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

Virulence Factors

PspA

PspC

Cel

l m

emb

ran

e

Cel

l w

all

Cap

sule

Pneumolysin

PsaA

• 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

Specific binding of

fluorescently labeled PPS

FIGURE.2.

Specific staining of PPS

hybridomas

Percentage of PPS specific CD19+

B cells pre- and post-vaccination

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

Phenotype of B lymphocytes

that respond to Pneumovax®

FIGURE.6.

A B

C D

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

Recombinant human immunoglobulin

expression vectors

IgG1

IgG2

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.

Questions?

Strange Matter - Antibiotic Resistance Recruitment by Nick D. Kim.

Phenotype of B lymphocytes

that respond to Pneumovax®

FIGURE.6.

A B

C D

B cell Culture Cocktail

• TRF

• IL-2

• IL-4

• PMA

• EL4-B5