Generation of a Peptide Vaccine Candidate against Falciparum Placental Malaria Based on a Discontinuous Epitope.

In pregnant women, Plasmodium falciparum-infected red blood cells adhere to the placenta via the parasite protein VAR2CSA. Two vaccine candidates based on VAR2CSA are currently in clinical trials; however, these candidates failed to elicit strain-transcending antibody responses. We previously showed that a cross-reactive monoclonal antibody (3D10) raised against the P. vivax antigen PvDBP targets epitopes in VAR2CSA. We now aim to design a peptide vaccine against VAR2CSA based on the epitope that generated 3D10. We mapped the epitope to subdomain 1 (SD1) of PvDBP and identified a peptide that contained the minimal sequence. However, this peptide did not elicit cross-reactive VAR2CSA antibodies in mice. When tested against a broader, overlapping peptide array spanning SD1, 3D10 in fact recognized a discontinuous epitope consisting of three segments of SD1. These findings presented the challenge to generate this larger structural epitope as a synthetic peptide since it is stabilized by two pairs of disulfide bonds. We overcame this using a synthetic scaffold to conformationally constrain the SD1 peptide and coupled it to keyhole limpet hemocyanin (KLH). The SD1-KLH conjugate elicited antibodies in mice that cross-reacted with VAR2CSA. This strategy successfully recapitulated a discontinuous epitope with a synthetic peptide and represents the first heterologous vaccine candidate against VAR2CSA.

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

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.

Antibodies to Cryptic Epitopes in Distant Homologues Underpin a Mechanism of Heterologous Immunity between Plasmodium vivax PvDBP and Plasmodium falciparum VAR2CSA.

Many pathogens evolve extensive genetic variation in virulence proteins as a strategy to evade host immunity. This poses a significant challenge for the host to develop broadly neutralizing antibodies. In Plasmodium falciparum, we show that a mechanism to circumvent this challenge is to elicit antibodies to cryptic epitopes that are not under immune pressure. We previously discovered that antibodies to the Plasmodium vivax invasion protein, PvDBP, cross-react with P. falciparum VAR2CSA, a distantly related virulence factor that mediates placental malaria. Here, we describe the molecular mechanism underlying this cross-species immunity. We identified an epitope in subdomain 1 (SD1) within the Duffy binding-like (DBL) domain of PvDBP that gives rise to cross-reactive antibodies to VAR2CSA and show that human antibodies affinity purified against a synthetic SD1 peptide block parasite adhesion to chondroitin sulfate A (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 recognized by vivax-derived SD1 antibodies (of human and mouse origin) are distinct from those recognized by VAR2CSA immune serum. We mapped two peptides in the DBL5ε domain of VAR2CSA that are recognized by SD1 antibodies. Both peptides map to regions outside the immunodominant sites, and antibodies to these peptides are not elicited following immunization with VAR2CSA or natural infection with P. falciparum in pregnancy, consistent with the cryptic nature of these target epitopes.IMPORTANCE In this work, we describe a molecular mechanism of heterologous immunity between two distant species of Plasmodium Our results suggest a mechanism that subverts the classic parasite strategy of presenting highly polymorphic epitopes in surface antigens to evade immunity to that parasite. This alternative immune pathway can be exploited to protect pregnant women from falciparum placental malaria by designing vaccines to cryptic epitopes that elicit broadly inhibitory antibodies against variant parasite strains.

Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA.

Background: In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. Methods: We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Results: Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. Conclusions: These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria.

Using Reference Quantitative Polymerase Chain Reaction to Assess the Clinical Performance of the Paracheck-Pf® Rapid Diagnostic Test in a Field Setting in Uganda.

Malaria rapid diagnostic tests (RDTs) are widely used in clinical and surveillance settings. However, the performance of most RDTs has not been characterized at parasite densities below detection by microscopy. We present findings from Uganda, where RDT results from 491 participants with suspected malaria were correlated with quantitative polymerase chain reaction (qPCR)-defined parasitemia. Compared with qPCR, the sensitivity and specificity of the RDT for Plasmodium falciparum mono-infections were 76% (95% confidence interval [CI]: 68-83%) and 95% (95% CI: 92-97%), respectively. The sensitivity of the RDT at parasite densities between 0.2 and 200 parasites/µL was surprisingly high (87%, 95% CI: 74-94%). The high sensitivity of the RDT is likely because of histidine-rich protein 2 from submicroscopic infections, gametocytes, or sequestered parasites. These findings underscore the importance of evaluating different RDTs in field studies against qPCR reference testing to better define the sensitivity and specificity, particularly at low parasite densities.

Achieving room temperature DNA amplification by dialling in destabilization.

The ability to amplify nucleic acid biomarkers at room temperature has remained elusive despite the great need of diagnostics suitable for the point of care. To exponentially amplify DNA within a wide range of ambient temperatures (18-26 °C), we explore combining two destabilizing elements in our isothermal lesion-induced DNA amplification system. We demonstrate rapid DNA amplification at the bench without a heat source.

Rapid, isothermal DNA self-replication induced by a destabilizing lesion.

You spin me round: Using a destabilizing abasic site and high concentration of ligase, rapid DNA self-replication in an isothermal ligase chain reaction (LCR) was produced. Both destabilization and rapid ligation are essential for proper LCR replication. This method also provides insight into prebiotic nucleotide replication and is a potential amplification method for biodiagnostics.