B C ELLS AND BLOOD STAGE MALARIA Sean Elias

M ALARIA L IFE C YCLE

A NTIGEN C ONSTRUCTS 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 D7-33 3D7-19 F VO-33 FVO-19 Glycine-Proline linker MSP-1 Glycine-Proline linker tPA3D7 FVO Ectodomai n FVO Terminus AMA-1

C LINICAL T RIAL S TRUCTURE VAC36VAC37 Adeno PrimeMVA Boost 8wk

C LINICAL T RIAL S TRUCTURE VAC39

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

M EMORY B C ELLS AND M ALARIA 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. falciparum in low transmission areas. Wipasa 2010 P. Yoelii Can 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

VAC37 : MSP-1 A NTIBODY D ATA A B C D Group 1AGroup 1B Group 2AGroup 2B+2C ELISA DATA: S. Draper

VAC37 : MSP-1 M EMORY B C ELL ELI SPOT Challenge Boost Prime Day of Diagnosis Prime Boost

VAC37 : MSP-1 M EMORY B C ELL ELI SPOT

Boost required for significant B cell response mBC’s correlate with Ab’s

VAC36 : AMA-1 M EMORY B C ELL ELI SPOT P= ELISA DATA: S. Draper, S. Biswas

VAC39 C ONTROLS 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

M EMORY B C ELL ELI SPOTS 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)

VAC39 : ASC ELI SPOT 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?

VAC39 : ASC ELI SPOT

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.

VAC44 : U PCOMING T RIAL

VAC44 : E XPERIMENTAL P LAN 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. mBC ELIspots: Can compare effect of boost regimes. Vectors/Protein. Effect of CPG adjuvant (mBC stimulator)

F UTURE W ORK 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?