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.     

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.     

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.     

Analysis of recombinant merozoite surface protein-1 of Plasmodium falciparum expressed in mammalian cells

Synthetic chimeric DNA constructs with a reduced A + T content coding for full-length merozoite surface protein-1 of Plasmodium falciparum (MSP1) and three fragments thereof were expressed in HeLa cells. To target the recombinant proteins to the surface of the host cell the DNA sequences coding for the N-terminal signal sequence and for the putative C-terminal recognition/attachment signal for the glycosyl-phosphatidyl-inositol (GPI)-anchor of MSP1 were replaced by the respective DNA sequences of the human decay-accelerating-factor (DAF). The full-length recombinant protein, hu-MSP1-DAF, was stably expressed and recognised by monoclonal antibodies that bind to the N-terminus or the C-terminus of the native protein, respectively. Its apparent molecular mass is higher as compared to the native protein and it is post-translationally modified by attachment of N-glycans whereas native MSP1 is not glycosylated. Immunofluorescence images of intact cells show a clear surface staining. After permeabilization hu-MSP1-DAF can be detected in the cytosol as well. As judged by protease treatment of intact cells 25% of recombinant MSP1 is located on the surface. This fraction of hu-MSP1-DAF can be cleaved off the cell membrane by phosphatidylinositol-specific phospholipase C indicating that the protein is indeed bound to the cell membrane via a GPI-anchor. Human erythrocytes do not adhere to the surface of mammalian cells expressing either of the constructs made in this study.     

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.     

The merozoite surface protein 6 gene codes for a 36 kDa protein associated with the Plasmodium falciparum merozoite surface protein-1 complex

A complex of non-covalently bound polypeptides is located on the surface of the merozoite form of the human malaria parasite Plasmodium falciparum. Four of these polypeptides are derived by proteolytic processing of the merozoite surface protein 1 (MSP-1) precursor. Two components, a 22 and a 36 kDa polypeptide are not derived from MSP-1. The N-terminal sequence of the 36 kDa polypeptide has been determined, the corresponding gene cloned, and the protein characterised. The 36 kDa protein consists of 211 amino acids and is derived from a larger precursor of 371 amino acids. The precursor merozoite surface protein 6 (MSP-6) has been designated, and the 36 kDa protein, MSP-6(36). Mass spectrometric analysis of peptides released from the polypeptide by tryptic digestion confirmed that the gene identified codes for MSP-6(36). Antibodies were produced to a recombinant protein containing the C-terminal 45 amino acid residues of MSP-6(36). In immunofluorescence studies these antibodies bound to antigen at the parasite surface or in the parasitophorous vacuole within schizonts, with a pattern indistinguishable from that of antibodies to MSP-1. MSP-6(36) was present in the MSP-1 complex immunoprecipitated from the supernatant of in vitro parasite cultures, but was also immunoprecipitated from this supernatant in a form not bound to MSP-1. Examination of the MSP-6 gene in three parasite lines detected no sequence variation. The sequence of MSP-6(36) is related to that of the previously described merozoite surface protein 3 (MSP-3). The MSP-6(36) amino acid sequence has 50% identity and 85% similarity with the C-terminal region of MSP-3. The proteins share a specific sequence pattern (ILGWEFGGG-[AV]-P) and a glutamic acid-rich region. The remainder of MSP-6 and MSP-3 are unrelated, except at the N-terminus. Both MSP-6(36) and MSP-3 are partially associated with the parasite surface and partially released as soluble proteins on merozoite release. MSP-6(36) is a hydrophilic negatively charged polypeptide, but there are two clusters of hydrophobic amino acids at the C-terminus, located in two amphipathic helical structures identified from secondary structure predictions. It was suggested that this 35 residue C-terminal region may be involved in MSP-6(36) binding to MSP-1 or other molecules; alternatively, based on the secondary structure and coil formation predictions, the region may form an intramolecular anti-parallel coiled-coil structure.     

Expression, extracellular secretion, and immunogenicity of the Plasmodium falciparum sporozoite surface protein 2 in Salmonella vaccine strains

Deleting transmembrane alpha-helix motifs from Plasmodium falciparum sporozoite surface protein (SSP-2) allowed its secretion from Salmonella enterica serovar Typhimurium SL3261 and S. enterica serovar Typhi CVD 908-htrA by the Hly type I secretion system. In mice immunized intranasally, serovar Typhimurium constructs secreting SSP-2 stimulated greater gamma interferon splenocyte responses than did nonsecreting constructs (P = 0.04).     

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.     

Characterization of conserved T- and B-cell epitopes in Plasmodium falciparum major merozoite surface protein 1

Vaccines for P. falciparum will need to contain both T- and B-cell epitopes. Conserved epitopes are the most desirable, but they are often poorly immunogenic. The major merozoite surface protein 1 (MSP-1) is currently a leading vaccine candidate antigen. In this study, six peptides from conserved or partly conserved regions of MSP-1 were evaluated for immunogenicity in B10 congenic mice. Following immunization with the peptides, murine T cells were tested for the ability to proliferate in vitro and antibody responses to MSP-1 were evaluated in vivo. The results showed that one highly conserved sequence (MSP-1#1, VTHESYQELVKKLEALEDAV; located at amino acid positions 20 to 39) and one partly conserved sequence (MSP-1#23, GLFHKEKMILNEEEITTKGA; located at positions 44 to 63) contained both T- and B-cell epitopes. Immunization of mice with these peptides resulted in T-cell proliferation and enhanced production of antibody to MSP-1 upon exposure to merozoites. MSP-1#1 stimulated T-cell responses in three of the six strains of mice evaluated, whereas MSP-1#23 was immunogenic in only one strain. Immunization with the other four peptides resulted in T-cell responses to the peptides, but none of the resulting peptide-specific T cells recognized native MSP-1. These results demonstrate that two sequences located in the N terminus of MSP-1 can induce T- and B-cell responses following immunization in a murine model. Clearly, these sequences merit further consideration for inclusion in a vaccine for malaria.     

Isolation and characterization of the 80000 dalton Plasmodium falciparum merozoite surface antigen

The 80 000 dalton merozoite surface protein, derived from the 185 000–195 000 dalton schizont precursor, was isolated from detergent extracts of naturally-released merozoites using chromatographic procedures. A rabbit antiserum raised against this antigen was used for characterizing the 80 000 dalton fragment. The antiserum did not inhibit merozoite invasion or parasite growth in vitro, suggesting that this merozoite surface antigen is not directly involved in the invasion process of the merozoite into the host erythrocyte.     

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.     

Antigenicity and immunogenicity of recombinant glutamate-rich protein of Plasmodium falciparum expressed in Escherichia coli.

A recombinant Plasmodium falciparum glutamate-rich protein (GLURP) was produced in Escherichia coli as a nearly full-length protein. In order to map immunodominant regions on GLURP, the nonrepetitive amino-terminal region (R0) as well as the central repeat region (R1) and the carboxy-terminal repeat region (R2) were also produced as separate products. All four purified gene products reacted specifically with serum samples from adults living in an area of Liberia where malaria is holoendemic. It appears that the human immune response against GLURP is primarily directed against the R2 region because 94% of the serum samples reacted with this region in an immunoassay. Antibody reactivity against the R0 region was also observed in 75% of the serum samples, while the R1 region showed only weak antibody-binding activity. When the nearly full-length GLURP molecule was adsorbed to Al(OH)3 it was found to be immunogenic in mice. In these experiments, the antibody response was almost exclusively directed against the R2 region. When anti-GLURP sera were obtained from rabbits immunized with the three regions, R0, R1, and R2, respectively, they recognized in immunoprecipitation experiments authentic GLURP from P. falciparum grown in vitro. These results demonstrate that GLURP produced in E. coli can induce a humoral immune response against GLURP derived from blood-stage parasites.     

Conformation and immunogenicity of engineered repeating segment of the circumsporozoite surface protein of Plasmodium falciparum

Peptide vaccines based on units of the immunodominant tetrapeptide repeats, Asn-Ala-Asn-Pro and Asn-Val-Asp-Pro, of the circumsporozoite surface protein of the parasite Plasmodium falciparum are presently being developed as potential malaria vaccines. The N-terminal fusion of a hydrophobic protein to units of the tetrapeptide repeat affected the immunogenicity and conformational stability of the peptide, and also induced a secondary structure in the peptide. Peptide antigenicity, as well as conformational stability, was significantly increased.     

Analysis of antibodies directed against merozoite surface protein 1 of the human malaria parasite Plasmodium falciparum

The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.     

Lack of sequence diversity in the gene encoding merozoite surface protein 5 of Plasmodium falciparum.

The gene encoding merozoite surface protein 5 (MSP5) of Plasmodium falciparum is situated between the genes encoding MSP2 and MSP4 on chromosome 2. Both MSP4 and MSP5 encode proteins that contain hydrophobic signal and glycosylphosphatidylinositol (GPI) attachment signals and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. The similar gene organization, location and similar structural features of the two genes suggest that they have arisen from a gene duplication event. In this study we provide further evidence for the merozoite surface location of MSP5 by demonstrating that MSP5 is present in isolated merozoites, partitions in the detergent-enriched phase following Triton X-114 fractionation and shows a staining pattern consistent with merozoite surface location by indirect immunofluorescence confocal microscopy. Analysis of antigenic diversity of MSP5 shows a lack of sequence variation between various isolates of P. falciparum from different geographical locations, a feature unusual for surface proteins of merozoites and one that may simplify vaccine formulation.