Two MSA 2 peptides that bind to human red blood cells are relevant to Plasmodium falciparum merozoite invasion.

Plasmodium falciparum merozoite membrane surface antigen 2 (MSA2) has been associated with the development of protective immunity against malaria. MSA2 antibodies were able to inhibit in vitro merozoite invasion. In our search for experimental evidence concerning the participation of MSA2 in merozoite invasion, 40 peptides were synthesized according to sequences reported for the CAMP and FC27 prototype Plasmodium strains. These peptides were purified, 125I-radiolabeled and tested for their ability to bind to erythrocytes. Two MSA2 synthetic peptides with high specific binding to human erythrocytes were found. The peptide coded 4044 (KNESKYSNTFINNAYNMSIR), located in the MSA2 N-terminal conserved region, has an affinity coefficient of 72 nM and showed a positive cooperativity for the receptor-ligand interaction. The other peptide, coded 4053 (NPNHKNAETNPKGKGEVQKP) and located in the central variable region of MSA2, has an affinity coefficient of 49nM and also showed a positive cooperativity for the receptor-ligand interaction. The binding capacity of these peptides is affected by erythrocytes treated with neuraminidase and trypsin, but it is not affected by chymotrypsin. Both of these sequences inhibit in vitro erythrocyte parasite invasion by up to 95% suggesting that they have an important role in the parasite's invasion process. Furthermore, as published previously [A. Saul et al. (1992) J. Immunol., 148, 208-211], a protective B epitope is included in the 4044 peptide sequence.     

Molecular cloning and sequencing of the merozoite surface antigen 2 gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in mycobacteria

Strain bacillus Calmette-Guerin (BCG) of Mycobacterium bovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-1/HN Plasmodium falciparum genome, sequenced, and expressed in M. bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.     

Relationship between Malaria Incidence and IgG Levels to Plasmodium falciparum Merozoite Antigens in Malian Children: Impact of Hemoglobins S and C

Heterozygous hemoglobin (Hb) AS (sickle-cell trait) and HbAC are hypothesized to protect against Plasmodium falciparum malaria in part by enhancing naturally-acquired immunity to this disease. To investigate this hypothesis, we compared antibody levels to four merozoite antigens from the P. falciparum 3D7 clone (apical membrane antigen 1, AMA1-3D7; merozoite surface protein 1, MSP1-3D7; 175 kDa erythrocyte-binding antigen, EBA175-3D7; and merozoite surface protein 2, MSP2-3D7) in a cohort of 103 HbAA, 73 HbAS and 30 HbAC children aged 3 to 11 years in a malaria-endemic area of Mali. In the 2009 transmission season we found that HbAS, but not HbAC, significantly reduced the risk of malaria compared to HbAA. IgG levels to MSP1 and MSP2 at the start of this transmission season inversely correlated with malaria incidence after adjusting for age and Hb type. However, HbAS children had significantly lower IgG levels to EBA175 and MSP2 compared to HbAA children. On the other hand, HbAC children had similar IgG levels to all four antigens. The parasite growth-inhibitory activity of purified IgG samples did not differ significantly by Hb type. Changes in antigen-specific IgG levels during the 2009 transmission and 2010 dry seasons also did not differ by Hb type, and none of these IgG levels dropped significantly during the dry season. These data suggest that sickle-cell trait does not reduce the risk of malaria by enhancing the acquisition of IgG responses to merozoite antigens.     

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.     

Protective immunization with invariant peptides of the Plasmodium falciparum antigen MSA2.

Three octapeptides from the N and C terminal C regions of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum elicit anti-MSA2 antibody when given as diphtheria toxoid conjugates. These antibodies also bind to the MSA2 homolog from the rodent malaria Plasmodium berghei. All mice vaccinated with these conjugates and challenged with an otherwise lethal inoculum of P. berghei showed substantial protection with most surviving. There was a inverse correlation between the development of the parasitemia and the antibody titer, with alum, algammulin, and CFA giving comparable results. These observations show that the conserved region of MSA2 could form the basis of a malaria vaccine when presented in a suitably immunogenic form, thus avoiding the problems of antigenic diversity [corrected].     

Atypical IgG subclass antibody responses to Plasmodium falciparum asexual stage antigens

The ability of Plasmodium falciparum to induce long-term immunity in the absence of continual restimulation has often been questioned. Recently it has been shown that, while a high proportion of individuals living in areas of high malaria endemicity have antibodies to merozoite surface antigen 2 (MSA2; MSP2) of P. falciparum, these antibodies are primarily of the IgG3 subclass. In this article, Antonio Ferrante and Christine Rzepczyk discuss how such atypical antibody responses may in part explain why immunity to malaria has been widely perceived to be short-lived.     

Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target

Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1.     

Evolutionary analysis of circumsporozoite surface protein and merozoite surface protein-1 (CSP and MSP-1) sequences of malaria parasites

Malaria, one of the world''s most common diseases, is caused by the intracellular protozoan parasite known as Plasmodium. In this study, we have determined the evolutionary relationship of two single-copy proteins, circumsporozoite protein (CSP) and merozoite surface protein-1 (MSP-1), among Plasmodium species using various bioinformatics tools and softwares. These two proteins are major blood stage antigens of Plasmodium species. This study demonstrates that the circumsporozoite protein of Plasmodium falciparum shows similarity with Plasmodium cynomolgi and Plasmodium knowlesi. The merozoite surface protein-1 of Plasmodium coatneyi forms a monophyletic group with Plasmodium knowlesi, demonstrating their close relationship and these two species also reveal similarity between the human malaria Plasmodium vivax. This Plasmodium phylogenetic arrangement is evidently crucial to identify shared derived characters as well as particular adaptation of plasmodium species from inside and between monophyletic groups.     

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.     

Allelic recombination and linkage disequilibrium within Msp-1 of Plasmodium falciparum, the malignant human malaria parasite

The C-terminal, cysteine-rich 19kDa domain of merozoite surface protein-1 (MSP-1) of Plasmodium falciparum is a target of the host's humoral immunity and thus a malaria vaccine candidate. Although variation in the 19kDa domain is limited among parasite isolates, tertiary structure-dependent intramolecular associations between the 19kDa domain and other parts of MSP-1 are suggested to be involved in immune evasion by allowing competitive binding of protective and non-protective antibodies directed to their epitopes, which are conformationally in close proximity but separated at the primary structure. Since allelic recombination can account for the major variability of the Msp-1 gene, we examined whether linkage disequilibrium occurs between polymorphic loci in the 5'- and the 3'-region, the latter encoding the 19kDa domain. From 184 Thai field isolates, we selected 69 isolates with a single allelic type in six variable blocks of Msp-1 as determined by PCR-based allelic typing. All the isolates showed no evidence of recombination in blocks 6 to 16, whereas recombination was apparent in blocks 2 to 6. Sequencing of the 3'-region revealed two potential recombination sites in block 17. Strong linkage disequilibrium was seen between polymorphic loci in the 5'- and 3'-regions. The strength of this disequilibrium did not correlate with distance between loci. We discuss the possible role of epistatic selection on particular association types (haplotypes) of Msp-1.     

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.     

Assessment of a vaccinia virus vectored multi-epitope live vaccine candidate for Plasmodium falciparum

We constructed a live recombinant vaccinia virus vaccine candidate containing a synthesised hybrid gene termed 'HGFSP' encoding circumsporozoite protein (CSP), major merozoite surface antigen-1(MSA1), major merozoite surface antigen-2 (MSA2), and ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum, interleukin-1 (IL-1) and tetanus toxin (TT) epitopes. Anti-recombinant vaccinia virus rabbit sera and IgG were tested in inhibition experiments in vitro. Results showed that the recombinant vaccinia virus had some capability to inhibit the growth of P. falciparum in vitro. The sera of rabbits, rats, and mice immunised with recombinant virus showed obvious IL-2 activity 4-6 weeks after immunisation. The interferon (IFN) level of sera from these animals 6 weeks after immunisation was significantly higher than before immunisation. These results indicate that the recombinant vaccinia virus can stimulate cell mediated responses (Th1 cell response) in immunised animals, and has the capability to inhibit multiplication of in vitro cultured P. falciparum. Thus this recombinant vaccinia virus is an appropriate vaccine candidate for further evaluation in Aotus monkey or human clinical trails.     

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.     

A Plasmodium vivax vaccine candidate displays limited allele polymorphism, which does not restrict recognition by antibodies.

BACKGROUND: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite. MATERIALS AND METHODS: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP1(19) described thus far. RESULTS: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein. CONCLUSIONS: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.     

Molecular characterisation of airport malaria: four cases in France during summer 1999

Four airport malaria cases have been observed in the vicinity of the Roissy-Charles-de-Gaulle International Airport, Paris, France. These cases were geographically very close to each other and clustered in a short period of time during the summer of 1999. The phenotype and genotype of the Plasmodium falciparum isolates obtained from these patients were determined in order to know whether a single mosquito could have infected more than one subject. The genomic characterisation of isolates was performed using the polymorphic markers merozoite surface protein 1 (Msp 1) and merozoite surface protein 2 (Msp 2) genes, the kappa and omega repeats domains of cg2 and the dihydrofolate reductase (DHFR) genotypes. Results showed identical genotypes for isolates 1, 2 and 4 whereas the genotype of isolate 3 differed at one locus. The molecular analysis was consistent with the hypothesis that all patients could have been bitten by the same mosquito and that patient 3, may have received a different clone and an additional species. In vitro susceptibility data did not confirm or rule out this hypothesis because isolates had the same profile of susceptibility to the tested drugs.     

Proteolytic processing and primary structure of Plasmodium falciparum apical membrane antigen-1

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a malaria merozoite integral membrane protein that plays an essential but poorly understood role in invasion of host erythrocytes. The PfAMA-1 ectodomain comprises three disulfide-constrained domains, the first of which (domain I) is preceded by an N-terminal prosequence. PfAMA-1 is initially routed to secretory organelles at the apical end of the merozoite, where the 83-kDa precursor (PfAMA-1(83)) is converted to a 66-kDa form (PfAMA-1(66)). At about the time of erythrocyte invasion, PfAMA-1(66) selectively translocates onto the merozoite surface. Here we use direct microsequencing and mass spectrometric peptide mass fingerprinting to characterize in detail the primary structure and proteolytic processing of PfAMA-1. We have determined the site at which processing takes place to convert PfAMA-1(83) to PfAMA-1(66) and have shown that both species possess a completely intact and unmodified transmembrane and cytoplasmic domain. Following relocation to the merozoite surface, PfAMA-1(66) is further proteolytically cleaved at one of two alternative sites, either between domains II and III, or at a membrane-proximal site following domain III. As a result, the bulk of the ectodomain is shed from the parasite surface in the form of two soluble fragments of 44 and 48 kDa. PfAMA-1 is not detectably modified by the addition of N-linked oligosaccharides.     

Evolution of Genetic Polymorphisms of Plasmodium falciparum Merozoite Surface Protein (PfMSP) in Thailand

Plasmodium falciparum malaria is a major public health problem in Thailand due to the emergence of multidrug resistance. The understanding of genetic diversity of malaria parasites is essential for developing effective drugs and vaccines. The genetic diversity of the merozoite surface protein-1 (PfMSP-1) and merozoite surface protein-2 (PfMSP-2) genes was investigated in a total of 145 P. falciparum isolates collected from Mae Sot District, Tak Province, Thailand during 3 different periods (1997-1999, 2005-2007, and 2009-2010). Analysis of genetic polymorphisms was performed to track the evolution of genetic change of P. falciparum using PCR. Both individual genes and their combination patterns showed marked genetic diversity during the 3 study periods. The results strongly support that P. falciparum isolates in Thailand are markedly diverse and patterns changed with time. These 2 polymorphic genes could be used as molecular markers to detect multiple clone infections and differentiate recrudescence from reinfection in P. falciparum isolates in Thailand.     

Plasmodium falciparum: IgG subclass antibody response to merozoite surface protein-1 among Amazonian gold miners, in relation to infection status and disease expression

The merozoite surface protein-1 (MSP-1) of Plasmodium falciparum comprises two major targets of antibody-mediated immunity: the polymorphic block 2 and the 19-kDa C-terminal domain MSP-1(19). Here, we measured antibodies to three block 2 variants and MSP-1(19) among Amazonian gold miners and examined the repertoire of block 2 variants in local parasites. Main findings were as follows: (1) Only seven different block 2 variants were found in 18 DNA sequences analyzed. (2) No major difference was observed in IgG subclass distribution of antibodies from symptomatic P. falciparum-infected patients, asymptomatic parasite carriers, and non-infected subjects. (3) Antibodies to all block 2 antigens, but not to MSP-1(19), were biased towards IgG3 across different strata of cumulative malaria exposure. (4) Similar proportions of symptomatic and asymptomatic subjects failed to recognize the block 2 variant expressed by infecting parasites. These negative results underscore the limits of conventional antibody assays to evaluate clinical immunity to malaria.     

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.