Immunogenicity of a chimeric Plasmodium falciparum merozoite surface protein vaccine in Aotus monkeys

Abstract

Background: The production of properly folded, recombinant sub-unit Plasmodium falciparum malaria vaccine candidates in sufficient quantities is often a challenge. Success in vaccine immunogenicity studies in small animal models does not always predict immunogenicity in non-human primates and/or human subjects. The aim of this study was to assess the immunogenicity of a chimeric blood-stage malaria vaccine in Aotus monkeys. This vaccine candidate includes the neutralizing B cell epitopes of P. falciparum merozoite surface protein 1 (rPfMSP119) genetically linked to a highly immunogenic, well-conserved P. falciparum merozoite surface protein 8 (rPfMSP8 (ΔAsn/Asp)) partner.

Methods: Aotus nancymaae monkeys were immunized with purified rPfMSP1/8 or rPfMSP8 (ΔAsn/Asp) formulated with Montanide ISA 720 as adjuvant, or with adjuvant alone. Antibody responses to MSP119 and MSP8 domains were measured by ELISA following primary, secondary and tertiary immunizations. The functionality of vaccine-induced antibodies was assessed in a standard P. falciparum blood-stage in vitro growth inhibition assay. Non-parametric tests with corrections for multiple comparisons when appropriate were used to determine the significance of differences in antigen-specific IgG titres and in parasite growth inhibition.

Results: The chimeric rPfMSP1/8 vaccine was shown to be well tolerated and highly immunogenic with boost-able antibody responses elicited to both PfMSP8 and PfMSP119 domains. Elicited antibodies were highly cross-reactive between FVO and 3D7 alleles of PfMSP119 and potently inhibited the in vitro growth of P. falciparum blood-stage parasites.

Conclusions: Similar to previous results with inbred and outbred mice and with rabbits, the PfMSP1/8 vaccine was shown to be highly effective in eliciting P. falciparum growth inhibitory antibodies upon immunization of non-human primates. The data support the further assessment of PfMSP1/8 as a component of a multivalent vaccine for use in human subjects. As important, the data indicate that rPfMSP8 (ΔAsn/Asp) can be used as a malaria specific carrier protein to: (1) drive production of antibody responses to neutralizing B cell epitopes of heterologous vaccine candidates and (2) facilitate production of properly folded, recombinant P. falciparum subunit vaccines in high yield.

Keywords: Aotus monkeys; Blood-stage malaria vaccine; Vaccine carrier protein.

Fig. 1

Purified recombinant chimeric rPfMSP1/8 and rPfMSP8 (ΔAsn/Asp) vaccine antigens. A Coomassie blue-stained 10 % SDS–polyacrylamide gel containing purified rPfMSP1/8 (3 μg, lanes 1 and 3) or rPfMSP8 (ΔAsn/Asp) (3 μg, lanes 2 and 4) was run under reducing (lanes q and 2) and non-reducing (lanes 3 and 4) conditions. Molecular weight markers in kilodaltons (kDa) are shown. Endotoxin levels and  % purity for each antigen preparation are also indicated

Fig. 2

Specificity of antibody response induced by immunization with rPfMSP8 (ΔAsn/Asp) versus rPfMSP1/8 vaccines. Antigen-specific IgG titres (mean ± standard deviation) in sera collected from Aotus monkeys (6 animals/group) immunized with a rPfMSP8 (ΔAsn/Asp) or b rPfMSP1/8 vaccines formulated with Montanide ISA 720 were determined by ELISA. Sera collected 2 weeks following primary, secondary and tertiary immunization were evaluated. ELISA plates were coated with either rPfMSP1/8, rPfMSP8 (ΔAsn/Asp), rGST-PfMSP1₁₉ (FVO), or rGST-PfMSP1₁₉ (3D7) as indicated. For each animal, reactivity of pre-immunization serum was subtracted as background

Fig. 3

In vitro inhibition of Plasmodium falciparum growth by Aotus IgG induced by immunization with rPfMSP8 (ΔAsn/Asp) versus rPfMSP1/8 vaccines. In vitro growth inhibitory activity of IgG from immunized Aotus monkeys for P. falciparum FVO blood-stage parasites was based on measurement of parasite lactate dehydrogenase levels. Purified IgG (10 mg/ml) from animals immunized three times with Montanide alone (n = 6), rPfMSP8 (ΔAsn/Asp) + Montanide (n = 5) or rPfMSP1/8 + Montanide (n = 5) was evaluated. Two immunized animals (1 rPfMSP8 (ΔAsn/Asp), 1 rPfMSP1/8) tested at less than 10 mg/ml of IgG were not included in the Figure. Per cent growth inhibition in the presence of Aotus IgG relative to controls in the absence of IgG is shown