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.     

Plasmodium falciparum: Rosettes do not protect merozoites from invasion-inhibitory antibodies

Rosetting is a parasite adhesion phenotype associated with severe malaria in African children. Why parasites form rosettes is unknown, although enhanced invasion or immune evasion have been suggested as possible functions. Previous work showed that rosetting does not enhance parasite invasion under standard in vitro conditions. We hypothesised that rosetting might promote invasion in the presence of host invasion-inhibitory antibodies, by allowing merozoites direct entry into the erythrocytes in the rosette and so minimising exposure to plasma antibodies. We therefore investigated whether rosetting influences invasion in the presence of invasion-inhibitory antibodies to MSP-1. We found no difference in invasion rates between isogenic rosetting and non-rosetting lines from two parasite strains, R29 and TM284, in the presence of MSP-1 antibodies (P = 0.62 and P = 0.63, Student's t test, TM284 and R29, respectively). These results do not support the hypothesis that rosettes protect merozoites from inhibitory antibodies during invasion. The biological function of rosetting remains unknown.     

Plasmodium falciparum: worldwide sequence diversity and evolution of the malaria vaccine candidate merozoite surface protein-2 (MSP-2)

We examined patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2) of Plasmodium falciparum, a major dimorphic malaria vaccine candidate antigen, by analyzing 448 msp-2 alleles from all continents. We describe several nucleotide replacements, insertion and deletion events, frameshift mutations, and proliferations of repeat units that generate the extraordinary diversity found in msp-2 alleles. We discuss the role of positive selection exerted by naturally acquired type- and variant-specific immunity in maintaining the observed levels of polymorphism and suggest that this is the most likely explanation for the significant excess of nonsynonymous nucleotide replacements found in dimorphic msp-2 domains. Hybrid sequences created by meiotic recombination between alleles of different dimorphic types were observed in few (3.1%) isolates, mostly from Africa. We found no evidence for an extremely ancient origin of allelic dimorphism at the msp-2 locus, predating P. falciparum speciation, in contrast with recent findings for other surface malarial antigens.     

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.     

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.     

Plasmodium falciparum: genetic diversity of C-terminal region of MSP-1 in isolates from Indian sub-continent

Malaria parasites exhibit sequence diversity for a number of stage specific antigens. Several studies have proved that merozoite surface protein-1 (MSP-1) is an effective target eliciting a protective immune response. The MSP-1(42) region comprising two EGF-like domains is involved in generating protective immune response in humans and other experimental animals. Searching for point mutations in this region is essential in view of vaccine development. We have investigated the sequence variations in Plasmodium falciparum MSP-1 carboxy terminal region in field isolates from different regions in India. Our study reveals the presence of eight variant types of MSP-1(19) in the Indian sub-continent, which comprise of E-TSR-L, Q-TSR-L, E-TSG-L, Q-KNG-L, Q-KNG-F, E-KNG-L, E-KNG-F, and E-KYG-F. The last named allele is a novel variant being reported for the first time.     

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.     

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.     

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.     

Antimalarial Activity of Cupredoxins: THE INTERACTION OF PLASMODIUM MEROZOITE SURFACE PROTEIN 119 (MSP119) AND RUSTICYANIN*

The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed, and the 19-kDa C-terminal fragment (MSP1₁₉) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth, MSP1₁₉ is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based on BiGGER is employed on MSP1₁₉-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP1₁₉ at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria treatment and provide valuable information for drug design.     

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.     

Distribution of IgG subclass antibodies specific for Plasmodium falciparum glutamate-rich-protein molecule in sickle cell trait children with asymptomatic infections

Polymorphism in the beta-globin gene (hemoglobin S) has been associated with protection against severe forms of malaria. In a cross-sectional study, 180 young Gabonese children with and without sickle cell trait and harboring asymptomatic Plasmodium falciparum infections, were assessed for the responses to recombinant protein containing the conserved region of glutamate-rich protein (GLURP). We reported increased age-dependence of antibody prevalence and levels of total IgG (p<0.0001), IgG1 (p=0.009), and IgG3 (p<0.03) antibodies to GLURP with a cut-off at 5 years of age. Whatever the hemoglobin type, cytophilic antibodies (IgG1 and IgG3) were prevalent, but GLURP-specific IgG4 antibodies were detected at significantly (p<0.05) lower levels in HbAS children. We showed that the distribution of non-cytophilic IgG antibodies differs according to the hemoglobin type and to the malaria antigens tested. This may have possible implication for the clearance of malaria parasites and for protection against severe malaria.     

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.     

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.     

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.     

Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.     

Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia

We have optimized a faster and cheaper real-time PCR and developed a conventional genus specific PCR based on 18S rRNA gene to detect malaria parasites in low-grade parasitemias. Additionally, we compared these PCRs to the OptiMAL-IT test. Since there is no consensus on choice of standard quantitative curve in real-time assays, we decided to investigate the performance of parasite DNA from three different sources: "genome", amplicon and plasmid. The amplicon curve showed the best efficiency in quantifying parasites. Both PCR assays detected 100% of the clinical samples tested; the sensitivity threshold was 0.5 parasite/mul and no PCR positive reaction occurred when malaria parasites were not present. Conversely, if OptiMAL-IT were employed for malaria diagnosis, 30% of false-negative results could be expected. We conclude that PCR assays have potential for detecting malaria parasites in asymptomatic infections, in evaluation of malaria vaccine molecule candidates, for screening blood donors, especially in endemic areas, or even in monitoring malaria therapy.     

Immunogenicity of C-terminus of Plasmodium falciparum merozoite surface protein 1 expressed as a non-glycosylated polypeptide in yeast

The C-terminal region of the merozoite surface protein 1 (MSP1_(19)) is one of the mostpromising vaccine candidates against the erythrocytic forms of malaria.In the present study,a gene encodingPlasmodium falciparum MSP1_(19) was expressed in yeast Pichia pastoris.A non-glycosylated form of therecombinant protein MSP1_(19) was purified from culture medium.This recombinant protein maintains itsantigenicity.Significant immune responses were seen in C57BL/6 mice after the second immunization.Moreover,the specific antibodies recognized the native antigens of P.falciparum,The prevailing isotypesof immunoglobulin (Ig)G associated with immunization were IgG1,IgG2a and IgG2b.The antibodiesisolated from mouse sera immunized with MSP1_(19) can inhibit parasite growth in vitro.Based on theseimmunological studies,we concluded that MSP1_(19) deserves further evaluation in pre-clinical immunizationsagainst P.falciparum.     

Sequence Diversity and Linkage Disequilibrium within the Merozoite Surface Protein-1 (Msp-1) Locus of Plasmodium falciparum: A Longitudinal Study in Brazil

ABSTRACT. The merozoite surface protein‐1 (MSP‐1) is a major vaccine candidate for the asexual blood stage of malaria. We examined both the extent of sequence diversity in block 17, the 3′end of Msp‐1 gene coding for a 19‐kDa polypeptide (MSP‐1₁₉) putatively involved in red blood cell binding, and the patterns of linkage disequilibrium between polymorphic sites throughout the Msp‐1 locus. The parasite population sample consisted of Plasmodium falciparum isolates collected between 1985 and 1998 in Rondônia. an area of hypoendemic malaria transmission in the southwestern Brazilian Amazon. Results were summarized as follows. (I) Seven block‐17 sequence variants or haplotypes were found among 130 isolates, including two new haplotypes (novel combinations of previously reported amino acid replacements), here named Brazil‐1 (E‐TSR‐F) and Brazil‐2 (Q‐TSR‐F). (2) As previously shown for other Msp‐1 polymorphisms, frequencies of block‐17 haplotypes displayed significant temporal variation. (3) Extensive linkage disequilibrium was demonstrated between neighboring dimorphic sites within block 17, as well as between polymorphisms at the 5′and 3′ends of Msp‐1 (map distance range: 3.83–4.99 kb). (4) The overall patterns of linkage disequilibrium within Msp‐1 remained stable over a period of nearly one decade, and examples of possible ‘epidemic’ expansion of parasites carrying particular Msp‐1 alleles were found in the 1980s and 1990s. These results are discussed in relation to the population biology of P. falciparum and the development of malaria vaccines based on MSP‐1.