A chimeric Plasmodium falciparum merozoite surface protein vaccine induces high titers of parasite growth inhibitory antibodies

Abstract

The C-terminal 19-kDa domain of Plasmodium falciparum merozoite surface protein 1 (PfMSP119) is an established target of protective antibodies. However, clinical trials of PfMSP142, a leading blood-stage vaccine candidate which contains the protective epitopes of PfMSP119, revealed suboptimal immunogenicity and efficacy. Based on proof-of-concept studies in the Plasmodium yoelii murine model, we produced a chimeric vaccine antigen containing recombinant PfMSP119 (rPfMSP119) fused to the N terminus of P. falciparum merozoite surface protein 8 that lacked its low-complexity Asn/Asp-rich domain, rPfMSP8 (ΔAsn/Asp). Immunization of mice with the chimeric rPfMSP1/8 vaccine elicited strong T cell responses to conserved epitopes associated with the rPfMSP8 (ΔAsn/Asp) fusion partner. While specific for PfMSP8, this T cell response was adequate to provide help for the production of high titers of antibodies to both PfMSP119 and rPfMSP8 (ΔAsn/Asp) components. This occurred with formulations adjuvanted with either Quil A or with Montanide ISA 720 plus CpG oligodeoxynucleotide (ODN) and was observed in both inbred and outbred strains of mice. PfMSP1/8-induced antibodies were highly reactive with two major alleles of PfMSP119 (FVO and 3D7). Of particular interest, immunization with PfMSP1/8 elicited higher titers of PfMSP119-specific antibodies than a combined formulation of rPfMSP142 and rPfMSP8 (ΔAsn/Asp). As a measure of functionality, PfMSP1/8-specific rabbit IgG was shown to potently inhibit the in vitro growth of blood-stage parasites of the FVO and 3D7 strains of P. falciparum. These data support the further testing and evaluation of this chimeric PfMSP1/8 antigen as a component of a multivalent vaccine for P. falciparum malaria.

Fig 1

Design, production, and analysis of a chimeric rPfMSP1/8 (FVO) vaccine antigen. (A) Cartoon of the chimeric PfMSP1/8 expression construct with sequences encoding the double EGF-like domains of PfMSP1₁₉ (stippled box), followed by a Gly-Ser spacer (filled box) and PfMSP8 (ΔAsn/Asp) including the C-terminal EGF-like domains (open and hatched boxes). Cysteine residues are indicated by vertical lines. (B) Coomassie blue-stained 10% SDS-polyacrylamide gel containing lysates of E. coli (reduced) expressing rPfMSP1/8 at the time of induction (T0) or 3 h postinduction (T3) and nickel-chelate affinity-purified rPfMSP1/8 (3 μg per lane) under reducing (R) and nonreducing (NR) conditions. (C) Immunoblot analysis of purified rPfMSP1/8 (0.1 μg per lane) under reducing (R) and nonreducing (NR) conditions probed with rabbit anti-rPfMSP1₁₉ (FVO), PfMSP1₁₉ (FVO)-specific monoclonal antibody (MAb 5.2), rabbit anti-rPfMSP8, or rabbit anti-rPfMSP8 (ΔAsn/Asp). Molecular weight markers (MW; in thousands) are indicated.

Fig 2

T cells induced by immunization with rPfMSP1/8 recognize epitopes within rPfMSP8 (ΔAsn/Asp). CB6F1/J mice (5 mice/group) were immunized three times with rPfMSP1/8 with Quil A as the adjuvant (black bars) or with Quil A alone (gray bars). Following the third immunization, splenocytes were harvested and stimulated with rPfMSP1/8, rPfMSP8 (ΔAsn/Asp), GST-PfMSP1₁₉ (FVO), GST, or ConA (A) or with overlapping synthetic peptides spanning the entire length of rPfMSP1/8 (B), as indicated on the x axes. See Table S1 in the supplemental material for sequence of individual peptides. After 4 days of culture, proliferation was quantitated by [³H]thymidine incorporation. The stimulation index was calculated as mean counts per minute in stimulated cultures/mean counts per minute in unstimulated cultures. Mean values ± standard deviations (SD) are shown with significant responses elicited by 4 peptides marked by an asterisk on the y axis (*, P < 0.05; **, P < 0.01).

Fig 3

Immunization of inbred CB6F1/J mice and outbred CD1 mice with rPfMSP1/8 elicits high-titer antibodies to both PfMSP1₁₉ and PfMSP8 (ΔAsn/Asp). Antigen-specific IgG titers (means ± standard deviations) in sera collected from CB6F1/J (A) and CD-1 (B) mice (10 mice/group) immunized with rPfMSP1/8 formulated with Quil A or M plus CpG were determined by ELISA on plates coated with either rPfMSP1/8, rPfMSP8 (ΔAsn/Asp), rGST-PfMSP1₁₉ (FVO), or rGST-PfMSP1₁₉ (3D7) as indicated. For each dilution, the mean absorbance values at A₄₀₅ of the pooled sera from adjuvant control mice (n = 5) was subtracted as the background.

Fig 4

Native P. falciparum blood-stage antigens boost PfMSP1₁₉- and PfMSP8 (ΔAsn/Asp)-specific antibody responses primed by rPfMSP1/8 immunization. CB6F1/J mice (5 mice per group) received a primary immunization with rPfMSP1/8 or adjuvant and then were boosted with either rPfMSP1/8 or a total antigen lysate (PfRBC) prepared from in vitro-cultured P. falciparum (FVO) blood-stage parasites. Antigens were formulated with Quil A as the adjuvant. Antibody titers specific for rPfMSP1/8, rPfMSP8 (ΔAsn/Asp), rGST-PfMSP1₁₉ (FVO), and rGST-PfMSP1₁₉ (3D7) were measured by ELISA following primary and secondary immunizations. Immunization groups are indicated on the x axis. *, P < 0.05.

Fig 5

Enhanced immunogenicity of the chimeric rPfMSP1/8 vaccine relative to rPfMSP1₄₂ vaccine formulations. CB6F1/J mice (5 mice per group) were immunized with rPfMSP1/8, PfMSP8 (ΔAsn/Asp), rPfMSP1₄₂, or a mixture of PfMSP8 (ΔAsn/Asp) and rPfMSP1₄₂, formulated with either Quil A or M plus CpG as the adjuvant. Antigen-specific IgG titers (means ± standard deviations) in sera collected following the third immunization were determined by ELISA on plates coated with either rPfMSP1/8, rPfMSP8 (ΔAsn/Asp), rGST-PfMSP1₁₉ (FVO), or rGST-PfMSP1₁₉ (3D7) as indicated. For each dilution, the mean absorbance values at A₄₀₅ of the pooled sera from adjuvant control mice (n = 5) was subtracted as the background. **, P < 0.01.

Fig 6

Rabbit anti-PfMSP1/8-dependent inhibition of P. falciparum growth is reversed in the presence of rGST-PfMSP1₁₉ (FVO) but not rPfMSP8 (ΔAsn/Asp). Inhibition of the in vitro growth of P. falciparum FVO blood-stage parasites was assessed by measuring parasite lactate dehydrogenase levels in the presence of rabbit anti-rPfMSP1/8 IgG (rabbits 1 to 3) or adjuvant control IgG. All purified rabbit IgG samples were tested at a constant concentration of 1.25 mg/ml. To test the specificity of growth inhibitory antibodies, anti-rPfMSP1/8 IgG was preincubated with rPfMSP1/8, rPfMSP8 (ΔAsn/Asp), or rGST-PfMSP1₁₉ (FVO) at an antigen concentration of 100 μg/ml. Residual growth inhibitory activity was then measured. No Ag, no antigen.