Merozoite surface protein 1 of Plasmodium vivax induces a protective response against Plasmodium cynomolgi challenge in rhesus monkeys

Abstract

The 42-kDa fragment of the merozoite surface protein 1 (MSP-1(42)) is a leading candidate for the development of a vaccine to control malaria. We previously reported a method for the production of Plasmodium vivax MSP-1(42) (PvMSP-1(42)) as a soluble protein (S. Dutta, L. W. Ware, A. Barbosa, C. F. Ockenhouse, and D. E. Lanar, Infect. Immun. 69:5464-5470, 2001). We report here a process to manufacture the same PvMSP-1(42) protein but as an insoluble inclusion body-derived protein which was then refolded in vitro. We compared the immunogenicity and protective efficacy of the soluble and refolded forms of PvMSP-1(42) protein by using a heterologous but closely related P. cynomolgi-rhesus monkey challenge model. As comparative controls we also expressed, purified, and immunized rhesus with the soluble and refolded forms of the P. cynomolgi MSP-1(42) (PcMSP-1(42)) proteins. All proteins induced equally high-titer, cross-reacting antibodies. Upon challenge with P. cynomolgi, none of the MSP-1(42)-vaccinated groups demonstrated sterile protection or a delay in the prepatent period. However, following an initial rise in parasitemia, all MSP-1-vaccinated animals had significantly lower parasite burdens as indicated by lower cumulative parasitemia, lower peak parasitemia, lower secondary peak parasitemia, and lower average daily parasitemia compared to the adjuvant control group (P < 0.05). Except the soluble PcMSP-1(42) group, monkeys in all other groups had fewer numbers of days with parasitemia of >10,000 parasites mm(-3). Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model. The soluble and refolded forms of PcMSP-1(42) and PvMSP-1(42) proteins also appeared to have a similar partially protective effect.

FIG. 1.

Top panel, Coomassie blue-stained SDS-PAGE gel showing ∼5 μg of the soluble (S) and refolded (R) forms of the P. vivax (Pv) and P. cynomolgi (Pc) MSP-1₄₂ products under nonreducing and reducing conditions. The MSP-1-specific bands at 42 and 33 kDa are indicated by arrows. Bottom panel, Western blot reactivity of the four proteins with conformation dependent PvMSP-1-specific mouse MAbs 5.14 and E9.14 and with a rabbit polyclonal serum against PvMSP-1₁₉. m, molecular mass marker lanes.

FIG. 2.

Group wise mean of the log ELISA titers (y axis) at each of the six bleeds (x axis). The immunization events are denoted by arrows at the bottom. Bleeds were collected at 2-week intervals, and immunizations were administered at 4-week intervals (arrows).

FIG. 3.

Daily parasitemia records of individual monkeys (parasites mm⁻³, y axis) plotted against days following the day of challenge (x axis). Groups shown from left to right in the top row are adjuvant control, P. cynomolgi MSP-1₄₂ soluble, and P. cynomolgi MSP-1₄₂ refolded; the bottom row shows P. vivax MSP-1₄₂ soluble and P. vivax MSP-1₄₂ refolded.

FIG. 4.

Group-wise average cumulative parasitemia (parasites mm⁻³) of the 35-day follow-up was plotted for all immunized groups. Only odd-day parasitemias were plotted, and the bars represent the standard errors calculated from the means.

FIG. 5.

Parameters chosen to represent parasite burden (open bars). Group-wise mean of primary peak parasitemia (A); secondary peak parasitemia (B); average daily parasitemia (C); number of days when >10,000 parasites mm⁻³ were recorded (D). Standard deviation within group is plotted as a line above each bar.