
cts
of in
tere
st: t
he p
rese
nter
is a
n em
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
An a nt i b o d y - b a s e d t e c h ni q u e f o r s t a b il i t y s tu d i e s o n a l u m a ds o r b e d b irc h p o l l e n a l l e rg o i d p r e p ara t i o n s
32
E. Kerkvliet, C. Franso, A. Segaar, R. van den Hout.
HAL Allergy BV, Leiden, The Netherlands.
Background IgE based potency assays for allergens are essential tests for monitoring stability in extracts. However, these IgE potency assays can not be used for potency determination of allergoid-based products, since they essentially lack IgE reactivity. Furthermore, aluminium can deteriorate binding of antibodies to proteins attached to a solid phase. We have developed and validated an IgG based inhibition assay that is suitable for determining IgG inhibition of an alum-adsorbed birch pollen allergoid (ALU-B). In order to find out whether the assay is stability indicating, temperature-stressed samples were analysed with the new method.
Method Rabbit IgG antibodies specific for birch allergoid are pre-incubated with various concentrations of ALU-B. The mix is then added to a birch allergoid coated microtiter plate to bind free allergoid-specific IgG, which is quantitatively measured by a donkey anti-rabbit HRP conjugate and stained with TMB. The extent to which the IgG antibodies bind the plate in presence of added inhibitor (ALU-B) is compared with the maximum amount of IgG antibodies that binds in absence of the inhibitor. Results are finally expressed as percentage inhibition relative to the uninhibited value and the concentration at 50% inhibi-tion is used as read out. An in house reference of birch allergoid is included as assay control and is used for factorizing the IgG inhibition values to reduce day to day variation.
Results Validation of the IgG inhibition assay showed that the assay is specific for birch preparations, linear, precise (CV=16%) and robust. Furthermore, the assay detected a two-fold decrease in IgG inhibition when two doses of ALU-B with a two-fold difference in strength were analysed. Storage of the ALU-B vaccine at various elevated temperatures resulted in increased 50% IgG inhibition values indicating a reduction of IgG binding to the epitopes.
Conclusion We have successfully developed and validated an assay that determines IgG inhibition of the alum-adsorbed birch pol-len vaccine. Moreover, the sensitivity of the assay is sufficient to at least detect a two-fold reduction in potency. Although the assay is still under evaluation whether the IgG inhibition assay can be used for stability studies of ALU-B, preliminary results showed reduced IgG binding to temperature stressed ALU-B vaccine.
EAACI, 16-20 June 2012, GenevaAbstract number: 572, Session date and time: Sunday 17 June; 12:00 - 13:30 Session title: Poster 13 - Immunotherapy: allergens and allergen-preparations
33
Poster Session 13 - Immunotherapy: allergens and allergen-preparations
An a nt i b o d y - b a s e d t e c h ni q u e f o r s t a b il i t y s tu d i e s o n a l u m a ds o r b e d b irc h p o l l e n a l l e rg o i d p r e p ara t i o n s
572
-An
antib
ody-
base
d Te
chni
que
for S
tabi
lity
Stud
ies
on
Alum
Ads
orbe
d B
irch
polle
n al
lerg
oid
prep
arat
ions
E. K
erkv
liet,
C. F
rans
o, A
. Seg
aar a
nd R
. van
den
Hou
tH
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds
Back
grou
nd a
nd a
im:
IgE
base
d po
tenc
y as
says
for
alle
rgen
s ar
e es
sent
ial t
ests
for
mon
itorin
g st
abilit
y in
ext
ract
s. H
owev
er,
thes
e Ig
Epo
tenc
y as
says
can
not
be
used
for p
oten
cy d
eter
min
atio
n of
alle
rgoi
d-ba
sed
prod
ucts
, si
nce
they
es
sent
ially
la
ck
IgE
reac
tivity
. Fu
rther
mor
e, a
lum
iniu
m c
an d
eter
iora
te b
indi
ng o
f ant
ibod
ies
to
prot
eins
atta
ched
to a
sol
id p
hase
. We
prev
ious
ly d
evel
oped
an
IgG
base
d in
hibi
tion
assa
y fo
r m
onito
ring
stab
ility
of a
lum
ad
sorb
ed m
ite p
repa
ratio
ns.
A sim
ilar
assa
y ha
s no
w be
en
deve
lope
d fo
r th
e de
term
inat
ion
of
IgG
inhi
bitio
n of
al
um
adso
rbed
birc
h po
llen
alle
rgoi
ds. T
his
assa
y ha
s be
en s
tudi
ed
for
use
as a
sta
bilit
y te
st a
nd w
heth
er t
he a
ssay
has
bee
n su
ffici
ent
sens
itive
to d
etec
t a
2 tim
es r
educ
tion.
Fin
ally,
the
assa
y wa
s va
lidat
ed a
ccor
ding
the
curre
nt g
uide
lines
.
Met
hods
:Th
e ne
wly
deve
lope
d Ig
Gin
hibi
tion
assa
y us
es Ig
Gan
tibod
ies
spec
ific
for
birc
h po
llen
alle
rgoi
dsth
at
were
ob
tain
ed
by
imm
uniza
tion
of r
abbi
ts w
ith g
luta
rald
ehyd
em
odifi
ed e
xtra
cts
(alle
rgoi
d) o
f birc
h po
llen.
The
ass
ay s
tarts
with
a p
re-in
cuba
tion
of a
mixt
ure
of a
nti-a
llerg
oid
seru
m a
nd d
iffer
ent c
once
ntra
tions
of
alu
m-a
dsor
bed
birc
h po
llen
alle
rgoi
d(A
LU-B
). Af
ter
pre-
incu
batio
n th
e m
ixtur
e is
add
ed t
o an
birc
h al
lerg
oid
coat
ed
mic
rotit
erpl
ate
to b
ind
free
alle
rgoi
d-sp
ecifi
c Ig
G. S
ubse
quen
tly,
boun
d Ig
Gis
quan
titat
ivel
y m
easu
red
by a
n HR
P co
njug
ate
and
stai
ned
with
TM
B. T
he e
xten
t to
whic
h th
e Ig
Gan
tibod
ies
bind
to
the
pla
te i
n pr
esen
ce o
f AL
U-B
is
co
mpa
red
with
the
m
axim
um a
mou
nt o
f Ig
Gan
tibod
ies
that
bin
ds in
abs
ence
of
ALU
-B (
E-m
ax).
Resu
lts a
re f
inal
ly ex
pres
sed
as p
erce
ntag
e in
hibi
tion
rela
tive
to t
he u
ninh
ibite
d va
lue.
The
50%
inhi
bitio
nva
lue
is us
ed a
s re
ad o
ut. A
n in
hou
se b
irch
alle
rgoi
dre
fere
nce
sam
ple
is u
sed
for d
ay-to
-day
cor
rect
ion
and
as a
ssay
con
trol.
Suita
bilit
y of
the
ass
ay w
as d
eter
min
ed w
ith d
iffer
ent
PUR-
B ba
tche
s an
d ba
tche
s st
ored
at e
leva
ted
tem
pera
ture
s.
Res
ults
:Th
e bi
rch
polle
n al
lerg
oid
prep
arat
ion
indu
ced
high
spe
cific
IgG
titer
s in
ra
bbits
, wh
ich
was
dem
onst
rate
d by
EL
ISA
and
imm
unob
lotti
ng(F
ig.
1).
Imm
unob
lotti
ngsh
ows
that
the
rab
bit
anti
seru
m re
cogn
ises
birc
h ex
tract
, birc
h al
lerg
oid
prep
arat
ions
an
d th
e re
leva
nt b
irch
alle
rgen
Bet
v1
(Fig
. 1B)
. Usi
ng d
iffer
ent
conc
entra
tions
of A
LU-B
mixe
d wi
th th
e ra
bbit
seru
m re
sulte
d in
do
se-re
spon
se c
urve
s on
birc
h al
lerg
oid
polle
n co
ated
micr
otite
rpl
ates
. By
test
ing
supe
rnat
ant o
f PUR
-B b
atch
es it
was
pro
ven
that
the
dos
e-re
spon
secu
rves
wer
e m
ainl
y ca
used
by
ALU
-B
and
not b
y un
boun
d m
ater
ial s
ince
sup
erna
tant
con
tain
s a
mor
e th
an 1
00 ti
mes
lowe
r sig
nal (
Fig
2). T
able
1 s
hows
a s
igni
fican
t in
crea
se in
the
50%
IgG
inhi
bitio
n va
lue
when
two
times
dilu
ted
batc
hes
are
anal
ysed
, ind
icat
ing
that
the
sens
itivity
of t
he a
ssay
is
suf
ficie
nt to
det
ect a
50%
redu
ctio
n in
act
ivity
. Sto
rage
of t
he
ALU
-B v
acci
ne a
t va
rious
ele
vate
d te
mpe
ratu
res
resu
lted
in
incr
ease
d 50
% I
gGin
hibi
tion
valu
es i
ndica
ting
a re
duct
ion
of
IgG
bind
ing
to t
he e
pito
pes
(Fig
3, T
able
2).
Valid
atio
n of
the
assa
y sh
owed
that
the
assa
y is
spe
cific
for b
irch,
has
a n
orm
al
dist
ribut
ion
of p
oint
s, s
hows
a g
ood
prec
isio
n an
d is
rob
ust
(Tab
le 3
). Th
e as
say
is s
till
unde
r ev
alua
tion
to e
stab
lish
whet
her
the
IgG
inhi
bitio
n EL
ISA
is s
uita
ble
for
long
itudi
nal
mon
itorin
g of
sta
bilit
y of
the
adso
rbed
pro
duct
Conc
lusi
on:
The
newl
y de
velo
ped
and
valid
ated
IgG
inhi
bitio
n as
say
is su
itabl
e fo
r m
easu
ring
IgG
bind
ing
to P
UR-B
bat
ches
and
is
able
to a
t lea
st d
etec
t a tw
o-fo
ld re
duct
ion
in p
oten
cy.
The
assa
y sh
owed
re
duce
d Ig
Gbi
ndin
g to
te
mpe
ratu
re
stre
ssed
ALU
-B v
acci
ne, s
ugge
stin
g th
at th
e as
say
is s
tabi
lity
indi
catin
g.
Figu
re 2
. IgG
inhi
bitio
n cu
rves
of 2
bat
ches
AL
U-B
(b
lue
curv
es)
and
IgG
inhi
bitio
n cu
rves
mea
sure
d in
the
ir su
pern
atan
t (re
d an
d or
ange
cur
ves)
.
Figu
re 3
. The
gra
ph re
pres
ents
IgG
inhi
bitio
n cu
rves
of a
PU
R-B
bat
ch s
tore
d at
5°C
, 25°
C,
37 °C
for 2
mon
ths
and
at 9
0°C
for 1
and
4
hrs.
The
dot
ted
line
show
s th
e 50
% in
hibi
tion
of th
e di
ffere
nt c
urve
s.
Res
ults
Valid
atio
npa
ram
eter
s
CV
=16
%In
ter-a
ssay
prec
isio
nTe
mpe
ratu
reco
nditio
nsar
e cr
ucia
lR
obus
tnes
s
CV
= 15
%In
tra-a
ssay
prec
isio
n
Nor
mal
dist
ribut
ion
of p
oint
s; a
ncho
r poi
nts
appr
oach
uppe
r an
d lo
wera
sym
ptot
eM
odel
Spec
ificfo
rALU
-B a
nd n
otto
oth
erPU
RET
HAL
pre
para
tions
Spec
ificity
Tabl
e 3.
Val
idat
ion
resu
lts o
f the
50%
IgG
inhi
bitio
n as
say
for A
LU-B
bat
ches
.
90T0
137
140
148
94 112
50%
IgG
inhi
bitio
nva
lue
in A
Ueq
/ml
% re
cove
rySt
orag
eco
nditi
ons
66 64 61
37° °°°C
, 2 m
onth
s
90° °°°C
, 1 h
our
90° °°°C
, 4 h
ours
96 80
5° °°°C
, 2 m
onth
s
25° °°°C
, 2 m
onth
s
Lane
1: M
arke
r; La
ne 2
: Bi
rch
polle
n ex
tract
; La
ne 3
-5: 3
diff
eren
t Bi
rch
polle
n al
lerg
oid
batc
hes.
The
box
in
dica
tes
the
Bet
v1
band
Figu
re 1
A. D
irect
ELI
SA o
f th
e ra
bbit
anti-
seru
m te
sted
on
Birc
h po
llen
extra
ct a
nd o
n al
lerg
oid.
The
ser
um s
hows
dos
e-re
spon
ses
to b
oth
prep
arat
ions
with
hig
her
affin
ity f
or
the
alle
rgoi
dpr
epar
atio
n.
Figu
re 1
B.I
mm
unob
loto
f rab
bit s
erum
im
mun
ized
with
birc
h po
llen
alle
rgoi
d. T
he
anti-
seru
m is
test
ed o
n bi
rch
polle
n ex
tract
an
d on
thre
e di
ffere
nt b
irch
polle
n al
lerg
oid
prep
arat
ions
..
Nor
mal
dose
½N
orm
aldo
se
Batc
h B
128
AU
eq/m
l
286
AU
eq/m
l
50%
IgG
inhi
bitio
nva
lue
CV
7.4%
5.5%
Nor
mal
dose
½N
orm
aldo
se
Batc
h A
128
AU
eq/m
l
294
AU
eq/m
l
50%
IgG
inhi
bitio
nva
lue
CV
1.5%
2.0%
Tabl
e 1.
50%
IgG
inhi
bitio
n va
lues
of
2 ba
tche
s AL
U-B
, al
so
test
ed
when
1:
1 di
lute
d in
form
ulat
ion
buffe
r.
12
34
51
23
45
0,0
1,0
2,0
3,0
4,0
5,0
1010
010
0010
000
1000
0010
0000
0
seru
m d
ilutio
nOD value (450 nm)
Birc
h po
llen
extra
ctBi
rch
polle
n al
lerg
oid
Tabl
e 2.
The
50%
IgG
inhi
bitio
n va
lues
of e
ach
curv
e ar
e pr
esen
ted
in th
e ta
ble.
The
dec
reas
e of
50%
IgG
inhi
bitio
n wa
s ca
lcula
ted
in re
latio
n to
the
ALU
-B s
ampl
e an
alys
ed a
t t0.
CV
of a
ll m
easu
rem
ents
is b
elow
10%
-100102030405060708090100
110
100
1000
1000
010
0000
ALU-
B in
AUe
q/m
l
IgG inhibition (%)
5°C
25°C
37°C
90°C
, 1 h
r 9
0°C
, 4 h
rs
020406080100
110
100
1000
1000
010
0000
ALU-
B in
AUe
q/m
l
IgG inhibition (%)
ALU-
B ba
tch
ASU
P AL
U-B
batc
h A
ALU-
B ba
tch
BSU
P AL
U-B
batc
h B
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
alor
perc
eive
dco
nflic
tsof
inte
rest
: th
e pr
esen
ter i
s an
empl
oyee
of H
AL A
llerg
y
572
-An
antib
ody-
base
d Te
chni
que
for S
tabi
lity
Stud
ies
on
Alum
Ads
orbe
d B
irch
polle
n al
lerg
oid
prep
arat
ions
E. K
erkv
liet,
C. F
rans
o, A
. Seg
aar a
nd R
. van
den
Hou
tH
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds
Back
grou
nd a
nd a
im:
IgE
base
d po
tenc
y as
says
for
alle
rgen
s ar
e es
sent
ial t
ests
for
mon
itorin
g st
abilit
y in
ext
ract
s. H
owev
er,
thes
e Ig
Epo
tenc
y as
says
can
not
be
used
for p
oten
cy d
eter
min
atio
n of
alle
rgoi
d-ba
sed
prod
ucts
, si
nce
they
es
sent
ially
la
ck
IgE
reac
tivity
. Fu
rther
mor
e, a
lum
iniu
m c
an d
eter
iora
te b
indi
ng o
f ant
ibod
ies
to
prot
eins
atta
ched
to a
sol
id p
hase
. We
prev
ious
ly d
evel
oped
an
IgG
base
d in
hibi
tion
assa
y fo
r m
onito
ring
stab
ility
of a
lum
ad
sorb
ed m
ite p
repa
ratio
ns.
A sim
ilar
assa
y ha
s no
w be
en
deve
lope
d fo
r th
e de
term
inat
ion
of
IgG
inhi
bitio
n of
al
um
adso
rbed
birc
h po
llen
alle
rgoi
ds. T
his
assa
y ha
s be
en s
tudi
ed
for
use
as a
sta
bilit
y te
st a
nd w
heth
er t
he a
ssay
has
bee
n su
ffici
ent
sens
itive
to d
etec
t a
2 tim
es r
educ
tion.
Fin
ally,
the
assa
y wa
s va
lidat
ed a
ccor
ding
the
curre
nt g
uide
lines
.
Met
hods
:Th
e ne
wly
deve
lope
d Ig
Gin
hibi
tion
assa
y us
es Ig
Gan
tibod
ies
spec
ific
for
birc
h po
llen
alle
rgoi
dsth
at
were
ob
tain
ed
by
imm
uniza
tion
of r
abbi
ts w
ith g
luta
rald
ehyd
em
odifi
ed e
xtra
cts
(alle
rgoi
d) o
f birc
h po
llen.
The
ass
ay s
tarts
with
a p
re-in
cuba
tion
of a
mixt
ure
of a
nti-a
llerg
oid
seru
m a
nd d
iffer
ent c
once
ntra
tions
of
alu
m-a
dsor
bed
birc
h po
llen
alle
rgoi
d(A
LU-B
). Af
ter
pre-
incu
batio
n th
e m
ixtur
e is
add
ed t
o an
birc
h al
lerg
oid
coat
ed
mic
rotit
erpl
ate
to b
ind
free
alle
rgoi
d-sp
ecifi
c Ig
G. S
ubse
quen
tly,
boun
d Ig
Gis
quan
titat
ivel
y m
easu
red
by a
n HR
P co
njug
ate
and
stai
ned
with
TM
B. T
he e
xten
t to
whic
h th
e Ig
Gan
tibod
ies
bind
to
the
pla
te i
n pr
esen
ce o
f AL
U-B
is
co
mpa
red
with
the
m
axim
um a
mou
nt o
f Ig
Gan
tibod
ies
that
bin
ds in
abs
ence
of
ALU
-B (
E-m
ax).
Resu
lts a
re f
inal
ly ex
pres
sed
as p
erce
ntag
e in
hibi
tion
rela
tive
to t
he u
ninh
ibite
d va
lue.
The
50%
inhi
bitio
nva
lue
is us
ed a
s re
ad o
ut. A
n in
hou
se b
irch
alle
rgoi
dre
fere
nce
sam
ple
is u
sed
for d
ay-to
-day
cor
rect
ion
and
as a
ssay
con
trol.
Suita
bilit
y of
the
ass
ay w
as d
eter
min
ed w
ith d
iffer
ent
PUR-
B ba
tche
s an
d ba
tche
s st
ored
at e
leva
ted
tem
pera
ture
s.
Res
ults
:Th
e bi
rch
polle
n al
lerg
oid
prep
arat
ion
indu
ced
high
spe
cific
IgG
titer
s in
ra
bbits
, wh
ich
was
dem
onst
rate
d by
EL
ISA
and
imm
unob
lotti
ng(F
ig.
1).
Imm
unob
lotti
ngsh
ows
that
the
rab
bit
anti
seru
m re
cogn
ises
birc
h ex
tract
, birc
h al
lerg
oid
prep
arat
ions
an
d th
e re
leva
nt b
irch
alle
rgen
Bet
v1
(Fig
. 1B)
. Usi
ng d
iffer
ent
conc
entra
tions
of A
LU-B
mixe
d wi
th th
e ra
bbit
seru
m re
sulte
d in
do
se-re
spon
se c
urve
s on
birc
h al
lerg
oid
polle
n co
ated
micr
otite
rpl
ates
. By
test
ing
supe
rnat
ant o
f PUR
-B b
atch
es it
was
pro
ven
that
the
dos
e-re
spon
secu
rves
wer
e m
ainl
y ca
used
by
ALU
-B
and
not b
y un
boun
d m
ater
ial s
ince
sup
erna
tant
con
tain
s a
mor
e th
an 1
00 ti
mes
lowe
r sig
nal (
Fig
2). T
able
1 s
hows
a s
igni
fican
t in
crea
se in
the
50%
IgG
inhi
bitio
n va
lue
when
two
times
dilu
ted
batc
hes
are
anal
ysed
, ind
icat
ing
that
the
sens
itivity
of t
he a
ssay
is
suf
ficie
nt to
det
ect a
50%
redu
ctio
n in
act
ivity
. Sto
rage
of t
he
ALU
-B v
acci
ne a
t va
rious
ele
vate
d te
mpe
ratu
res
resu
lted
in
incr
ease
d 50
% I
gGin
hibi
tion
valu
es i
ndica
ting
a re
duct
ion
of
IgG
bind
ing
to t
he e
pito
pes
(Fig
3, T
able
2).
Valid
atio
n of
the
assa
y sh
owed
that
the
assa
y is
spe
cific
for b
irch,
has
a n
orm
al
dist
ribut
ion
of p
oint
s, s
hows
a g
ood
prec
isio
n an
d is
rob
ust
(Tab
le 3
). Th
e as
say
is s
till
unde
r ev
alua
tion
to e
stab
lish
whet
her
the
IgG
inhi
bitio
n EL
ISA
is s
uita
ble
for
long
itudi
nal
mon
itorin
g of
sta
bilit
y of
the
adso
rbed
pro
duct
Conc
lusi
on:
The
newl
y de
velo
ped
and
valid
ated
IgG
inhi
bitio
n as
say
is su
itabl
e fo
r m
easu
ring
IgG
bind
ing
to P
UR-B
bat
ches
and
is
able
to a
t lea
st d
etec
t a tw
o-fo
ld re
duct
ion
in p
oten
cy.
The
assa
y sh
owed
re
duce
d Ig
Gbi
ndin
g to
te
mpe
ratu
re
stre
ssed
ALU
-B v
acci
ne, s
ugge
stin
g th
at th
e as
say
is s
tabi
lity
indi
catin
g.
Figu
re 2
. IgG
inhi
bitio
n cu
rves
of 2
bat
ches
AL
U-B
(b
lue
curv
es)
and
IgG
inhi
bitio
n cu
rves
mea
sure
d in
the
ir su
pern
atan
t (re
d an
d or
ange
cur
ves)
.
Figu
re 3
. The
gra
ph re
pres
ents
IgG
inhi
bitio
n cu
rves
of a
PU
R-B
bat
ch s
tore
d at
5°C
, 25°
C,
37 °C
for 2
mon
ths
and
at 9
0°C
for 1
and
4
hrs.
The
dot
ted
line
show
s th
e 50
% in
hibi
tion
of th
e di
ffere
nt c
urve
s.
Res
ults
Valid
atio
npa
ram
eter
s
CV
=16
%In
ter-a
ssay
prec
isio
nTe
mpe
ratu
reco
nditio
nsar
e cr
ucia
lR
obus
tnes
s
CV
= 15
%In
tra-a
ssay
prec
isio
n
Nor
mal
dist
ribut
ion
of p
oint
s; a
ncho
r poi
nts
appr
oach
uppe
r an
d lo
wera
sym
ptot
eM
odel
Spec
ificfo
rALU
-B a
nd n
otto
oth
erPU
RET
HAL
pre
para
tions
Spec
ificity
Tabl
e 3.
Val
idat
ion
resu
lts o
f the
50%
IgG
inhi
bitio
n as
say
for A
LU-B
bat
ches
.
90T0
137
140
148
94 112
50%
IgG
inhi
bitio
nva
lue
in A
Ueq
/ml
% re
cove
rySt
orag
eco
nditi
ons
66 64 61
37° °°°C
, 2 m
onth
s
90° °°°C
, 1 h
our
90° °°°C
, 4 h
ours
96 80
5° °°°C
, 2 m
onth
s
25° °°°C
, 2 m
onth
s
Lane
1: M
arke
r; La
ne 2
: Bi
rch
polle
n ex
tract
; La
ne 3
-5: 3
diff
eren
t Bi
rch
polle
n al
lerg
oid
batc
hes.
The
box
in
dica
tes
the
Bet
v1
band
Figu
re 1
A. D
irect
ELI
SA o
f th
e ra
bbit
anti-
seru
m te
sted
on
Birc
h po
llen
extra
ct a
nd o
n al
lerg
oid.
The
ser
um s
hows
dos
e-re
spon
ses
to b
oth
prep
arat
ions
with
hig
her
affin
ity f
or
the
alle
rgoi
dpr
epar
atio
n.
Figu
re 1
B.I
mm
unob
loto
f rab
bit s
erum
im
mun
ized
with
birc
h po
llen
alle
rgoi
d. T
he
anti-
seru
m is
test
ed o
n bi
rch
polle
n ex
tract
an
d on
thre
e di
ffere
nt b
irch
polle
n al
lerg
oid
prep
arat
ions
..
Nor
mal
dose
½N
orm
aldo
se
Batc
h B
128
AU
eq/m
l
286
AU
eq/m
l
50%
IgG
inhi
bitio
nva
lue
CV
7.4%
5.5%
Nor
mal
dose
½N
orm
aldo
se
Batc
h A
128
AU
eq/m
l
294
AU
eq/m
l
50%
IgG
inhi
bitio
nva
lue
CV
1.5%
2.0%
Tabl
e 1.
50%
IgG
inhi
bitio
n va
lues
of
2 ba
tche
s AL
U-B
, al
so
test
ed
when
1:
1 di
lute
d in
form
ulat
ion
buffe
r.
12
34
51
23
45
0,0
1,0
2,0
3,0
4,0
5,0
1010
010
0010
000
1000
0010
0000
0
seru
m d
ilutio
n
OD value (450 nm)
Birc
h po
llen
extra
ctBi
rch
polle
n al
lerg
oid
Tabl
e 2.
The
50%
IgG
inhi
bitio
n va
lues
of e
ach
curv
e ar
e pr
esen
ted
in th
e ta
ble.
The
dec
reas
e of
50%
IgG
inhi
bitio
n wa
s ca
lcula
ted
in re
latio
n to
the
ALU
-B s
ampl
e an
alys
ed a
t t0.
CV
of a
ll m
easu
rem
ents
is b
elow
10%
-100102030405060708090100
110
100
1000
1000
010
0000
ALU-
B in
AUe
q/m
l
IgG inhibition (%)
5°C
25°C
37°C
90°C
, 1 h
r 9
0°C
, 4 h
rs
020406080100
110
100
1000
1000
010
0000
ALU-
B in
AUe
q/m
l
IgG inhibition (%)
ALU-
B ba
tch
ASU
P AL
U-B
batc
h A
ALU-
B ba
tch
BSU
P AL
U-B
batc
h B
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
alor
perc
eive
dco
nflic
tsof
inte
rest
: th
e pr
esen
ter i
s an
empl
oyee
of H
AL A
llerg
y
572
-An
antib
ody-
base
d Te
chni
que
for S
tabi
lity
Stud
ies
on
Alum
Ads
orbe
d B
irch
polle
n al
lerg
oid
prep
arat
ions
E. K
erkv
liet,
C. F
rans
o, A
. Seg
aar a
nd R
. van
den
Hou
tH
AL A
llerg
y BV
, Lei
den,
The
Net
herla
nds
Back
grou
nd a
nd a
im:
IgE
base
d po
tenc
y as
says
for
alle
rgen
s ar
e es
sent
ial t
ests
for
mon
itorin
g st
abilit
y in
ext
ract
s. H
owev
er,
thes
e Ig
Epo
tenc
y as
says
can
not
be
used
for p
oten
cy d
eter
min
atio
n of
alle
rgoi
d-ba
sed
prod
ucts
, si
nce
they
es
sent
ially
la
ck
IgE
reac
tivity
. Fu
rther
mor
e, a
lum
iniu
m c
an d
eter
iora
te b
indi
ng o
f ant
ibod
ies
to
prot
eins
atta
ched
to a
sol
id p
hase
. We
prev
ious
ly d
evel
oped
an
IgG
base
d in
hibi
tion
assa
y fo
r m
onito
ring
stab
ility
of a
lum
ad
sorb
ed m
ite p
repa
ratio
ns.
A sim
ilar
assa
y ha
s no
w be
en
deve
lope
d fo
r th
e de
term
inat
ion
of
IgG
inhi
bitio
n of
al
um
adso
rbed
birc
h po
llen
alle
rgoi
ds. T
his
assa
y ha
s be
en s
tudi
ed
for
use
as a
sta
bilit
y te
st a
nd w
heth
er t
he a
ssay
has
bee
n su
ffici
ent
sens
itive
to d
etec
t a
2 tim
es r
educ
tion.
Fin
ally,
the
assa
y wa
s va
lidat
ed a
ccor
ding
the
curre
nt g
uide
lines
.
Met
hods
:Th
e ne
wly
deve
lope
d Ig
Gin
hibi
tion
assa
y us
es Ig
Gan
tibod
ies
spec
ific
for
birc
h po
llen
alle
rgoi
dsth
at
were
ob
tain
ed
by
imm
uniza
tion
of r
abbi
ts w
ith g
luta
rald
ehyd
em
odifi
ed e
xtra
cts
(alle
rgoi
d) o
f birc
h po
llen.
The
ass
ay s
tarts
with
a p
re-in
cuba
tion
of a
mixt
ure
of a
nti-a
llerg
oid
seru
m a
nd d
iffer
ent c
once
ntra
tions
of
alu
m-a
dsor
bed
birc
h po
llen
alle
rgoi
d(A
LU-B
). Af
ter
pre-
incu
batio
n th
e m
ixtur
e is
add
ed t
o an
birc
h al
lerg
oid
coat
ed
mic
rotit
erpl
ate
to b
ind
free
alle
rgoi
d-sp
ecifi
c Ig
G. S
ubse
quen
tly,
boun
d Ig
Gis
quan
titat
ivel
y m
easu
red
by a
n HR
P co
njug
ate
and
stai
ned
with
TM
B. T
he e
xten
t to
whic
h th
e Ig
Gan
tibod
ies
bind
to
the
pla
te i
n pr
esen
ce o
f AL
U-B
is
co
mpa
red
with
the
m
axim
um a
mou
nt o
f Ig
Gan
tibod
ies
that
bin
ds in
abs
ence
of
ALU
-B (
E-m
ax).
Resu
lts a
re f
inal
ly ex
pres
sed
as p
erce
ntag
e in
hibi
tion
rela
tive
to t
he u
ninh
ibite
d va
lue.
The
50%
inhi
bitio
nva
lue
is us
ed a
s re
ad o
ut. A
n in
hou
se b
irch
alle
rgoi
dre
fere
nce
sam
ple
is u
sed
for d
ay-to
-day
cor
rect
ion
and
as a
ssay
con
trol.
Suita
bilit
y of
the
ass
ay w
as d
eter
min
ed w
ith d
iffer
ent
PUR-
B ba
tche
s an
d ba
tche
s st
ored
at e
leva
ted
tem
pera
ture
s.
Res
ults
:Th
e bi
rch
polle
n al
lerg
oid
prep
arat
ion
indu
ced
high
spe
cific
IgG
titer
s in
ra
bbits
, wh
ich
was
dem
onst
rate
d by
EL
ISA
and
imm
unob
lotti
ng(F
ig.
1).
Imm
unob
lotti
ngsh
ows
that
the
rab
bit
anti
seru
m re
cogn
ises
birc
h ex
tract
, birc
h al
lerg
oid
prep
arat
ions
an
d th
e re
leva
nt b
irch
alle
rgen
Bet
v1
(Fig
. 1B)
. Usi
ng d
iffer
ent
conc
entra
tions
of A
LU-B
mixe
d wi
th th
e ra
bbit
seru
m re
sulte
d in
do
se-re
spon
se c
urve
s on
birc
h al
lerg
oid
polle
n co
ated
micr
otite
rpl
ates
. By
test
ing
supe
rnat
ant o
f PUR
-B b
atch
es it
was
pro
ven
that
the
dos
e-re
spon
secu
rves
wer
e m
ainl
y ca
used
by
ALU
-B
and
not b
y un
boun
d m
ater
ial s
ince
sup
erna
tant
con
tain
s a
mor
e th
an 1
00 ti
mes
lowe
r sig
nal (
Fig
2). T
able
1 s
hows
a s
igni
fican
t in
crea
se in
the
50%
IgG
inhi
bitio
n va
lue
when
two
times
dilu
ted
batc
hes
are
anal
ysed
, ind
icat
ing
that
the
sens
itivity
of t
he a
ssay
is
suf
ficie
nt to
det
ect a
50%
redu
ctio
n in
act
ivity
. Sto
rage
of t
he
ALU
-B v
acci
ne a
t va
rious
ele
vate
d te
mpe
ratu
res
resu
lted
in
incr
ease
d 50
% I
gGin
hibi
tion
valu
es i
ndica
ting
a re
duct
ion
of
IgG
bind
ing
to t
he e
pito
pes
(Fig
3, T
able
2).
Valid
atio
n of
the
assa
y sh
owed
that
the
assa
y is
spe
cific
for b
irch,
has
a n
orm
al
dist
ribut
ion
of p
oint
s, s
hows
a g
ood
prec
isio
n an
d is
rob
ust
(Tab
le 3
). Th
e as
say
is s
till
unde
r ev
alua
tion
to e
stab
lish
whet
her
the
IgG
inhi
bitio
n EL
ISA
is s
uita
ble
for
long
itudi
nal
mon
itorin
g of
sta
bilit
y of
the
adso
rbed
pro
duct
Conc
lusi
on:
The
newl
y de
velo
ped
and
valid
ated
IgG
inhi
bitio
n as
say
is su
itabl
e fo
r m
easu
ring
IgG
bind
ing
to P
UR-B
bat
ches
and
is
able
to a
t lea
st d
etec
t a tw
o-fo
ld re
duct
ion
in p
oten
cy.
The
assa
y sh
owed
re
duce
d Ig
Gbi
ndin
g to
te
mpe
ratu
re
stre
ssed
ALU
-B v
acci
ne, s
ugge
stin
g th
at th
e as
say
is s
tabi
lity
indi
catin
g.
Figu
re 2
. IgG
inhi
bitio
n cu
rves
of 2
bat
ches
AL
U-B
(b
lue
curv
es)
and
IgG
inhi
bitio
n cu
rves
mea
sure
d in
the
ir su
pern
atan
t (re
d an
d or
ange
cur
ves)
.
Figu
re 3
. The
gra
ph re
pres
ents
IgG
inhi
bitio
n cu
rves
of a
PU
R-B
bat
ch s
tore
d at
5°C
, 25°
C,
37 °C
for 2
mon
ths
and
at 9
0°C
for 1
and
4
hrs.
The
dot
ted
line
show
s th
e 50
% in
hibi
tion
of th
e di
ffere
nt c
urve
s.
Res
ults
Valid
atio
npa
ram
eter
s
CV
=16
%In
ter-a
ssay
prec
isio
nTe
mpe
ratu
reco
nditio
nsar
e cr
ucia
lR
obus
tnes
s
CV
= 15
%In
tra-a
ssay
prec
isio
n
Nor
mal
dist
ribut
ion
of p
oint
s; a
ncho
r poi
nts
appr
oach
uppe
r an
d lo
wera
sym
ptot
eM
odel
Spec
ificfo
rALU
-B a
nd n
otto
oth
erPU
RET
HAL
pre
para
tions
Spec
ificity
Tabl
e 3.
Val
idat
ion
resu
lts o
f the
50%
IgG
inhi
bitio
n as
say
for A
LU-B
bat
ches
.
90T0
137
140
148
94 112
50%
IgG
inhi
bitio
nva
lue
in A
Ueq
/ml
% re
cove
rySt
orag
eco
nditi
ons
66 64 61
37° °°°C
, 2 m
onth
s
90° °°°C
, 1 h
our
90° °°°C
, 4 h
ours
96 80
5° °°°C
, 2 m
onth
s
25° °°°C
, 2 m
onth
s
Lane
1: M
arke
r; La
ne 2
: Bi
rch
polle
n ex
tract
; La
ne 3
-5: 3
diff
eren
t Bi
rch
polle
n al
lerg
oid
batc
hes.
The
box
in
dica
tes
the
Bet
v1
band
Figu
re 1
A. D
irect
ELI
SA o
f th
e ra
bbit
anti-
seru
m te
sted
on
Birc
h po
llen
extra
ct a
nd o
n al
lerg
oid.
The
ser
um s
hows
dos
e-re
spon
ses
to b
oth
prep
arat
ions
with
hig
her
affin
ity f
or
the
alle
rgoi
dpr
epar
atio
n.
Figu
re 1
B.I
mm
unob
loto
f rab
bit s
erum
im
mun
ized
with
birc
h po
llen
alle
rgoi
d. T
he
anti-
seru
m is
test
ed o
n bi
rch
polle
n ex
tract
an
d on
thre
e di
ffere
nt b
irch
polle
n al
lerg
oid
prep
arat
ions
..
Nor
mal
dose
½N
orm
aldo
se
Batc
h B
128
AU
eq/m
l
286
AU
eq/m
l
50%
IgG
inhi
bitio
nva
lue
CV
7.4%
5.5%
Nor
mal
dose
½N
orm
aldo
se
Batc
h A
128
AU
eq/m
l
294
AU
eq/m
l
50%
IgG
inhi
bitio
nva
lue
CV
1.5%
2.0%
Tabl
e 1.
50%
IgG
inhi
bitio
n va
lues
of
2 ba
tche
s AL
U-B
, al
so
test
ed
when
1:
1 di
lute
d in
form
ulat
ion
buffe
r.
12
34
51
23
45
0,0
1,0
2,0
3,0
4,0
5,0
1010
010
0010
000
1000
0010
0000
0
seru
m d
ilutio
n
OD value (450 nm)
Birc
h po
llen
extra
ctBi
rch
polle
n al
lerg
oid
Tabl
e 2.
The
50%
IgG
inhi
bitio
n va
lues
of e
ach
curv
e ar
e pr
esen
ted
in th
e ta
ble.
The
dec
reas
e of
50%
IgG
inhi
bitio
n wa
s ca
lcula
ted
in re
latio
n to
the
ALU
-B s
ampl
e an
alys
ed a
t t0.
CV
of a
ll m
easu
rem
ents
is b
elow
10%
-100102030405060708090100
110
100
1000
1000
010
0000
ALU-
B in
AUe
q/m
l
IgG inhibition (%)
5°C
25°C
37°C
90°C
, 1 h
r 9
0°C
, 4 h
rs
020406080100
110
100
1000
1000
010
0000
ALU-
B in
AUe
q/m
l
IgG inhibition (%)
ALU-
B ba
tch
ASU
P AL
U-B
batc
h A
ALU-
B ba
tch
BSU
P AL
U-B
batc
h B
In re
latio
nto
this
pres
enta
tion,
I de
clare
the
follo
win
g, re
alor
perc
eive
dco
nflic
tsof
inte
rest
: th
e pr
esen
ter i
s an
empl
oyee
of H
AL A
llerg
yIn
rela
tion
to th
is p
rese
ntat
ion,
I de
clar
e th
e fo
llow
ing,
real
or p
erce
ived
con
flict
s of
inte
rest
: the
pre
sent
er is
an
empl
oyee
of H
AL A
llerg
y.EA
ACI C
ongr
ess
2012
B i o c h e mic a l c h ara c t e ri z a t i o n o f c h e mic a l l y m o d i f i e d Ara h 2 is o f o r ms; s tru c tu r e - f u n c t i o n r e l a t i o n s h i p s
34
D. Luykx (1) , D. Apostolovic (1), H. Warmenhoven (1), D. Verbart (1), G.A.H. de Jong (2), S.J. Koppelman (1).
(1) HAL Allergy BV, Leiden, The Netherlands, (2) TNO, Zeist, The Netherlands.
BackgroundModified forms of major peanut allergens Ara h2 and Ara h6 were shown to be less allergenic than their native counterparts, making them potentially suitable for peanut allergy immunotherapy. Two isoforms of Ara h2 are known: heavy isoform of appr. 20 kDa and a light isoform of appr. 17 kDa. Furthermore, Ara h6 is an allergen of appr. 15 kDa, homologous to Ara h2. This study aims to describe the biochemical characteristics and IgE-binding potency of the modified allergens Ara h2-heavy isoform, Ara h2-light isoform, and Ara h6 in comparison with their native counterparts.
MethodsAra h2-heavy isoform, Ara h2-light isoform, and Ara h6 were purified to homogeneity to obtain the native allergens. To obtain the modified allergens, the native allergens were modified by reduction of the disulfide bonds and subsequent alkylation of the free Cys residues. The modified allergens as well as their native counter parts were analysed for change in molecular weight, secondary structure, and allergenicity, using SDS-PAGE, far-UV CD spectroscopy, and an IgE-binding assay respectively.
ResultsThe modification of the allergens was optimized by investigating the kinetics, resulting in a consistent procedure for modification. All three allergens showed an increase in apparent molecular weight and unfolding of protein structure upon reduction and alkylation. Consequently, the IgE-binding potency was decreased about a hundred-fold for each of the tested allergens.
ConclusionsUnderstanding the biochemical nature of the modification of the major peanut allergens Ara h2 and Ara h6 allows to optimize the process to reduce their allergenicity.
EAACI, 16-20 June 2012, GenevaAbstract number: 1448, Session date and time: Tuesday 19 June; 12:00 - 13:30 Session title: Poster 71 - Clinical and experimental applications of molecular allergology
35
Poster Session 71 - Clinical and experimental applications of molecular allergology
B i o c h e mic a l c h ara c t e ri z a t i o n o f c h e mic a l l y m o d i f i e d Ara h 2 is o f o r ms; s tru c tu r e - f u n c t i o n r e l a t i o n s h i p s
Figu
re 2
: Far
UV
CD
spe
ctra
of n
ativ
e (b
lack
) and
mod
ified
(blu
e) A
rah2
-hea
vy is
ofor
m(A
), Ar
ah2
-ligh
t iso
form
(B) a
nd A
rah6
(C).
1448
-Bio
chem
ical
cha
ract
eriz
atio
n of
che
mic
al m
odifi
catio
n of
Ara
h2
isof
orm
s: s
truc
ture
-func
tion
rela
tions
hips
H. W
arm
enho
ven1
& D
. Apo
stol
ovic
1 , D
. Luy
kx1 ,
D. V
erba
rt1, A
. de
Jong
2 , T.
Cirk
ovic
Vel
icko
vic3
, S. K
oppe
lman
1
1 HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s, 2 TN
O Q
ualit
y of
Life
, Zei
st, T
he N
ethe
rland
s, 3 U
nive
rsity
of B
elgr
ade,
Bel
grad
e, S
erbi
a
Figu
re 1
: SD
S-P
AGE
patte
rns
of A
rah2
-hea
vy is
ofor
m(A
), Ar
ah2
-ligh
t iso
form
(B),
and
Ara
h6 (C
) afte
r red
ucin
g th
e al
lerg
ens
with
0.5
or 5
.0 m
MD
TT fo
r diff
eren
t per
iods
of
time
(0-6
0 m
in).
Mol
ecul
ar w
eigh
t mar
ker (
M).
Figu
re 3
: Int
rinsi
c flu
ores
cenc
e sp
ectr
a of
nat
ive
(bla
ck) a
nd m
odifi
ed (r
ed) A
rah2
-he
avy
isof
orm
(A),
Ara
h2-li
ght i
sofo
rm(B
) and
Ara
h6 (C
).
C.
M
0
0
.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0 6
0 m
in
0.5
mM
DTT
5 m
MD
TT
M
0
0.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0
60 m
in
M
0
0
.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0 6
0 m
in
B.
A.
B
.
C.
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
A.
B.
C
.
010203040506070
290
310
330
350
370
390
Wav
eleng
th (nm
)
Fluorescence intensity (105 LU)
010203040
290
310
330
350
370
390
Wav
eleng
th (nm
)
Fluorescence intensity (105 LU)
Bac
kgro
und
& A
im:
Mod
ified
form
s of
maj
or p
eanu
t alle
rgen
s Ar
ah2
and
Ara
h6 w
ere
show
n to
be
less
alle
rgen
ic th
an th
eir
nativ
e co
unte
rpar
ts. I
n th
is w
ay th
ey a
re
pote
ntia
lly s
uita
ble
for
pean
ut a
llerg
y im
mun
othe
rapy
. Tw
o is
ofor
ms
of
Ara
h2 a
re k
now
n: a
hea
vy is
ofor
mof
~20
kD
aan
d a
light
isof
orm
of ~
17
kDa.
Ara
h6 w
ith a
mol
ecul
ar w
eigh
t of ~
15 k
Da
is h
omol
ogou
s w
ith A
rah2
. Th
is s
tudy
aim
s to
inv
estig
ate
the
bioc
hem
ical
cha
ract
eris
tics
of
redu
ced
and
alky
late
dAr
ah2
isof
orm
sin
com
paris
on w
ith t
heir
nativ
e co
unte
rpar
ts a
nd to
stu
dy th
e ki
netic
s of
the
mod
ifica
tion.
The
refo
re, t
he
thre
e m
odifi
ed a
llerg
ens
wer
e an
alyz
ed f
or a
cha
nge
in m
olec
ular
wei
ght,
prot
ein
stru
ctur
e an
d al
lerg
enic
ityby
usi
ng S
DS-
PAG
E, m
ass
spec
trom
etry
(M
S),
CD
and
flu
ores
cenc
e sp
ectro
scop
y, a
nd a
n Ig
E-bi
ndin
g as
say.
Met
hods
:M
odifi
catio
n: A
rah2
-hea
vy i
sofo
rm,
Ara
h2-li
ght
isof
orm
and
Ara
h6
wer
e m
odifi
ed b
y re
duct
ion
of t
he d
isul
phid
e bo
nds
with
0.5
or
5m
MD
TT
and
subs
eque
nt
alky
latio
n of
th
e fre
e C
ysre
sidu
es
with
io
doac
etam
ide
(50
mM
).
SDS-
PAG
E: N
on-re
duce
d, p
artia
lly r
educ
ed a
nd f
ully
red
uced
sam
ples
w
ere
appl
ied
onto
14%
pol
yacr
ylam
ide
gels
and
sta
ined
with
Coo
mas
sie
Blue
.
Mas
s sp
ectro
met
ry:
Tryp
ticpe
ptid
es o
btai
ned
from
the
sam
ples
wer
e se
para
ted
via
liqui
d ch
rom
atog
raph
y be
fore
ele
ctro
spra
yio
nisa
tion
and
ente
ring
the
LTQ
Orb
itrap
XL h
ybrid
mas
s sp
ectro
met
er.
The
ioni
sed
pept
ides
wer
e fra
gmen
ted
to re
veal
the
(mod
ified
) am
ino
acid
seq
uenc
e fo
r ide
ntity
. MS
was
als
o ap
plie
d fo
r int
act n
ativ
e an
d m
odifi
edal
lerg
ens.
CD
: Far
-UV
CD
spe
ctra
wer
e re
cord
ed fr
om 1
90 to
260
nm
.
Fluo
resc
ence
spe
ctro
scop
y: F
luor
esce
nce
spec
tra w
ere
reco
rded
fro
m
290
to 3
90 n
m w
ith e
xcita
tion
at 2
80 n
m.
IgE
inhi
bitio
n as
say:
Th
e m
odifi
ed
alle
rgen
s co
mpe
te
with
th
eir
corre
spon
ding
nat
ive
form
coa
ted
to E
LISA
pla
tes
for
bind
ing
to s
erum
Ig
E. Res
ults
:Th
e ki
netic
s of
the
pean
ut a
llerg
en m
odifi
catio
n w
as s
tudi
ed w
ith S
DS-
PAG
E as
an
incr
ease
of m
olec
ular
mas
s w
as e
xpec
ted
durin
g re
duct
ion
and
alky
latio
n (F
ig. 1
). By
usi
ng 0
.5 m
MD
TT th
e th
ree
alle
rgen
s w
ere
tota
lly m
odifi
ed a
fter 3
0 m
in w
here
as 5
min
was
nee
ded
for m
odifi
catio
n w
hen
usin
g 5
mM
DTT
. Ac
cord
ing
to t
he S
DS-
PAG
E pa
ttern
s th
e in
crea
se o
f the
mol
ecul
ar m
ass
for t
he m
odifi
ed a
llerg
ens
appe
ars
to b
e ~4
kD
a. A
ccor
ding
to M
S, a
mas
s in
crea
se o
f 465
.6 D
aw
as d
eter
min
ed
for
Ara
h2.
Con
side
ring
the
pres
ence
of
8 C
ysre
sidu
es in
Ara
h2 a
nd
the
alky
latio
n (w
hich
add
s 57
Da
per
Cys
resi
due)
, th
is l
atte
r m
ass
shou
ld b
e co
rrect
. Th
e ov
eres
timat
ed m
ass
incr
ease
for
the
mod
ified
al
lerg
ens
obta
ined
with
SD
S-PA
GE
can
be e
xpla
ined
by
thei
r pr
otei
n st
ruct
ures
. Far
-UV
CD
spe
ctra
indi
cate
typi
cal
-hel
ix s
truct
ures
for
the
nativ
e al
lerg
ens
(i.e.
, el
liptic
ityat
208
and
222
nm
)w
here
as t
ypic
al
defo
lded
prot
ein
stru
ctur
es w
ere
obse
rved
for
the
mod
ified
alle
rgen
s (F
ig.
2).
Def
olde
dan
d th
eref
ore
stre
tche
d pr
otei
ns m
igra
te r
elat
ivel
y sl
ow t
hrou
gh t
he S
DS-
PAG
E ge
l in
com
paris
on t
o fo
lded
pro
tein
s re
sulti
ng in
ban
ds th
at c
orre
spon
d to
rela
tivel
y hi
gher
mol
ecul
ar m
asse
s th
an e
xpec
ted.
The
def
olde
dpr
otei
n st
ruct
ures
of t
he m
odifi
ed a
llerg
ens
are
supp
orte
d by
fluo
resc
ence
spe
ctro
scop
y (F
ig. 3
). Af
ter f
ull r
educ
tion
of th
e th
ree
alle
rgen
s, th
e in
trins
ic fl
uore
scen
ce in
tens
ity d
ecre
ases
and
a
smal
l shi
ft of
the
fluor
esce
nce
emis
sion
max
imum
(Em
ax) i
s ob
serv
ed
for
the
Ara
h2-li
ght i
sofo
rm a
nd A
ra h
6 (i.
e., 3
and
8 n
m, r
espe
ctiv
ely)
. Th
ese
findi
ngs
indi
cate
that
the
Trp
and
Tyr r
esid
ues
in A
rah2
, and
Tyr
re
sidu
es i
n Ar
ah6
(Ar
ah6
doe
s no
t co
ntai
n Tr
p) a
re g
ettin
g m
ore
expo
sed
to t
he b
uffe
r. Th
is o
ccur
s du
ring
defo
ldin
gof
pro
tein
s. T
he
defo
lded
prot
ein
stru
ctur
es
of
the
pean
ut
alle
rgen
s re
sult
in
a co
nsid
erab
le d
ecre
ase
of a
llerg
enic
ityas
the
rela
tive
rest
pot
ency
(Ig
E)
of m
odifi
ed A
rah2
-hea
vy is
ofor
m, A
rah2
-ligh
t iso
form
and
Ara
h6 w
as
dete
rmin
ed to
0.0
1, 0
.02
and
0, re
spec
tivel
y.
Con
clus
ion:
Red
uctio
n an
d al
kyla
tion
of A
rah2
and
Ara
h6 r
esul
ts i
n co
mpl
ete
mod
ifica
tion
of a
ll is
ofor
ms
resu
lting
in u
nfol
ded
prot
ein
stru
ctur
es.
A.
0102030
290
310
330
350
370
390
Wave
length
(nm)
Fluorescence intensity (105 LU)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy
EAAC
I Con
gres
s 20
12
Figu
re 2
: Far
UV
CD
spe
ctra
of n
ativ
e (b
lack
) and
mod
ified
(blu
e) A
rah2
-hea
vy is
ofor
m(A
), Ar
ah2
-ligh
t iso
form
(B) a
nd A
rah6
(C).
1448
-Bio
chem
ical
cha
ract
eriz
atio
n of
che
mic
al m
odifi
catio
n of
Ara
h2
isof
orm
s: s
truc
ture
-func
tion
rela
tions
hips
H. W
arm
enho
ven1
& D
. Apo
stol
ovic
1 , D
. Luy
kx1 ,
D. V
erba
rt1, A
. de
Jong
2 , T.
Cirk
ovic
Vel
icko
vic3
, S. K
oppe
lman
1
1 HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s, 2 TN
O Q
ualit
y of
Life
, Zei
st, T
he N
ethe
rland
s, 3 U
nive
rsity
of B
elgr
ade,
Bel
grad
e, S
erbi
a
Figu
re 1
: SD
S-P
AGE
patte
rns
of A
rah2
-hea
vy is
ofor
m(A
), Ar
ah2
-ligh
t iso
form
(B),
and
Ara
h6 (C
) afte
r red
ucin
g th
e al
lerg
ens
with
0.5
or 5
.0 m
MD
TT fo
r diff
eren
t per
iods
of
time
(0-6
0 m
in).
Mol
ecul
ar w
eigh
t mar
ker (
M).
Figu
re 3
: Int
rinsi
c flu
ores
cenc
e sp
ectr
a of
nat
ive
(bla
ck) a
nd m
odifi
ed (r
ed) A
rah2
-he
avy
isof
orm
(A),
Ara
h2-li
ght i
sofo
rm(B
) and
Ara
h6 (C
).
C.
M
0
0
.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0 6
0 m
in
0.5
mM
DTT
5 m
MD
TT
M
0
0.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0
60 m
in
M
0
0
.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0 6
0 m
in
B.
A.
B
.
C.
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
A.
B.
C
.
010203040506070
290
310
330
350
370
390
Wav
eleng
th (nm
)
Fluorescence intensity (105 LU)
010203040
290
310
330
350
370
390
Wav
eleng
th (nm
)
Fluorescence intensity (105 LU)
Bac
kgro
und
& A
im:
Mod
ified
form
s of
maj
or p
eanu
t alle
rgen
s Ar
ah2
and
Ara
h6 w
ere
show
n to
be
less
alle
rgen
ic th
an th
eir
nativ
e co
unte
rpar
ts. I
n th
is w
ay th
ey a
re
pote
ntia
lly s
uita
ble
for
pean
ut a
llerg
y im
mun
othe
rapy
. Tw
o is
ofor
ms
of
Ara
h2 a
re k
now
n: a
hea
vy is
ofor
mof
~20
kD
aan
d a
light
isof
orm
of ~
17
kDa.
Ara
h6 w
ith a
mol
ecul
ar w
eigh
t of ~
15 k
Da
is h
omol
ogou
s w
ith A
rah2
. Th
is s
tudy
aim
s to
inv
estig
ate
the
bioc
hem
ical
cha
ract
eris
tics
of
redu
ced
and
alky
late
dAr
ah2
isof
orm
sin
com
paris
on w
ith t
heir
nativ
e co
unte
rpar
ts a
nd to
stu
dy th
e ki
netic
s of
the
mod
ifica
tion.
The
refo
re, t
he
thre
e m
odifi
ed a
llerg
ens
wer
e an
alyz
ed f
or a
cha
nge
in m
olec
ular
wei
ght,
prot
ein
stru
ctur
e an
d al
lerg
enic
ityby
usi
ng S
DS-
PAG
E, m
ass
spec
trom
etry
(M
S),
CD
and
flu
ores
cenc
e sp
ectro
scop
y, a
nd a
n Ig
E-bi
ndin
g as
say.
Met
hods
:M
odifi
catio
n: A
rah2
-hea
vy i
sofo
rm,
Ara
h2-li
ght
isof
orm
and
Ara
h6
wer
e m
odifi
ed b
y re
duct
ion
of t
he d
isul
phid
e bo
nds
with
0.5
or
5m
MD
TT
and
subs
eque
nt
alky
latio
n of
th
e fre
e C
ysre
sidu
es
with
io
doac
etam
ide
(50
mM
).
SDS-
PAG
E: N
on-re
duce
d, p
artia
lly r
educ
ed a
nd f
ully
red
uced
sam
ples
w
ere
appl
ied
onto
14%
pol
yacr
ylam
ide
gels
and
sta
ined
with
Coo
mas
sie
Blue
.
Mas
s sp
ectro
met
ry:
Tryp
ticpe
ptid
es o
btai
ned
from
the
sam
ples
wer
e se
para
ted
via
liqui
d ch
rom
atog
raph
y be
fore
ele
ctro
spra
yio
nisa
tion
and
ente
ring
the
LTQ
Orb
itrap
XL h
ybrid
mas
s sp
ectro
met
er.
The
ioni
sed
pept
ides
wer
e fra
gmen
ted
to re
veal
the
(mod
ified
) am
ino
acid
seq
uenc
e fo
r ide
ntity
. MS
was
als
o ap
plie
d fo
r int
act n
ativ
e an
d m
odifi
edal
lerg
ens.
CD
: Far
-UV
CD
spe
ctra
wer
e re
cord
ed fr
om 1
90 to
260
nm
.
Fluo
resc
ence
spe
ctro
scop
y: F
luor
esce
nce
spec
tra w
ere
reco
rded
fro
m
290
to 3
90 n
m w
ith e
xcita
tion
at 2
80 n
m.
IgE
inhi
bitio
n as
say:
Th
e m
odifi
ed
alle
rgen
s co
mpe
te
with
th
eir
corre
spon
ding
nat
ive
form
coa
ted
to E
LISA
pla
tes
for
bind
ing
to s
erum
Ig
E. Res
ults
:Th
e ki
netic
s of
the
pean
ut a
llerg
en m
odifi
catio
n w
as s
tudi
ed w
ith S
DS-
PAG
E as
an
incr
ease
of m
olec
ular
mas
s w
as e
xpec
ted
durin
g re
duct
ion
and
alky
latio
n (F
ig. 1
). By
usi
ng 0
.5 m
MD
TT th
e th
ree
alle
rgen
s w
ere
tota
lly m
odifi
ed a
fter 3
0 m
in w
here
as 5
min
was
nee
ded
for m
odifi
catio
n w
hen
usin
g 5
mM
DTT
. Ac
cord
ing
to t
he S
DS-
PAG
E pa
ttern
s th
e in
crea
se o
f the
mol
ecul
ar m
ass
for t
he m
odifi
ed a
llerg
ens
appe
ars
to b
e ~4
kD
a. A
ccor
ding
to M
S, a
mas
s in
crea
se o
f 465
.6 D
aw
as d
eter
min
ed
for
Ara
h2.
Con
side
ring
the
pres
ence
of
8 C
ysre
sidu
es in
Ara
h2 a
nd
the
alky
latio
n (w
hich
add
s 57
Da
per
Cys
resi
due)
, th
is l
atte
r m
ass
shou
ld b
e co
rrect
. Th
e ov
eres
timat
ed m
ass
incr
ease
for
the
mod
ified
al
lerg
ens
obta
ined
with
SD
S-PA
GE
can
be e
xpla
ined
by
thei
r pr
otei
n st
ruct
ures
. Far
-UV
CD
spe
ctra
indi
cate
typi
cal
-hel
ix s
truct
ures
for
the
nativ
e al
lerg
ens
(i.e.
, el
liptic
ityat
208
and
222
nm
)w
here
as t
ypic
al
defo
lded
prot
ein
stru
ctur
es w
ere
obse
rved
for
the
mod
ified
alle
rgen
s (F
ig.
2).
Def
olde
dan
d th
eref
ore
stre
tche
d pr
otei
ns m
igra
te r
elat
ivel
y sl
ow t
hrou
gh t
he S
DS-
PAG
E ge
l in
com
paris
on t
o fo
lded
pro
tein
s re
sulti
ng in
ban
ds th
at c
orre
spon
d to
rela
tivel
y hi
gher
mol
ecul
ar m
asse
s th
an e
xpec
ted.
The
def
olde
dpr
otei
n st
ruct
ures
of t
he m
odifi
ed a
llerg
ens
are
supp
orte
d by
fluo
resc
ence
spe
ctro
scop
y (F
ig. 3
). Af
ter f
ull r
educ
tion
of th
e th
ree
alle
rgen
s, th
e in
trins
ic fl
uore
scen
ce in
tens
ity d
ecre
ases
and
a
smal
l shi
ft of
the
fluor
esce
nce
emis
sion
max
imum
(Em
ax) i
s ob
serv
ed
for
the
Ara
h2-li
ght i
sofo
rm a
nd A
ra h
6 (i.
e., 3
and
8 n
m, r
espe
ctiv
ely)
. Th
ese
findi
ngs
indi
cate
that
the
Trp
and
Tyr r
esid
ues
in A
rah2
, and
Tyr
re
sidu
es i
n Ar
ah6
(Ar
ah6
doe
s no
t co
ntai
n Tr
p) a
re g
ettin
g m
ore
expo
sed
to t
he b
uffe
r. Th
is o
ccur
s du
ring
defo
ldin
gof
pro
tein
s. T
he
defo
lded
prot
ein
stru
ctur
es
of
the
pean
ut
alle
rgen
s re
sult
in
a co
nsid
erab
le d
ecre
ase
of a
llerg
enic
ityas
the
rela
tive
rest
pot
ency
(Ig
E)
of m
odifi
ed A
rah2
-hea
vy is
ofor
m, A
rah2
-ligh
t iso
form
and
Ara
h6 w
as
dete
rmin
ed to
0.0
1, 0
.02
and
0, re
spec
tivel
y.
Con
clus
ion:
Red
uctio
n an
d al
kyla
tion
of A
rah2
and
Ara
h6 r
esul
ts i
n co
mpl
ete
mod
ifica
tion
of a
ll is
ofor
ms
resu
lting
in u
nfol
ded
prot
ein
stru
ctur
es.
A.
0102030
290
310
330
350
370
390
Wave
length
(nm)
Fluorescence intensity (105 LU)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy
EAAC
I Con
gres
s 20
12
Figu
re 2
: Far
UV
CD
spe
ctra
of n
ativ
e (b
lack
) and
mod
ified
(blu
e) A
rah2
-hea
vy is
ofor
m(A
), Ar
ah2
-ligh
t iso
form
(B) a
nd A
rah6
(C).
1448
-Bio
chem
ical
cha
ract
eriz
atio
n of
che
mic
al m
odifi
catio
n of
Ara
h2
isof
orm
s: s
truc
ture
-func
tion
rela
tions
hips
H. W
arm
enho
ven1
& D
. Apo
stol
ovic
1 , D
. Luy
kx1 ,
D. V
erba
rt1, A
. de
Jong
2 , T.
Cirk
ovic
Vel
icko
vic3
, S. K
oppe
lman
1
1 HAL
Alle
rgy
BV, L
eide
n, T
he N
ethe
rland
s, 2 TN
O Q
ualit
y of
Life
, Zei
st, T
he N
ethe
rland
s, 3 U
nive
rsity
of B
elgr
ade,
Bel
grad
e, S
erbi
a
Figu
re 1
: SD
S-P
AGE
patte
rns
of A
rah2
-hea
vy is
ofor
m(A
), Ar
ah2
-ligh
t iso
form
(B),
and
Ara
h6 (C
) afte
r red
ucin
g th
e al
lerg
ens
with
0.5
or 5
.0 m
MD
TT fo
r diff
eren
t per
iods
of
time
(0-6
0 m
in).
Mol
ecul
ar w
eigh
t mar
ker (
M).
Figu
re 3
: Int
rinsi
c flu
ores
cenc
e sp
ectr
a of
nat
ive
(bla
ck) a
nd m
odifi
ed (r
ed) A
rah2
-he
avy
isof
orm
(A),
Ara
h2-li
ght i
sofo
rm(B
) and
Ara
h6 (C
).
C.
M
0
0
.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0 6
0 m
in
0.5
mM
DTT
5 m
MD
TT
M
0
0.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0
60 m
in
M
0
0
.5
1
2
5
10 3
0 6
0 0
.5
1
2
5 1
0 3
0 6
0 m
in
B.
A.
B
.
C.
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
-40-200204060
260
250
240
230
220
210
200
190
Wave
lengt
h (nm
)
Ellipticity (mDeg)
A.
B.
C
.
010203040506070
290
310
330
350
370
390
Wav
eleng
th (nm
)
Fluorescence intensity (105 LU)
010203040
290
310
330
350
370
390
Wav
eleng
th (nm
)
Fluorescence intensity (105 LU)
Bac
kgro
und
& A
im:
Mod
ified
form
s of
maj
or p
eanu
t alle
rgen
s Ar
ah2
and
Ara
h6 w
ere
show
n to
be
less
alle
rgen
ic th
an th
eir
nativ
e co
unte
rpar
ts. I
n th
is w
ay th
ey a
re
pote
ntia
lly s
uita
ble
for
pean
ut a
llerg
y im
mun
othe
rapy
. Tw
o is
ofor
ms
of
Ara
h2 a
re k
now
n: a
hea
vy is
ofor
mof
~20
kD
aan
d a
light
isof
orm
of ~
17
kDa.
Ara
h6 w
ith a
mol
ecul
ar w
eigh
t of ~
15 k
Da
is h
omol
ogou
s w
ith A
rah2
. Th
is s
tudy
aim
s to
inv
estig
ate
the
bioc
hem
ical
cha
ract
eris
tics
of
redu
ced
and
alky
late
dAr
ah2
isof
orm
sin
com
paris
on w
ith t
heir
nativ
e co
unte
rpar
ts a
nd to
stu
dy th
e ki
netic
s of
the
mod
ifica
tion.
The
refo
re, t
he
thre
e m
odifi
ed a
llerg
ens
wer
e an
alyz
ed f
or a
cha
nge
in m
olec
ular
wei
ght,
prot
ein
stru
ctur
e an
d al
lerg
enic
ityby
usi
ng S
DS-
PAG
E, m
ass
spec
trom
etry
(M
S),
CD
and
flu
ores
cenc
e sp
ectro
scop
y, a
nd a
n Ig
E-bi
ndin
g as
say.
Met
hods
:M
odifi
catio
n: A
rah2
-hea
vy i
sofo
rm,
Ara
h2-li
ght
isof
orm
and
Ara
h6
wer
e m
odifi
ed b
y re
duct
ion
of t
he d
isul
phid
e bo
nds
with
0.5
or
5m
MD
TT
and
subs
eque
nt
alky
latio
n of
th
e fre
e C
ysre
sidu
es
with
io
doac
etam
ide
(50
mM
).
SDS-
PAG
E: N
on-re
duce
d, p
artia
lly r
educ
ed a
nd f
ully
red
uced
sam
ples
w
ere
appl
ied
onto
14%
pol
yacr
ylam
ide
gels
and
sta
ined
with
Coo
mas
sie
Blue
.
Mas
s sp
ectro
met
ry:
Tryp
ticpe
ptid
es o
btai
ned
from
the
sam
ples
wer
e se
para
ted
via
liqui
d ch
rom
atog
raph
y be
fore
ele
ctro
spra
yio
nisa
tion
and
ente
ring
the
LTQ
Orb
itrap
XL h
ybrid
mas
s sp
ectro
met
er.
The
ioni
sed
pept
ides
wer
e fra
gmen
ted
to re
veal
the
(mod
ified
) am
ino
acid
seq
uenc
e fo
r ide
ntity
. MS
was
als
o ap
plie
d fo
r int
act n
ativ
e an
d m
odifi
edal
lerg
ens.
CD
: Far
-UV
CD
spe
ctra
wer
e re
cord
ed fr
om 1
90 to
260
nm
.
Fluo
resc
ence
spe
ctro
scop
y: F
luor
esce
nce
spec
tra w
ere
reco
rded
fro
m
290
to 3
90 n
m w
ith e
xcita
tion
at 2
80 n
m.
IgE
inhi
bitio
n as
say:
Th
e m
odifi
ed
alle
rgen
s co
mpe
te
with
th
eir
corre
spon
ding
nat
ive
form
coa
ted
to E
LISA
pla
tes
for
bind
ing
to s
erum
Ig
E. Res
ults
:Th
e ki
netic
s of
the
pean
ut a
llerg
en m
odifi
catio
n w
as s
tudi
ed w
ith S
DS-
PAG
E as
an
incr
ease
of m
olec
ular
mas
s w
as e
xpec
ted
durin
g re
duct
ion
and
alky
latio
n (F
ig. 1
). By
usi
ng 0
.5 m
MD
TT th
e th
ree
alle
rgen
s w
ere
tota
lly m
odifi
ed a
fter 3
0 m
in w
here
as 5
min
was
nee
ded
for m
odifi
catio
n w
hen
usin
g 5
mM
DTT
. Ac
cord
ing
to t
he S
DS-
PAG
E pa
ttern
s th
e in
crea
se o
f the
mol
ecul
ar m
ass
for t
he m
odifi
ed a
llerg
ens
appe
ars
to b
e ~4
kD
a. A
ccor
ding
to M
S, a
mas
s in
crea
se o
f 465
.6 D
aw
as d
eter
min
ed
for
Ara
h2.
Con
side
ring
the
pres
ence
of
8 C
ysre
sidu
es in
Ara
h2 a
nd
the
alky
latio
n (w
hich
add
s 57
Da
per
Cys
resi
due)
, th
is l
atte
r m
ass
shou
ld b
e co
rrect
. Th
e ov
eres
timat
ed m
ass
incr
ease
for
the
mod
ified
al
lerg
ens
obta
ined
with
SD
S-PA
GE
can
be e
xpla
ined
by
thei
r pr
otei
n st
ruct
ures
. Far
-UV
CD
spe
ctra
indi
cate
typi
cal
-hel
ix s
truct
ures
for
the
nativ
e al
lerg
ens
(i.e.
, el
liptic
ityat
208
and
222
nm
)w
here
as t
ypic
al
defo
lded
prot
ein
stru
ctur
es w
ere
obse
rved
for
the
mod
ified
alle
rgen
s (F
ig.
2).
Def
olde
dan
d th
eref
ore
stre
tche
d pr
otei
ns m
igra
te r
elat
ivel
y sl
ow t
hrou
gh t
he S
DS-
PAG
E ge
l in
com
paris
on t
o fo
lded
pro
tein
s re
sulti
ng in
ban
ds th
at c
orre
spon
d to
rela
tivel
y hi
gher
mol
ecul
ar m
asse
s th
an e
xpec
ted.
The
def
olde
dpr
otei
n st
ruct
ures
of t
he m
odifi
ed a
llerg
ens
are
supp
orte
d by
fluo
resc
ence
spe
ctro
scop
y (F
ig. 3
). Af
ter f
ull r
educ
tion
of th
e th
ree
alle
rgen
s, th
e in
trins
ic fl
uore
scen
ce in
tens
ity d
ecre
ases
and
a
smal
l shi
ft of
the
fluor
esce
nce
emis
sion
max
imum
(Em
ax) i
s ob
serv
ed
for
the
Ara
h2-li
ght i
sofo
rm a
nd A
ra h
6 (i.
e., 3
and
8 n
m, r
espe
ctiv
ely)
. Th
ese
findi
ngs
indi
cate
that
the
Trp
and
Tyr r
esid
ues
in A
rah2
, and
Tyr
re
sidu
es i
n Ar
ah6
(Ar
ah6
doe
s no
t co
ntai
n Tr
p) a
re g
ettin
g m
ore
expo
sed
to t
he b
uffe
r. Th
is o
ccur
s du
ring
defo
ldin
gof
pro
tein
s. T
he
defo
lded
prot
ein
stru
ctur
es
of
the
pean
ut
alle
rgen
s re
sult
in
a co
nsid
erab
le d
ecre
ase
of a
llerg
enic
ityas
the
rela
tive
rest
pot
ency
(Ig
E)
of m
odifi
ed A
rah2
-hea
vy is
ofor
m, A
rah2
-ligh
t iso
form
and
Ara
h6 w
as
dete
rmin
ed to
0.0
1, 0
.02
and
0, re
spec
tivel
y.
Con
clus
ion:
Red
uctio
n an
d al
kyla
tion
of A
rah2
and
Ara
h6 r
esul
ts i
n co
mpl
ete
mod
ifica
tion
of a
ll is
ofor
ms
resu
lting
in u
nfol
ded
prot
ein
stru
ctur
es.
A.
0102030
290
310
330
350
370
390
Wave
length
(nm)
Fluorescence intensity (105 LU)
In re
latio
nto
this
pres
enta
tion,
I de
clar
eth
e fo
llow
ing,
real
orpe
rcei
ved
conf
licts
of in
tere
st:
the
pres
ente
r is
anem
ploy
ee o
f HAL
Alle
rgy
EAAC
I Con
gres
s 20
12
In re
latio
n to
this
pre
sent
atio
n, I
decl
are
the
follo
win
g, re
al o
r per
ceiv
ed c
onfli
cts
of in
tere
st: t
he p
rese
nter
is a
n em
ploy
ee o
f HAL
Alle
rgy.
EAAC
I Con
gres
s 20
12
Su m m ar y
Pro duct information .
SUBLIVAC ®
VenomenHAL ®
Composition: SUBLIVAC ®, sublingual drops, contains allergen extracts that are specifically prescribed for the patient on the basis of a diagnostic examination. Indication: Treatment of IgE-mediated allergy in patients with symptoms of allergic rhinitis, allergic conjunctivitis and/or allergic asthma, caused by allergens. Dosage and administration: The treatment is started with a daily dose of one drop. This dose is then increased every day with one drop until the highest daily dose of five drops is reached. The treatment is continued with five drops. The drops are to be kept under the tongue for at least 1 minute (preferably 2 - 3 minutes) before they are swallowed. The treatment should be continued for 3 to 5 years in order to remain as symptom-free as possible after the completion of the course of treatment. Contraindications: Acute infections of the eye, airways or organs involved in the immune system, secondary changes in the lungs(e.g. lung emphysema or bronchi-ectasie), severe immunopathological diseases or malignancies (e.g. autoimmune diseases of the kidneys, thyroid glands or the nervous system, rheumatism, tuberculosis and HIV), immunodeficiencies (including as a consequence of immunosuppressants), severe uncontrolled asthma with FEV
1 under 70%, or hypersensitivity
to any of the excipients. Warnings and precautions: If the treatment with pollen extracts is started during the pollen season, there is an increased risk of side effects. Take special care in case of pregnancy, inflammations in the mouth and after removal teeth or molars. Side effects: Local reactions in the mouth and throat, stomach upset and diarrhoea. Worsening of allergic reactions such as rhinitis, conjunctivitis, coughing and atopic eczema. Intensified systemic reactions (such as shortness of breath, generalised urticaria or Quincke’s oedema) can develop in rare cases. Intensified allergic reactions can particularly develop in very hypersensitive patients. These symptoms generally arise within 30 minutes after intake of the drops. Conditions for storage: Store in a refrigerator (2°C - 8°C).When the product is in use, it can be stored for a maximum of 6 months below 25 °C. Package: A SUBLIVAC ® Initial and maintenance treatment set consists of one bottle with dropper containing 24 ml fluid. The complete product information is available upon request. HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: April 2011
36
Composition: VenomenHAL® contains 120 micrograms/vial freeze-dried bee or wasp venom; powder and solvent for solution for injection.Indications: Diagnostic use (prick-testing and intracutaneous testing) and for causal treatment of patients with IgE-mediated insect venom allergy who have suffered a systemic reaction after a bee or wasp sting. Dosage and administration: The 120 µg freeze-dried insect venom should be dissolved with 1.2 ml solvent to 100 µg/ml. The dilution series are prepared by diluting 0.5 ml dissolved venom with 4.5 ml solution. Skin testing: The skin tests are performed by means of end point titration. It always begins with the skin-prick test to determine the patient’s level of sensibility. The intracutaneous test determines the individual starting concentration for treatment. Immunotherapy: Usually the initial treatment is carried out in hospital by administering several subcutaneous injections with increasing doses daily, starting with 0.1 ml of 0.0001 µg/ml until after day 5 the maintenance dose of 1 ml of 100 µg/ml is reached. If in-patient treatment is not possible, immunotherapy may be performed in outpatients, with one injection every 7 days at slowly increasing doses and concentrations. Treatment with the maintenance dose is continued as an outpatient procedure, the injection interval being extended gradually from 7 to 14, 21 and finally 28 days. It is advised to carry out the treatment over a period of 3 - 5 successive years. Contraindications: Skin testing: Secondary infections of the skin, hypersensitivity to any of the excipients. Relative: Acute severe allergic reactions, treatment with ß-blockers, pregnancy, cardiovascular diseases with increased risk if using adrenalin. Immunotherapy: Acute inflammatory diseases/feverish infection at the target organ; active tuberculosis; secondary alterations of the target organ (emphysema, bronchi-ectasis and others); autoimmune disorders e.g. of the kidneys, thyroid gland, nervous system and rheumatic diseases); immune deficiencies (e.g. that caused by immunosuppresants); severe uncontrolled asthma, particularly with an FEV
1 persistently below 70%; cardiovascular diseases
with increased risk if using adrenalin; use of ß-blockers or ACE inhibitors, clinical active malignant tumor, hypersensitivity to any of the excipients. Special warnings and special precautions for use: Treatment with immunotherapy injections should only be performed by physicians qualified in allergology. Appropriate emergency treatment for shock must be immediately available during and after every injection. The patient must remain under medical supervision after the injection for 30 minutes. Side effects: Especially in the case of patients with a high degree of sensitization, intensified allergic reactions may occur. These symptoms generally arise within 30 minutes of receiving the injection. Intensified local reactions at the injection site. Reappearance of patient specific allergic symptoms as mild systemic reactions (itching of eyes, sneezing, coughing, atopic eczema), intensified systemic reactions (shortness of breath, generalized urticaria, Quincke’s Oedema), in extreme rare cases also anaphylactic shock. Occasionally delayed reactions occur (type III and IV). Package: A VenomenHAL® set consists of 6 vials with each 120 µg freezed-dried bee or wasp venom and 6 vials with each with 1.2 ml solvent. A DILUENT set contains 10 vials with each 4.5 ml diluent. The complete product information is available upon request. HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: May 2011
PUReTHAL ®
Composition: PUReTHAL ® contains 20,000 AUM/ml modified pollen allergen extracts, 20,000 AUeq/ml of modified mites allergen extract respectively adsorbed onto aluminium hydroxide; suspension for subcutaneous injection. Indications: Treatment of immediate type allergic disorders (IgE-mediated), such as allergic rhinitis, allergic conjunctivitis and allergic bronchial asthma, which are triggered by allergenic substances. Dosage and administration: The therapy is started with a subcutaneous injection of 0.05 ml. After the first injection the dosage is increased stepwise to a maximum dose of 0.5 ml that is finally administered in monthly intervals. Always check by aspiration that the injection needle has not entered a blood vessel. Do not exceed the maximum dose of 0.5 ml. It is advised to carry out the treatment over a period of 3 - 5 successive years. Contraindications: Acute inflammatory diseases/feverish infection at the target organ, secondary changes of the target organ (emphysema, bronchi-ectasia and others), autoimmune disorders (e.g. of the kidneys, thyroid gland, nervous system and rheumatic diseases), immune deficiencies (e.g. that caused by immuno-suppressants), severe uncontrolled asthma particularly with an FEV
1 persistency below 70%, cardiovascular failure
with increased risk if using adrenalin, clinical active malignant tumor, hypersensitivity to any of the excipients. Special warnings and special precautions for use: Treatment with immunotherapy injections should only be performed by physicians qualified in allergology. Appropriate emergency treatment for shock must be immediately available during and after every injection. The patient must remain under medical supervision after the injection for 30 minutes. Special care should be taken in case of treatment with β-blockers, pregnancy and lactation, use of the product in children below the age of 5. Prophylactic immunization should be carried out no sooner than 7 days after the last injection. Side effects: Especially in the case of patients with a high degree of sensitization, intensified allergic reactions may occur. These symptoms generally arise within 30 minutes of receiving the injection. Intensified local reactions at the injection site. Reappearance of patient specific allergic symptoms as mild systemic reactions (itching of eyes, sneezing, coughing, atopic eczema), intensified systemic reactions (shortness of breath, generalized urticaria, Quincke’s oedema), in extreme rare cases also anaphylactic shock. After use subcutaneous knots and swellings (granulomas) at the injection site may be observed. Package: PUReTHAL ® is delivered in a 6 ml multidose vial with stopper and sealed with an aluminium cap. The complete product information is available on request. HAL Allergy BV, P.O. Box 1204, 2302 BE Leiden, The Netherlands. Date: May 2011
SUBLIVAC ®
VenomenHAL ®
Basisinformationen SUBLIVAC ® FIX / SUBLIVAC ®
Zusammensetzung: Lösungen zur sublingualen Immuntherapie. SUBLIVAC ® enthält Allergenextrakte nach individueller ärztlicher Rezeptur. Sonstige Bestandteile: Glycerol, Wasser, 6-Aminohexansäure (ε-Amino-Capronsäure/EACA), Dinatrium-hydrogenphosphat, Natrium dihydrogenphosphat, Pfefferminzöl. Anwendungsgebiete: Spezifische Immuntherapie allergischer Erkrankungen vom Soforttyp (IgE-vermittelt). Gegenanzeigen, absolute: Akute Entzündungsprozesse/Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); schwere Autoimmunerkrankungen; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Immundefekte (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV1 unter 70% Sollwert; Sensibilisierung gegenüber einem der sonstigen Bestandteile; relative: Schwangerschaft und Stillzeit; Infektionen des Mund-/Rachenraumes; nach zahnärztlicher Behandlung (z.B. Zahnentfernung). Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Fachinformation. Nebenwirkungen: Exazerbation der patientenspezifischen allergischen Symptomatik. Allergische Lokal- und/oder Allgemeinreaktionen; Bauchschmerzen und Durchfall; Exazerbation eines atopischen Ekzems. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Fachinformation. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012
Basisinformationen VenomenHAL® Biene / VenomenHAL® WespeZusammensetzung: VenomenHAL® Biene, Wirkstoff: Bienengift. VenomenHAL® Wespe, Wirkstoff: Wespengift. 6 Durchstechflaschen enthalten jeweils 120 µg reines, gefriergetrocknetes Bienen- bzw. Wespengift. Sonstige Bestandteile: HSA (humanes Serum Albumin) und Mannitol. Lösungsmittel: 6 Durchstechflaschen enthalten jeweils 1,2 ml einer Lösung von Natriumchlorid, Phenol, HSA und Wasser zur Injektion. Anwendungsgebiete: Hauttestung und Therapie von Patienten mit IgE - vermittelter Insektengiftallergie, bei denen systemische Reaktionen nach Bienen- oder Wespenstich aufgetreten sind. Gegenanzeigen: Akute Entzündungsprozesse / Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); Autoimmunerkrankungen; aktive Tuberkulose; Immundefekte (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV1 unter 70 % Sollwert; Herz- und Kreislauferkrankungen mit erhöhtem Risiko bei der Anwendung von Adrenalin; Behandlung mit ß-Blockern und mit ACE-Hemmern; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Sensibilisierung gegenüber einem der sonstigen Bestandteile des Arzneimittels. Während der Schwangerschaft ist von der Einleitung einer Behandlung mit VenomenHAL® Biene oder VenomenHAL® Wespe abzusehen. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Gebrauchs- und Fachinformation. Ferner gelten die bekannten Gegenanzeigen für die Durchführung von Hauttestungen (siehe Gebrauchs- und Fachinformation). Die genannten Kontraindikationen sollten gegen die Gefährdung des Patienten durch einen eventuellen Insektenstich abgewogen werden. Nebenwirkungen: Lokal- und/oder Allgemeinreaktionen bis hin zum anaphylaktischen Schock. Patienten nach der Injektion mindestens 30 Minuten überwachen, eine Schockapotheke muss bereitgestellt sein. Gelegentlich treten auch Reaktionen vom verzögerten Typ (Typ III und IV nach Coombs und Gell) auf. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Gebrauchs- und Fachinformation. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. In seltenen Fällen kann nach der Injektion leichte Müdigkeit auftreten, was beim Führen von Kraftfahrzeugen oder beim Bedienen von Maschinen zu berücksichtigen ist. Warnhinweise: Hyposensibilisierungsimpfstoffe zur Injektion dürfen nur durch allergologisch weitergebildete bzw. allergologisch erfahrene Ärzte verschrieben und angewendet werden. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012
PUReTHAL ®
Basisinformationen PUReTHAL ®
Zusammensetzung: Suspensionen zur subkutanen Injektion, enthalten an Aluminiumhydroxid adsorbierte, mit Glutaraldehyd chemisch modifizierte allergene Substanzen aus Pollen (20.000 AUM/ml) oder Milben (20.000 AUeq/ml). Sonstige Bestandteile: NaCl, Phenol, Aluminiumhydroxid, Wasser zur Injektion. Anwendungsgebiete: Spezifische Immuntherapie allergischer Erkrankungen vom Soforttyp (IgE-vermittelt). Gegenanzeigen, absolute: Akute Entzündungsprozesse/Infektionskrankheiten am Reaktionsorgan; Sekundärveränderungen am Reaktionsorgan (z.B. Emphysem, Bronchiektasen); Autoimmunerkrankungen; Immundefekte (auch durch Immunsuppressiva induziert); schweres, unkontrollierbares Asthma bronchiale, insbesondere bei einem persistierenden FEV1 unter 70% Sollwert; maligne Tumorerkrankungen mit aktuellem Krankheitswert; Erkrankungen mit Kontraindikationen gegen die Anwendung von Adrenalin; Sensibilisierung gegenüber einem der sonstigen Bestandteile; relative: Schwangerschaft und Stillzeit; nicht für Kinder unter 5 Jahren; Behandlung mit ß-Blockern. Zum zeitlichen Intervall zu Schutzimpfungen und für weiterführende Informationen siehe Fachinformation. Nebenwirkungen: Exazerbation der patientenspezifischen allergischen Symptomatik. Lokal- und/oder Allgemeinreaktionen bis hin zum anaphylaktischen Schock. In Einzelfällen Granulombildung am Injektionsort, Schwäche, Schwindel, Konzentrationsstörungen, Kopfschmerzen, Magen-Darm-Beschwerden, Gelenkschmerzen, Fieber. Patienten nach der Injektion mindestens 30 Minuten überwachen, eine Schockapotheke muss bereitgestellt sein. Nebenreaktionen können auch noch zu einem späteren Zeitpunkt auftreten. Für weiterführende Informationen und zur Behandlung von Nebenwirkungen siehe Fachinformation. Hinweise: Verschreibungspflichtig. Arzneimittel für Kinder unzugänglich aufbewahren. In seltenen Fällen kann nach der Injektion leichte Müdigkeit auftreten, was beim Führen von Kraftfahrzeugen oder beim Bedienen von Maschinen zu berücksichtigen ist. Warnhinweise: Hyposensibilisierungsimpfstoffe zur Injektion dürfen nur durch allergologisch weitergebildete bzw. allergologisch erfahrene Ärzte verschrieben und angewendet werden. HAL Allergie GmbH, Poststraße 5-6, D-40213 Düsseldorf. Datum: Mai 2012
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N o t e s
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