The Case for Exploiting Cross-Species Epitopes in Malaria Vaccine Design

Abstract

The infection dynamics between different species of Plasmodium that infect the same human host can both suppress and exacerbate disease. This could arise from inter-parasite interactions, such as competition, from immune regulation, or both. The occurrence of protective, cross-species (heterologous) immunity is an unlikely event, especially considering that strain-transcending immunity within a species is only partial despite lifelong exposure to that species. Here we review the literature in humans and animal models to identify the contexts where heterologous immunity can arise, and which antigens may be involved. From the perspective of vaccine design, understanding the mechanisms by which exposure to an antigen from one species can elicit a protective response to another species offers an alternative strategy to conventional approaches that focus on immunodominant antigens within a single species. The underlying hypothesis is that certain epitopes are conserved across evolution, in sequence or in structure, and shared in antigens from different species. Vaccines that focus on conserved epitopes may overcome the challenges posed by polymorphic immunodominant antigens; but to uncover these epitopes requires approaches that consider the evolutionary history of protein families across species. The key question for vaccinologists will be whether vaccines that express these epitopes can elicit immune responses that are functional and contribute to protection against Plasmodium parasites.

Keywords: Plasmodium; cross-species; epitopes; heterologous; immunity; malaria; vaccines.

FIGURE 1

Putative cross-species vaccine candidates at different stages of the parasite life cycle. Arrowheads indicate the direction of cross-reactivity and double arrowheads show reciprocal cross-reactivity. Gray arrows denote immunological cross-reactivity, but unknown functional activity; purple arrows denote that heterologous function was not demonstrated; blue arrows denote that heterologous function was demonstrated, and green arrows denote cross-boosting following heterologous vaccination. The box indicates heterologous cross-stage reactivity (antibodies to the merozoite antigen recognize an iRBC surface antigen). Spz(Pf) = P. falciparum sporozoites. Subscript letters denote route of exposure to parasite or antigen; C = Controlled human malaria infections (CHMI); V = exposure through vaccination; N = natural infection. *Antigen recognition was blocked by heterologous antigen in a subset of samples from co-exposed individuals. Created with Biorender.com.

FIGURE 2

Non-reciprocal, cross-species immunity mediated by conserved domains in functionally distinct proteins from P. vivax and P. falciparum. (i) Antibodies to the P. vivax merozoite protein PvDBP that arise from natural infection in humans or by vaccination with the recombinant protein in mice recognize epitopes within the DBL domain of PvDBP (ii). A subset of antibodies (green) that recognize subdomain 1 (SD1; blue) also cross-react with the DBL domains of P. falciparum VAR2CSA (iii), a protein that mediates sequestration of parasites in the placenta. Although PvDBP is not thought to play a role in P. vivax pregnancy-associated malaria, antibodies against the SD1 region of PvDBP can block P. falciparum parasites from binding in vitro to CSA, the placental ligand (iv). The recognition sites of these cross-reactive antibodies in one of the DBL domains of VAR2CSA, DBL5ε, map to two non-overlapping peptides, P20 (orange) and P23 (red) (v). These epitopes are spatially distinct from the immunodominant epitopes recognized by sera from multigravid women from Tanzania (green). Concordantly, these epitopes are cryptic; P20 and P23 are not recognized by sera from multigravid women from Uganda, nor by sera from rabbits immunized with VAR2CSA. As observed in other studies of human and mouse malaria, the immune recognition of these proteins is non-reciprocal as antibodies elicited through natural exposure to VAR2CSA in pregnant women or through immunization of animals with recombinant VAR2CSA did not recognize SD1. The cross-reactivity of antibodies to PvDBP and VAR2CSA exemplifies a mechanism of immune recognition to functionally distinct proteins in different Plasmodium species that is mediated by structurally conserved domains. Modified from (79). Created with Biorender.com.

FIGURE 3

Proposed strategy to identify cross-species vaccine candidates based on cross-reactive B-cell epitopes. Given that cross-species immunity is a rare event in naturally exposed populations, a large number of samples from endemic populations will need to be screened (e.g. by IFA or flow cytometry) to identify sera that recognize heterologous parasites. Antibody function against the heterologous species should then be confirmed (e.g. invasion, cytoadherence, transmission-blocking assays), and the antigen that mediates this functional cross-reactivity identified. This can be achieved through a variety of methods, including depletion or competition experiments. Antigen-specific antibodies can be affinity-purified from sera, or monoclonal antibodies generated using PBMCs from naturally exposed individuals or from animals vaccinated with the antigen. Functional analysis of these antibodies can then be used to down-select candidate antigens before applying a variety of empirical approaches to map the cross-reactive epitope. Phage and peptide libraries can be screened with the cross-reactive antibodies. Mutagenesis techniques, such as site-directed mutagenesis or alanine scanning of recombinant proteins can map residues that are critical for antibody binding. Physical mapping, such as co-crystallization of the antigen-antibody complex, is another powerful approach to map the contact residues within the epitope. These experimental tools can be integrated with computational analysis of the antigens from each species. Once a putative cross-reactive epitope is identified, the next step is to generate a recombinant protein or synthetic peptide that recapitulates this epitope, raise epitope-specific antibodies in animals, and test for cross-reactivity and function in vitro. It is important to note that the process of identifying and refining the epitope is iterative and each approach can complement and inform the other to yield potent, functional cross-species antibodies. Created with Biorender.com, including crystal structures PDB accession numbers 1SME and 6R2S.