Immunity to recombinant plasmodium falciparum merozoite surface protein 1 (MSP1): protection in Aotus nancymai monkeys strongly correlates with anti-MSP1 antibody titer and in vitro parasite-inhibitory activity

Abstract

A number of malarial blood-stage candidate vaccines are currently being tested in human clinical trials, but our understanding of the relationship between clinical immunity and data obtained from in vitro assays remains inadequate. An in vitro assay which could reliably predict protective immunity in vivo would facilitate vaccine development. Merozoite surface protein1 (MSP1) is a leading blood-stage malaria vaccine candidate, and anti-MSP1 antibodies from individuals that are clinically immune to malaria inhibit the invasion of Plasmodium merozoites into erythrocytes in vitro. Using expression in Escherichia coli and subsequent refolding, we have produced two allelic forms of MSP1(42) (FVO and 3D7). Aotus nancymai monkeys were immunized with MSP1(42)-FVO, MSP1(42)-3D7, or a combination of FVO and 3D7 allelic forms, (MSP1(42)-C1) and were subsequently challenged with Plasmodium falciparum FVO parasites. Sera obtained prior to challenge were tested by standardized enzyme-linked immunosorbent assay (ELISA) to determine antibody titer, and immunoglobulin G (IgG) fractions were also obtained from the same sera; the IgG fractions were tested in an in vitro growth inhibition (GI) assay to evaluate biological activity of the antibodies. Regardless of the immunogen used, all monkeys that had >200,000 ELISA units against MSP1(42)-FVO antigen before challenge controlled their infections. By contrast, all monkeys whose purified IgGs gave <60% inhibition activity in an in vitro GI assay with P. falciparum FVO required treatment for high parasitemia after challenge. There is a strong correlation between ELISA units (Spearman rank correlation of greater than 0.75) or GI activity (Spearman rank correlation of greater than 0.70) and protective immunity judged by various parameters (e.g., cumulative parasitemia or day of patency). These data indicate that, in this monkey model, the ELISA and GI assay values can significantly predict protective immunity induced by a blood-stage vaccine, and they support the use of these assays as part of evaluation of human clinical trials of MSP1-based vaccines.

FIG. 1.

Daily parasitemia in A. nancymai monkeys after challenge. Monkeys were vaccinated three times with 50 μg/dose of Pfs25H (a), 50 μg/dose of MSP1₄₂-FVO (b), 50 μg/dose of MSP1₄₂-3D7 (c), or 100 μg/dose of MSP1₄₂-C1 (d). Seventeen days after the final vaccination, they were challenged with 5 × 10⁴ P. falciparum FVO parasitized red blood cells. Parasitemia was monitored daily by inspection of Giemsa-stained thin blood smears until treatment and was calculated based on examination of approximately 2,000 RBCs; if no parasites were seen, then 40 more high-power fields were examined. Monkeys were treated with a curative dose of mefloquine when parasitemia reached 5%, when their hematocrit fell below 25%, or at day 28. T, monkey was treated for high parasitemia; H, monkey was treated for anemia; C, monkey was treated at day 28. D, one monkey in the MSP1₄₂-C1 group was found dead on day 23; the hematocrit on day 21 was 39%, and the parasitemia on day 22 was 0.19%.

FIG. 2.

Correlation between ELISA units and protection data. Monkeys were vaccinated and challenged with P. falciparum as described in the legend to Fig. 1. The rank of protection data using various outcome measures is plotted against the rank of anti-MSP1₄₂-FVO ELISA units. (a) Ranking by parasitemia. Monkeys were ranked based on the parasitemia on the day that the first monkey was treated for anemia (15 days after challenge). There is a significant correlation between the ranks of ELISA and of parasitemia (Spearman rank correlation, 0.79; P < 0.001). (b) Ranking by day of patency. Monkeys were ranked based on the day that parasites were first observed in Giemsa-stained blood smears. There is a significant positive correlation between the rank of ELISA and of patency (Spearman rank correlation, 0.85; P < 0.001). (c) Ranking by treatment. Monkeys were ranked based on the day of treatment. There is a significant positive correlation between the rank of ELISA and day of treatment (Spearman rank correlation, 0.75; P < 0.001).

FIG. 3.

Correlation between ELISA units and growth inhibition activity in vitro. From the individual monkey serum on 17 days after the final immunization (just before parasite challenge), IgG was purified using protein G columns. The antibody titers of the purified IgGs were determined by ELISA, and the GI activities were determined by a standardized GI assay with P. falciparum FVO parasites in vitro. The antibody units in the GI assay wells as determined by ELISA units against MSP1₄₂ FVO antigen were plotted on the abscissa and the percent inhibition in the GI assay on the ordinate. There is a significant positive correlation between the ELISA units and the percent inhibition by GI assay (Spearman rank correlation, 0.864; P = 0.0003). The line is the best fit for the hyperbolic function.