Structure of the C-terminal domains of merozoite surface protein-1 from Plasmodium knowlesi reveals a novel histidine binding site

The protozoan parasite Plasmodium causes malaria, with hundreds of millions of cases recorded annually. Protection against malaria infection can be conferred by antibodies against merozoite surface protein (MSP)-1, making it an attractive vaccine candidate. Here we present the structure of the C-terminal domains of MSP-1 (known as MSP-1(19)) from Plasmodium knowlesi. The structure reveals two tightly packed epidermal growth factor-like domains oriented head to tail. In domain 1, the molecule displays a histidine binding site formed primarily by a highly conserved tryptophan. The protein carries a pronounced overall negative charge primarily due to the large number of acidic groups in domain 2. To map protein binding surfaces on MSP-1(19), we have analyzed the crystal contacts in five different crystal environments, revealing that domain 1 is highly preferred in protein-protein interactions. A comparison of MSP-1(19) structures from P. knowlesi, P. cynomolgi, and P. falciparum shows that, although the overall protein folds are similar, the molecules show significant differences in charge distribution. We propose the histidine binding site in domain 1 as a target for inhibitors of protein binding to MSP-1, which might prevent invasion of the merozoite into red blood cells.     

Identifying and characterising the Plasmodium falciparum merozoite surface protein 10 Plasmodium vivax homologue

Plasmodium vivax malaria is one of the most prevalent parasitic diseases in Asia and Latin-America. The difficulty of maintaining this parasite culture in vitro has hampered identifying and characterising proteins implied in merozoite invasion of red blood cells. We have been able to identify an open reading frame in P. vivax encoding the Plasmodium falciparum merozoite surface protein 10 homologous protein using the partial sequences from this parasite's genome reported during 2004. This new protein contains 479 amino-acids, two epidermal growth factor-like domains, hydrophobic regions at the N- and C-termini, being compatible with a signal peptide and a glycosylphosphatidylinositol anchor site, respectively. The protein is expressed during the parasite's asexual stage and is recognised by polyclonal sera in parasite lysate using Western blot. P. vivax-infected patients' sera highly recognised recombinant protein by ELISA.     

Construction and immunogenicity in mice of attenuated Salmonella typhi expressing Plasmodium falciparum merozoite surface protein 1 (MSP-1) fused to tetanus toxin fragment C

One strategy to develop a multi-antigen malaria vaccine is to employ live vectors to carry putative protective Plasmodium falciparum antigens to the immune system. The 19 kDa carboxyl terminus of P. falciparum merozoite surface protein 1 (MSP-1), which is essential for erythrocyte invasion and is a leading antigen for inclusion in a multivalent malaria vaccine, was genetically fused to fragment C of tetanus toxin and expressed within attenuated Salmonella typhi CVD 908. Under conditions in the bacterial cytoplasm, the fragment C-MSP-1 fusion did not form the epidermal growth factor (EGF)-like domains of MSP-1; monoclonal antibodies failed to recognize these conformational domains in immunoblots of non-denatured protein extracted from live vector sonicates. The MSP-1 was nevertheless immunogenic. One month following intranasal immunization of BALB/c mice with the live vector construct, four out of five mice exhibited > or =four-fold rises in anti-MSP-1 by ELISA (GMT=211); a single intranasal booster raised titers further (GMT=1280). Post-immunization sera recognized native MSP-1 on merozoites as determined by indirect immunofluorescence. These data encourage efforts to optimize MSP-1 expression in S. typhi (e.g. as a secreted protein), so that the EGF-like epitopes, presumably necessary for stimulating protective antibodies, can form.     

Antibodies and Plasmodium falciparum merozoites

There is considerable interest in using merozoite proteins in a vaccine against falciparum malaria. Observations that antibodies to merozoite surface proteins block invasion are a basis for optimism. This article draws attention to important and varied aspects of how antibodies to Plasmodium falciparum merozoites affect red blood cell invasion.     

Merozoite surface protein 1, immune evasion, and vaccines against asexual blood stage malaria

There is an urgent need for a vaccine against malaria and proteins on the surface of the merozoite are good targets for development as vaccine candidates because they are exposed to antibody. However, it is possible that the parasite has evolved mechanisms to evade a protective immune response to these proteins. Merozoite surface protein 1 (MSP-1) is a candidate for vaccine development and its C-terminal sequence is the target of protective antibody. MSP-1 is cleaved by proteases in two processing steps, the second step releases the bulk of the protein from the surface and goes to completion during successful red blood cell invasion. Antibodies binding to the C-terminus of Plasmodium falciparum MSP-1 can inhibit both the processing and erythrocyte invasion. Other antibodies that bind to either the C-terminal sequence or elsewhere in the molecule are 'blocking' antibodies, which on binding prevent the binding of the inhibitory antibodies. Blocking antibodies are a mechanism of immune evasion, which may be based on antigenic conservation rather than diversity. This mechanism has a number of implications for the study of protective immunity and the development of malaria vaccines, emphasising the need for appropriate functional assays and careful design of the antigen.     

Plasmodium yoelii: experimental evidences for the conserved epitopes between mouse and human malaria parasite, Plasmodium falciparum

Bioinformatic analyses of gene homologues have revealed functionally conserved epitopes between human and rodent malaria parasites. Here, we present experimental evidence for the presence of functionally and antigenically conserved domains between Plasmodium falciparum and Plasmodium yoelii asexual blood-stages. Merozoite released soluble proteins (MRSPs) from both P. falciparum and P. yoelii bound to heterologous mouse or human red blood cells, respectively. The presence of conserved antigenic epitopes between the two species of parasites was evident by the inhibitory effect of antibodies, developed against P. yoelii in convalescent mice, on P. falciparum growth and merozoite reinvasion in vitro. Furthermore, mice immunized with P. falciparum MRSPs were protected from infection by a P. yoelii challenge. These data indicate that different species of Plasmodium contain antigenically conserved interspecies domains, which are immunogenic and, thus constitute a potential novel antigen source for vaccine development and testing using a mouse model.     

The merozoite surface protein 1 complex of human malaria parasite Plasmodium falciparum: interactions and arrangements of subunits

The major protein component at the surface of merozoites, the infectious form of blood stage malaria parasites, is the merozoite surface protein 1 (MSP-1) complex. In the human malaria parasite Plasmodium falciparum, this complex is generated by proteolytic cleavage of a 190-kDa glycosylphosphatidylinositol-anchored precursor into four major fragments, which remain non-covalently associated. Here, we describe the in vitro reconstitution of the MSP-1 complex of P. falciparum strain 3D7 from its heterologously produced subunits. We provide evidence for the arrangement of the subunits within the complex and show how they interact with each other. Our data indicate that the conformation assumed by the reassembled complex as well as by the heterologously produced 190-kDa precursor corresponds to the native one. Based on these results we propose a first structural model for the MSP-1 complex. Together with access to faithfully produced material, this information will advance further structure-function studies of MSP-1 that plays an essential role during invasion of erythrocytes by the parasite and that is considered a promising candidate for a malaria vaccine.     

A common cross-species function for the double epidermal growth factor-like modules of the highly divergent plasmodium surface proteins MSP-1 and MSP-8

An understanding of structural and functional constraints on the C-terminal double epidermal growth factor (EGF)-like modules of merozoite surface protein (MSP)-1 and related proteins is of importance to the development of these molecules as malaria vaccines and drug targets. Using allelic replacement, we show that Plasmodium falciparum parasites can invade erythrocytes and grow efficiently in the absence of an MSP-1 protein with authentic MSP-1 EGF domains. In this mutant parasite line, the MSP-1 EGFs were replaced by the corresponding double EGF module from P. berghei MSP-8, the sequence of which shares only low identity with its MSP-1 counterpart. Hence, the C-terminal EGF domains of at least some Plasmodium surface proteins appear to perform the same function in asexual blood-stage development. Mapping the surface location of the few residues that are common to these functionally complementary EGF modules revealed the presence of a highly conserved pocket of potential functional significance. In contrast to MSP-8, an even more divergent double EGF module, that from the sexual stage protein PbS25, was not capable of complementing MSP-1 EGF function. More surprisingly, two chimeric double EGF modules comprising hybrids of the EGF domains from P. falciparum and P. chabaudi MSP-1 were also not capable of replacing the P. falciparum MSP-1 EGF module. Together, these data suggest that although the MSP-1 EGFs can accommodate extensive sequence diversity, there appear to be constraints that may restrict the simple accumulation of point mutations in the face of immune pressure in the field.     

Acquired immune responses to Plasmodium falciparum merozoite surface protein-1 in the human fetus.

Infants born in areas of stable malaria transmission are relatively protected against severe morbidity and high density Plasmodium falciparum blood-stage infection. This protection may involve prenatal sensitization and immunologic reactivity to malaria surface ligands that participate in invasion of red cells. We examined cord blood T and B cell immunity to P. falciparum merozoite surface protein-1 (MSP-1) in infants born in an area of stable malaria transmission in Kenya. T cell cytokine responses to the C-terminal 19-kDa fragment of MSP-1 (MSP-1(19)) were detected in 24 of 92 (26%) newborns (4-192 IFN-gamma and 3-88 IL-4-secreting cells per 10(6)/cord blood lymphocytes). Peptide epitopes in the N-terminal block 3 region of MSP-1 also drove IFN-gamma and/or IL-13 production. There was no evidence of prenatal T cell sensitization to liver-stage Ag-1. A total of 5 of 86 (6%) newborns had cord blood anti-MSP-1(19) IgM Abs, an Ig isotype that does not cross the placenta and is therefore of fetal origin. The frequency of neonatal B cell sensitization was higher than that indicated by serology alone, as 5 of 27 (18%) cord blood samples contained B cells that produced IgG when stimulated with MSP-1(19) in vitro. Neonatal B cell IgG responses were restricted to the Q-KNG allele of MSP-1(19), the major variant in this endemic area, whereas T cells responded to all four MSP-1(19) alleles evaluated. In utero sensitization to MSP-1 correlated with the presence of malaria parasites in cord blood (chi(2) = 20, p < 0.0001). These data indicate that prenatal sensitization to blood-stage Ags occurs in infants born in malaria endemic areas.     

Plasmodium falciparum: variations in the C-terminal cysteine-rich region of the merozoite surface protein-1 in field samples among Indian isolates.

The cysteine-rich C-terminal region of the merozoite surface protein-1, MSP-119, of Plasmodium falciparum has been the most promising vaccine target antigen to date, based on protective immunization studies with recombinant proteins in mice and monkey models. To be further developed as a vaccine candidate, it is essential to study its sequence heterogeneity in field isolates from diverse geographical areas. We have analyzed the DNA sequences encoding the C-terminal region of P. falciparum MSP-1 (1526-1744 aa, corresponding to part of the 16th and all of the 17th blocks) of 16 isolates from different regions in India. The PNG-MAD20 type of MSP-1 sequence predominated in this subcontinent. The MSP-119 region as usual was found to be highly conserved, with amino acid variations at four positions. Based on these variations, only three MSP-119 forms (Q-KNG, E-KNG, and E-TSG, a novel variant) were detected among these isolates. The two MSP-119 variant forms (Q-KNG and E-TSG) were expressed in Escherichia coli as histidine-tagged polypeptides and were characterized immunologically to corroborate the sequence data.     

Evidence that invasion-inhibitory antibodies specific for the 19-kDa fragment of merozoite surface protein-1 (MSP-1 19) can play a protective role against blood-stage Plasmodium falciparum infection in individuals in a malaria endemic area of Africa

The C-terminal 19-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP-1(19)) is a target of protective Abs against blood-stage infection and a leading candidate for inclusion in a human malaria vaccine. However, the precise role, relative importance, and mechanism of action of Abs that target this protein remain unclear. To examine the potential protective role of Abs to MSP-1(19) in individuals naturally exposed to malaria, we conducted a treatment time to infection study over a 10-wk period in 76 residents of a highland area of western Kenya during a malaria epidemic. These semi-immune individuals were not all equally susceptible to reinfection with P. falciparum following drug cure. Using a new neutralization assay based on transgenic P. falciparum expressing the P. chabaudi MSP-1(19) orthologue, individuals with high-level MSP-1(19)-specific invasion-inhibitory Abs (>75th percentile) had a 66% reduction in the risk of blood-stage infection relative to others in the population (95% confidence interval, 3-88%). In contrast, high levels of MSP-1(19) IgG or IgG subclass Abs measured by enzyme immunoassay with six different recombinant MSP-1(19) Ags did not correlate with protection from infection. IgG Abs measured by serology and functional invasion-inhibitory activity did not correlate with each other. These findings implicate an important protective role for MSP-1(19)-specific invasion inhibitory Abs in immunity to blood-stage P. falciparum infection, and suggest that the measurement of MSP-1(19) specific inhibitory Abs may serve as an accurate correlate of protection in clinical trials of MSP-1-based vaccines.     

Prenatal malaria immune experience affects acquisition of Plasmodium falciparum merozoite surface protein-1 invasion inhibitory antibodies during infancy

African infants are often born of mothers infected with malaria during pregnancy. This can result in fetal exposure to malaria-infected erythrocytes or their soluble products with subsequent fetal immune priming or tolerance in utero. We performed a cohort study of 30 newborns from a malaria holoendemic area of Kenya to determine whether T cell sensitization to Plasmodium falciparum merozoite surface protein-1 (MSP-1) at birth correlates with infant development of anti-MSP-1 Abs acquired as a consequence of natural malaria infection. Abs to the 42- and 19-kDa C-terminal processed fragments of MSP-1 were determined by serology and by a functional assay that quantifies invasion inhibition Abs against the MSP-1(19) merozoite ligand (MSP-1(19) IIA). Infants had detectable IgG and IgM Abs to MSP-1(42) and MSP-1(19) at 6 mo of age with no significant change by age 24-30 mo. In contrast, MSP-1(19) IIA levels increased from 6 to 24-30 mo of age (16-29%, p < 0.01). Infants with evidence of prenatal exposure to malaria (defined by P. falciparum detection in maternal, placental, and/or cord blood compartments) and T cell sensitization at birth (defined by cord blood lymphocyte cytokine responses to MSP-1) showed the greatest age-related increase in MSP-1(19) IIA compared with infants with prenatal exposure to malaria but who lacked detectable T cell MSP-1 sensitization. These data suggest that fetal sensitization or tolerance to MSP-1, associated with maternal malaria infection during pregnancy, affects the development of functional Ab responses to MSP-1 during infancy.     

A conserved region of the MSP-1 surface protein of Plasmodium falciparum contains a recognition sequence for erythrocyte spectrin.

The major surface protein MSP-1 of Plasmodium falciparum blood-stage malaria parasites contains notably conserved sequence blocks with unknown function. The recombinant protein 190L, which represents such a block, exhibits a high affinity for red blood cell membranes. We demonstrate that both 190L and native MSP-1 protein bind to the inner red blood cell membrane skeleton protein spectrin. By using overlapping peptides covering the 190L molecule, we show that the spectrin contact site of 190L is included in a linear sequence of 30 amino acid residues. Association of 190L with naturally occurring spectrin deficient red blood cells is drastically reduced. In the same cells parasite invasion is normal, but the intracellular parasite development arrests late in the trophozoite stage. A similar situation arises when synthetic peptides covering the spectrin recognition sequence of 190L are added to P.falciparum cultures. These data and the cellular localization of MSP-1 suggest the possibility that MSP-1 associates with spectrin under natural conditions.     

Passive immunoprotection of Plasmodium falciparum-infected mice designates the CyRPA as candidate malaria vaccine antigen

An effective malaria vaccine could prove to be the most cost-effective and efficacious means of preventing severe disease and death from malaria. In an endeavor to identify novel vaccine targets, we tested predicted Plasmodium falciparum open reading frames for proteins that elicit parasite-inhibitory Abs. This has led to the identification of the cysteine-rich protective Ag (CyRPA). CyRPA is a cysteine-rich protein harboring a predicted signal sequence. The stage-specific expression of CyRPA in late schizonts resembles that of proteins known to be involved in merozoite invasion. Immunofluorescence staining localized CyRPA at the apex of merozoites. The entire protein is conserved as shown by sequencing of the CyRPA encoding gene from a diverse range of P. falciparum isolates. CyRPA-specific mAbs substantially inhibited parasite growth in vitro as well as in a P. falciparum animal model based on NOD-scid IL2Rγ(null) mice engrafted with human erythrocytes. In contrast to other P. falciparum mouse models, this system generated very consistent results and evinced a dose-response relationship and therefore represents an unprecedented in vivo model for quantitative comparison of the functional potencies of malaria-specific Abs. Our data suggest a role for CyRPA in erythrocyte invasion by the merozoite. Inhibition of merozoite invasion by CyRPA-specific mAbs in vitro and in vivo renders this protein a promising malaria asexual blood-stage vaccine candidate Ag.     

Broadly reactive antibodies specific for Plasmodium falciparum MSP-119 are associated with the protection of naturally exposed children against infection

ABSTRACT: BACKGROUND: The 19 kDa C-terminal region of Plasmodium falciparum Merozoite Surface Protein-1 is a known target of naturally acquired humoral immunity and a malaria vaccine candidate. MSP- 119 has four predominant haplotypes resulting in amino acid changes labelled EKNG, QKNG, QTSR and ETSR. IgG antibodies directed against all four variants have been detected, but it is not known if these variant specific antibodies are associated with haplotype-specific protection from infection. METHODS: Blood samples from 201 healthy Kenyan adults and children who participated in a 12-week treatment time-to-infection study were evaluated. Venous blood drawn at baseline (week 0) was examined for functional and serologic antibodies to MSP-119 and MSP-142 variants. MSP-119 haplotypes were detected by a multiplex PCR assay at baseline and weekly throughout the study. Generalized linear models controlling for age, baseline MSP-119 haplotype and parasite density were used to determine the relationship between infecting P. falciparum MSP-119 haplotype and variant-specific antibodies. RESULTS: A total of 964 infections resulting in 1,533 MSP-119 haplotypes detected were examined. The most common haplotypes were EKNG and QKNG, followed by ETSR and QTSR. Children had higher parasite densities, greater complexity of infection (>1 haplotype), and more frequent changes in haplotypes over time compared to adults. Infecting MSP-119 haplotype at baseline (week 0) had no influence on haplotypes detected over the subsequent 11 weeks among children or adults. Children but not adults with MSP-119 and some MSP-142 variant antibodies detected by serology at baseline had delayed time-to-infection. There was no significant association of variant-specific serology or functional antibodies at baseline with infecting haplotype at baseline or during 11 weeks of follow up among children or adults. CONCLUSIONS: Variant transcending IgG antibodies to MSP-119 are associated with protection from infection in children, but not adults. These data suggest that inclusion of more than one MSP-119 variant may not be required in a malaria blood stage vaccine.     

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species

Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.     

How many pathways for invasion of the red blood cell by the malaria parasite?

Merozoites of the malaria parasite Plasmodium falciparum use several receptors for cellular engagement when they invade human red blood cells. Recently, a merozoite erythrocyte-binding protein, EBA-140, has been identified that specifically binds to glycophorin C on red blood cells. Up to 50% of Melanesians have a deletion in this gene, and the resultant Gerbich-negative red blood cells are relatively resistant to invasion. While discovery of multiple pathways for invasion could confound the search for suitable vaccine targets, they could also be considered in the design of therapeutic interventions that prevent malaria parasites entering red blood cells.     

Human antibodies to the polymorphic block 2 domain of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) exhibit a highly skewed, peptide-specific light chain distribution

Antibodies to polymorphic block 2 of the Plasmodium falciparum merozoite surface protein 1 (MSP-1) present a paradoxical association with acquired protection against clinical malaria, while showing restricted and fixed specificity, reminiscent of antigenic sin. We report here that these antibodies present a highly imbalanced, peptide-specific light chain distribution. This was not observed with several other parasite-derived peptides or antigens. These data point to a skewed immune response to MSP-1 block 2 that is constrained both in specificity and chain usage. This is the first report of a biased response to polymorphic epitopes of a surface antigen in malaria parasites.     

Merozoite surface protein 2 allelic variation influences the specific antibody response during acute malaria in individuals from a Brazilian endemic area

The antibody response to Plasmodium falciparum parasites of naturally infected population is critical to elucidate the role of polymorphic alleles in malaria. Thus, we evaluated the impact of antigenic diversity of repetitive and family dimorphic domains of the merozoite surface protein 2 (MSP-2) on immune response of 96 individuals living in Peixoto de Azevedo (MT-Brazil), by ELISA using recombinant MSP-2 proteins. The majority of these individuals were carrying FC27-type infections. IgG antibody responses were predominantly directed to FC27 parasites and were correlated to the extension of polymorphism presented by each MSP-2 region. This finding demonstrated the impact of the genetic polymorphism on antibody response and therefore, its importance on malaria vaccine efficacy.     

MSP-1 malaria pseudopeptide analogs: biological and immunological significance and three-dimensional structure

Merozoite Surface Protein-1 (MSP-1) has been considered as a malaria vaccine candidate. It is processed during the Plasmodium falciparum invasion process of red blood cells (RBCs). A conserved MSP-1 C-terminal peptide was identified as a high-activity erythrocyte-binding peptide (HAEBP) termed 1585. Since conserved HAEBPs are neither antigenic nor immunogenic we decided to assess the significance of a single peptide bond replacement in 1585. Thus, two pseudopeptides were obtained by introducing a Y[CH2-NH] reduced amide isoster into the 1585 critical binding motif. The pseudopeptides bound to different HLA-DR alleles, suggesting that backbone modifications affect MHC-II binding patterns. Pseudopeptide-antibodies inhibit in vitro parasite RBC invasion by recognizing MSP-1. Each pseudopeptide-induced antibody shows distinct recognition patterns. 1H-NMR studies demonstrated that isoster bonds modulate the pseudopeptides' structure and thus their immunological properties, therefore representing a possible subunit malaria vaccine component.