ICT role in 21st century education and its challenges
Ociit conference copy
1. Immune Response to Pneumococcal
Polysaccharides in Elderly and Young
Rebecca Thompson
OCIIT Forum
2. Streptococcus Pneumoniae
– Gram-positive bacteria
– Colonizes the nasopharynx
– Over 90 identified
serotypes
• Serotypes are determined by
capsular polysaccharide
– Vaccines induce antibodies
against capsular
polysaccharide
– These 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. Vaccines
– 7-Valent Pneumococcal Conjugate Vaccine
• Prevnar®
– Contains 7 purified capsular polysaccharide covalently conjugated to
CRM197, a diptheria toxoid
• Recommended for young children
– 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
• 80% protective efficacy in healthy young adults
• However despite normal antibody levels efficacy in elderly is
drastically reduced. This drop in efficacy is thought to be linked
to antibody structure.
5. Previous Research
– Several research groups have studied this structure-function
relationship of anti-pneumococcal antibodies.
– Hybridomas and combinatorial libraries have been used to
express recombinant anti-pneumococcal antibodies.
– The bulk of this information has come from combinatorial
libraries.
• Reverse transcription PCR of expressed immunoglobulin genes
• Cloning of random VH and VL chains into an expression system.
• Assumption that these pairings are representative of a natural
repertoire.
6. Zhou et al. Recurrent variable region gene usage and somatic mutation in the
human antibody response to the capsular polysaccharide of Streptococcus
pneumoniae type 23F. Infect Immun (2002) vol. 70 (8) pp. 4083-91
7. • Zhou’s combinatorial library possesses a clear
over-representation of certain VL genes
– Of 30 PPS23F specific clones
• 15 clones L6 VL gene
• 12 clones A23 VL gene
• Is this a result of random pairing producing the
most avid clones? Or is this the true
representation of natural pairings of VH and VL in
response to S. pneumoniae?
• We hypothesized that PPS-specific VH could
recombine with various VL chains (i.e. not
restricted).
8. Methodology
• Vaccinate healthy young volunteers with Pneumovax®
– Ages 18-30
– Age group with highest vaccine efficacy
– Allows us to establish a natural antibody repertoire in response to PPS
• Draw blood at day 7 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 specific B cells
• Harvest and lyse cells
• Make cDNA and PCR variable heavy (VH) and variable light (VL)
immunoglobulin genes
• Ligate paired VH and VL into rhuIg expression plasmids and
transfect human cells
9. Baxendale et al. Immunogenetic analysis of the immune response to pneumococcal
polysaccharide. Eur J Immunol (2000) vol. 30 (4) pp. 1214-23
– To this purpose we obtained a well characterized
human mono-specific 23F mAb, CbE2
• Reverse transcriptase PCR of CbE2 VH
• Ligated CbE2 VH into a human rIg plasmid along with a
random VL chain also in a separate human rIg plasmid
– Light chains obtained by selecting B cells with a variety of
different PPS
10. Variable Light Chains
Clone Isolating Antigen VL CDR3
21d3 PPS14 L2 QQYNNRPRT
14F2 PPS4 B3 QQYYSTPVT
23d3 PPS14 B3 QQYYSTPAT
24e8 PPS14 B3 QQYYSTPYT
25b4 PPS14 A27 QQYGSSPPWT
31b5 PPS23 A27 QQYDRSPLT
31F7 PPS23 L6 QQRSNWPPLLT
31E2 PPS23 A20 QKYNGAPFT
32E8 PPS23 O12 QRSSGGPIS
• Transient transfection into HEK293 cells
• On day 3, culture supernatant was collected from transfected cells
• Samples were tested by ELISA for human Ig, PPS6B, PPS14 and
PPS23F
17. Summary
• As demonstrated by the promiscuity of the CbE2
VH chain to pair with multiple non-specific light
chains, combinatorial libraries do not represent
specific binding to PPS23F with exclusive VL
clones.
• Therefore combinatorial libraries do not
characterize the natural repertoire of anti-
pneumococcal antibodies and natural VH/VL
pairings are crucial for the accurate understanding
of the immune response to S. pneumoniae.
18. Future Work
• Stable transfections
– Allow for purification of rhuIg
• Subsequent avidity studies
– Competition assays
– Surface plasmon resonance
• Enzymatic digestion of antibodies
– Isolate individual antibody regions to determine
areas of importance in response to PPS
19. Acknowledgements
• Major Advisor • Collaborators
– Dr. Julie Westerink, MD – Dr. Baxendale
• Lab Members • Committee Members
– Jason Mosakowski – Dr. Dignam
– Jieying Wang – Dr. Malhotra
– Dr. Louise Smithson, PhD – Dr. Ruch
• Funding – Dr. Wooten
– NIH RO1
