Date post: | 16-Jul-2015 |
Category: |
Technology |
Upload: | bek1826 |
View: | 508 times |
Download: | 0 times |
Polyreactive and antigen-specific
B-cell antibody response to
Streptococcus pneumoniae
Rebecca Thompson
March 29, 2012
Streptococcus pneumoniae
• Colonizes the nasopharynx
• Over 90 identified
serotypes
• Serotypes are determined by capsular polysaccharide (PPS)
• Vaccines induce protective antibodies against PPS
Streptococcus pneumoniae in sputum smear. Gram stain (1200X) LeBeau 2009
0
40
80
120
160
200
<1 1 2-4 3-17 18-34 35-49 50-64 ≥ 65 Total
Ra
te p
er 1
00,0
00
per
son
s
Age (in years)
Incidence of Invasive Pneumococcal Disease in U.S.
2000
2010
dum
my
Epidemiology
• Streptococcus pneumoniae is responsible for considerable morbidity and mortality worldwide.
• Invasive pneumococcal disease (IPD) exhibits a characteristic age distribution with the majority of cases occurring
• Very young (<2 years old)• Elderly (>65 years old)
• Other high risk populations • Immune systems in these populations are defective in response to pneumococcal
infection
Data adapted from www.cdc.gov/abcs
Mortality rate* per 100,000 children under five years of age due to
Streptococcus pneumoniae, 2000
http://www.who.int/nuvi/pneumococcus/decision_implementation/en/index.html
10- <100 100-<300 300-<500 >500
<10
* HIV+ve deaths excluded
Pathogenesis
1. Colonization
• Inhalation of S. pneumoniae
in aerosolized particles
• Natural carriage
2. Otitis Media
3. Pneumonia
4. Meningitis/Encephalitis
• Innate
• Complement mediated
• Not antibody specific
• Adaptive
• Antibody mediated
• Antibodies against surface molecules
• Role of these in clearance of bacteria are yet to be determined
• Antigen specific
Immune Response
Pneumococcal Therapies
• Anti-serum
• 1891 serum protected from pneumococcal disease
• Antibiotics
• Mid-20th century, penicillin was successful in treating
infections with gram-positive organisms
• Mutations soon lead to penicillin resistant pneumococcus
• Purified pneumococcal polysaccharide vaccine
• 1977 14 valent purified pneumococcal polysaccharide vaccine
(PPV) was licensed
• 1983 updated to 23 valent
• Pneumococcal conjugate vaccine
• In 2000, a 7 valent pneumococcal conjugate vaccine (PCV)
• The most effective form of prevention is
vaccination
• The 23-valent PPV has an 80% protective
efficacy in healthy young adults
• Administration of the PCV decreased the
incidence of IPD in children under the age
of 5 by 80%
Prevention
• We analyzed the B-cell response to PPS
in healthy young volunteers two ways
• Developed a method to directly label anti-
PPS cells
• Characterization of polyreactive anti-PPS
antibodies
Purpose
• Anti-PPS B cells which respond to PPV are
IgM memory cells
• Human polyreactive anti-PPS antibodies are
low avidity but elicit protection from
pneumococcal challenge
Hypotheses
7 days post-
vaccination
Isolation of
B cells
Flow cytometry with
fluorescently labeled
PPS to identify PPS-
specific B cells
Testing of single B
cell culture
supernatant by
ELISA
• Vaccinate volunteer with Pneumovax®
• Draw blood at day 0, 7 and 28
• Analyze B cells at day 0 and 7• Single cell sort PPS
binding B cells from day 7
• Analyze antibody titers at day 0 and 28
• Opsonophagocyticassay day 0 and 28
Isolation of PPS B cells
Healthy immune response
after vaccinationIg
G le
ve
l (m
g/
ml)
IgG
le
vel
(mg
/ml)
Ig
M l
ev
el
(mg
/ml)
Ig
A l
eve
l (m
g/m
l)
IgG
le
vel
(mg
/ml)
Ig
M l
ev
el
(mg
/ml)
Ig
A l
eve
l (m
g/m
l)
p< 0.0001
p< 0.0001
NS
p< 0.0001
p< 0.001
p< 0.03
Pre-immunization
Post-immunization PPS23F PPS14
Healthy immune response
after vaccination
PPS14 PPS23FPre-immunization
Post-immunization
Op
so
no
ph
ag
oc
yti
c In
de
x
Op
so
no
ph
ag
oc
yti
c In
de
x
p<0.0001 p<0.0001
Pre-immunization Post-immunization Pre-immunization Post-immunization
Phenotype of B lymphocytes
that respond to Pneumovax®
Pre-vaccination Post-vaccination
*
*
* = p<0.0001
Phenotype of B lymphocytes
that respond to Pneumovax®
PPS14 - Selected PPS23F - Selected
*
*
**
##
#
##
# = p<0.002, ## = p<0.0001 * = p<0.05, ** = p<0.0001
Summary
• Developed fluorescently labeled PPS to identify
PPS-specific B cells using flow cytometry
• CD27+ IgM+ B cells increased after PPV in
healthy young volunteers
• Antibody titers and opsonophagocytic activity
also correlated with the increase in CD27+ IgM+
memory B cells
• These cells play a crucial role in the immune
response to PPS
Natural Antibodies
• Natural Antibodies
• Direct neutralization of
pathogen
• Activation of
complement
• Complement mediated
lysis
Immunology Today, Volume 21, Issue 12, 1 December 2000, Pages 624-630 Adrian F Ochsenbein, Rolf M Zinkernagel
B1 cell
IL-5
Pneumococcal
polysaccharide
• Antibodies have two functional regions
• Variable
• Capable of specific recognition
and binding to epitope
• Constant
• Classically thought to
contribute to effector functions
• Complement activation
• Mediation of immune
phagocytosis
• Antibody-dependent
cytotoxicity
Antibody structure
Variable region
Constant region
C
H
2
C
H
2
C
H
3
C
H
3
Hinge region
IgG1 vs. IgG2
• IgG1 is more flexible than IgG2• 2 vs 4 disulfide bonds in hinge region
• More amino acids in constant region
• Recognized by all FcΥR on effector cells
• IgG2 is a poor activator of complement
• Low capacity to bind C1q due to decreased flexibility
• Recognized by FcΥRII which is a low affinity receptor
Fab arm
waving Fc tail
wagging
Fab elbow
bendingFab
arm
rotation
43 107
117
IgG1
3299
127
IgG2
The Journal of Immunology October 1, 1997 vol. 159 no. 7 3372-3382
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
IgG1 IgG2 IgG1 IgG2 IgG1 IgG2 IgG1 IgG2 IgG1 IgG2 IgG1 IgG2
21B2 24F5 33G8 32E8 31E2 31B5
OD
49
0n
m
Polyreactive Antibodies binding to Multiple PPS
PPS4
PPS6B
PPS14
PPS23F
Polyreactive antibodies
Opsonophagocytic activity of
polyreactive antibodies
0
10
20
30
40
50
60
70
80
90
100
33g8 32e8 31e2 31b5 21b2 24f5
Ma
xim
um
per
cen
t k
illi
ng
Opsonophagocytic Activity PPS23F
IgG1
IgG2
0
10
20
30
40
50
60
70
80
90
100
33g8 32e8 31e2 31b5 21b2 24f5
Ma
xim
um
per
cen
t k
illi
ng
Opsonophagocytic Activity PPS14
IgG1
IgG2
Sequences of polyreactive
pneumococcal antibodies
Clone VH CDR3 # of Mutations % Homology VH Gene JH Gene
34C10 AKDSRGSTPRAFDP 12 97 3-23 JH5b
33G8 ARDGRQQWLVRPYYYGMDV 5 99 1-18 JH6b
32E8 TKWRWQQSEFDY 29 93 3-7 JH4b
31E2 AKDSRGSTPRAFDP 36 88 3-66 JH5b
31B5 AKKAFSGYSPFDY 20 94 3-23 JH4b
21B2 ARNGGVGATDPPYYYYGMDV 5 99 3-30 JH6b
24F5 AKDRSLREYSSSWYYPFYYYGMDV 4 99 3-23 JH6b
33E2 AREEYYGSGFDP 4 99 3-48 JH5b
Clone Vk CDR3 # of Mutations % Homology Vk Gene Jk Gene
34C10 QKYNSAPFT 12 99 A20 JK3
33G8 QQANSFSLT 10 99 L5 JK4
32E8 QRSSGGPIS 35 90 O12 JK5
31E2 QKYNGAPFT 14 97 A20 JK3
31B5 QQYDRSPLT 15 98 A27 JK4
21B2 QRSSGGPIS 37 89 O12 JK5
24F5 QKYNGAPFT 15 93 A20 JK3
33E2 QQHYNTPT 14 98 B3 JK5
Polyreactive vs. specific
PPS antibodies
0
5
10
15
20
25
VH CDR3 Vk CDR3
# o
f am
ino a
cid
s
Average CDR3 Length
Polyreactive
PPS3
PPS6B
PPS14
PPS23F
Series6 80
82
84
86
88
90
92
94
96
98
100
VH CDR3 Vk CDR3
% H
om
olo
gy
Homology to Germline
Polyreactive
PPS-specific
dumm
y
Polyreactive vs. specific
PPS antibodies
0
1
2
3
4
5
6
7
Flexible AA - RWY Positive AA - RHK Negative AA - DE
Av
erag
e n
um
ber
of
amin
o a
cids
Average Number of Amino Acid Groups in Variable
Heavy Chain CDR3
Polyreactive
PPS3
PPS6B
PPS14
PPS23F
Series12
***
0
1
2
3
4
5
6
7
Flexible AA - RWY Positive AA - RHK Negative AA - DE
Av
erag
e n
um
ber
of
amin
o a
cids
Average Number of Amino Acid Groups in Variable
Light Chain CDR3
Polyreactive
PPS3
PPS6B
PPS14
PPS23F
Series6
Surface Plasmon Resonance
Anti-human antibodies
Pneumococcal
polysaccharide
Human polyreactive anti-
pneumococcal antibodies
Laser
Prism
Plotted data
Time
mR
IU
Gold chip
Polyreactive antibody avidity
IgG1 vs. IgG2
33G8 32E8 31B5 21B2 24F5
IgG1 1.7 x E-6 0.42 x E-6 1.2 x E-6 0.29 x E-6 0.51 x E-6
IgG2 0.73 x E-6 0.302 x E-6 1.4 x E-6 1.5 x E-6 1.005 x E-6
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
KD
uM
Binding to PPS23F
33G8 32E8 31B5 21B2 24F5
IgG1 1.02 x E-6 0.57 x E-6 0.67 x E-6 0.76 x E-6 0.28 x E-6
IgG2 0.12 x E-6 0.97 x E-6 0.99 x E-6 5.3 x E-6 1.4 x E-6
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
KD
uM
Binding to PPS14
Polyreactive antibody avidity
IgG1 vs. IgG2
F(ab)’2
fragment
Pepsin
digestion
KD PPS14 PPS23F
Clone IgG1 F(ab)’2 IgG2 F(ab)’2 IgG1 F(ab)’2 IgG2 F(ab)’2
33G8 0.49492 uM 0.5001 uM 0.500 uM 0.4989 uM
21B2 0.5359 uM 1.35362 uM 0.69445 uM 0.80091 uM
24F5 1.07 uM 1.16921 uM 0.98 uM 1.00 uM
Summary
• All but one Mab VL CDR3 length – 9 AA
• VH CDR3
• Various CDR3 lengths
• Large range of mutations
• Mostly VH3
• Significantly higher number of flexible amino
• For both PPS14 and 23F, IgG1 had an overall higher avidity
Conclusion
1. Developed unique tool – labeled PPS
• Established IgM memory B cells crucial in PPV response
• Helps to identify individuals at risk
• Further understanding of cell surface expression
2. Characterized polyreactive B cells
• Express high % of flex AA
3. IgG1 may be preferable isotype in immune response to PPS
• Important for future vaccine and adjuvant development
Future Studies
• Analyze PPS immune response in the elderly and HIV+
• Studies have shown both populations have• Polyclonal activation
• Hypergammaglobulinemia
• Activation of resting B cells
• Decrease naïve B cells
• Decrease CD27+IgM+ B cells
• By understanding the impaired immune response in these populations guidelines can be established to improve the immune response to vaccination
• Establish a healthy control response to PPS to aid in identifying immune dysfunction in HIV+ and elderly
• Compare phenotype of PPS-specific B cells
• Compare VH3 usage
• Compare anti-PPS antibody titers and opsonophagocytic activity
• When is it best to vaccinate?
• Is there a benefit to vaccination every 5 years?
Clinical significance
Thank you
• Major advisor, Dr. M. A. Julie Westerink M.D.
• Academic advisory committee• J. David Dignam, Ph.D.
• Dr. Deepak Malhotra, M.D., Ph.D.
• Randall Ruch, Ph.D.
• R. Mark Wooten, Ph.D.
• Noor Khaskhely, M.D.,Ph.D. for all of this help with flow cytometry and B cell acquisition
• The present and past members of the Westerink lab for their support and help• Noor Khaskhely, M.D., Ph.D, Kristin
Malhotra, David Leggat, M.S., Anita Iyer, M.S., Jason Mosakowski, JieyingWang, Chris Selleck and S. Louise Smithson, Ph.D.
• Dr. Gary McLean for providing the recombinant human expression vectors
• Dr. Sandra Romero-Steiner for serotypes of S. pneumoniae used in opsonophagocytic assays
• Pfizer, Inc. for donating hybridomasspecific for PPS14 and PPS23F
• Dr. Sadik Khuder for statistical analysis
• The volunteers who have participated in our study
• These studies were supported by National Institute of Health grants AG081558 and AG05978.