Investigation of the immunological cross-reactivity between Plasmodium vivax Duffy Binding Protein and Plasmodium falciparum VAR2CSA for vaccine development

  • Author / Creator
    Mitran, Catherine J
  • Worldwide, an estimated 30 million pregnancies are at risk of malaria infection each year. In pregnancy, Plasmodium falciparum parasites express a protein called VAR2CSA that mediates adherence of infected red blood cells (iRBCs) to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are acquired during infection in pregnancy and are associated with protection from placental malaria, which can cause pre-term birth, low birth weight, spontaneous abortion, maternal anemia and even infant and maternal mortality. We previously observed that men and children in Colombia had antibodies to VAR2CSA, but the origin of these antibodies was unknown. We hypothesized that infection with P. vivax elicited antibodies to epitopes that are conserved across the structurally related Duffy Binding-like (DBL) domains found in the P. vivax antigen, PvDBP, and VAR2CSA.
    We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against the DBL domain of PvDBP (DBPII). We found that over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified DBPII human antibodies and a DBPII monoclonal antibody, called 3D10, recognized VAR2CSA, showing that PvDBP exposure can give rise to cross-reactive antibodies. Importantly 3D10 also inhibited parasite binding to CSA, which is the primary in vitro correlate of protection against placental malaria. These data suggest that PvDBP induces antibodies that recognize VAR2CSA and can block parasite adhesion to CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.
    Next, we investigated the cross-reactive epitopes in PvDBP and VAR2CSA. We determined that the epitope which elicits cross-reactive antibodies in PvDBP is contained within the subdomain 1 (SD1) region. Notably, human antibodies affinity purified against a synthetic SD1 peptide blocked parasite adhesion to CSA in vitro. The epitope in SD1 is subdominant and highly conserved in PvDBP, and in turn, SD1 antibodies target cryptic epitopes in P. falciparum VAR2CSA. The epitopes in VAR2CSA that are recognized by vivax-derived cross-reactive antibodies are distinct from those recognized by VAR2CSA immune serum.
    We then attempted to exploit this cross-reactivity to develop a peptide vaccine against against VAR2CSA based on the epitope that generated 3D10. We mapped the minimal binding epitope for 3D10 to a peptide in SD1. However, this peptide did not elicit cross-reactive VAR2CSA antibodies in mice. When we tested 3D10 against a broader, overlapping peptide array spanning SD1, we found that it recognized a discontinuous epitope consisting of three segments of SD1. To preserve the native structure of the epitope as much as possible, we used a synthetic scaffold to conformationally-constrain the SD1 peptide and coupled it to a carrier protein. Immunizations with this peptide, SD1CLIPS, elicited antibodies in mice and a rabbit that cross-reacted with VAR2CSA. However, the cross-reactive antibodies were at low titre in the sera and bound to VAR2CSA with low avidity. We then affinity purified the cross-reactive IgG from the anti-SD1CLIPS rabbit serum on a column coated with VAR2CSA. The cross-reactive IgG recognized native VAR2CSA on the surface of RBCs infected with three different strains of P. falciparum. However, the antibodies did not block parasite adhesion to CSA or mediate opsonic phagocytosis of iRBCs, in vitro.
    We then tried several approaches to enhance the cross-reactive antibody response elicited by SD1CLIPS immunizations. First, we compared three different adjuvants and found that formulation with GLA-SE elicited a higher titre of cross-reactive antibodies in mice, compared to either GLA-LSQ or Complete Freund’s Adjuvant. Next, we employed a heterologous prime/boost strategy, in which mice received a boost with recombinant VAR2CSA following immunizations with SD1CLIPS. However, compared to control mice, a VAR2CSA boost did not enhance the cross-reactive antibody response. We also tested whether exposure to native VAR2CSA on the surface of iRBCs would boost the cross-reactive antibodies generated through SD1CLIPS immunizations. But, similar to the heterologous recombinant protein boost, injection with iRBCs expressing VAR2CSA did not boost the cross-reactive antibodies in mice.
    SD1CLIPS is not a viable vaccine candidate in its current state. However, this work has laid a foundation for further immunization studies with second generation SD1CLIPS-based vaccine candidates. The fact that immunizations with a peptide from PvDBP elicited antibodies in a rabbit that cross-reacted with native VAR2CSA from three different strains of P. falciparum, shows the potential of targeting conserved epitopes to elicit strain-transcending immune responses.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.