This document discusses the role that antibody constant regions play in binding to pneumococcal polysaccharides. It summarizes that the constant region determines the antibody isotype, which can influence avidity and fine specificity. The author aims to express identical variable regions from human antibodies targeting pneumococcal polysaccharides with either IgG1 or IgG2 constant regions. Surface plasmon resonance will then be used to compare binding of the two isotypes to determine if constant region impacts affinity. Understanding this relationship could help design vaccines that direct the immune response towards the optimal isotype.

1. Role of Constant Regions in Antibody
Binding to Pneumococcal
Polysaccharide
Rebecca Thompson

2. Streptococcus pneumoniae
• Gram-positive bacteria
• Colonizes the nasopharynx
– Over 90 identified serotypes
• Serotypes are determined
by capsular polysaccharide
• These antibodies against
capsular pneumococcal
polysaccharide (PPS) are
protective
• However antibodies are
specific to each serotype
and are not protective http://contanatura.com
against other serotypes

3. Pneumococcal Disease
• Pathogenesis
– Pneumonia
– Otitis media
– Meningitis
www.mayoclinicproceedings.com
– Bacteremia
• High risk groups include the very young (<5
years old), the very old (<65 years), the
immunocompromised and the asplenic
• Responsible for >40,000 deaths annually in
the United States

4. Vaccines in U.S.
• 23-Valent Pneumococcal Polysaccharide Vaccine
– Pneumovax®
• Contains 23 purified capsular polysaccharides
– Serotypes responsible for >85% of invasive disease in the
U.S.
– Recommended for adults >65
• 7-Valent Pneumococcal Conjugate Vaccine
– Prevnar®
• Contains 7 purified capsular polysaccharide covalently conjugated to
CRM197, a diptheria toxoid
– Recommended for young children

5. Antibody Structure
• 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.
• Recent studies suggest that the
F(ab')2 may influence effector
functions such as antibody-
dependent cytotoxicity while
antibody fine specificity can be a
function of isotype/subclass (Fc).
Janeway’s Immunobiology, 2001

6. Characteristics of Subclass
IgG1 IgG2
Heavy chain γ1 γ2
Molecular weight 146 146
kDa
Mean serum level 9 3
(mean adult mg/mL)
Half life (days) 21 20
Classical pathway of ++ +
complement
activation
Binding to + -
macrophages and
other phagocytes
Dimer formation - +
Janeway’s Immunobiology, 2001

7. Subclass Distribution in Reaction to
Pneumococcal Polysaccharide
• Subclass distribution is age-related
– Children <5 years of age show predominately
IgG1 antibodies
– Adults elicit a predominate IgG2 response
• Ratio of IgG1:IgG2 continues to decrease
with age
– Lottenbach et al showed this ratio is conserved
regardless of vaccine (conjugate or PPS)

8. Previous Studies
• McLean et al. expressed the VL and VH of mAb specific for the
capsular polysaccharide of Cryptococcus neoformans with
human constant regions μ, γ1, γ2, γ3, γ4 and α1. The the mouse
human antibodies IgM, IgG3 and IgG4 antibodies clearly showed
differences in fine specificity despite identical variable region
sequences suggesting that the constant region can affect
conformation of the variable region affecting fine specificity and
possibly avidity.
• Pritsch et al. studied the influence of constant region on the
avidity of recombinant human antibodies sharing identical VH
and VL to tubulin. Immunoglobulin G and A antibodies and their
Fab fragments with identical variable regions bound tubulin with
different avidity.
• Torres et al. murine IgG3 antibodies noncovalently associate
allowing for a concentration dependent increase in antigen
binding.

9. Summary
• Isotype distribution of IgG1 and IgG2 in
response to PPS is clearly age related
• Constant region, which determines antibody
isotype, appears to play a role in antibody
avidity and fine specificity

10. Why is isotype so important?
• Newly developed adjuvants allow for specific stimulation of
TH1 versus TH2 branches of the immune system
– For example alum skews the immune system towards a TH2
response
• Secretion of IL-4, IL-5
• Generation of IgG1 and IgE isotypes
– CpG and MPL are novel adjuvant that promote TH1 response
• Secretion of IFN-γ, TNF-α, IL-12
• IgG2
• Therefore we can direct either IgG1 or IgG2 production
• Thus it is crucial that we first define the most desirable
reaction to PPS

11. Current Project
• What role does the constant region have in
antibody binding?
– Paired VH VL with specificity to PPS regions will
be subcloned into expression vector
• Isolated from single human PPS specific B cells
– Expressed in mammalian cells
– Measure binding to PPS using SPR
• Thus comparing IgG isotypes with identical
variable regions

12. McLean Vectors
• Mammalian expression vectors
- Express a recombinant human IgG antibody
• The antibody expressed will have either
a IgG1 or IgG2 constant region
• Variable chain pairs will be cloned into
each expression vector and analyzed
- Here the variable region is conserved in both
recombinant subclasses of Ig allowing for true
interpretation of antibody binding to
pneumococcal polysaccharide

13. Surface Plasmon Resonance
• Recombinant antibodies will be analyzed by
surface plasmon resonance (SPR).
- SPR allows for real-time data collection of the
interaction between antibody and antigen
- Surface of sensor chip is coated with antigen
(pneumococcal polysaccharide)
- Antibody is passed over the chip’s surface in liquid
phase
- Interactions between the recombinant antibody
and pneumococcal polysaccharide are recorded
by the SPR equipment

14. RU
40
35
30
25
20
15
R
es 10
p.
Di
ff. 5
0
-5
0 50 100 15 0 200 250 3 00
T ime s
• Bivalent analyte model
• Overlay sensorgrams at different
concentrations to determine KD

15. Analysis of Antibody Affinity
• After antibody SPR data collection, IgG1 and
IgG2 values will be compared
• If there is a statistical difference between
IgG1 and IgG2 affinity, Fabs will be created
to determine the molecular basis of binding

16. Clinical significance
• If there is an observed difference between
IgG1 and IgG2 constant region in fine
specificity of antibody binding, then it would
be advantageous to design vaccines that
allow for the direction of immune response
to either IgG1 or IgG2
• Adding adjuvants to such vaccines would
allow for the stimulation of IgG1 or IgG2
antibodies improving the protective ability
of the vaccine

17. Current Status
• Successfully cloned PPS specific naturally
paired VH VL chains into rhIgG expression
vectors
• Transient transfection into human kidney
cells
• ELISA to test for antibody binding

18. Binding to PPS23

19. Future Work
• Presently purifying antibodies 113G1 and
113G2 for analysis by SPR
• Once this system is optimized several other
VH and VL pairs are ready for cloning into
the McLean expression vectors

20. Acknowledgements
• Dr. Westerink
• Dr. Smithson
• Jason Mosakowski
• McLean Lab
• Dr. Dignam