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.     

Sequence comparison of allelic forms of the Plasmodium falciparum merozoite surface antigen MSA2

MSA2 is a strain variable blood-stage merozoite surface antigen of Plasmodium falciparum. We have derived the MSA2 nucleotide sequence for four cloned parasite isolates. Comparison with three other published sequences suggests that variation may be limited, and that the architecture of the gene can be conveniently described by segregation into four distinct regions. The N and C terminal regions (Regions 1 and 4) are highly conserved in all seven genes. Six of these seven MSA2 genes can be grouped in a single family, within which variation is largely limited to a region characterized by the presence of tandem repeats (Region 2). We have observed two new forms of repeat in a Gly, Ser, Ala-rich block, and noted the absence of repeat in this block of the CAMP strain. The region downstream of the repeat region (Region 3) is highly conserved within this family. Immunochemical analysis reveals that MSA2 is one of the antigens recognized by immune antibodies eluted from intact merozoites. Regions 2 and 3, expressed as recombinant proteins, are recognized by these antibodies, suggesting that these regions are exposed at the surface of the intact merozoite.     

Differential recognition of Plasmodium falciparum merozoite surface protein 2 variants by antibodies from malaria patients in Brazil

Four variants of merozoite surface protein 2 (MSP-2) of Plasmodium falciparum were used in serology to examine whether changes in repeat units affect its recognition by antibodies during infection with parasites of known MSP-2 types. The results indicate that variation in MSP-2 repeats may represent a mechanism for parasite immune evasion.     

Extensive antigenic polymorphism within the repeat sequence of the Plasmodium falciparum merozoite surface protein 1 block 2 is incorporated in a minimal polyvalent immunogen

Polymorphism in pathogen antigens presents a complex challenge for vaccine design. A prime example is the N-terminal block 2 region of the Plasmodium falciparum merozoite surface protein 1 (MSP1), to which allele-specific antibodies have been associated with protection from malaria. In a Zambian population studied here, 49 of 91 alleles sampled were of the K1-like type (the most common of three block 2 types in all African populations), and most of these had unique sequences due to variation in tri- and hexapeptide repetitive motifs. There were significant negative correlations between allelic sequence lengths of different regions of the repeats, so the complete repeat sequence had less length variation than its component parts, suggesting a constraint on overall length. Diverse epitopes recognized by three murine monoclonal antibodies and 24 individual human sera were then mapped by using a comprehensive panel of synthetic peptides, revealing epitopes in all regions of the repeats. To incorporate these different epitopes in a single molecule, a composite sequence of minimal overall length (78 amino acids) was then designed and expressed as a recombinant antigen. More human immune sera reacted with this "K1-like Super Repeat" antigen than with proteins consisting of single natural allelic sequences, and immunization of mice elicited antibodies that recognized a range of five cultured parasite lines with diverse K1-like MSP1 block 2 repeat sequences. Thus, complex allelic polymorphism was deconstructed and a minimal composite polyvalent antigen was engineered, delivering a designed candidate sequence for inclusion in a malaria vaccine.     

Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum

An integral membrane protein associated with the merozoite surface of Plasmodium falciparum termed merozoite surface antigen 2 (the 45-kDa merozoite surface antigen), occurs in antigenically diverse forms. Here we report the sequences of the MSA 2 gene from two other isolates of P. falciparum. The 43 N-terminal residues and the 74 C-terminal residues of all three MSA 2 sequences are highly conserved, but between these conserved regions there are dramatic differences among the alleles. Instead of the two copies of a 32-amino-acid repeat present in the MSA 2 of isolate FC27, MSA 2 from clone 3D7 and isolate Indochina 1 contain 5 and 12 copies respectively of the four amino acid sequence Gly Gly Ser Ala. The sequences flanking the repeats also differ among the three antigens. The repeats in MSA 2 appear to be immunodominant during natural infection, and antibodies to the repeat regions of different alleles react with a restricted number of parasite isolates.     

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.     

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.     

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.     

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.     

Roles of conserved and allelic regions of the major merozoite surface protein (gp195) in immunity against Plasmodium falciparum.

The Plasmodium falciparum major merozoite surface protein gp195 is a candidate antigen for a vaccine against human malaria. The significance of allelism and polymorphism in vaccine-induced immunity to gp195 was investigated in this study. Rabbits were immunized with each of two allelic forms of gp195 that were affinity purified from the FUP and FVO parasite isolates. gp195-specific antibodies raised against one allelic form of gp195 cross-reacted extensively with the gp195 of the heterologous allele in enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays. Competitive binding ELISAs with homologous and heterologous gp195s confirmed that a majority of the anti-gp195 antibodies produced against either allelic protein were cross-reactive. Moreover, the biological activities of the gp195 antibody responses were also highly cross-reactive, since anti-gp195 sera inhibited the in vitro growth of the homologous and heterologous parasites with equal efficiency. The degree of cross-reactivity with strain-specific and allele-specific determinants of gp195 in the ELISA was low. These results suggest that the immunological cross-reactivity between the two gp195 proteins is due to recognition of conserved determinants. They also suggest that a gp195-based vaccine may be effective against blood-stage infection with a diverse array of parasite isolates.     

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.     

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??.     

Antibody responses to infections with strains of Plasmodium falciparum expressing diverse forms of merozoite surface protein 2

Individuals living in areas where Plasmodium falciparum is endemic experience numerous episodes of infection. These episodes may or may not be symptomatic, with the outcome depending on a combination of parasite and host factors, several of which are poorly understood. One factor is believed to be the particular alleles of several parasite proteins to which the host is capable of mounting protective immune responses. We report a study examining antibody responses to MSP2 in 15 semi-immune teenagers and adults living in the Khanh-Hoa area of southern-central Vietnam, where P. falciparum is highly endemic; subjects were serially infected with multiple strains of P. falciparum. The MSP2 alleles infecting these subjects were determined by nucleotide sequencing. A total of 62 MSP2 genes belonging to both dimorphic families were identified, of which 33 contained distinct alleles, with 61% of the alleles being detected once. Clear changes in the repertoire occurred between infections. Most infections contained a mixture of parasites expressing MSP2 alleles from both dimorphic families. Two examples of reinfection with a strain expressing a previously encountered allele were detected. Significant changes in antibody levels to various regions of MSP2 were detected over the course of the experiment. There was no clear relation between the infecting form of MSP2 and the ensuing antibody response. This study highlights the complexity of host-parasite relationship for this important human pathogen.     

Allelic diversity and antibody recognition of Plasmodium falciparum merozoite surface protein 1 during hypoendemic malaria transmission in the Brazilian amazon region.

The polymorphic merozoite surface protein (MSP-1) of Plasmodium falciparum is a major asexual blood-stage malaria vaccine candidate. The impact of allelic diversity on recognition of MSP-1 during the immune response remains to be investigated in areas of hypoendemicity such as the Brazilian Amazon region. In this study, PCR was used to type variable regions, blocks 2, 4, and 10, of the msp-1 gene and to characterize major gene types (unique combinations of allelic types in variable blocks) in P. falciparum isolates collected across the Amazon basin over a period of 12 years. Twelve of the 24 possible gene types were found among 181 isolates, and 68 (38%) of them had more than one gene type. Temporal, but not spatial, variation was found in the distribution of MSP-1 gene types in the Amazon. Interestingly, some gene types occurred more frequently than expected from random assortment of allelic types in different blocks, as previously found in other areas of endemicity. We also compared the antibody recognition of polymorphic (block 2), dimorphic (block 6), and conserved (block 3) regions of MSP-1 in Amazonian malaria patients and clinically immune Africans, using a panel of recombinant peptides. Results were summarized as follows. (i) All blocks were targeted by naturally acquired cytophilic antibodies of the subclasses IgG1 and IgG3, but the balance between IgG1 and IgG3 depended on the subjects' cumulative exposure to malaria. (ii) The balance between IgG1 and IgG3 subclasses and the duration of antibody responses differed in relation to distinct MSP-1 peptides. (iii) Antibody responses to variable blocks 2 and 6 were predominantly type specific, but variant-specific antibodies that target isolate-specific repetitive motifs within block 2 were more frequent in Amazonian patients than in previously studied African populations.     

Comparative Immunogenicities of full-length Plasmodium falciparum merozoite surface protein 3 and a 24-kilodalton N-terminal fragment

Recombinant Plasmodium falciparum merozoite surface protein 3 (PfMSP3F) and a 24-kDa fragment from its N terminus (MSP3N) that includes the essential conserved domain, which elicits the maximum antibody (Ab)-dependent cellular inhibition (ADCI), were expressed as soluble proteins in Escherichia coli. Both proteins were found to be stable in both soluble and lyophilized forms. Immunization with MSP3F and MSP3N formulated separately with two human-compatible adjuvants, aluminum hydroxide (Alhydrogel) and Montanide ISA 720, produced significant antibody responses in mice and rabbits. Polyclonal Abs against both antigens recognized native MSP3 in the parasite lysate. These two Abs also recognized two synthetic peptides, previously characterized to possess B cell epitopes from the N-terminal region. Antibody depletion assay showed that most of the IgG response is directed toward the N-terminal region of the full protein. Anti-MSP3F and anti-MSP3N rabbit antibodies did not inhibit merozoite invasion or intraerythrocytic development but significantly reduced parasitemia in the presence of human monocytes. The ADCI demonstrated by anti-MSP3N antibodies was comparable to that exhibited by anti-MSP3F antibodies (both generated in rabbit). These results suggest that the N-terminal fragment of MSP3 can be considered a vaccine candidate that can form part of a multigenic vaccine against malaria.