Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1

Plasmodium vivax is one of the most widely distributed human malaria parasites and due to drug-resistant strains, its incidence and prevalence has increased, thus an effective vaccine against the parasites is urgently needed. One of the major constraints in developing P. vivax vaccine is the lack of suitable in vivo models for testing the protective efficacy of the vaccine. P. vivax and P. cynomolgi bastianelli are the two closely related malaria parasites and share a similar clinical course of infection in their respective hosts. The merozoite surface protein-1 (MSP-1) of these parasites has found to be protective in a wide range of host-parasite systems. P. vivax MSP-1 is synthesized as 200 kDa polypeptide and processed just prior to merozoite release from the erythrocytes into smaller fragments. The C- terminal 42 kDa cleavage product of MSP-1 (MSP-1(42)) is present on the surface of merozoites and a major candidate for blood stage malaria vaccine. In the present study, we have biochemically and immunologically characterized the soluble and refolded 42 kDa fragment of MSP-1 of P. vivax (PvMSP-1(42)) and P. cynomolgi B (PcMSP-1(42)). SDS-PAGE analysis showed that both soluble and refolded E. coli expressed P. vivax and P. cynomolgi B MSP-1(42) proteins were homogenous in nature. The soluble and refolded MSP-1(42) antigens of both parasites showed high reactivity with protective monkey sera and conformation-specific monoclonal antibodies against P. cynomolgi B and P. vivax MSP-1(42) antigens. Immunization of BALB/c mice with these antigens resulted in the production of high titres of cross-reactive antibodies primarily against the conformational epitopes of MSP-1(42) protein. The immune sera from rhesus monkeys. immunized with soluble and refolded MSP-1(42) antigens of both parasites also showed high titered cross-reactive antibodies against MSP-1(42) conformational epitopes. These results suggested that the soluble and refolded forms of E. coli expressed P. vivax MSP-1(42) antigens were highly immunogenic and thus a viable candidate for vaccine studies.     

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.     

A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.     

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.     

Expression and purification of Plasmodium falciparum MSP-1(42): A malaria vaccine candidate

The C-terminal 42.10(3) Da portion of the merozoite surface protein (MSP-1) of the human malaria parasite Plasmodium falciparum is of interest, not only because it may constitute an essential part of a future anti-malaria vaccine, but also due to its role during the infection of erythrocytes by the parasite. We have cloned and expressed two synthetic DNA sequences encoding the two prototypic MSP-1(42) variants in E. coli. When over-produced, both proteins form insoluble aggregates which were isolated in high purity and yield. After solubilisation and refolding in vitro, both proteins were purified to homogeneity by a three-step procedure applying Ni-chelate, size exclusion and immuno-affinity chromatography. After purification, both proteins meet key criteria of preparations for clinical use. First, conformational studies suggest proper folding of the proteins, particularly in the region containing two EGF-like domains. Polyclonal serum raised against E. coli produced MSP-1(42) recognizes native MSP-1 in Plasmodium infected erythrocytes as shown by immunofluorescence.     

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.     

Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system

We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.     

Differential antibody recognition of four allelic variants of the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum

The merozoite surface protein-2 (MSP-2) is a major vaccine candidate for the asexual blood stage of Plasmodium falciparum. MSP-2 is essentially dimorphic, and allelic families are named after the representative isolates FC27 and IC1. The polymorphic central region contains immunodominant repeats, which vary in number, length, and sequence within and between allelic families. We have examined the antibody recognition of repeat regions from both MSP-2 allelic families expressed as recombinant fusion peptides. The results are summarized as follows. (1) Immunization of mice with the fusion peptides elicited IgG antibodies that cross-reacted with the native MSP-2 molecule in an allelic family-specific manner. (2) These mouse antibodies recognized the recombinant proteins in both a variant-specific and a family-specific manner, as shown in inhibition immunoassays. Antibodies raised against the peptide FC27 seemed to be essentially variant-specific, since the soluble form of the S20 antigen (a member of FC27 family) had relatively little inhibitory effect on them. (3) The overall pattern of human IgG antibody responses to MSP-2 in Karitiana Indians, a population continuously exposed to hypoendemic malaria in the Brazilian Amazon Region, differs from that described in hyperendemic areas in Africa and Papua New Guinea in two important features: there was no clear age-dependent increase in the prevalence and mean concentration of specific IgG antibodies, and there was no skewing towards the IgG3 subclass in antibody responses. (4) The relatively poor correlation between concentrations of IgG antibodies that are specific for members of the same allelic family suggests that recognition of MSP-2 peptides by naturally acquired antibodies was largely variant-specific in this population. The potential role of naturally acquired variant-specific antibodies in immune evasion, by selecting mutant parasites carrying insertions or deletions of repeat sequences, is briefly discussed.     

Health and reproductive profiles of malaria antigen-producing transgenic goats derived by somatic cell nuclear transfer

Nuclear transfer (NT) using transfected primary cells is an efficient approach for the generation of transgenic goats. However, reprogramming abnormalities associated with this process might result in compromised animals. We examined the health, reproductive performance, and milk production of four transgenic does derived from somatic cell NT. Goats were derived from two fetal cell lines, each transfected with a transgene expressing a different version of the MSP-1(42) malaria antigen, either glycosylated or non-glycosylated. Two female kids were produced per cell line. Health and growth of these NT animals were monitored and compared with four age-matched control does. There were no differences in birth and weaning weights between NT and control animals. The NT does were bred and produced a total of nine kids. The control does delivered five kids. The NT does expressing the glycosylated antigen lactated only briefly, probably as a result of over-expression of the MSP-1(42) protein. However, NT does expressing the non-glycosylated antigen had normal milk yields and produced the recombinant protein. These data demonstrated that the production of healthy transgenic founder goats by somatic cell NT is readily achievable and that these animals can be used successfully for the production of a candidate Malaria vaccine.     

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.     

Glycosylation and proteolytic processing of 70 kDa C-terminal recombinant polypeptides of Plasmodium falciparum merozoite surface protein 1 expressed in mammalian cells

The cDNAs that encode the 70 kDa C-terminal portion of Plasmodium falciparum merozoite surface protein 1 (MSP-1), with or without an N-terminal signal peptide sequence and C-terminal glycosylphosphatidylinositol (GPI) signal sequence of MSP-1, were expressed in mammalian cell lines via recombinant vaccinia virus. The polypeptides were studied with respect to the nature of glycosylation, localization, and proteolytic processing. The polypeptides derived from the cDNAs that contained the N-terminal signal peptide were modified with N -linked high mannose type structures and low levels of O -linked oligosaccharides, whereas the polypeptides from the cDNAs that lacked the signal peptide were not glycosylated. The GPI anchor moiety is either absent or present at a very low level in the polypeptide expressed from the cDNA that contained both the signal peptide and GPI signal sequences. Together, these data establish that whereas the signal peptide of MSP-1 is functional, the GPI anchor signal is either nonfunctional or poorly functional in mammalian cells. The polypeptides expressed from the cDNAs that contained the signal peptide were proteolytically cleaved at their C-termini, whereas the polypeptides expressed from the cDNAs that lacked the signal peptide were uncleaved. While the polypeptide expressed from the cDNA containing both the signal peptide and GPI anchor signal was truncated by approximately 14 kDa at the C-terminus, the polypeptide derived from the cDNA with only the signal peptide was processed to remove approximately 6 kDa, also from the C-terminus. Furthermore, the polypeptides derived from cDNAs that lacked the signal peptide were exclusively localized intra-cellularly, the polypeptides from cDNAs that contained the signal peptide were predominantly intracellular, with low levels on the cell surface; none of the polypeptides was secreted into the culture medium to a detectable level.These results suggest that N -glycosylation alone is not sufficient for the efficient extracellular transport of the recombinant MSP-1 polypeptides through the secretory pathway in mammalian cells.     

Mapping the anatomy of a Plasmodium falciparum MSP-1 epitope using pseudopeptide-induced mono- and polyclonal antibodies and CD and NMR conformation analysis

Antigen structure modulation represents an approach towards designing subunit malaria vaccines. A specific epitope's alpha carbon stereochemistry, as well as its backbone topochemistry, was assessed for obtaining novel malarial immunogens. A variety of MSP-1(38-61) Plasmodium falciparum epitope pseudopeptides derived were synthesised, based on solid-phase pseudopeptide chemistry strategies; these included all-L, all-D, partially-D substituted, all-Psi-[NH-CO]-Retro, all-Psi-[NH-CO]-Retro-inverso, and Psi-[CH2NH] reduced amide surrogates. We demonstrate that specific recombinant MSP-1(34-469) fragment binding to red blood cells (RBCs) is specifically inhibited by non-modified MSP-1(42-61), as well as by its V52-L53, M51-V52 reduced amide surrogates and partial-D substitutions in K48 and E49. In vivo tests revealed that reduced amide pseudopeptide-immunised Aotus monkeys induced neutralising antibodies specifically recognising the MSP-1 N-terminus region. These findings support the role of molecular conformation in malaria vaccine development.     

恶性疟裂殖子表面蛋白1合成基因在毕赤酵母中的表达

恶性疟原虫裂殖子表面蛋白1是当今疟疾疫苗主要的候选抗原。由于天然MSP1基因AT含量异常高(为74%),使得克隆全长天然基因无法实现。本文已全合成了msp1基因(4940bp),解决了该天然基因在异源系统中不稳定的问题。为制备大量msp1重组蛋白进行疫苗有效性试验,本研究建立了msp1基因在毕赤酵母中的表达,将合成的msp1基因克隆到毕赤酵母胞内表达载体pPIC3.5,构建了重组质粒pPIC3.5/msp1,用电击转化毕赤酵母得到重组转化子,经PCR证实msp1基因已整合于毕赤酵母染色体中。含有重组表达质粒的毕赤酵母菌经甲醇诱导后表达出全长msp1重组蛋白。表达产物能与识别MSP1分子二硫键依赖构象表位的特异性单抗发生很强的反应,表明msp1重组蛋白至少在该表位构象上与天然蛋白一致。从毕赤酵母中分离得到大量msp1为开展该蛋白的结构与功能,特别是测定其疟疾保护性免疫提供可能。     

Plasmodium falciparum: sequence diversity and antibody recognition of the Merozoite surface protein-2 (MSP-2) in Brazilian Amazonia

The merozoite surface protein-2 (MSP-2) of Plasmodium falciparum comprises repeats flanked by dimorphic domains defining the allelic families FC27 and IC1. Here, we examined sequence diversity at the msp-2 locus in Brazil and its impact on MSP-2 antibody recognition by local patients. Only 25 unique partial sequences of msp-2 were found in 61 isolates examined. The finding of identical msp-2 sequences in unrelated parasites, collected 6-13 years apart, suggests that no major directional selection is exerted by variant-specific immunity in this malaria-endemic area. To examine antibody cross-reactivity, recombinant polypeptides derived from locally prevalent and foreign MSP-2 variants were used in ELISA. Foreign IC1-type variants, such as 3D7 (currently tested for human vaccination), were less frequently recognized than FC27-type and local IC1-type variants. Antibodies discriminated between local and foreign IC1-type variants, but cross-recognized structurally different local IC1-type variants. The use of evolutionary models of MSP-2 is suggested to design vaccines that minimize differences between local parasites and vaccine antigens.     

A malaria vaccine based on the polymorphic block 2 region of MSP-1 that elicits a broad serotype-spanning immune response

Polymorphic parasite antigens are known targets of protective immunity to malaria, but this antigenic variation poses challenges to vaccine development. A synthetic MSP-1 Block 2 construct, based on all polymorphic variants found in natural Plasmodium falciparum isolates has been designed, combined with the relatively conserved Block 1 sequence of MSP-1 and expressed in E.coli. The MSP-1 Hybrid antigen has been produced with high yield by fed-batch fermentation and purified without the aid of affinity tags resulting in a pure and extremely thermostable antigen preparation. MSP-1 hybrid is immunogenic in experimental animals using adjuvants suitable for human use, eliciting antibodies against epitopes from all three Block 2 serotypes. Human serum antibodies from Africans naturally exposed to malaria reacted to the MSP-1 hybrid as strongly as, or better than the same serum reactivities to individual MSP-1 Block 2 antigens, and these antibody responses showed clear associations with reduced incidence of malaria episodes. The MSP-1 hybrid is designed to induce a protective antibody response to the highly polymorphic Block 2 region of MSP-1, enhancing the repertoire of MSP-1 Block 2 antibody responses found among immune and semi-immune individuals in malaria endemic areas. The target population for such a vaccine is young children and vulnerable adults, to accelerate the acquisition of a full range of malaria protective antibodies against this polymorphic parasite antigen.     

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.     

Plasmodium vivax: Comparison of the immune responses between oral and parenteral immunization of rPv54 in BALB/c mice

The merozoite surface protein-1 (MSP-1) from Plasmodium vivax was evaluated as an oral vaccine candidate by cloning and expressing the interspecies conserved block 10 (ICB10) of the MSP-1 from a Korean isolate in Escherichia coli. The expressed fusion protein contained ICB10 and a maltose-binding protein (MBP), rPv54, has a molecular weight of approximately 54 kDa as determined by SDS-PAGE analysis. IgG against rPv54 was successfully produced in BALB/c mice by oral immunization and sustained for more than 4 months. IgG2b was dominantly produced in both oral and parenteral immunizations. The rPv54 increased the frequency of NK, NKT, CD4+ T, CD8+ T, and B cells in both immunizations. IL-5 and TNF-α were increased in both significantly. In conclusion, rPv54 might be a valuable potential vaccine candidate for the oral and parenteral immunization against vivax malaria.     

人乳头瘤病毒16L1-减毒志贺氏杆菌为载体疫苗的构建及免疫效应初步鉴定

为预防高危型人乳头瘤病毒16型(HPV16)诱发宫颈癌,制备以减毒志贺氏杆菌为载体的HPV16预防疫苗,以期载体可介导机体产生粘膜免疫反应,达到预防HPVl6感染的目的。为此构建了以HPV16L1为免疫原的减毒志贺氏杆菌苗,并初步鉴定候选疫苗的减毒特性和免疫效果。利用基于志贺氏杆菌virG／icsA基因的表达载体(pHS3199),将HPV16L1基因插入后构成pHS3199-hpv16L1质粒,电穿孔法将其转入减毒志贺氏杆菌sh42,经筛选获得重组减毒sh42-HPV16L1工程菌。用免疫印迹法检测HPV16L1蛋白表达,连续传代法确定其传代和目的蛋白表达的稳定性;豚鼠角膜巩膜炎症试验检测细菌的毒力和菌苗的免疫效果;小鼠红细胞凝集抑制试验检测免疫血清对病毒样颗粒(VLP)的中和活性。免疫印迹检测证实,重组菌株sh42-HPV16L1可稳定表达HPV16L1;豚鼠角膜巩膜炎症试验证实,该候选菌苗无致病性。减毒sh42-HPV16L1经结膜囊途径免疫豚鼠,可以产生特异性体液免疫应答,免疫动物体内的血清、肠道、阴道分泌物中抗HPV16L1 VLPIgG、IgA含量显著高于对照组,并且sh42-HPV16L1免疫动物血清可明显抑制HPV16L1 VLP引起的小鼠红细胞凝集。因而sh42-HPV16L1将是一种潜在的HPV16候选预防疫苗。     

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.     

Genetic diversity in merozoite surface protein (MSP)-1 and MSP-2 genes of Plasmodium falciparum in a major endemic region of Iran

Merozoite surface protein-1 (MSP-1) and merozoite surface protein-2 (MSP-2) were used to develop vaccines and to investigate the genetic diversity in Plasmodium falciparum malaria in Iran. Nested polymerase chain reaction amplification was used to determine polymorphisms of block 2 of the MSP-1 and the central domain of MSP-2 genes. A total of 67 microscopically positive P. falciparum infected individuals from a major endemic region, southeast Iran, were included in this trial. Nine alleles of MSP-1 and 11 alleles of MSP-2 were identified. The results showed that amplified product from these surface antigen genes varied in size and there was specific pattern for each isolate. Besides, regarding this pattern, 23 multiple infections with at least 2 alleles were observed. While the endemic regions of malaria in Iran is classified in low to moderate group, but extensive polymorphism was observed for each marker and the MSP-2 central repeat was the most diverse that could be considered in designing malaria vaccine.