The immunogenicity and protective efficacy of a baculovirus recombinant polypeptide based on the Plasmodium falciparum merozoite surface protein 1 (MSP-1) has been evaluated in Aotus lemurinus griseimembra monkeys. The MSP-1-based polypeptide, BVp42, corresponds to the 42-kDa C-terminal processing fragment of the precursor molecule. Immunization of Aotus monkeys with BVp42 in complete Freund's adjuvant resulted in high antibody titers against the immunogen as well as parasite MSP-1. Fine specificity studies indicated that major epitopes recognized by these antibodies localize to conserved determinants of the 19-kDa C-terminal fragment derived from cleavage of the 42-kDa processing fragment. Effective priming of MSP-1-specific T cells was also demonstrated in lymphocyte proliferation assays. All three Aotus monkeys immunized with BVp42 in complete Freund's adjuvant showed evidence of protection against blood-stage challenge with P. falciparum. Two animals were completely protected, with only one parasite being detected in thick blood films on a single day after infection. The third animal had a modified course of infection, controlling its parasite infection to levels below detection by thick blood smears for an extended period in comparison with adjuvant control animals. All vaccinated, protected Aotus monkeys produced antibodies which inhibited in vitro parasite growth, indicating that this assay may be a useful correlate of protective immunity and that immunity induced by BVp42 immunization is mediated, at least in part, by a direct effect of antibodies against the MSP-1 C-terminal region. The high level of protection obtained in these studies supports further development of BVp42 as a candidate malaria vaccine.

The roles of allelic and conserved epitopes in vaccine-induced immunity to the C-terminal 42-kDa fragment of the Plasmodiumfalc4ium merozoite surface protein 1 (MSP1) were investigated. The C-terminal fragment of MSP1 was expressed as a baculovirus recombinant protein, BVp42. Rabbits were immunized with BVp42, and antibodies were tested for reactivity to MSP1s of the homologous and heterologous allelic forms, represented by the FUP, FVO, FC27, and Honduras parasite isolates, by enzyme-linked immunosorbent assay and indirect immunofluorescence antibody assay. Despite the fact that allelic sequences accounted for * Corresponding author.

We have shown that conserved B epitopes were immunodominant in animals hyperimmunized with parasitepurified or recombinant merozoite surface protein MSP1 of Plasmodium falciparum. Cross-priming studies also suggested that a conserved T-helper epitope(s) is efficient in inducing the anti-MSP1 antibody response. In this study, we determined whether a similar profile of immune responses was induced during live P. falciparum infections. Naive Aotus monkeys were infected by blood-stage challenge with either one of the two dimorphic MSP1 alleles represented by the FUP and FVO parasites. Sera collected after parasite clearance were analyzed by enzyme-linked immunosorbent assays (ELISAs). Monkeys infected with parasites carrying one allelic form of MSP1 had antibodies that were equally reactive with homologous or heterologous MSP1s. This preferential recognition of conserved epitopes of MSP1 was confirmed by competitive binding ELISAs. Studies with Plasmodium yoelii and P. falciparum show that the C-terminal 19-kDa fragment of MSP1, MSP1 19 , is the target of protective immunity. Thus, monkey sera were assayed for recognition with recombinant MSP1 19 s expressing variant and conserved B epitopes. Results of direct and competitive binding ELISAs showed that the anti-MSP1 19 antibodies were also directed primarily against conserved determinants. The similarities between vaccine-or infection-induced antibody responses suggest a possible reciprocal enhancement of the two populations of anti-MSP1 antibodies when a subunit MSP1 vaccine is introduced into populations living in areas where malaria is endemic. This together with previous observations that conserved determinants are important in MSP1-mediated immunity provides an optimistic outlook that a subunit MSP1 vaccine may be effective and practical for field applications in malaria-exposed populations.

The immunogenicity of the C-terminal 19-kDa fragment of Plasmodium falciparum MSP1 expressed in yeast as a nonfusion product, YMSP1(19), was studied. Immunization with YMSP1(19) in rabbits induced high titers of Abs specific for native conformational epitopes on parasite MSP1. In mice, immunogenicity was dependent on the mouse strain and the adjuvant formulation. This suggests that different adjuvants may alter the immunogenicity of MSP1(19) in a genetically diverse population. Although YMSP1(19) induced anti-MSP1 Abs, they did not inhibit in vitro parasite growth. This contrasts with the strong inhibitory activities of Abs produced against a recombinant MSP1(42) (BVp42), which contains the entire MSP1(19) coding sequence. Further analyses showed that YMSP1(19) was the target of the inhibitory, anti-BVp42 Abs because YMSP1(19) could completely block binding of anti-BVp42 Abs to parasite MSP1 or BVp42. Moreover, depletion of YMSP1(19)-specific Abs completely abolished the parasite inhibitory activities of anti-BVp42 sera. Anti-YMSP1(19) sera did not block the inhibitory activities of anti-BVp42 sera, suggesting that inhibitory epitopes were not in close structural proximity with noninhibitory epitopes. The finding that YMSP1(19) possessed inhibitory epitopes but induced anti-MSP1 Abs that were not inhibitory suggests that although the T-epitope(s) produced by immunization with YMSP1(19) could provide help for Ab production, it did not induce an effective inhibitory Ab response. We hypothesize that the nature/specificity of T helper epitopes on MSP1 may be crucial in efficient induction of biologically relevant and/or protective Abs.

The merozoite surface protein-1 (MSP1) of Plasmodium falciparum possesses intervening conserved and nonconserved sequences. The relative importance of these sequences in providing T cell help for Ab production was investigated in a series of cross-priming studies using homologous and heterologous parasite MSP1 proteins. Cross-priming with heterologous MSP1s was as efficient as homologous immunizations in inducing anti-MSP1 Abs. Similar to homologous immunization, cross-priming with heterologous MSP1s induced primarily Abs to conserved epitopes. The specificities of the Abs were also similar for the two immunization regimens. Studies were also performed with use of the C-terminal p42 fragment of MSP1 expressed in baculovirus (BVp42). When BVp42 was used either as the priming Ag followed by boosting with homologous (or heterologous) MSP1 or as the booster Ag after priming with homologous (or heterologous) MSP1, much lower anti-BVp42 Ab titers were produced compared with priming/boosting with homologous or heterologous MSP1s or BVp42 alone. Thus, immunization with the complete parasite MSP1 induced a dominant, conserved Th epitope(s) specific for anti-p42 Ab production, and such determinant(s) was either located outside the p42 region or was not provided by the BVp42 because of possible differences in the processing of parasite MSP1 vs BVp42. Our data provided a strong rationale to identify and include conserved Th epitope(s) in MSP1 vaccines. Furthermore, a MSP1 vaccine on the basis of the C-terminal p42 fragment may benefit by the inclusion of additional Th epitopes to achieve effective boosting in the field.