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Ociit 2011
1. Analysis of Human Polyreactive
Pneumococcal Antibodies
Rebecca Thompson
OCIIT 2011
2. Streptococcus Pneumoniae
– Gram-positive bacteria
– Colonizes the nasopharynx
– Over 90 identified
serotypes
• Serotypes are determined by
capsular polysaccharide
– Antibodies against capsular
pneumococcal polysaccharide
(PPS) are protective
3. Pneumococcal Disease
– Pathogenesis
• Pneumonia
• Otitis media
• Meningitis
• Bacteremia
– High risk groups include the very young (<5 years
old), the elderly (>65 years), the
immunocompromised and the asplenic
– Responsible for >40,000 deaths annually in the
United States
4. Natural Antibodies
– First line of defense against pneumococcal
invasive disease
– Also aid in the clearance of pneumococcus if an
infection is already present
– Weakly polyreactive
– In murine studies, polyreactive antibodies are
produced by B1 cells
– In humans, the origin and characteristics of these
antibodies are not well characterized
5. Methodology
• Draw blood from healthy young volunteers and isolate B cells
• Single cell sort B cells into a 96 well plate
• Expand B cells in culture
• Test culture supernatants by ELISA
– Identify Ig secreting B cells and PPS-binding B cells
• Harvest and lyse cells
• Make cDNA and PCR variable heavy (VH) and variable light (VL)
immunoglobulin genes
– Natural VH/VL pairing is maintained
– True representation of antibody repertoire
• Ligate paired VH and VL into rhuIg expression plasmids and
transfect human cells
• Harvest recombinant human antibodies, perform avidity
measurements on whole and F(ab)’2 fragments
15. Summary
• After analysis of our polyreactive antibodies, there
are multiple features limited to this group of
antibodies.
– Conservation of Vk CDR3 length
– Increased number of flexible (arginine, tyrosine and
tryptophan) amino acids in the VH CDR3
– Longer VH CDR3 length when compared to PPS-specific
antibodies
• In general, the IgG1 isotype bound more avidity to
PPS14 and PPS23F than IgG2
• When antibodies were digested into F(ab)’2
fragments the difference in avidity between isotypes
was largely diminished
16. Future Work
• Cloning of isolated PPS-specific antibodies
• Comparison of PPS binding kinetics
– Polyreactive vs PPS-specific
17. Acknowledgements
• Major Advisor • Committee Members
– Dr. Julie Westerink, MD – Dr. Dignam
• Lab Members – Dr. Malhotra
– David Leggat – Dr. Ruch
– Dr. Noor Khaskhely – Dr. Wooten
• Funding
– NIH RO1
Editor's Notes
Our lab studies the human antibody response to Streptococcus pneumoniae Serotypes are determined by the structure of the pneumococcal capsular polysaccharide
The lab’s two RO1 grants are aimed to study the elderly and HIV positive populations
While our lab focuses on the specific antibody response to pneumococcal polysaccharides there are also natural antibodies
Identified during screening
These antibodies did not have a striking trend in gene usage. PPS-specific antibodies classically express only one or two genes. For example, reported PPS23F specific antibodies variable light chain is restricted to L6 and A23.
Analysis of these polyreactive clones revealed several unique trends
Went on to analyze amino acid groups that would influence binding to pneumococcal polysaccharide Greater number of flexible amino acids in the VH CDR3
After observing these trends in polyreactive antibodies we wanted to compare these findings to PPS-specific antibodies Analyzed all reported human PPS-specific antibodies
Went on to determine specific avidity to PPS14 and PPS23F using SPR Polyreactive clones were expressed as both IgG1 and IgG2
Mention reversed y-axis Smaller number the better A specific monoclonal antibody measured a avidity of 70pM Generally, IgG1 bound both polysaccharides more avidity than IgG2
Upon cleavage of the constant region, the differences between antibody avidity was no longer observed
