A new rodent model to assess blood stage immunity to the Plasmodium falciparum antigen merozoite surface protein 119 reveals a protective role for invasion inhibitory antibodies

Abstract

Antibodies capable of inhibiting the invasion of Plasmodium merozoites into erythrocytes are present in individuals that are clinically immune to the malaria parasite. Those targeting the 19-kD COOH-terminal domain of the major merozoite surface protein (MSP)-119 are a major component of this inhibitory activity. However, it has been difficult to assess the overall relevance of such antibodies to antiparasite immunity. Here we use an allelic replacement approach to generate a rodent malaria parasite (Plasmodium berghei) that expresses a human malaria (Plasmodium falciparum) form of MSP-119. We show that mice made semi-immune to this parasite line generate high levels of merozoite inhibitory antibodies that are specific for P. falciparum MSP-119. Importantly, protection from homologous blood stage challenge in these mice correlated with levels of P. falciparum MSP-119-specific inhibitory antibodies, but not with titres of total MSP-119-specific immunoglobulins. We conclude that merozoite inhibitory antibodies generated in response to infection can play a significant role in suppressing parasitemia in vivo. This study provides a strong impetus for the development of blood stage vaccines designed to generate invasion inhibitory antibodies and offers a new animal model to trial P. falciparum MSP-119 vaccines.

Figure 1.

Schematic representation of P. berghei and P. falciparum MSP-1 chimeras. The MSP-1 sequences of P. berghei (gray), P. falciparum (red), and P. chabaudi (blue) are represented. The arrows indicate the MSP-1 secondary cleavage site.

Figure 2.

Generation of P. berghei chimera lines containing either P. berghei or P. falciparum MSP-1 ₁₉. (A) Schematic diagram of the P. berghei MSP-1 locus, the transfection vector (pPb-PfM19) used to replace the endogenous MSP-1₁₉ molecule, and the predicted MSP-1 locus of the Pb-PfM19 chimeric line after integration. The gray box represents endogenous P. berghei MSP-1 ₁₉ sequence and the black box represents P. falciparum MSP-1 ₁₉ sequence (or P. berghei MSP-1 ₁₉ sequence for creation of the Pb-PbM19 chimeric line). The solid lines in pPb-PfM19 depict targeting sequence. TgDHFR-TS, selectable marker cassette; 3′, HSP86 3′ UTR. The expected sizes of fragments resulting from digestion with either HincII (H) or PstI (P) are shown. (B) Southern blot analysis of digested genomic DNA from P. berghei wild-type and chimeric lines.

Figure 3.

Phenotypic analysis of P. berghei chimeric lines. (A) Western blot analysis of late stage parasite extracts using rabbit αPbM19 or αPfM19 antibodies (both diluted 1/4,000) detecting both full-length MSP-1 (∼200 kD) and MSP-1₁₉ (∼19 kD). (B) Localization of MSP-1₁₉ by IFA. Schizont stage parasites were incubated with a mixture of rabbit αPbM19 (1/1,000) and 4H9/19 (αPfM19; 1/100) antibodies, followed by a mixture of FITC-conjugated anti–rabbit and rhodamine-conjugated anti–mouse immunoglobulins (both diluted 1/200). (C) Course of blood parasitemia in mice after infection at day 0 with P. berghei wild-type, Pb-PbM19, or Pb-PfM19. Shown is the mean ± SD of the parasitemia observed in five mice.

Figure 4.

Mice repeatedly infected with P. berghei transfectants elicit MSP-1₁₉–specific inhibitory antibodies. (A) Reciprocal anti–MSP-1₁₉ antibody endpoint titres of serum from Pb-PfM19 and Pb-PbM19 immune mice against recombinant P. falciparum and P. berghei MSP-1₁₉–GST fusion proteins. (B) Invasion inhibition assay of D10-PfM3′ and D10-PcMEGF parasite lines in the presence of individual serum from Pb-PfM19 and Pb-PbM19 immune mice. Invasion is expressed as a percentage of the invasion observed in parasites cultured in human nonimmune sera (HNIS). The numbers shown represent the P. falciparum MSP-1₁₉–specific invasion inhibitory activity of a given serum, calculated by subtracting the invasion rate of D10-PfM3′ from that of D10-PcMEGF.

Figure 5.

Evidence that MSP-1₁₉–specific inhibitory antibodies control a blood stage infection. MSP-1₁₉–specific invasion inhibitory activity of serum from individual Pb-PfM19 immune mice plotted against peak parasitemia attained after challenging corresponding mice with Pb-PfM19.