Antibodies against PfEMP1, RIFIN, MSP3 and GLURP are acquired during controlled Plasmodium falciparum malaria infections in naïve volunteers

Abstract

Antibodies to polymorphic antigens expressed during the parasites erythrocytic stages are important mediators of protective immunity against P. falciparum malaria. Therefore, polymorphic blood stage antigens like MSP3, EBA-175 and GLURP and variant surface antigens PfEMP1 and RIFIN are considered vaccine candidates. However, to what extent these antibodies to blood stage antigens are acquired during naive individuals' first infections has not been studied in depth. Using plasma samples collected from controlled experimental P. falciparum infections we show that antibodies against variant surface antigens, PfEMP1 and RIFIN as well as MSP3 and GLURP, are acquired during a single short low density P. falciparum infection in non-immune individuals including strain transcendent PfEMP1 immune responses. These data indicate that the immunogenicity of the variant surface antigens is similar to the less diverse merozoite antigens. The acquisition of a broad and strain transcendent repertoire of PfEMP1 antibodies may reflect a parasite strategy of expressing most or all PfEMP1 variants at liver release optimizing the likelihood of survival and establishment of chronic infections in the new host.

Figure 1. Proportion of malaria naïve volunteers acquiring IgG against GLURP, MSP3 and EBA175 antigens after controlled experimental P. falciparum infections.

The percentage of malaria infected volunteers with a measurable IgG response against GLURP R0, R1, R2, MSP3 and EBA175 malaria antigens on day 21 (N = 34) and/or day 35, 42 or 90 (N = 30) post infection in descending order. In total 44 volunteers were included (Table S1) and the IgG response was measured by bead-based Luminex technology.

Figure 2. Proportion of malaria naïve volunteers acquiring IgG against PfEMP1 antigens after controlled experimental P. falciparum infections.

The percentage of malaria infected volunteers with a measurable IgG response against 79 PfEMP1 malaria antigens on day 21 (N = 34) and/or day 35, 42 or 90 (N = 30) post infection in descending order. The PfEMP1 domain classification was according to Rask et al . 25 DBL and CIDR domains were omitted from the figure as there was no IgG recognition of these domains (see Table S2). In total 44 volunteers were included (Table S1) and the IgG response was measured by bead-based Luminex technology.

Figure 3. Acquisition of IgG to 104 PfEMP1 domains, eight RIFINs, and GLURP, MSP3, and EBA-175 antigens of Plasmodium falciparum in malaria naïve individuals exposed to controlled experimental P. falciparum infections.

The IgG reactivity pattern of the six groups represented by their acquisition pattern of IgG to blood stage antigens observed in the 44 examined volunteers after exposure of a short P. falciparum infection: Volunteers with no acquisition of antibodies to any of the tested malaria antigens (n = 3); volunteers who acquired antibodies to GLURP only (n = 11); and volunteers who acquired antibodies to 1, 2, 3 or >3 PfEMP1 domains, respectively, as well as one or more merozoite antigens (n = 12, 5, 4 and 9). Results are expressed as median fluorescent intensity (MFI): Red >2000 MFI; Green >1000 MFI; Yellow >500 MFI (cut-off value).

Figure 4. Proportion of malaria naïve volunteers acquiring IgG against RIFIN antigens after controlled experimental P. falciparum infections.

The percentage of malaria infected volunteers with a measurable IgG response against eight RIFIN malaria antigens on day 21 (N = 34) and/or day 35, 42 or 90 (N = 30) post infection in descending order. The grouping of RIFINs was according to Wang et al. . V2: Variable domain 2, the putative extracellular polymorphic region defined by Cheng et al. . In total 44 volunteers were included (Table S1) and the IgG response was measured by bead-based Luminex technology.

Figure 5. Acquisition of IgG to GLURP R2 during a liver stage immunization study.

The anti-GLURP R2 IgG levels in 44 volunteers experimentally infected with Plasmodium falciparum (indicated by a thick arrow day one) and in 10 volunteers immunized by three exposures to P. falciparum while treated with chloroquine killing blood stage parasites (indicated by thin blue arrows day one, 33 and 61) and after challenge without a drug cover (thick blue arrow day 118). “35+” represents plasma samples taken 35, 42 or 90 days after infection. CHMI: controlled human malaria infection.

Figure 6. Acquisition of IgG Abs to 104 PfEMP1 domains, eight RIFINs, and GLURP, MSP3, and EBA-175 antigens in two immunized volunteers.

The two volunteers, 1 and 2, were selected to represent volunteers with acquisition of antibodies to PfEMP1 and merozoite antigens (n = 3) and volunteers with acquisition of antibodies to the merozoite antigens only (n = 7). The letters A, B, C, D and E correspond to 32, 60, 116, 151 and 256 days after first immunization. Parasite challenges were at day 118 . The results are expressed as median fluorescent intensity (MFI): Red >2000 MFI; Green > 1000 MFI; Yellow > 500 MFI (cut-off value).