Protective immune responses to the 42-kilodalton (kDa) region of Plasmodium yoelii merozoite surface protein 1 are induced by the C-terminal 19-kDa region but not by the adjacent 33-kDa region.

Vaccination of mice with the 42-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP1(42)) or its 19-kDa C-terminal processing product (MSP1(19)) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP1(33)) of MSP1(42) also induces protection, the gene segment encoding MSP1(33) was expressed as a glutathione S-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP1(33) and subsequently challenged with the lethal P. yoelii YM blood stage parasite. GST-MSP1(33) failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP1(19) were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP1(33) had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP1(19). The finding that P. yoelii MSP1(33) elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based on Plasmodium falciparum or Plasmodium vivax MSP1(42).     

The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites

A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.     

Immunogenicity and efficacy in aotus monkeys of four recombinant Plasmodium falciparum vaccines in multiple adjuvant formulations based on the 19-kilodalton C terminus of merozoite surface protein 1

The immunogenicity and protective efficacy of four versions of recombinant C-terminal 19-kDa epidermal growth factor-like region of the major surface protein 1 (rMSP1(19)) of Plasmodium falciparum was studied in Aotus monkeys. Vaccination with each of the four rMSP1(19) constructs elicited high levels of antibodies to MSP1(19) but only one construct, the 19-kDa fragment expressed as a secreted fusion protein from Saccharomyces cerevisiae (yP30P2MSP1(19)), induced a high degree of protective immunity in Aotus nancymai against lethal P. falciparum challenge. Protective formulation required Freund's adjuvant; vaccination with yP30P2MSP1(19) in six other adjuvants that are suitable for human use induced lower levels of antibody response and no protection. These results emphasize the need to continue the search for an adjuvant that is comparable to Freund's adjuvant in potency and is safe for use in humans.     

Efficacy of two alternate vaccines based on Plasmodium falciparum merozoite surface protein 1 in an Aotus challenge trial

In an attempt to produce a more defined, clinical-grade version of a vaccine based on Plasmodium falciparum merozoite surface protein 1 (MSP1), we evaluated the efficacy of two recombinant forms of MSP1 in an Aotus nancymai challenge model system. One recombinant vaccine, bvMSP1(42), based on the 42-kDa C-terminal portion of MSP1, was expressed as a secreted protein in baculovirus-infected insect cells. A highly pure baculovirus product could be reproducibly expressed and purified at yields in excess of 8 mg of pure protein per liter of culture. This protein, when tested for efficacy in the Aotus challenge model, gave significant protection, with only one of seven monkeys requiring treatment for uncontrolled parasitemia after challenge with P. falciparum. The second recombinant protein, P30P2MSP1(19), has been used in previous studies and is based on the smaller, C-terminal 19-kDa portion of MSP1 expressed in Saccharomyces cerevisiae. Substantial changes were made in its production process to optimize expression. The optimum form of this vaccine antigen (as judged by in vitro and in vivo indicators) was then evaluated, along with bvMSP1(42), for efficacy in the A. nancymai system. The new formulation of P30P3MSP1(19) performed significantly worse than bvMSP1(42) and appeared to be less efficacious than we have found in the past, with four of seven monkeys in the vaccinated group requiring treatment for uncontrolled parasitemia. With both antigens, protection was seen only when high antibody levels were obtained by formulation of the vaccines in Freund's adjuvant. Vaccine formulation in an alternate adjuvant, MF59, resulted in significantly lower antibody titers and no protection.     

The 22 kDa component of the protein complex on the surface of Plasmodium falciparum merozoites is derived from a larger precursor, merozoite surface protein 7

The gene coding for merozoite surface protein 7 has been identified and sequenced in three lines of Plasmodium falciparum. The gene encodes a 351 amino acid polypeptide that is the precursor of a 22-kDa protein (MSP7(22)) on the merozoite surface and non-covalently associated with merozoite surface protein 1 (MSP1) complex shed from the surface at erythrocyte invasion. A second 19-kDa component of the complex (MSP7(19)) was shown to be derived from MSP7(22) and the complete primary structure of this polypeptide was confirmed by mass spectrometry. The protein sequence contains several predicted helical and two beta elements, but has no similarity with sequences outside the Plasmodium databases. Four sites of sequence variation were identified in MSP7, all within the MSP7(22) region. The MSP7 gene is expressed in mature schizonts, at the same time as other merozoite surface protein genes. It is proposed that MSP7(22) is the result of cleavage by a protease that may also cleave MSP1 and MSP6. A related gene was identified and cloned from the rodent malaria parasite, Plasmodium yoelii YM; at the amino acid level this sequence was 23% identical and 50% similar to that of P. falciparum MSP7.     

Processing of the Plasmodium falciparum major merozoite surface protein-1: identification of a 33-kilodalton secondary processing product which is shed prior to erythrocyte invasion.

We have previously shown that only a single 19-kDa fragment of the Plasmodium falciparum major merozoite surface protein (MSP1) is carried with an invading merozoite into the infected red cell. This fragment (MSP1(19] is derived from the C-terminal membrane-bound end of a major product, MSP1(42), of the primary stage of MSP1 proteolytic processing. Using a monoclonal antibody mapped to an epitope within the N-terminal region of MSP1(42), we have shown that a soluble 33-kDa polypeptide (MSP1(33) corresponding to the N-terminal region of MSP1(42) is shed into culture supernatants during merozoite release and erythrocyte invasion. These observations provide further evidence that the secondary processing of MSP1(42) involves a highly site-specific proteolytic activity.     

Merozoite surface protein 4/5 provides protection against lethal challenge with a heterologous malaria parasite strain

Immunization with merozoite surface protein 4/5 (MSP4/5), the murine malaria homologue of Plasmodium falciparum MSP4 and MSP5, has been shown to protect mice against challenge by parasites expressing the homologous form of the protein. The gene encoding MSP4/5 was sequenced from a number of Plasmodium yoelii isolates in order to assess the level of polymorphism in the protein. The gene was found to be highly conserved among the 13 P. yoelii isolates sequenced, even though many of the same isolates showed pronounced variability in their MSP1(19) sequences. Nonsynonymous mutations were detected only for the isolates Plasmodium yoelii nigeriensis N67 and Plasmodium yoelii killicki 193L and 194ZZ. Immunization and challenge of BALB/c mice showed that the heterologous MSP4/5 proteins were able to confer a level of protection against lethal Plasmodium yoelii yoelii YM challenge infection similar to that induced by immunization with the homologous MSP4/5 protein. To explore the limits of heterologous protection, mice were immunized with recombinant MSP4/5 protein from Plasmodium berghei ANKA and Plasmodium chabaudi adami DS and challenged with P. y. yoelii YM. Interestingly, significant protection was afforded by P. berghei ANKA MSP4/5, which shows 81% sequence identity with P. y. yoelii YM MSP4/5, but it was abolished upon reduction and alkylation. Significant protection was not observed for mice immunized with recombinant P. c. adami DS MSP4/5, which shows 55.7% sequence identity with P. y. yoelii YM MSP4/5. This study demonstrates the robustness of MSP4/5 in conferring protection against variant forms of the protein in a murine challenge system, in contrast to the situation found for other asexual-stage proteins, such as MSP1(19) and AMA1.     

Biochemical and immunological characterization of bacterially expressed and refolded Plasmodium falciparum 42-kilodalton C-terminal merozoite surface protein 1

Protection against Plasmodium falciparum can be induced by vaccination in animal models with merozoite surface protein 1 (MSP1), which makes this protein an attractive vaccine candidate for malaria. In an attempt to produce a product that is easily scaleable and inexpensive, we expressed the C-terminal 42 kDa of MSP1 (MSP1(42)) in Escherichia coli, refolded the protein to its native form from insoluble inclusion bodies, and tested its ability to elicit antibodies with in vitro and in vivo activities. Biochemical, biophysical, and immunological characterization confirmed that refolded E. coli MSP1(42) was homogeneous and highly immunogenic. In a formulation suitable for human use, rabbit antibodies were raised against refolded E. coli MSP1(42) and tested in vitro in a P. falciparum growth invasion assay. The antibodies inhibited the growth of parasites expressing either homologous or heterologous forms of P. falciparum MSP1(42). However, the inhibitory activity was primarily a consequence of antibodies directed against the C- terminal 19 kDa of MSP1 (MSP1(19)). Vaccination of nonhuman primates with E. coli MSP1(42) in Freund's adjuvant protected six of seven Aotus monkeys from virulent infection with P. falciparum. The protection correlated with antibody-dependent mechanisms. Thus, this new construct, E. coli MSP1(42), is a viable candidate for human vaccine trials.     

A recombinant baculovirus 42-kilodalton C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 protects Aotus monkeys against malaria.

The immunogenicity and protective efficacy of 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 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 days after injection. 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.     

Oral immunization with a combination of Plasmodium yoelii merozoite surface proteins 1 and 4/5 enhances protection against lethal malaria challenge

Oral immunization of mice with Escherichia coli-expressed Plasmodium yoelii merozoite surface protein 4/5 or the C-terminal 19-kDa fragment of merozoite surface protein 1 induced systemic antibody responses and protected mice against lethal malaria infection. A combination of these two proteins administered orally conferred improved protection compared to that conferred by either protein administered alone.     

Enhanced protection against malaria by a chimeric merozoite surface protein vaccine

The 42-kDa processed fragment of Plasmodium falciparum merozoite surface protein 1 (MSP-1(42)) is a prime candidate for a blood-stage malaria vaccine. Merozoite surface protein 8 contains two C-terminal epidermal growth factor (EGF)-like domains that may function similarly to those of MSP-1(42). Immunization with either MSP-1 or MSP-8 induces protection that is mediated primarily by antibodies against conformation-dependent epitopes. In a series of comparative immunogenicity and efficacy studies using the Plasmodium yoelii rodent model, we tested the ability of recombinant P. yoelii MSP-8 (rPyMSP-8) to complement rPyMSP-1-based vaccines. Unlike MSP-1, PyMSP-8-dependent protection required immunization with the full-length protein and was not induced with recombinant antigens that contained only the C-terminal EGF-like domains. Unlike PyMSP-8, the immunogenicity of the PyMSP-1 EGF-like domains was low when present as part of the rPyMSP-1(42) antigen. Immunization with a mixture of rPyMSP-1(42) and rPyMSP-8 further inhibited the antibody response to protective epitopes of rPyMSP-1(42) and did not improve vaccine efficacy. To improve PyMSP-1 immunogenicity, we produced a chimeric antigen containing the EGF-like domains of PyMSP-1 fused to the N terminus of PyMSP-8. Immunization with the chimeric rPyMSP-1/8 antigen induced high and comparable antibody responses against the EGF-like domains of both PyMSP-1 and PyMSP-8. This enhanced MSP-1-specific antibody response and the concurrent targeting of MSP-1 and MSP-8 resulted in improved, nearly complete protection against lethal P. yoelii 17XL malaria. Unexpectedly, immunization with rPyMSP-1/8 failed to protect against challenge infection with reticulocyte-restricted P. yoelii 17X parasites. Overall, these data establish an effective strategy to improve the efficacy of P. falciparum MSP-based vaccines.     

Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys.

We have shown that conserved B epitopes were immunodominant in animals hyperimmunized with parasite-purified 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.     

Analysis of immunological nonresponsiveness to the 19-kilodalton fragment of merozoite surface Protein 1 of Plasmodium yoelii: rescue by chemical conjugation to diphtheria toxoid (DT) and enhancement of immunogenicity by prior DT vaccination

The Plasmodium merozoite surface protein 1 (MSP1) is a leading vaccine candidate for protecting against the blood stage of malaria. Previous studies have shown that the 19-kDa carboxyl terminus of this protein is able to induce protective immunity in some monkey and mouse strains. We show that immunization with the recombinant Plasmodium yoelii 19-kDa fragment of MSP1 (MSP1(19)) expressed in Saccharomyces cerevisiae (yMSP1(19)) can induce protective antibodies in several inbred mouse strains and one outbred mouse strain. However, mice expressing the H-2(s) major histocompatibility complex haplotype are unable to generate yMSP1(19)-specific antibodies. While synthetic peptides derived from MSP1(19) are immunogenic in B10.S mice, they cannot function as helper epitopes, and immunization with yMSP1(19) does not induce T cells that recognize the recombinant protein or synthetic peptides corresponding to its sequence. Nonresponsiveness could be overcome by using chemical linkers to conjugate yMSP1(19) to diphtheria toxoid (DT), resulting in immunogens capable of inducing protective yMSP1(19)-specific antibodies in both MSP1(19)-responsive and otherwise nonresponsive mouse strains. The ability of sera from mice immunized with the conjugate to inhibit binding of a protective monoclonal antibody (MAb 302) to yMSP1(19) correlated strongly with a delay in the prepatent period. Chemical conjugation of yMSP1(19) to DT may be a preferred method to enhance immunogenicity, as carrier priming experiments demonstrated that an existing immune response to DT enhanced a subsequent antibody response to yMSP1(19) after vaccination with yMSP1(19)-DT. These results have important implications for the development of a malaria vaccine to protect a population with diverse HLAs.     

Linkage of exogenous T-cell epitopes to the 19-kilodalton region of Plasmodium yoelii merozoite surface protein 1 (MSP1(19)) can enhance protective immunity against malaria and modulate the immunoglobulin subclass response to MSP1(19)

The degree of protection against Plasmodium yoelii asexual blood stages induced by immunization of mice with the 19-kDa region of merozoite surface protein 1 (MSP1(19)) is H-2 dependent. As a strategy to improve the protection, mouse strains with disparate H-2 haplotypes were immunized with glutathione S-transferase (GST)-MSP1(19) proteins including either a universal T-cell epitope from tetanus toxin (P2) or an I-A(k)-restricted T-cell epitope (P8) from Plasmodium falciparum Pf332. In H-2(k) mice which are poorly protected following immunization with GST-MSP1(19), GST-P2-MSP1(19) significantly improved the protection. In mice partially (H-2(k/b)) or well protected by GST-MSP1(19) (H-2(d) and H-2(b)), P2 did not further increase the protection. However, the protection of H-2(k/b) mice and to some extent H-2(k) mice was improved by immunization with GST-P8-MSP1(19). The magnitudes of immunoglobulin G1 (IgG1) and IgG2a responses in mice immunized with the GST-MSP1(19) variants correlated with low peak parasitemia, indicating a protective capacity of these IgG subclasses. In H-2(k) mice immunized with GST-P2-MSP1(19), both IgG1 and IgG2a responses were significantly enhanced. The epitope P2 appeared to have a general ability to modulate the IgG subclass response since all four mouse strains displayed elevated IgG2a and/or IgG2b levels after immunization with GST-P2-MSP1(19). In contrast, GST-P8-MSP1(19) induced a slight enhancement of IgG responses in H-2(k/b) and H-2(k) mice without any major shift in IgG subclass patterns. The ability to improve the protective immunity elicited by P. yoelii MSP1(19) may have implications for improvement of human vaccines based on P. falciparum MSP1(19).     

Immunological cross-reactivity of the C-terminal 42-kilodalton fragment of Plasmodium falciparum merozoite surface protein 1 expressed in baculovirus.

The roles of allelic and conserved epitopes in vaccine-induced immunity to the C-terminal 42-kDa fragment of the Plasmodium falciparum 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 approximately 50% of the BVp42 molecule, anti-BVp42 antibodies cross-reacted extensively with parasites carrying heterologous MSP1 alleles. Enzyme-linked immunosorbent inhibition assays confirmed that an overwhelming majority of the anti-BVp42 antibodies were cross-reactive, suggesting that determinants within conserved block 17 are dominant B-cell epitopes in the anti-BVp42 response. Moreover, the BVp42 polypeptide could inhibit (> 90%) the cross-reactivity of anti-MSP1 antibodies in animals immunized with the complete native MSP1 protein. Anti-BVp42 antibodies were equally effective in inhibiting the in vitro growth of parasites carrying homologous or heterologous MSP1 alleles. Serotyping by monoclonal antibodies indicated that the immunological and biological cross-reactivities were not caused by identical variant-specific amino acid substitutions within conserved block 17. These results should provide the impetus to develop a vaccine based on the C-terminal conserved region(s) of MSP1 against parasites of diverse genetic makeup.     

Nature and specificity of the required protective immune response that develops postchallenge in mice vaccinated with the 19-kilodalton fragment of Plasmodium yoelii merozoite surface protein 1

Immunity induced by the 19-kDa fragment of Plasmodium yoelii merozoite surface protein 1 (MSP1(19)) is dependent on high titers of specific antibodies present at the time of challenge and a continuing active immune response postinfection. However, the specificity of the active immune response postinfection has not been defined. In particular, it is not known whether anti-MSP1(19) antibodies that arise following infection alone are sufficient for protection. We developed systems to investigate whether an MSP1(19)-specific antibody response alone both prechallenge and postchallenge is sufficient for protection. We were able to exclude antibodies with other specificities, as well as any contribution of MSP1(19)-specific CD4(+) T cells acting independent of antibody, and we concluded that an immune response focused solely on MSP1(19)-specific antibodies is sufficient for protection. The data imply that the ability of natural infection to boost an MSP1(19)-specific antibody response should greatly improve vaccine efficacy.     

Truncation of Plasmodium berghei merozoite surface protein 8 does not affect in vivo blood-stage development

Merozoite surface protein 8 (MSP8) has shown promise as a vaccine candidate in the Plasmodium yoelii rodent malaria model and has a proposed role in merozoite invasion of erythrocytes. However, the temporal expression and localisation of MSP8 are unusual for a merozoite antigen. Moreover, in Plasmodium falciparum the MSP8 gene could be disrupted with no apparent effect on invitro growth. To address the invivo function of full-length MSP8, we truncated MSP8 in the rodent parasite Plasmodium berghei. PbDeltaMSP8 disruptant parasites displayed a normal blood-stage growth rate but no increase in reticulocyte preference, a phenomenon observed in P. yoelii MSP8 vaccinated mice. Expression levels of erythrocyte surface antigens were similar in P. berghei wild-type and PbDeltaMSP8-infected erythrocytes, suggesting that a parasitophorous vacuole function for MSP8 does not involve global trafficking of such antigens. These data demonstrate that a full-length membrane-associated form of PbMSP8 is not essential for blood-stage growth.     

Evaluation of the immunogenicity and vaccine potential of recombinant Plasmodium falciparum merozoite surface protein 8

The C-terminal 19-kDa domain of merozoite surface protein 1 (MSP1??) is the target of protective antibodies but alone is poorly immunogenic. Previously, using the Plasmodium yoelii murine model, we fused P. yoelii MSP1?? (PyMSP1??) with full-length P. yoelii merozoite surface protein 8 (MSP8). Upon immunization, the MSP8-restricted T cell response provided help for the production of high and sustained levels of protective PyMSP1??- and PyMSP8-specific antibodies. Here, we assessed the vaccine potential of MSP8 of the human malaria parasite, Plasmodium falciparum. Distinct from PyMSP8, P. falciparum MSP8 (PfMSP8) contains an N-terminal asparagine and aspartic acid (Asn/Asp)-rich domain whose function is unknown. Comparative analysis of recombinant full-length PfMSP8 and a truncated version devoid of the Asn/Asp-rich domain, PfMSP8(ΔAsn/Asp), showed that both proteins were immunogenic for T cells and B cells. All T cell epitopes utilized mapped within rPfMSP8(ΔAsn/Asp). The dominant B cell epitopes were conformational and common to both rPfMSP8 and rPfMSP8(ΔAsn/Asp). Analysis of native PfMSP8 expression revealed that PfMSP8 is present intracellularly in late schizonts and merozoites. Following invasion, PfMSP8 is found distributed on the surface of ring- and trophozoite-stage parasites. Consistent with a low and/or transient expression of PfMSP8 on the surface of merozoites, PfMSP8-specific rabbit IgG did not inhibit the in vitro growth of P. falciparum blood-stage parasites. These studies suggest that the further development of PfMSP8 as a malaria vaccine component should focus on the use of PfMSP8(ΔAsn/Asp) and its conserved, immunogenic T cell epitopes as a fusion partner for protective domains of poor immunogens, including PfMSP1??.     

Long-lasting protective immune response to the 19-kilodalton carboxy-terminal fragment of Plasmodium yoelii merozoite surface protein 1 in mice

Merozoite surface protein 1 (MSP1) is the major protein on the surface of the plasmodial merozoite, and its carboxy terminus, the 19-kDa fragment (MSP1(19)), is highly conserved and effective in induction of a protective immune response against malaria parasite infection in mice and monkeys. However, the duration of the immune response has not been elucidated. As such, we immunized BALB/c mice with a standard four-dose injection of recombinant Plasmodium yoelii MSP1(19) formulated with Montanide ISA51 and CpG oligodeoxynucleotide (ODN) and monitored the MSP1(19)-specific antibody levels for up to 12 months. The antibody titers persisted constantly over the period of time without significant waning, in contrast to the antibody levels induced by immunization with Freund's adjuvant, where the antibody levels gradually declined to significantly lower levels 12 months after immunization. Investigation of immunoglobulin G (IgG) subclass longevity revealed that only the IgG1 antibody level (Th2 type-driven response) decreased significantly by 6 months, while the IgG2a antibody level (Th1 type-driven response) did not change over the 12 months after immunization, but the boosting effect was seen in the IgG1 antibody responses but not in the IgG2a antibody responses. After challenge infection, all immunized mice survived with negligibly patent parasitemia. These findings suggest that protective immune responses to MSP1(19) following immunization using oil-based Montanide ISA51 and CpG ODN as an adjuvant are very long-lasting and encourage clinical trials for malaria vaccine development.     

Immunization of Aotus nancymai with recombinant C terminus of Plasmodium falciparum merozoite surface protein 1 in liposomes and alum adjuvant does not induce protection against a challenge infection.

Merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is an antimalarial vaccine candidate. The highly conserved 19-kDa C-terminal processing fragment of MSP-1 (MSP-1(19)) is of particular interest since it contains epitopes recognized by monoclonal antibodies which inhibit the invasion of erythrocytes in vitro. The presence of naturally acquired anti-MSP-1(19) antibodies in individuals exposed to malaria has been correlated with reduced morbidity, and immunization with an equivalent recombinant P. yoelii antigen induces substantial protection against this parasite in mice. We have expressed P. falciparum MSP-1(19) in Escherichia coli as a correctly folded protein and immunized Aotus nancymai monkeys by using the protein incorporated into liposomes and adsorbed to alum. After vaccination, the sera from these animals contained anti-MSP-1(19) antibodies, some of which competed for binding to MSP-1(19) with monoclonal antibodies that inhibit parasite invasion of erythrocytes in vitro. However, after challenge with either a homologous or a heterologous strain of parasite, all animals became parasitemic and required treatment. The immunization did not induce protection in this animal model.