Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: a malaria vaccine candidate

Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.     

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts.  Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.       

A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.     

Structures of phage-display peptides that bind to the malarial surface protein,apical membrane antigen 1, and block erythrocyte invasion

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum is synthesized by schizont stage parasites and has been implicated in merozoite invasion of host erythrocytes. Phage-display techniques have recently been used to identify two 15-residue peptides, F1 and F2, which bind specifically to P. falciparum AMA1 and inhibit parasite invasion of erythrocytes [Li, F., et al. (2002) J. Biol. Chem. 277, 50303-50310]. We have synthesized F1, F2, and three peptides with high levels of sequence identity, determined their relative binding affinities for P. falciparum AMA1 with a competition ELISA, and investigated their solution structures by NMR spectroscopy. The strongest binding peptide, F1, contains a beta-turn that includes residues identified via an alanine scan as being critical for binding to AMA1 and inhibition of merozoite invasion of erythrocytes. The three F1 analogues include a 10-residue analogue of F1 truncated at the C-terminus (tF1), a partially scrambled 15-mer (sF1), and a disulfide-constrained 14-mer (F1tbp) which is related to F1 but has a sequence identical to that of a disulfide-constrained loop in the first epidermal growth factor module of the latent transforming growth factor-beta binding protein. tF1 and F1tbp bound competitively with F1 to AMA1, and all three contain a type I beta-turn encompassing key residues involved in F1 binding. In contrast, sF1 lacked this structural motif, and did not compete for binding to AMA1 with F1; rather, sF1 contained a type III beta-turn involving a different part of the sequence. Although F2 was able to bind to AMA1, it was unstructured in solution, consistent with its weak invasion inhibitory effects. Thus, the secondary structure elements observed for these peptides in solution correlate well with their potency in binding to AMA1 and inhibiting merozoite invasion. The structures provide a valuable starting point for the development of peptidomimetics as antimalarial antagonists directed at AMA1.     

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.     

Functional conservation of the malaria vaccine antigen MSP-119across distantly related Plasmodium species

The C-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP-119) is at present a leading malaria vaccine candidate. Antibodies against the epidermal growth factor-like domains of MSP-1 19are associated with immunity to P. falciparum and active immunization with recombinant forms of the molecule protect against malaria challenge in various experimental systems. These findings, with the knowledge that epidermal growth factor-like domains in other molecules have essential binding functions, indicate the importance of this protein in merozoite invasion of red blood cells. Despite extensive molecular epidemiological investigations, only limited sequence polymorphism has been identified in P. falciparum MSP-119 (refs. 9-11). This indicates its sequence is functionally constrained, and is used in support of the use of MSP-119 as a vaccine. Here, we have successfully complemented the function of most of P. falciparum MSP-119 with the corresponding but highly divergent sequence from the rodent parasite P. chabaudi. The results indicate that the role of MSP-119 in red blood cell invasion is conserved across distantly related Plasmodium species and show that the sequence of P. falciparum MSP-119 is not constrained by function.     

Stage-specific sensitivity of Plasmodium falciparum to antifolates

Highly synchronous cultures of Plasmodium falciparum were exposed to therapeutic concentrations of sulfadoxine or pyrimethamine at different developmental stages to investigate the effect on subsequent growth. Morphological observations showed that schizont formation from uninuclear trophozoites was the only process inhibited by the drugs. Segmentation of mature schizonts, merozoite invasion and development of the ring stage remained unaffected. These results support earlier reports suggesting that DNA synthesis is most pronounced in 32-42 h old trophozoites. The possible relevance of our results to the metabolism of P. falciparum is discussed.     

Acquisition of growth-inhibitory antibodies against blood-stage Plasmodium falciparum

Background

Antibodies that inhibit the growth of blood-stage Plasmodium falciparum may play an important role in acquired and vaccine-induced immunity in humans. However, the acquisition and activity of these antibodies is not well understood.

Methods

We tested dialysed serum and purified immunoglobulins from Kenyan children and adults for inhibition of P. falciparum blood-stage growth in vitro using different parasite lines. Serum antibodies were measured by ELISA to blood-stage parasite antigens, extracted from P. falciparum schizonts, and to recombinant merozoite surface protein 1 (42 kDa C-terminal fragment, MSP1-42).

Results

Antibodies to blood-stage antigens present in schizont protein extract and to recombinant MSP1-42 significantly increased with age and were highly correlated. In contrast, growth-inhibitory activity was not strongly associated with age and tended to decline marginally with increasing age and exposure, with young children demonstrating the highest inhibitory activity. Comparison of growth-inhibitory activity among samples collected from the same population at different time points suggested that malaria transmission intensity influenced the level of growth-inhibitory antibodies. Antibodies to recombinant MSP1-42 were not associated with growth inhibition and high immunoglobulin G levels were poorly predictive of inhibitory activity. The level of inhibitory activity against different isolates varied.

Conclusions

Children can acquire growth-inhibitory antibodies at a young age, but once they are acquired they do not appear to be boosted by on-going exposure. Inhibitory antibodies may play a role in protection from early childhood malaria.     

Stage-dependent inhibition of chloroquine on Plasmodium falciparum in vitro

Morphological observation of the life cycle of the malaria parasite, Plasmodium falciparum, in highly synchronous cultures after an exposure to therapeutic concentrations of chloroquine in ring, trophozoite and schizont stages, respectively, were carried out in order to determine the influence of chloroquine on the growth of the different stages of the malarial parasites. It was found that chloroquine could not affect merozoite invasion of the erythrocytes; the ring stage was more sensitive to chloroquine than the trophozoite and schizont stages; and chloroquine in therapeutic concentrations prevented only the transformation of rings to trophozoites and could not affect the transformations of trophozoites to schizonts and schizonts to new rings. The determination of the IC50 of chloroquine showed that the IC50 of trophozoites was about 6 times as high as that of rings.     

Immunization of monkeys with a 140 kilodalton merozoite surface protein of Plasmodium knowlesi malaria: appearance of alternate forms of this protein

The merozoite is the invasive stage of the malaria parasite which is released by rupture of the schizont-infected erythrocyte. A monoclonal antibody against a 140 kilodalton (kDa) merozoite surface antigen of Plasmodium knowlesi was used to characterize and to purify this antigen. It was shown by pulse-chase metabolic labeling of mature schizonts that the 140 kDa merozoite antigen was the processed product of a 143 kDa schizont component, and that processing occurred at the time of erythrocyte rupture. Antiserum, prepared by immunizing a rabbit with the 143/140 kDa antigen purified by immunoaffinity chromatography with the monoclonal antibody, strongly inhibited invasion of erythrocytes in vitro; Fab fragments prepared from purified rabbit IgG were inactive at blocking invasion, suggesting that agglutination of merozoites was the mechanism of invasion inhibition. The purified 143/140 kDa antigen was used in Freund's adjuvant to immunize four rhesus monkeys. Two of the immunized animals developed fulminating infections on challenge with 10(4) schizonts, as did the three control animals. The remaining two immunized animals controlled their infections and developed chronic low-grade parasitemias. The animals which were partially protected were those that had developed anti-143/140 kDa antibodies capable of blocking invasion in vitro. Parasites were isolated from the chronic stage of infection (V5 population) and were compared with the original parasite population used for challenge (P population). Inhibition of invasion, immunofluorescence, and immunoprecipitation with anti-143/140 kDa monoclonal antibody, with immune rabbit, and with monkey sera showed that the 143/140 kDa surface antigen had been replaced by multiple cross-reacting alternate antigenic forms of the molecule in the V population. Thus, specific immune response directed against a purified merozoite surface antigen resulted in the replacement of this antigen by variant or mutant forms.     

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.     

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.     

Proteome analysis reveals a large merozoite surface protein-1 associated complex on the Plasmodium falciparum merozoite surface

Plasmodium merozoite surface protein-1 (MSP-1) is an essential antigen for the merozoite invasion of erythrocytes. A key challenge to the development of an effective malaria vaccine that can block the erythrocyte invasion is to establish the molecular interaction(s) among the parasite surface proteins as well as with the host cell encoded receptors. In the present study, we applied molecular interactions and proteome approaches to identify PfMSP-1 associated complex on the merozoite surface. Proteomic analysis identified a major malaria surface protein, PfRhopH3 interacting with PfMSP-1(42). Pull-down experiments with merozoite lysate using anti-PfMSP-1 or anti-PfRhopH3 antibodies showed 16 bands that when identified by tandem mass spectrometry corresponded to11 parasite proteins: PfMSP-3, PfMSP-6, PfMSP-7, PfMSP-9, PfRhopH3, PfRhopH1, PfRAP-1, PfRAP-2, and two RAP domain containing proteins. This MSP-1 associated complex was specifically seen at schizont/merozoite stages but not the next ring stage. We could also identify many of these proteins in culture supernatant, suggesting the shedding of the complex. Interestingly, the PfRhopH3 protein also showed binding to the human erythrocyte and anti-PfRhopH3 antibodies blocked the erythrocyte invasion of the merozoites. These results have potential implications in the development of PfMSP-1 based blood stage malaria vaccine.     

A merozoite receptor protein from Plasmodium knowlesi is highly conserved and distributed throughout Plasmodium

The 66-kDa merozoite surface antigen (PK66) of Plasmodium knowlesi, a simian malaria, possesses vaccine-related properties that are thought to originate from a receptor-like role in parasite invasion of erythrocytes. We report the complete sequence of PK66 which allowed the demonstration that highly conserved analogues exist throughout Plasmodium including a recently reported gene from P. falciparum (Peterson, M. G., Marshall, V. M., Smythe, J. A., Crewther, P. E., Lew, A., Silva, A., Anders, R. F., and Kemp, D. J. (1989) Mol. Cell. Biol. 9, 3151-3155). These analogues are highly promising vaccination candidates. The distribution of PK66 changes after schizont rupture in a coordinate manner associated with merozoite invasion. The protein is concentrated at the apical end prior to rupture, following which it can distribute itself entirely across the surface of the free merozoite. During invasion, immunofluorescence studies suggest that, PK66 is excluded from the erythrocyte at, and behind, the invasion interface.     

Monoclonal antibody characterization of Plasmodium falciparum antigens in immune complexes formed when schizonts rupture in the presence of immune serum

When Plasmodium falciparum parasites are cultured with some immune sera, merozoites are agglutinated by antibodies to form immune clusters of merozoites and prevent their invasion into erythrocytes. Within these immune clusters of merozoites, several antigens that are normally found in the soluble fraction after detergent extraction accumulate in relatively insoluble immune complexes. From mice immunized with these immune complexes, we obtained hybridomas secreting monoclonal antibodies (mAb) that react with various immune clusters of merozoites antigens, including mAb 3D5, which recognizes a 101-kDa antigen (p101) and mAb, 5E3, which recognizes a 113-kDa antigen (p113). Both mAb reacted with antigens at the surface of schizonts, in the vacuolar space, and at the surface of merozoites before their release from schizont-infected cells. Both p101 and p113 were synthesized by mature trophozoites and young schizonts. In pulse-chase experiments, p113 was processed to 100-, 70-, 55-, and 50-kDa products. Both p101 and p113 appeared in the culture medium when schizont rupture occurred in normal culture medium but were found in immune complexes when schizont rupture occurred in the presence of immune serum. Antibodies in immune complexes, when dissociated with acid and used to probe immunoblots, reacted with affinity-purified p101 and p113. Antigens such as these, which are accessible at the parasite surface and react with antibodies present in immune serum that inhibits parasite invasion, are logical candidates to study in the search for a vaccine against the erythrocytic stages of malaria.     

Monoclonal auto-antibodies and sera of autoimmune patients react with Plasmodium falciparum and inhibit its in vitro growth

The relationship between autoimmunity and malaria is not well understood. To determine whether autoimmune responses have a protective role during malaria, we studied the pattern of reactivity to plasmodial antigens of sera from 93 patients with 14 different autoimmune diseases (AID) who were not previously exposed to malaria. Sera from patients with 13 different AID reacted against Plasmodium falciparum by indirect fluorescent antibody test with frequencies varying from 33-100%. In addition, sera from 37 AID patients were tested for reactivity against Plasmodium yoelii 17XNL and the asexual blood stage forms of three different P. falciparum strains. In general, the frequency of reactive sera was higher against young trophozoites than schizonts (p < 0.05 for 2 strains), indicating that the antigenic determinants targeted by the tested AID sera might be more highly expressed by the former stage. The ability of monoclonal auto-antibodies (auto-Ab) to inhibit P. falciparum growth in vitro was also tested. Thirteen of the 18 monoclonal auto-Ab tested (72%), but none of the control monoclonal antibodies, inhibited parasite growth, in some cases by greater than 40%. We conclude that autoimmune responses mediated by auto-Ab may present anti-plasmodial activity.     

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.