47
Vector vaccines and immunity Thierry Van den Berg Coda Cerva, Belgium
Vector vaccines and
immunity
Thierry Van den BergCoda Cerva,
Belgium
Overview of the presentation
o Theory
• Background
• Classical vaccines
• New technology vaccines
• Chicken immunology
o Laboratory
• Tools for measurement of chicken immunity
• Humoral
• Cell-mediated
• Local (or mucosal): HALT, BALT, GALT & RALT
o Practice
• NDV
o Conclusions
17/04/2013 2
3
Industrial poultry vaccination=
Mass application
Up to 50.000 birds in a single space
Need for effective methods
Aim = vaccinating high enoughproportion of birds in the flock
Proportion will depend on
Type of infectious agent involved
Current epidemiological situation
Epizootic diseases (AI-ND): maximized protection
Many other situations: minimizeeconomic impact of disease
MDA
S
A
F
E
T
Y
P
O
T
E
N
C
Y
Transfert of passive immunity in birds
Pros and cons of classical vaccines
Attenuated Inactivated
Administration Varied routes (mass) SC, IM
Safety Residual virulence
Risk of reversion
Lower < 2 wk-old
OK >2 wk-old
Immunity Broad (hum., cell., muc.)
Quick onset
Humoral mainly
Slow onset
Field strains match Not always Yes
Efficacy Sometimes too attenuated
MDA interference
Low < 2wk-old
Production Low dose High dose
Quality risk Extraneous agents Bad inactivation
Why new types of vaccines ?
To further improve classical vaccines
New technology vaccines need to show
clear advantages over classical vaccines
And fill the gaps !!
e.g. attenuation impossible or large scale production difficult, interference of MDA, mass vaccination, DIVA…
Need to correlate protection parameters with vaccine performances
8
Ideal vaccine
• Quality
• Safety
• Efficacy
• Ease of production
• Ease of administration
• Duration
• Interference of MDA
Evolution to reach the « ideal vaccine »
Live vector vaccine – definition
Recombinant vector vaccines are
bioengineered vaccines similar to DNA
vaccines, but they use an attenuated
virus to introduce DNA to cells of the
body where it will be expressed.
3 steps:
o Clone the protective gene
o Insert the protective gene into the
genome of the vector (herpesvirus,
poxvirus, adenovirus…)
o Produce the vector = vaccine
Being intracellular, they are able to
induce a strong cellular immunity
against the inserted product and
prime a humoral response! 9
Slower immune responsesLow/late humoral immunityStrong B & T cell memory
Advantages of live vector vaccines
o Safety = vector dependent• Live attenuated (no reversion to virulence)• No need for adjuvant• Early age (day-old or in ovo)
o Efficacy = insert and vector dependent• Broad immunity (cellular and humoral)• Rapid onset after one administration• Interference with maternal antibodies• Bivalent vaccine if vector is a vaccine
o Production = vector dependent
o Differential diagnostic (DIVA) = insert dependent
Incubatorregularly turning
Hatching trayno turning
ED0 ED18 Hatching
Transport
Opportunity to immunise each individual chickwithout additional stress of manipulation
“In ovo vaccination”
At ED18 : - development of the immune system is nearly complete- active immune response possible.
What is in ovo vaccination ?
NDV+
IBDV+
ED16
ED18
ED19
ED20
ED21
Day 1
Day 2
Day 3
NDV-specific IgG
IBDV-specific Ig
Transfer of maternal antibodies from the yolk sac to the chick
In ovo vaccination :
low titres of maternal antibodies
virus replication possible
low vaccination doses evoke strong, long-lasting immune responses provided that the vaccine is not pathogenic for the embryo
A window of opportunity exists for avoiding interference of maternal antibodies with vaccination
IBDV
NDV
o Primary organs : the bursa of Fabricius and the thymus regulatethe development of the humoral and cellular compartments of the immune system, respectively.
o Peripheral organs : spleen, gut-, bronchus- and head-associatedlymphoid tissue (GALT, BALT and HALT, respectively).
o Chickens do not have lymph nodes, the primary site of antigenpresentation in mammals.
Thymus
Spleen
Meckel’diverticulum
Bursa of Fabricius
Caecaltonsils
Harderianglands
Lymphoïd organs in chickens
Immune response in chickens
Local immunity in the digestive tract
Local immunity in the respiratory tract
Local immunity in the head
Humoral immunity
Cell-mediatedimmunity
Local immunity in the reproductive tract
Immune response in chickens
Chickens respond to infection or vaccination by developing humoral, cellular and mucosal immune responses
o Humoral immunity :
• B-cells
• Measured by detection of specific antibodies in sera (ELISA, HI, SN)
o Cell-mediated immunity :
• T-cells (Th1 and Th2 responses)
• Measured by proliferation tests (3H uptake, WST1) and cytokines production (ChIFNg) after specific recall (ex vivo) on splenic and peripheral blood lymphocytes
o Mucosal immunity :
• Immunity at mucosal surfaces : major route of entry for many infectious agents into the body : HALT, BALT and GALT + reproductive tract
• Local antibody- and cell-mediated immunity = first line of defence against pathogens
• Viral excretion is reduced by local Ab
• Difficulties and labor-intensiveness of collecting samples
• Poorly investigated in poultry
17
Assessment of the immune responses
• Humoral immunity :
– Development of virus specific ELISAs to detect IgG, IgM and IgA specific [Rauw et
al., 2009, 2010]
• Cell mediated immunity:
– New tools to measure CMI: production of ChIFNg after recall stimulation
• In the spleen [Lambrecht et al., 2004, Rauw et al., 2009, 2010]
• In the peripheral blood [Rauw et al., 2010]
• In the digestive tract [Rauw et al., 2010]
• In the respiratory tract [Rauw et al., 2011]
• Local (mucosal) immunity :
– Live vaccines are inoculated by mucosal route (spray, drinking water)
– New tools to measure lachrymal, respiratory and digestive immunity [Rauw et al.,
2009, 2010a, 2010b]
NDV – AIV – IBDV
Immune response in the chickens
Humoral immunity
Serum
NDV-specific IgG ELISA
Purified whole NDV (2 µg/ml) Saturation (2,5 % casein)
Mouse anti-Chicken IgG - BIOT
NDV-specific IgG
TMB substrat
A450nm
Streptavidine-POD
*
*
** *
* **
Day-old SPF chickens (n = 5)
1 dose of rHVT-NDSubcutaneous inoculation
*
* * **
**
NDV-specific IgM ELISA
NDV-specific IgM
Purified whole NDV
Mouse IgG1 anti-NDV - BIOT
Mouse anti-Chicken IgM
Saturation
*
**
*
** *
*
*
Day-old SPF chickens (n = 5)
1 dose of rHVT-NDSubcutaneous inoculation
TMB substrat
A450nm
Streptavidine-POD
*
**
*
NDV-specific IgA ELISA
NDV-specific IgA
Purified whole NDV
Mouse IgG1 anti-NDV - BIOT
Mouse anti-Chicken IgA
TMB substrat
A450nm
Streptavidine-POD
Saturation
*
* *
* ** * *
* **
**
*
*Day-old SPF chickens
(n = 5)
1 dose of rHVT-NDSubcutaneous inoculation
Immune response in chickens
Cell-mediatedimmunity
Spleen
Peripheralblood
Cell-mediated immunity (CMI)
Cytokines production Proliferation
In vivo
Specific T-lymphocytes
Ex vivo
Cytokines production Proliferation
Pathogensproteins
+
Ag recallactivation
RECALL
Isolation
T-lymphocytes
Lymphocytes
• Spleen
• Blood
• Digestive tract
• Respiratory tract
Pathogens
T-lymphocytes
Specific T-lymphocytes
CYTOKINES PRODUCTION
ELISAEnzyme-linked
immunosorbent assay
H3 uptakeCell harvester and
cpm assay
PROLIFERATION
NDV-specific CMI in the spleen
**
*
Day-old MDA+ chickens (n = 5)
1 dose rHVT-NDSubcutaneous inoculation
Rauw F. et al. (2009). Vaccine, 27 : 3631-3642
BUT :• Chickens sacrifice
• No information about circulation and response of specific T-lymphocytes in periphery
PROTOCOL :
• Day-old SPF chicks
• 107 c/ml, RPMI 2 % FCSi
• 72h
• Flat-bottomed plate
• prot-NDV : 1 µg/ml
NDV-specific CMI in the blood (I)
Blood fromjugular vein
White blood cells : PBMC
Anticoagulant :
• Heparin
• Alsever solution
• Sedimentation
• Low speed centrifugation
• Ficoll HistoPaque (1083 – 1077)
Activation :
• 48 or 72h
• medium (serum %)
• plate (bottom)
ChIFNg production by T-lymphocytes
PBL
NDV-specific CMI in the blood (II)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Alsever Heparin Alsever Heparin Alsever Heparin Alsever Heparin Alsever Heparin Alsever Heparin
1 µg/ml 5 µg/ml 10 µg/ml 1 µg/ml 5 µg/ml 10 µg/ml
48h 72h
Anticoagulant, gp-NDV concentration and culture conditions
O.D
.
Negative
Enterotrocpic live ND vaccine
Enterotropic live ND vaccine and challenge
0.0
0.5
1.0
1.5
2.0
2.5
3.0
2 % ChSi 10 % ChSi
PMA/Iono (1 µg/ml)
PROTOCOL :
• Sedimentation (4h)
• 107 c/ml, RPMI 10 % ChSi
• Heparin
• 72h
• Round-bottomed plate
• prot-NDV : 1 µg/ml
Rauw F. et al. (2010). Veterinary Immunology and Immunopathology, 134 : 249-258
Day-old SPF chickens (n = 5)
1 dose rHVT-NDSubcutaneous inoculation
AIV-specific CMI
10-day-old SPF chickens (n = 5)
106 EID50 LP H5N2Oculo/nasal inoculation
Day-old SPF chickens (n = 5)
1 dose rHVT-AISubcutaneous inoculation
Very low AIV-specific
CMI
Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : 27-37
Spleen Blood
Immune response in chickens
Local immunity in the head
Tears
Antibody
Lachrymal antibody-mediatedimmunity specific to NDV
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
1 week 2 weeks 3 weeks 4 weeks 5 weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks
NDV specific IgG NDV specific IgA NDV specific IgM
Time post-vaccination and Ig isotypes
O.D
.
Negative
Tracheotropic live ND vaccine
Enterotropic live ND vaccine
Centrifugation -20°C NDV-specificIgG/M/A ELISA
Day-old SPF chickens (n = 5)
1 dose Cevac Vitapest L or Cevac UNI L
Oculo/nasal inoculation
Rauw F. et al. (2010). Vaccine, 28 : 823-833
Immune response in chickens
Local immunity in the digestive tract
Duodenum
AntibodyCMI
Bile
Antibody
Digestive antibody-mediatedimmunity specific to NDV
Rauw F. et al. (2010). Vaccine, 28 : 823-833
Ex vivotissues culture (72h)
Centrifugation -20°C NDV-specificIgG/M/A ELISA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
1 week 2 weeks 3 weeks 4 weeks 5 weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks
NDV specific IgG NDV specific IgA NDV specific IgM
Time post-vaccination and Ig isotypes
O.D
.
Negative
Tracheotropic live ND vaccine
Enterotropic live ND vaccine
Day-old SPF chickens (n = 5)
1 dose Cevac Vitapest L or Cevac UNI L
Oculo/nasal inoculation
GALT CMI specific to NDV
Cuts (5 mm) of duodenal tissues
37 °C
37 °C
HBSS 1 mM EDTA
HBSS with 0,4 U/ml of Liberase Blendzyme 3
(ROCHE)
30 min
30 min
Washes
LPL : Lamina Propria Lymphocytes
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4 5 6 7 4 5 6 7
1 µg/ml gp-NDV 5 µg/ml gp-NDV
Weeks post-vaccination (challenge at 4 weeks p.v.)
O.D
.
Negative
Enterotropic live ND vaccine
Enterotropic live ND vaccine with challenge
Rauw F. et al. (2010). Veterinary Immunology and Immunopathology, 134 : 249-258
PROTOCOL :
• 107 c/ml, RPMI 10 % FCSi
• Flat-bottomed plate
• 72 h
• prot-NDV : 1 – 5 µg/ml
Immune response in chickens
Local immunity in the respiratory tract
Trachea Lung
AntibodyCMI
Respiratory antibody-mediatedimmunity specific to NDV
-20°C NDV-specificIgG/M/A ELISAEuthanasia by
i.p injection of Nembutal
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
3 5 3 5 3 5
IgG IgA IgM
Isotypes and weeks post-vaccination
O.D
.
Negative
Tracheotropic live ND vaccine
Enterotropic live ND vaccine
Rauw F. et al. (2010). Vaccine, 28 : 823-833
PROTOCOL :Holt et al. (2005). Avian Pathology, 34, 396-398Personnal communications of B. Kaspers (Institut für Tierphysiologie, Munich, Germany)
Day-old SPF chickens (n = 5)
1 dose Cevac Vitapest L or Cevac UNI L
Occulo/nasal inoculation
BALT CMI specific to AIV
Cuts (5 mm) of lung
37 °C
2 h
HBSS with 0,2 % (w/v) of Protease XIV (Sigma)
Ficoll HistoPaque1083 gradient
Erythrocytes
Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : 27-37
10-day-old SPF chickens (n = 5)
106 EID50 LP H5N2Oculo/nasal inoculation
PROTOCOL :
• 107 c/ml, RPMI 10 % FCSi
• Flat-bottomed plate
• 72 h
• prot-H5N2 : 1 – 5 µg/ml
Tracheal CMI specific to AIV
37 °C
2 h
HBSS with 0,2 % (w/v) of Protease XIV
(Sigma) + 0,02 % (w/v) of DNase I (Sigma)
Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : 27-37
Cuts (5 mm) of trachea
PROTOCOL :
• 107 c/ml, RPMI 5 % FCSi
• Flat-bottomed plate
• 72 h
• prot-H5N2 : 1 – 5 µg/ml
10-day-old SPF chickens (n = 5)
106 EID50 LP H5N2Oculo/nasal inoculation
37
Field situation: Goal of vaccination
Infection
Clinical signs, mortality, egg drop, growth retardation..
Excretion (oral and cloacal)
Resistance to infection
Reduction of shedding
Clinical protection
38
Egg dropHITitre
Mortality
≤ 100 % mortality_22
_
0 % egg drop in layers
_28_
_29
_
_27
_
_211
__210
_
Clinical signs
0 % mortality
_23
_
_24
_
Allan et al. (1978)_211
_
Clinical
protection
Inactivated vaccine
_25
_
_26
_
_28_
_23
_
_27
_
_24
_
Excretion ≤ 10 days
_29
_
_210
_
Excretion ≤ 3 days
_22
_
Excretion ≈ 14 days
Shedding
reduction
Westbury et al. (1984)
_25
_
_26
_
0 % symptoms (resp., nerv., dig.)
Clinical signs
NDV
0,0
0,5
1,0
1,5
2,0
2,5
2 3 4 5
Weeks post- vaccination
O.D
.
Negative (SPF)
Negative (Conv)
Cevac Vitapest L ND (SPF)
Cevac Vitapest L ND (Conv)
rHVT-ND / Cevac Vitapest L ND (Conv)
0
5
10
15
20
25
30
35
40
45
50
2 3 4 5
Weeks post- vaccination
O.D
.
Negative (SPF)
Negative (Conv)
Cevac Vitapest L ND (SPF)
Cevac Vitapest L ND (Conv)
rHVT-ND / Cevac Vitapest L ND (Conv)
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2 3 4 5
Weeks post- vaccination
O.D
.
Negative (SPF)
Negative (Conv)
Cevac Vitapest L ND (SPF)
Cevac Vitapest L ND (Conv)
rHVT-ND / Cevac Vitapest L ND (Conv)
Humoral immunity
CMI (spleen)
Local immunity in the digestive tract
1) NDV vaccination: 1st experiment
o Day-old
o SPF or MDA positive
o Vitapest or Vitapest + HVT-ND
o Follow-up of immune responses from to 2 to 5 week of age
2d experiment on conventional layer chickens (MDA+)
• Eggs from Isa Brown 38-week-old breeder flock which received the following ND vaccination shedule :
– Nobilis Clone 30 (Intervet) at 4 and 8-week-old
– Nobilis Newcavac (Intervet) at 16-week-old
• 4 groups :
– Negative (non-vaccinated)
– Live ND
– rHVT-ND in ovo
– rHVT-ND/live ND
18 ED
rHVT-ND vaccine
Vectormune®ND
One-day-old
Live ND vaccineCevac Vitapest
L
(+)
2 weeks
Immunity
Humoral immunity
• Presence of MDA until the 4th week of age
• Active humoral immunity well established :– At the 4th week pv in live ND and rHVT-ND/live ND groups
– At the 5th week pv in rHVT-ND group
• No statistical difference between vaccinated groups at 5 weeks pv
2
4
6
8
10
12
14
16
18
20
2 weeks post-
challenge
2 3 4 5 7
Times
HI
titr
e (
log
2)
Negative Live ND rHVT-ND rHVT-ND/live ND
HI titre
0,0
0,5
1,0
1,5
2,0
2,5
3,0
2 weeks post-
challenge
2 3 4 5 7
Weeks post-vaccination
O.D
.Negative Live ND rHVT-ND rHVT-ND/live ND
NDV specific IgG ELISA
AAA
BC
AB
C
B
A
A A
A
B
AB
A
A
B
AA
Cell-mediated immunity
• Cell-mediated immunity from the 3rd week pv
• No statistical difference between vaccinated groups
0
10
20
30
40
50
60
70
80
90
2 weeks post-
challenge
2 3 4 5 7
Weeks post-vaccination
S.I
.
Negative ND live rHVT-ND rHVT-ND/live ND
A
A
A
A
A
B B
HALT
• Presence of MDA in tears until the 4th week of age
• Active lachrymal immunity well established :
– At the 4th week pv in live ND and rHVT-ND/live ND groups
– At the 5th week pv in rHVT-ND group
0,0
0,5
1,0
1,5
2,0
2,5
3,0
2 3 4 5
Weeks post-vaccination
O.D
.
Negative Live ND rHVT-ND rHVT-ND/live ND
NDV specific IgG in tears
A
A AA
B
B
B
A
GALT
• Presence of MDA in digestive tract until the 4th week of age
• Earlier and stronger local antibody-mediated immunity in rHVT-ND/live ND group (P < 0.05) since the 4th week pv
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
2 3 4 5
Times
O.D
.
Negative Live ND rHVT-ND rHVT-ND/live ND
A
A
B
A
B
ANDV specific IgG in duodenum
Conclusions
• Most classical attenuated/inactivated vaccines are
satisfactory in laboratory conditions but have some
limitations in the field
• Improved immunological parameters can correlate with
improved protection
• New technology vaccines may solve issues related to
classical vaccines w/o negative effect (efficacy, safety,
production, mass administration, interference of MDA,
DIVA)
• This should allow the development on purpose of safer and
more efficient vaccination programmes in the future
Thanks !
2003 2007 2012
CELOVAC project MUCOVAC project HVTIMMUNOVAC project
2015
10 years
CEVA-CERVA collaboration
Vilmos Palya
Yannick Gardin
Bénédicte Lambrecht
Fabienne Rauw
Scientific promoters
Technical staff Eva Ngabirano
Sophie Lemaire
Martine Gonze PhD
PUBLICATIONS :• Rauw F. et al. (2007). Journal of Interferon and Cytokines, 27 : 11-118.• Rauw F. et al. (2009). Vaccine, 27 : 3631-3642.• De Vriese et al. (2010). Avian diseases, 54, 246-252.• Rauw F. et al. (2010). Veterinary Immunology and Immunopathology, 134 : 249-258.• Rauw F. et al. (2010). Vaccine, 28 : 823-833.• Rauw F. et al. (2011). Vaccine, 29 ; 2590-2600.• Rauw F. et al. (2011). Veterinary Immunology and Immunopathology, 143 : 27-37• Rauw F. et al. (2012). Avian Diseases, 56: 913-922 .
Thank you for your attention
