Plasmodium falciparum Merozoite Surface Protein 1 (MSP-1)-MSP-3 Chimeric Protein: Immunogenicity Determined with Human-Compatible Adjuvants and Induction of Protective Immune Response

A chimeric gene, MSP-Fu₂₄, was constructed by genetically coupling immunodominant, conserved regions of the two leading malaria vaccine candidates, Plasmodium falciparum merozoite surface protein 1 (C-terminal 19-kDa region [PfMSP-1₁₉]) and merozoite surface protein 3 (11-kDa conserved region [PfMSP-3₁₁]). The recombinant MSP-Fu₂₄ protein was produced in Escherichia coli cells and purified to homogeneity by a two-step purification process with a yield of ∼30 mg/liter. Analyses of conformational properties of MSP-Fu₂₄ using PfMSP-1₁₉-specific monoclonal antibody showed that the conformational epitopes of PfMSP-1₁₉ that may be critical for the generation of the antiparasitic immune response remained intact in the fusion protein. Recombinant MSP-Fu₂₄ was highly immunogenic in mice and in rabbits when formulated with two different human-compatible adjuvants and induced an immune response against both PfMSP-1₁₉ and PfMSP-3₁₁. Purified anti-MSP-Fu₂₄ antibodies showed invasion inhibition of P. falciparum 3D7 and FCR parasites, and this effect was found to be dependent on antibodies specific for the PfMSP-1₁₉ component. The protective potential of MSP-Fu₂₄ was demonstrated by in vitro parasite growth inhibition using an antibody-dependent cell inhibition (ADCI) assay with anti-MSP-Fu₂₄ antibodies. Overall, the antiparasitic activity was mediated by a combination of growth-inhibitory antibodies generated by both the PfMSP-1₁₉ and PfMSP-3₁₁ components of the MSP-Fu₂₄ protein. The antiparasitic activities elicited by anti-MSP-Fu₂₄ antibodies were comparable to those elicited by antibodies generated with immunization with a physical mixture of two component antigens, PfMSP-1₁₉ and PfMSP-3₁₁. The fusion protein induces a protective immune response with human-compatible adjuvants and may form a part of a multicomponent malaria vaccine.Malaria is among the major parasitic diseases in tropical and subtropical countries. With as many as 300 to 500 million new cases each year, malaria accounts for the death of over 2 million people globally each year, and most are children (41). Among the four species of Plasmodium that infect humans, the most threatening is Plasmodium falciparum. The extensive spread of drug-resistant P. falciparum strains as well as the insecticide-resistant mosquito necessitates the development of a malaria vaccine on an urgent basis. Collectively, the major objective of the ongoing vaccine effort in this field is to develop a multistage, multivalent vaccine against P. falciparum (34).The blood-stage cycle of the parasite is responsible for malaria pathogenesis. Intervention at this stage of the parasite''s development through vaccination is likely to reduce malaria-related clinical symptoms. As a major interface between host and pathogen, the merozoite surface is an obvious target for the development of a malaria vaccine. A number of potential vaccine candidate antigens identified so far are located on or associated with the surface of the merozoite or in apical organelles. These include merozoite surface protein 1 (MSP-1), MSP-2, MSP-3, MSP-4, MSP-5, MSP-8, RAP1/2, AMA-1, and EBA-175, which are implicated in the process of merozoite invasion of the erythrocyte (23).MSP-1 is one of the most extensively studied proteins of P. falciparum (18). It is synthesized as a ∼200-kDa precursor and then processed in two steps: the primary processing step produces a complex of four fragments that are present on the merozoite surface, and the secondary processing step at invasion results in the shedding of the complex from the surface, except for the C-terminal 19-kDa domain (MSP-1₁₉), which remains anchored to the parasite surface by a glycosylphosphatidylinositol (GPI) moiety (2). The C-terminal 19-kDa fragment of MSP-1 is well conserved among P. falciparum isolates and contains two epidermal growth factor (EGF)-like domains that play a role in merozoite invasion. Substantial data from studies with P. falciparum MSP-1 and in vivo immunization studies of mice with Plasmodium yoelii and Plasmodium chabaudi indicate that the protective immune responses are directed against the C-terminal 19-kDa domain (10, 12, 15, 20, 27, 35). The inhibition of MSP-1 processing by conformation-specific antibodies (Abs) was previously proposed to be one of the possible mechanism for the inhibition of merozoite invasion (1).Another merozoite surface protein, MSP-3, was also shown to be the target of the protective immune responses in humans (29). The PfMSP-3 protein contains three blocks of four tandem heptad repeats based on the AXXAXX motif at the N terminus, a glutamic acid-rich domain, and a putative leucine zipper sequence at the C terminus (25). Although a clear surface localization of PfMSP-3 is known, it lacks any transmembrane domain or glycosylphosphatidylinositol (GPI) anchor site (24, 25) and is therefore considered to be loosely associated with the merozoite surface by interactions with other merozoite surface proteins. PfMSP-3 was identified as a candidate vaccine antigen by an antibody-dependent cellular inhibition (ADCI) assay using human immune sera (28). The potential of PfMSP-3 as a vaccine candidate was further illustrated by ADCI using mice antibodies and was further confirmed by the suppression of P. falciparum growth in an immunocompromised mouse after the passive transfer of human antibodies purified on MSP-3 peptides together with human monocytes (28, 40, 42). The immunization of Aotus and Saimiri monkeys with recombinant PfMSP-3 or its fragments provided protection against parasite challenge (6, 16). A 70-amino-acid-long conserved domain of PfMSP-3, referred to here as the PfMSP-3₁₁ region, was identified as the target of protective antibodies in human immune responses (40). The presence of high titers of cytophilic antibodies, IgG3, against this conserved region of MSP-3 has been correlated with protection against the parasite. In addition, immunization of humans with a synthetic peptide corresponding to this region was previously shown to induce antiparasitic antibodies that suppress parasite growth in an ADCI assay (11).It is generally believed that a combination vaccine for malaria is likely to be more effective than vaccines based on a single antigen, and attempts are being made to develop a malaria vaccine by using a mixture of more than one antigen or by combining immunologically relevant proteins of the target antigens as fusion proteins (31, 43, 45). In the present study, we have constructed a fusion chimera (MSP-Fu₂₄) consisting of PfMSP-1₁₉ and PfMSP-3₁₁ and produced the corresponding recombinant MSP-Fu₂₄ protein in Escherichia coli cells. The two individual components, PfMSP-1₁₉ and PfMSP-3₁₁, were also expressed and purified separately; the immunological properties of MSP-Fu₂₄ were compared with a physical mixture of the two individual components. MSP-Fu₂₄ retained the native conformation of the PfMSP-1₁₉ component and was highly immunogenic in small animals. The anti-MSP-Fu₂₄ antibodies inhibited parasite invasion into host red blood cells (RBCs) and also inhibited parasite growth in a monocyte-dependent manner, suggesting the potential of the fusion protein as a malaria vaccine candidate.