A protective glycosylphosphatidylinositol-anchored membrane protein of Plasmodium yoelii trophozoites and merozoites contains two epidermal growth factor-like domains

Using sera from mice immunized and protected against Plasmodium yoelii malaria, we identified a novel blood-stage antigen gene, pypag-2. The 2.1-kb pypag-2 cDNA contains a single open reading frame that encodes a 409-amino-acid protein with a predicted molecular mass of 46.8 kDa. Unlike many characterized plasmodial antigens, blocks of tandemly repeated amino acids are lacking in the pypAg-2 protein sequence. Recombinant pypAg-2, comprising the full-length protein minus the predicted N-terminal signal and C-terminal anchor sequences, was produced and used to raise a high-titer polyclonal rabbit antiserum. This antiserum was used to identify and characterize the native protein through immunoblotting, immunoprecipitation and immunofluorescence assays. Consistent with the presence of a glycosylphosphatidylinositol anchor, pypAg-2 fractionated with the detergent phase of Triton X-114-solubilized proteins and could be metabolically labeled with [(3)H]palmitic acid. By immunofluorescence, pypAg-2 expression was localized to both the trophozoite and merozoite membranes. Similar to Plasmodium falciparum merozoite surface protein 1, pypAg-2 contains two C-terminal epidermal growth factor (EGF)-like domains. Most importantly, immunization with recombinant pypAg-2 protected mice against lethal P. yoelii malaria. Thus, pypAg-2 is a target of protective immune responses and represents a novel addition to the family of merozoite surface proteins that contain one or more EGF-like domains.     

A second merozoite surface protein (MSP-4) of Plasmodium falciparum that contains an epidermal growth factor-like domain.

Merozoite surface proteins of Plasmodium falciparum play a critical role in the invasion of human erythrocytes by the malaria parasite. Here we describe the identification of a novel protein with a molecular mass of 40 kDa that is found on the merozoite surface of P. falciparum. We call this protein merozoite surface protein 4 (MSP-4). Evidence for the surface location of MSP-4 includes (i) a staining pattern that is consistent with merozoite surface location in indirect immunofluorescent studies of cultured parasites, (ii) localization of MSP-4 in the detergent phase in Triton X-114 partitioning studies, and (iii) nucleotide sequencing studies which predict the presence of an N-terminal signal sequence and a hydrophobic C-terminal sequence in the protein. Immunoprecipitation studies of biosynthetically labelled parasites with [3H] myristic acid indicated that MSP-4 is anchored on the merozoite surface by a glycosylphosphatidylinositol moiety. Of considerable interest is the presence of a single epidermal growth factor-like domain at the C terminus of the MSP-4 protein, making it the second protein with such a structure to be found on the merozoite surface.     

Proteolytic processing of the Plasmodium falciparum merozoite surface protein-1 produces a membrane-bound fragment containing two epidermal growth factor-like domains.

The amino-terminal sequence has been obtained for 2 fragments of the Plasmodium falciparum T9/94 merozoite surface protein precursor (PfMSP1) and these have been compared with the sequence predicted from the gene. These data define the position of these fragments in the precursor and indicate that the C-terminal sequence which is carried into the red cell during invasion consists of 2 epidermal growth factor (EGF)-like domains. A homologous cleavage sequence and domain structure can be identified in the MSP1 molecules of other malarial species. In addition the results suggest that the smaller fragment is not N-glycosylated.     

Naturally acquired human antibodies which recognize the first epidermal growth factor-like module in the Plasmodium falciparum merozoite surface protein 1 do not inhibit parasite growth in vitro.

Merozoite surface protein 1, one of the major surface proteins of the invasive blood stage of the malaria parasite, is a prime candidate for the development of a vaccine against the human disease. Previously, monoclonal antibodies which both inhibited the growth of Plasmodium falciparum in vitro and bound to the first of two epidermal growth factor-like modules located near the carboxy terminus of the protein had been identified. In this study, we have used affinity chromatography on a recombinant fusion protein corresponding to the first epidermal growth factor-like module in P. falciparum merozoite surface protein 1 to prepare antibody induced by natural infection. The antibody was purified from the total immunoglobulin G fraction of adult West African donors, shown to passively confer immunity against falciparum malaria. Such affinity-purified antibodies were shown to recognize the native protein by a number of separate criteria and to block the binding of an inhibitory monoclonal antibody, but they failed to inhibit parasite invasion in an in vitro growth assay. These results indicate that antibody alone is not sufficient to interfere with erythrocyte invasion.     

MSP8 is a non-essential merozoite surface protein in Plasmodium falciparum

MSP8 is a recently identified merozoite surface protein that shares similar structural features with the leading vaccine candidate MSP1. Both proteins contain two C-terminal epidermal growth factor (EGF)-like domains, a glycosylphosphatidylinositol (GPI) anchor attachment sequence and undergo proteolytic processing. By double recombination, we have disrupted the MSP8 gene in P. falciparum 3D7 parasites, and confirmed integration by southern hybridisation and PCR. Western blot analysis of lysates from asynchronous cultures and isolated merozoites demonstrated the absence of MSP8 in two cloned knockout lines. There was no significant difference in growth rate observed between 3D7 and the cloned DeltaMSP8 lines. Thus, unlike MSP1, MSP8 is not required for asexual stage parasite growth and replication in vitro. Further analysis of the cloned lines showed that loss of MSP8 had no effect on the levels of expression of other merozoite surface proteins including MSP1-5, 7 and 10. Stage-specific immunoblots showed that MSP8 expression commences in late rings and extends throughout the rest of the erythrocytic life cycle in the 3D7 parent line, but is absent from all stages in the DeltaMSP8 transfectants.     

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4

Microneme proteins are secreted from apicomplexan parasites during invasion of host cells and they play crucial roles in parasite-host cell adhesion. EtMIC4 is a 240 kDa transmembrane protein from Eimeria tenella that contains 31 tandemly arranged epidermal growth factor (EGF), like repeats within its extracellular domain. The majority of these repeats have calcium binding (cb) consensus sequences. Little is known about cbEGFs in apicomplexan parasites but their presence in microneme proteins suggests that they may contribute to parasite-host interactions. To investigate the potential role of cbEGFs we have expressed and correctly refolded a cbEGF triplet from EtMIC4 (cbEGF7-9) and demonstrated that this triplet binds calcium. Circular dichroism spectroscopic analysis of cbEGF7-9 demonstrates that the molecule undergoes a gradual change in conformation with increasing levels of calcium. In the presence of calcium, the triplet becomes resistant to proteolytic degradation by a variety of proteases, a characteristic feature of cbEGF repeats from higher eukaryotic proteins, such as fibrillin, suggesting that calcium binding induces the formation of a rigid conformation. Moreover, mass spectrometric mapping of the cleavage sites that are protected by calcium shows that these sites are located both close to and distant from the calcium binding sites, indicating that protection is not due to steric hindrance by calcium ions, but rather due to the overall conformation adopted by the triplet in the presence of calcium. Thus, the tandemly-arranged cbEGF repeats within EtMIC4 provide a mechanism whereby, in the calcium-rich extracellular environment, the molecule could adopt a protease-resistant, rigid structure that could favour its interaction with host cell ligands.     

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??.     

Temporal Variation of the Merozoite Surface Protein-2 Gene of Plasmodium falciparum

Extensive polymorphism of key parasite antigens is likely to hamper the effectiveness of subunit vaccines against Plasmodium falciparum infection. However, little is known about the extent of the antigenic repertoire of naturally circulating strains in different areas where malaria is endemic. To address this question, we conducted a study in which blood samples were collected from parasitemic individuals living within a small hamlet in Western Irian Jaya and subjected to PCR amplification using primers that would allow amplification of the gene encoding merozoite surface protein-2 (MSP2). We determined the nucleotide sequence of the amplified product and compared the deduced amino acid sequences to sequences obtained from samples collected in the same hamlet 29 months previously. MSP2 genes belonging to both major allelic families were observed at both time points. In the case of the FC27 MSP2 family, we observed that the majority of individuals were infected by parasites expressing the same form of MSP2. Infections with parasites expressing 3D7 MSP2 family alleles were more heterogeneous. No MSP2 alleles observed at the earlier time point were detectable at the later time point, either for the population as a whole or for individuals who were assayed at both time points. We examined a subset of the infected patients by using blood samples taken between the two major surveys. In no patients could we detect reinfection by a parasite expressing a previously encountered form of MSP2. Our results are consistent with the possibility that infection induces a form of strain-specific immune response against the MSP2 antigen that biases against reinfection by parasites bearing identical forms of MSP2.The development of a host-protective immune response against Plasmodium falciparum takes several years and many episodes of infection, at least for children living in areas where malaria is endemic. One of the reasons for this is believed to be the large number of distinct parasite strains circulating within an area of endemicity and the assumption that exposure must occur to a sufficiently large sample of these before lasting immunity is induced. However, the detailed epidemiology of endemic malaria infection remains poorly understood at the molecular level, and there is surprisingly little nucleotide sequence data to support the concept of a large repertoire of antigenically distinct strains.There are at least six antigenically diverse proteins of the asexual stage that are known to be the target of potentially protective host responses. The definition of antigenically distinct strains involves identification of the allelic form expressed at all antigenically diverse loci—the extended antigenic haplotype. The loci would include merozoite surface protein-1 to -3 (3), apical membrane antigen-1 (17), S-antigen (6), and P. falciparum erythrocyte membrane protein-1 (PfEMP-1) (5). Such a complete molecular definition of infecting parasites is a highly ambitious task, particularly in the case of blood samples collected from patients harboring mixed infections. Accordingly, most studies focus on one or other of the antigenically diverse antigens. We have elected to study merozoite surface protein-2 (MSP2) (27), a 45- to 50-kDa glycoprotein anchored in the merozoite surface by a glycosylphosphatidylinositol anchor. This surface protein is a promising candidate for inclusion in a malaria subunit vaccine, as both in vitro and in vivo studies have demonstrated the ability of immune responses to MSP2 to inhibit parasite multiplication (23, 25). However, the efficacy of any subunit vaccine containing a single form of MSP2 may be limited by the presence of antigenically distinct parasite strains within an area of endemicity. We will adopt the recently proposed convention for parasite genes and gene products of denoting the gene sequence as MSP2 and the protein as MSP2.Sequence polymorphism has been described for MSP2 genes of both laboratory-maintained isolates (29) and field isolates (14, 16, 19, 30). Comparison of MSP2 gene sequences from these isolates reveals highly conserved 5′ and 3′ sequences that flank a central variable region. This central region is composed of repeats flanked by nonrepetitive sequences. The nonrepetitive sequences are one or other of two distinct forms that define two allelic families, FC27 and IC-1/3D7 (29). The central repeats are more variable and define the individual alleles of MSP2. There is a correlation between the general form of the central repeat sequence and the allelic family. For example, FC27 family members have variants of a central 96-bp pair sequence that may be present in one to four copies followed by a 21-bp partial repeat and a variably represented 36-bp sequence that may be present in one to five copies. In contrast, alleles belonging to the 3D7 family show a central repeat region made up of variable numbers of 12- to 24-bp repeats separated by repeating 6-bp sequences.Field studies aimed at defining the antigenic diversity of MSP2 have approached the problem by determining MSP2 gene structure by various forms of PCR. The rationale for this is that P. falciparum is haploid and MSP2 has been shown to be present in all laboratory and field isolates examined (8–10, 15). Most studies examining the distribution and frequency of different allelic forms of MSP2 have enumerated the presence of the allelic families (11, 12, 14). Whereas a skewed distribution of predominantly 3D7 family alleles exists among laboratory-adapted strains, in the field a more even distribution of FC27 and 3D7 alleles occurs. Often FC27 family alleles are more prevalent than 3D7 alleles, and novel FC27 and 3D7 family alleles have been found in field malaria strains (14, 16). Some field studies examining recurrent MSP2 infections have been performed, but these have classified MSP2 alleles on the basis of family and length of the central repeat region (7, 11, 14). This makes it difficult to form conclusions about the repertoire of repeat sequences in the circulating pool of parasites and to infer the possible action of immune responses to MSP2 repeats. We were interested to examine the sequences of MSP2 alleles circulating in an area of endemicity over time and to determine the persistence of various MSP2 alleles within a localized area. This study describes MSP2 genotypes from malaria-infected inhabitants of the Oksibil region of Irian Jaya and allows comparison of the variation in MSP2 sequences seen over a 2.5-year period within the region as a whole and in particular individuals.     

Merozoite surface protein 1 of Plasmodium vivax induces a protective response against Plasmodium cynomolgi challenge in rhesus monkeys

The 42-kDa fragment of the merozoite surface protein 1 (MSP-1(42)) is a leading candidate for the development of a vaccine to control malaria. We previously reported a method for the production of Plasmodium vivax MSP-1(42) (PvMSP-1(42)) as a soluble protein (S. Dutta, L. W. Ware, A. Barbosa, C. F. Ockenhouse, and D. E. Lanar, Infect. Immun. 69:5464-5470, 2001). We report here a process to manufacture the same PvMSP-1(42) protein but as an insoluble inclusion body-derived protein which was then refolded in vitro. We compared the immunogenicity and protective efficacy of the soluble and refolded forms of PvMSP-1(42) protein by using a heterologous but closely related P. cynomolgi-rhesus monkey challenge model. As comparative controls we also expressed, purified, and immunized rhesus with the soluble and refolded forms of the P. cynomolgi MSP-1(42) (PcMSP-1(42)) proteins. All proteins induced equally high-titer, cross-reacting antibodies. Upon challenge with P. cynomolgi, none of the MSP-1(42)-vaccinated groups demonstrated sterile protection or a delay in the prepatent period. However, following an initial rise in parasitemia, all MSP-1-vaccinated animals had significantly lower parasite burdens as indicated by lower cumulative parasitemia, lower peak parasitemia, lower secondary peak parasitemia, and lower average daily parasitemia compared to the adjuvant control group (P < 0.05). Except the soluble PcMSP-1(42) group, monkeys in all other groups had fewer numbers of days with parasitemia of >10,000 parasites mm(-3). Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model. The soluble and refolded forms of PcMSP-1(42) and PvMSP-1(42) proteins also appeared to have a similar partially protective effect.     

Plasmodium falciparum merozoite surface protein 6 is a dimorphic antigen

Merozoite surface protein 1 (MSP1) is a highly polymorphic Plasmodium falciparum merozoite surface protein implicated in the invasion of human erythrocytes during the asexual cycle. It forms a complex with MSP6 and MSP7 on the merozoite surface, and this complex is released from the parasite around the time of erythrocyte invasion. MSP1 and many other merozoite surface proteins contain dimorphic elements in their protein structures, and here we show that MSP6 is also dimorphic. The sequences of eight MSP6 genes indicate that the alleles of each dimorphic form of MSP6 are highly conserved. The smaller 3D7-type MSP6 alleles are detected in parasites from all malarious regions of the world, whereas K1-type MSP6 alleles have only been detected in parasites from mainland Southeast Asia. Cleavage of MSP6, which produces the p36 fragment in 3D7-type MSP6 and associates with MSP1, also occurs in K1-type MSP6 but at a different site in the protein. Anti-3D7 MSP6 antibodies weakly inhibited erythrocyte invasion by homologous 3D7 merozoites but did not inhibit a parasite line expressing the K1-type MSP6 allele. Antibodies from hyperimmune individuals affinity purified on an MSP3 peptide cross-reacted with MSP6; therefore, MSP6 may also be a target of antibody-dependent cellular inhibition.     

Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family

The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of cytoadherent proteins has a central role in disease from malaria infection. This highly diverse gene family is involved in binding interactions between infected erythrocytes and host cells and is expressed in a clonally variant pattern at the erythrocyte surface. We describe by sequence analysis the structure and domain organization of 20 PfEMP1 from the GenBank database. Four domains comprise the majority of PfEMP1 extracellular sequence: the N-terminal segment (NTS) located at the amino terminus of all PfEMP1, the C2, the Cysteine-rich Interdomain Region (CIDR) and the Duffy Binding-like (DBL) domains. Previous work has shown that CIDR and DBL domains can possess adhesive properties. CIDR domains grouped as three distinct sequence classes (, β, and γ) and DBL domains as five sequence classes (, β, γ, δ, and ). Consensus motifs are described for the different DBL and CIDR types. Whereas the number of DBL and CIDR domains vary between PfEMP1, PfEMP1 domain architecture is not random in that certain tandem domain associations — such as DBLCIDR, DBLδCIDRβ, and DBLβC2 — are preferentially observed. This conservation may have functional significance for PfEMP1 folding, transport, or binding activity. Parasite binding phenotype appears to be a determinant of infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome. The sequence classification of DBL and CIDR types may have predictive value for identifying PfEMP1 domains with a particular binding property. This information might be used to develop interventions targeting parasite binding variants that cause disease.     

Plasmodium falciparum merozoite surface protein 8 is a ring-stage membrane protein that localizes to the parasitophorous vacuole of infected erythrocytes

To date, the following seven glycosylphosphatidylinositol (GPI)-anchored merozoite antigens have been described in Plasmodium falciparum: merozoite-associated surface protein 1 (MSP-1), MSP-2, MSP-4, MSP-5, MSP-8, MSP-10, and the rhoptry-associated membrane antigen. Of these, MSP-1, MSP-8, and MSP-10 possess a double epidermal growth factor (EGF)-like domain at the C terminus, and these modules are considered potential targets of protective immunity. In this study, we found that surprisingly, P. falciparum MSP-8 is transcribed and translated in the ring stage and is absent from the surface of merozoites. MSP-8 is the only GPI-anchored protein known to be expressed at this time. It is synthesized as a mature 80-kDa protein which is rapidly processed to a C-terminal 17-kDa species that contains the double EGF module. As determined by a combination of immunofluorescence and membrane purification approaches, it appears likely that MSP-8 initially localizes to the parasite plasma membrane in the ring stage. Although the C-terminal 17-kDa fragment is present in more mature stages, at these times it is found in the food vacuole. We successfully disrupted the MSP-8 gene in P. falciparum, a process that validated the specificity of the antibodies used in this study and also demonstrated that MSP-8 does not play a role essential to maintenance of the erythrocyte cycle. This finding, together with the observation that MSP-8 is exclusively intracellular, casts doubt over the viability of this antigen as a vaccine. However, it is still possible that MSP-8 is involved in an early parasitophorous vacuole function that is significant for pathogenesis in the human host.     

Sequential Processing of Merozoite Surface Proteins during and after Erythrocyte Invasion by Plasmodium falciparum

Plasmodium falciparum causes malaria disease during the asexual blood stages of infection when merozoites invade erythrocytes and replicate. Merozoite surface proteins (MSPs) are proposed to play a role in the initial binding of merozoites to erythrocytes, but precise roles remain undefined. Based on electron microscopy studies of invading Plasmodium merozoites, it is proposed that the majority of MSPs are cleaved and shed from the surface during invasion, perhaps to release receptor-ligand interactions. In this study, we demonstrate that there is not universal cleavage of MSPs during invasion. Instead, there is sequential and coordinated cleavage and shedding of proteins, indicating a diversity of roles for surface proteins during and after invasion. While MSP1 and peripheral surface proteins such as MSP3, MSP7, serine repeat antigen 4 (SERA4), and SERA5 are cleaved and shed at the tight junction between the invading merozoite and erythrocyte, the glycosylphosphatidylinositol (GPI)-anchored proteins MSP2 and MSP4 are carried into the erythrocyte without detectable processing. Following invasion, MSP2 rapidly degrades within 10 min, whereas MSP4 is maintained for hours. This suggests that while some proteins that are shed upon invasion may have roles in initial contact steps, others function during invasion and are then rapidly degraded, whereas others are internalized for roles during intraerythrocytic development. Interestingly, anti-MSP2 antibodies did not inhibit invasion and instead were carried into erythrocytes and maintained for approximately 20 h without inhibiting parasite development. These findings provide new insights into the mechanisms of invasion and knowledge to advance the development of new drugs and vaccines against malaria.     

Antigenic Characterization of an Intrinsically Unstructured Protein,Plasmodium falciparum Merozoite Surface Protein 2

Merozoite surface protein 2 (MSP2) is an abundant glycosylphosphatidylinositol (GPI)-anchored protein of Plasmodium falciparum, which is a potential component of a malaria vaccine. As all forms of MSP2 can be categorized into two allelic families, a vaccine containing two representative forms of MSP2 may overcome the problem of diversity in this highly polymorphic protein. Monomeric recombinant MSP2 is an intrinsically unstructured protein, but its conformational properties on the merozoite surface are unknown. This question is addressed here by analyzing the 3D7 and FC27 forms of recombinant and parasite MSP2 using a panel of monoclonal antibodies raised against recombinant MSP2. The epitopes of all antibodies, mapped using both a peptide array and by nuclear magnetic resonance (NMR) spectroscopy on full-length recombinant MSP2, were shown to be linear. The antibodies revealed antigenic differences, which indicate that the conserved N- and C-terminal regions, but not the central variable region, are less accessible in the parasite antigen. This appears to be an intrinsic property of parasite MSP2 and is not dependent on interactions with other merozoite surface proteins as the loss of some conserved-region epitopes seen using the immunofluorescence assay (IFA) on parasite smears was also seen on Western blot analyses of parasite lysates. Further studies of the structural basis of these antigenic differences are required in order to optimize recombinant MSP2 constructs being evaluated as potential vaccine components.     

Structural and antigenic properties of merozoite surface protein 4 of Plasmodium falciparum

Merozoite surface protein 4 (MSP4) of Plasmodium falciparum is a glycosylphosphatidylinositol-anchored integral membrane protein of 272 residues that possesses a single epidermal growth factor (EGF)-like domain near the carboxyl terminus. We have expressed both full-length MSP4 and a number of fragments in Escherichia coli and have used these recombinant proteins to raise experimental antisera. All recombinant proteins elicited specific antibodies that reacted with parasite-derived MSP4 by immunoblotting. Antibody reactivity was highly dependent on the protein conformation. For example, reduction and alkylation of MSP4 almost completely abolished the reactivity of several antibody preparations, including specificities directed to regions of the protein that do not contain cysteine residues and are far removed from the cysteine-containing EGF-like domain. This indicated the presence of conformation-dependent epitopes in MSP4 and demonstrated that proper folding of the EGF-like domain influenced the antigenicity of the entire molecule. The recombinant proteins were used to map epitopes recognized by individuals living in areas where malaria is endemic, and at least four distinct regions are naturally antigenic during infection. Binding of human antibodies to the EGF-like domain was essentially abrogated after reduction of the recombinant protein, indicating the recognition of conformational epitopes by the human immune responses. This observation led us to examine the importance of conformation dependence in responses to other integral membrane proteins of asexual stages. We analyzed the natural immune responses to a subset of these antigens and demonstrated that there is diminished reactivity to several antigens after reduction. These studies demonstrate the importance of reduction-sensitive structures in the maintenance of the antigenicity of several asexual-stage antigens and in particular the importance of the EGF-like domain in the antigenicity of MSP4.     

Development of two monoclonal antibodies against Plasmodium falciparum sporozoite surface protein 2 and mapping of B-cell epitopes.

The Plasmodium yoelii sporozoite surface protein 2 (PySSP2) is the target of protective cellular immunity. Cytotoxic T cells specific for the Plasmodium falciparum analog PfSSP2, also known as thrombospondin-related anonymous protein (TRAP), are induced in human volunteers immunized with irradiated sporozoites. PfSSP2 is an important candidate antigen for a multicomponent malaria vaccine. We generated and characterized three monoclonal antibodies (MAbs) specific for PfSSP2/TRAP. The MAbs PfSSP2.1 (immunoglobulin G1 [IgG1]), PfSSP2.2 (IgG2a), and PfSSP2.3 (IgM) were species specific and identified three distinct B-cell epitopes containing sequences DRYI, CHPSDGKC, and TRPHGR, respectively. PfSSP2.1 partially inhibited P. falciparum liver-stage parasite development in human hepatocyte cultures (42 and 86% in two experiments at 100 microg/ml). Mice immunized with vaccinia virus expressing full-length PfSSP2 protein produced antibodies to (DRYIPYSP)3, and humans living in malaria-endemic areas (Indonesia and Kenya), who have lifelong exposure and partial clinical immunity to malaria, had antibodies to both (DRYIPYSP)3 and (CHPSDGKCN)2. Mice immunized with multiple antigen peptides MAP4 (DRYIPYSP)3P2P30 and MAP4 (CHPSDGKCN)3P2P30 in TiterMax developed antibodies to sporozoites that partially inhibited sporozoite invasion of human hepatoma cells (39 to 71% at a serum dilution of 1:50 in three different experiments). The modest inhibitory activities of the MAbs and the polyclonal antibodies to PfSSP2/TRAP epitopes do not suggest that a single-component vaccine designed to induce antibodies against PfSSP2/TRAP will be protective. Nonetheless, the MAbs directed against PfSSP2, and the peptides recognized by these MAbs, will be essential reagents in the development of PfSSP2/TRAP as a component of a multivalent P. falciparum human malaria vaccine.     

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.