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B Cells and blood stage malaria

B Cells and blood stage malaria. Sean Elias. Malaria Life Cycle. Antigen Constructs. FVO Ectodomain. tPA. 3D7. FVO Terminus. F VO-33 FVO-19. MSP-1. MSP-1: Merozoite surface protein. 19 fragment contains major B cell epitopes (anchored to RBCs during invasion)

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B Cells and blood stage malaria

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  1. B Cells and blood stage malaria Sean Elias

  2. Malaria Life Cycle

  3. Antigen Constructs FVO Ectodomain tPA 3D7 FVO Terminus F VO-33 FVO-19 MSP-1 • MSP-1: Merozoite surface protein. • 19 fragment contains major B cell epitopes (anchored to RBCs during invasion) • Preclinical association of 19 antibodies with protection • 33 fragment contains Th cell epitopes • AMA-1: Apical membrane antigen. • Required for RBC invasion • Preclinical association of antibodies with protection 1 3 5 12 3D7-33 3D7-19 Glycine-Proline linker Glycine-Proline linker AMA-1

  4. Clinical Trial Structure VAC36 VAC37 8wk Adeno Prime MVA Boost

  5. Clinical Trial Structure VAC39

  6. Vac1 AdCh63 Vac1 AdCh63 Vac1 AdCh63 = Blood test AdCh63 = AdCh63 AMA-1 (VAC36), MSP-1 (VAC37) • MVA = MVA AMA1 (VAC36), MSP-1 (VAC37) S = Screening S S S D0 D2 D28 D56 D14 D0 D63 D84 D90 D28 D2 D14 D14 D28 D56 D58 D140 D2 D0 D63 C-1 D56 D58 C+21 / DoD Vac 2 MVA Vac 2 MVA Volunteer Bleeds C+7 C+11 C+35 C+90

  7. Memory B Cells and Malaria • What is the effect of challenge/infection on mBC expansion? • The P. falciparum-specific human memory B cell compartment expands gradually with repeated malaria infections. Weiss 2010 • Long-lived antibody and B cell memory to P. falciparumin low transmission areas. Wipasa 2010 • P. YoeliiCan Ablate Vaccine-Induced Long Term protection in mice. Wykes 2005 • Kenyan children exposed to malaria may develop antibody but not memory to blood stage antigens (AMA-1 more so than MSP-1) Dorfman 2005 • Decreases in memory and naive B cell subsets in Kenyan children with acute P. falciparum infection. Asito 2008 • Atypical memory B cells (‘exhausted’) are greatly expanded in individuals living in a malaria-endemic area. Weiss 2009 • A Positive Correlation between Atypical Memory B Cells and Plasmodium falciparum Transmission Intensity in Cross-Sectional Studies in Peru and Mali Weiss 2011

  8. VAC37 : MSP-1 Antibody Data A B Group 1A Group 1B C D Group 2A Group 2B+2C ELISA DATA: S. Draper

  9. VAC37 : MSP-1 Memory B Cell ELIspot Challenge Boost Boost Prime Prime Day of Diagnosis

  10. VAC37 : MSP-1 Memory B Cell ELIspot

  11. VAC37 : MSP-1 Memory B Cell ELIspot • Boost required for significant B cell response • mBC’s correlate with Ab’s

  12. VAC36 : AMA-1 Memory B Cell ELIspot P= 0.0962 ELISA DATA: S. Draper, S. Biswas

  13. VAC39 Controls • 3/5 controls had mBC response to MSP-1 at 35 days after challenge • No response to AMA-1 • Appears to be rough correlation with Ab titre for MSP-1. Needs more power to confirm relationship. • Only 2/5 volunteers showed low Ab titre to AMA-1 at C+35 • Kenyan children exposed to malaria may develop antibody but not memory to blood stage antigens (AMA-1 more so than MSP-1) Dorfman 2005

  14. Memory B Cell ELIspots • Conclusions: • Ad/MVA prime boost regime induces mBC formation to both MSP-1 & AMA-1 • Following this regime with parasite exposure further boosts mBCs to levels much higher than seen through natural exposure (up to x10?) • mBC derived B cells appear to correlate with Ab titre • Activation of mBC by malaria antigens does not appear to activate other mBC’s (Diphtheria) by bystander effect • Blood stage parasitemia appears to deplete circulating mBC’s but this doesn’t effect boosting when patients drug cured at cut-off peak parasitemia. • Will be interesting to see boost effect of challenge on vaccinees on AMA-1 given lack of seroconversion and mBC’s in controls. • VAC39 aims: • Replicate data for challenged volunteers (Group 1) • Look for effect of challenge on mBC’s for AMA-1 (Group 2) • Study effect of dual antigen vaccination on mBC formation (Group 3)

  15. VAC39 : ASC ELIspot • Ahmed 2008: Showed that for flu vaccination there was a rapid and robust influenza specific IgG+ antibody secreting plasma cell (ASC) response that peaked at approximately day 7 after boosting. • Questions: • What time after boosting can we see a peak? Limited to D63 with current protocol. • What % of cells detected are antigen specific. • When during challenge can such cells be seen? • Is there a correlation with Antibody production?

  16. VAC39 : ASC ELIspot

  17. VAC39 : ASC ELIspot • Answers?: • Peak likely to be somewhere between day 5 and 8. • Harder to calculate total IgG secreting cells compared to mBC assay. • No ASC’s detected during challenge (C+7, C+11) • No ASC’s detected in protected volunteer at C+21 • No significant correlation with Antibody production Day 63.

  18. VAC44 : Upcoming Trial

  19. VAC44 : Experimental Plan • ASC ELIspots: • Additional timepoints for detection (1, 4 & 7 days post boost) • More regulation on visit windows so these time points more accurate • Can test whether successive boosts shorten time for peak response • Can compare effect of boost regimes. Vectors/Protein/Adjuvant. • mBCELIspots: • Can compare effect of boost regimes. Vectors/Protein. • Effect of CPG adjuvant (mBC stimulator)

  20. Future Work • B cell phenotyping • Atypical memory B cells (‘exhausted’): Are they induced in our vaccination regimes or during controlled challenge? • B cell clonality • Clonal dissection of mBC repertoire (Pinna 2009). • Clonal burst size • Antibody production on clonal level • B cell receptor (BCR) variable region genes will be cloned and sequenced to assess the antibody / BCR repertoire induced by different priming and boosting vaccinations • Does successive vaccinations/challenge give a more directed, higher affinity response?

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