Serum antibodies from malaria immune donors can inhibit merozoite dispersal by forming immune complexes through surface-accessible regions of membrane associated antigens. Such merozoite forms are referred to as immune clusters of merozoites (ICM). Antibodies dissociated from ICM of Plasmodium falciparum identify a restricted subset of antigens, including merozoite surface protein-1 (MSP-1). We performed epitope mapping by comparing the reactivity of whole immune sera and ICM-derived antibodies in immunoblotting assays, using fourteen overlapping recombinant MSP-1 fragments, and by ELISA, using each of the 1720 octapeptides encoded within MSP-1. Antibodies in immune sera reacted with thirteen recombinant fragments and hundreds of octapeptides, but antibodies derived from ICM reacted with only six recombinant fragments and twenty octapeptides. Recombinant fragment recognition by ICM-derived antibodies was delimited to three regions 150-200 residues long, with seven of the octapeptide epitopes also mapping to these regions. The octapeptides recognized most strongly by antibodies in whole serum corresponded to the degenerate repeats near the N-terminus of MSP-1, however, neither recombinant fragments, nor octapeptides containing these degenerate repeats, were recognized by ICM-derived antibodies. Compared to reactions with recombinant fragments, the reactions observed with octapeptides were weak and may represent low-affinity mimetopes or cross-reactions. Alternatively, they may represent reactions with a portion of an epitope assembled from more than one non-contiguous peptide. These results suggest that ICM-derived antibodies can be used to map surface-accessible epitopes on MSP-1 and that the recombinant fragments with which they react are appropriate candidates for further evaluation as components of a malaria vaccine.

A complex of non-covalently bound polypeptides is located on the surface of the merozoite form of the human malaria parasite Plasmodium falciparum. Four of these polypeptides are derived by proteolytic processing of the merozoite surface protein 1 (MSP-1) precursor. Two components, a 22 and a 36 kDa polypeptide are not derived from MSP-1. The N-terminal sequence of the 36 kDa polypeptide has been determined, the corresponding gene cloned, and the protein characterised. The 36 kDa protein consists of 211 amino acids and is derived from a larger precursor of 371 amino acids. The precursor merozoite surface protein 6 (MSP-6) has been designated, and the 36 kDa protein, MSP-6(36). Mass spectrometric analysis of peptides released from the polypeptide by tryptic digestion confirmed that the gene identified codes for MSP-6(36). Antibodies were produced to a recombinant protein containing the C-terminal 45 amino acid residues of MSP-6(36). In immunofluorescence studies these antibodies bound to antigen at the parasite surface or in the parasitophorous vacuole within schizonts, with a pattern indistinguishable from that of antibodies to MSP-1. MSP-6(36) was present in the MSP-1 complex immunoprecipitated from the supernatant of in vitro parasite cultures, but was also immunoprecipitated from this supernatant in a form not bound to MSP-1. Examination of the MSP-6 gene in three parasite lines detected no sequence variation. The sequence of MSP-6(36) is related to that of the previously described merozoite surface protein 3 (MSP-3). The MSP-6(36) amino acid sequence has 50% identity and 85% similarity with the C-terminal region of MSP-3. The proteins share a specific sequence pattern (ILGWEFGGG-[AV]-P) and a glutamic acid-rich region. The remainder of MSP-6 and MSP-3 are unrelated, except at the N-terminus. Both MSP-6(36) and MSP-3 are partially associated with the parasite surface and partially released as soluble proteins on merozoite release. MSP-6(36) is a hydrophilic negatively charged polypeptide, but there are two clusters of hydrophobic amino acids at the C-terminus, located in two amphipathic helical structures identified from secondary structure predictions. It was suggested that this 35 residue C-terminal region may be involved in MSP-6(36) binding to MSP-1 or other molecules; alternatively, based on the secondary structure and coil formation predictions, the region may form an intramolecular anti-parallel coiled-coil structure.

The merozoite of Plasmodium vivax possesses a high molecular mass surface protein called Pv-merozoite surface protein 1, PvMSP-1, which exhibits antigenic diversity among isolates. In this study, the extent of sequence variation in the polymorphic region and the flanking interspecies conserved blocks (ICBs) 5 and 6 of the PvMSP-1 gene was analyzed using the polymerase chain reaction to amplify the DNA fragment encompassing these regions, followed by sequencing. Twenty different alleles were obtained from 15 Thai isolates. Results revealed five distinct sequence types of the polymorphic region, two of which were newly identified in this study: one probably generated by intragenic recombination at a site different from that previously reported and the other by duplication of a 30 nucleotide (nt) sequence at the 3' end of the region. On the other hand, almost all nucleotide substitutions in the flanking regions, ICB5 and ICB6, were dimorphic, creating microheterogeneity in the region. Furthermore, stretches of nucleotide substitutions were found to be linked in ICB6, suggesting the potential recombination sites between these stretches. It is also noted that extensive sequence variation in the PvMSP-1 gene and coinfection with different PvMSP-1 alleles occurred among the P. vivax population in the endemic areas of Thailand.

MSP8 is a recently identified merozoite surface protein that shares similar structural features with the leading vaccine candidate MSP1. Both proteins contain two C-terminal epidermal growth factor (EGF)-like domains, a glycosylphosphatidylinositol (GPI) anchor attachment sequence and undergo proteolytic processing. By double recombination, we have disrupted the MSP8 gene in P. falciparum 3D7 parasites, and confirmed integration by southern hybridisation and PCR. Western blot analysis of lysates from asynchronous cultures and isolated merozoites demonstrated the absence of MSP8 in two cloned knockout lines. There was no significant difference in growth rate observed between 3D7 and the cloned DeltaMSP8 lines. Thus, unlike MSP1, MSP8 is not required for asexual stage parasite growth and replication in vitro. Further analysis of the cloned lines showed that loss of MSP8 had no effect on the levels of expression of other merozoite surface proteins including MSP1-5, 7 and 10. Stage-specific immunoblots showed that MSP8 expression commences in late rings and extends throughout the rest of the erythrocytic life cycle in the 3D7 parent line, but is absent from all stages in the DeltaMSP8 transfectants.

The genes encoding two merozoite surface proteins of Plasmodium vivax that are related to PvMSP3 [1] are reported. One of these genes was identified within P. vivax lambdagt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum. The insert DNA of this clone was used as a probe to isolate the complete gene from a P. vivax lambdaDASH genomic (g) DNA library. Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots. The gene, however, encodes a protein with a calculated size of 75677 Da, and 3' and 5' RACE analyses were employed to confirm the size of the gene and its coding region. The second related P. vivax gene was isolated by hybridization of a fragment of an orthologous P. knowlesi gene. The encoded proteins of all three related P. vivax genes have putative signal peptides, large central domains that contain >20% alanine residues bound by charged regions, are predicted to form alpha-helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite. Although the overall identity in amino acid alignment among the three encoded proteins is low (<40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3alpha, Pvmsp-3beta, and Pvmsp-3gamma. We further demonstrate that this family also includes related proteins from P. knowlesi and P. falciparum.

Multiple genetically distinct strains of a pathogen circulate and compete for dominance within populations of animal reservoir hosts. Understanding the basis for genotypic strain structure is critical for predicting how pathogens respond to selective pressures and how shifts in pathogen population structure can lead to disease outbreaks. Evidence from related Apicomplexans such as Plasmodium, Toxoplasma, Cryptosporidium and Theileria suggests that various patterns of population dynamics exist, including but not limited to clonal, oligoclonal, panmictic and epidemic genotypic strain structures. In Babesia bovis, genetic diversity of variable merozoite surface antigen (VMSA) genes has been associated with disease outbreaks, including in previously vaccinated animals. However, the extent of VMSA diversity within a defined population in an endemic area has not been examined. We analyzed genotypic diversity and temporal change of MSA-1, a member of the VMSA family, in individual infected animals within a reservoir host population. Twenty-eight distinct MSA-1 genotypes were identified within the herd. All genotypically distinct MSA-1 sequences clustered into three groups based on sequence similarity. Two thirds of the animals tested changed their dominant MSA-1 genotypes during a 6-month period. Five animals within the population contained multiple genotypes. Interestingly, the predominant genotypes within those five animals also changed over the 6-month sampling period, suggesting ongoing transmission or emergence of variant MSA-1 genotypes within the herd. This study demonstrated an unexpected level of diversity for a single copy gene in a haploid genome, and illustrates the dynamic genotype structure of B. bovis within an individual animal in an endemic region. Co-infection with multiple diverse MSA-1 genotypes provides a basis for more extensive genotypic shifts that characterizes outbreak strains.

The present study aimed to examine the genetic diversity of human malaria parasites (i.e., P. falciparum, P. vivax and P. knowlesi) in Malaysia and southern Thailand targeting the 19-kDa C-terminal region of Merozoite Surface Protein-1 (MSP-119). This region is essential for the recognition and invasion of erythrocytes and it is considered one of the leading candidates for asexual blood stage vaccines. However, the genetic data of MSP-119 among human malaria parasites in Malaysia is limited and there is also a need to update the current sequence diversity of this gene region among the Thailand isolates. In this study, genomic DNA was extracted from 384 microscopy-positive blood samples collected from patients who attended the hospitals or clinics in Malaysia and malaria clinics in Thailand from the year 2008 to 2016. The MSP-119 was amplified using PCR followed by bidirectional sequencing. DNA sequences identified in the present study were subjected to Median-joining network analysis with sequences of MSP-119 obtained from GenBank. DNA sequence analysis revealed that PfMSP-119 of Malaysian and Thailand isolates was not genetically conserved as high number of haplotypes were detected and positive selection was prevalent in PfMSP-119, hence questioning its suitability to be used as a vaccine candidate. A novel haplotype (Q/TNG/L) was also detected in Thailand P. falciparum isolate. In contrast, PvMSP-119 was highly conserved, however for the first time, a non-synonymous substitution (A1657S) was reported among Malaysian isolates. As for PkMSP-119, the presence of purifying selection and low nucleotide diversity indicated that it might be a potential vaccine target for P. knowlesi.

The genes encoding merozoite surface protein 4/5 (MSP4/5) from Plasmodium berghei and Plasmodium yoelii have been cloned and completely sequenced. Comparisons of the predicted protein sequences with those of Plasmodium chabaudi MSP4/5 and Plasmodium falciparum MSP4 and MSP5 show general structural similarities. All predicted proteins contain hydrophobic signal sequences, potential GPI attachment sequences and a single epidermal growth factor (EGF)-like domain at the C-terminus. The amino acid sequence of the EGF-like motif is highly conserved in rodent malaria species and also shows a considerable degree of similarity with the EGF-like domains found in the P. falciparum proteins. Both the P. yoelii and P. berghei genes show evidence of both spliced and unspliced mRNA at steady state. This phenomenon is similar to that seen for the P. chabaudi MSP4/5 gene, and is believed to be involved in regulation of protein expression. We describe here the construction of clones expressing full length recombinant protein. Antibodies directed against recombinant MSP4/5 proteins recognize a single polypeptide on parasite material and show crossreactivity between MSP4/5 from different murine malaria species, but do not crossreact with either MSP4 or MSP5 from P. falciparum. The various antisera show reactivity against reduction sensitive epitopes as well as reduction insensitive epitopes.

The gene coding for merozoite surface protein 7 has been identified and sequenced in three lines of Plasmodium falciparum. The gene encodes a 351 amino acid polypeptide that is the precursor of a 22-kDa protein (MSP7(22)) on the merozoite surface and non-covalently associated with merozoite surface protein 1 (MSP1) complex shed from the surface at erythrocyte invasion. A second 19-kDa component of the complex (MSP7(19)) was shown to be derived from MSP7(22) and the complete primary structure of this polypeptide was confirmed by mass spectrometry. The protein sequence contains several predicted helical and two beta elements, but has no similarity with sequences outside the Plasmodium databases. Four sites of sequence variation were identified in MSP7, all within the MSP7(22) region. The MSP7 gene is expressed in mature schizonts, at the same time as other merozoite surface protein genes. It is proposed that MSP7(22) is the result of cleavage by a protease that may also cleave MSP1 and MSP6. A related gene was identified and cloned from the rodent malaria parasite, Plasmodium yoelii YM; at the amino acid level this sequence was 23% identical and 50% similar to that of P. falciparum MSP7.

Plasmodium merozoites are covered with a palisade layer of proteins that are arranged as organized bundles or appear as protruding spikes by electron microscopy. Here we present a third Plasmodium vivax merozoite surface protein, PvMSP-3, which is associated with but not anchored in the merozoite membrane. Serum from a P. vivax immune squirrel monkey was used to screen a lambdagt11 P. vivax genomic DNA (gDNA) library. Plaque-selected antibodies from clone no. 6.1, and rabbit antisera against its encoded protein, produced a pattern in immunofluorescence assays (IFAs) that is consistent with a localization at the surface of mature schizonts and free merozoites. Specific antisera also agglutinated merozoites and recognized a protein of 150 000 Da by SDS-PAGE. The complete msp-3 gene and flanking sequences were cloned from a P. vivax lambda Dash II gDNA library and also partly characterized by RACE (rapid amplification of cDNA ends). The immediate upstream sequence contains non-coding repeats and a putative protein encoding open reading frame (ORF), which are also present on the msp-3 5'RACE gene product. Pvmsp-3 encodes a protein with a calculated mass of 89 573 Da, which has a potential signal peptide and a major central alanine-rich domain (31%) that exhibits largely alpha-helical secondary structure and is flanked by charged regions. The protein does not have a putative transmembrane domain or a consensus sequence for a glycosylphosphatidylinositol (GPI) anchor modification. However, the alanine-rich domain has heptad repeats that are predicted to form coiled-coil tertiary structures, which mediate protein-protein interactions. PvMSP-3 is structurally related to P. falciparum MSP-3 and the 140000 Da MSP of P. knowlesi. Characterization of PvMSP-3, thus, also begins to define a new interspecies family of evolutionarily related Plasmodium merozoite proteins.

Merozoite surface protein 1 (MSP 1) of Plasmodium falciparum has a major allelic dimorphism in the majority of its sequence, the origin and significance of which is obscure. Here, the cloning and sequencing of the msp1 gene from P. reichenowi (a chimpanzee parasite that is the nearest relative of P. falciparum) and P. gallinaceum (a malaria parasite of birds) is reported. P. reichenowi msp1 is most closely related to one allelic type (K1) of P. falciparum. The other P. falciparum major allelic type (MAD20) is very divergent from these sequences, although not as divergent as msp1 of P. gallinaceum. Assuming a date of 6 million years ago (mya) for the divergence of the P. falciparum K1 and the P. reichenowi msp1 genes (on the basis of previous estimates for these parasite species as well as host divergence times), the most recent common ancestor of the dimorphic region of msp1 would date to approximately 27mya. Thus, the P. falciparum msp1 dimorphism is confirmed as one of the oldest polymorphisms known with the exception of self-incompatibility S genes in Solanaceae. In contrast with the major allelic dimorphism, the polymorphisms present in the relatively conserved C terminus of P. falciparum msp1 appear to have arisen since the divergence of the P. falciparum and P. reichenowi msp1 genes.

In Plasmodium falciparum, merozoite surface protein 7 (MSP7) was originally identified as a 22kDa protein on the merozoite surface and associated with the MSP1 complex shed during erythrocyte invasion. MSP7 is synthesised in schizonts as a 351-amino acid precursor that undergoes proteolytic processing. During biosynthesis the MSP1 and MSP7 precursors form a complex that is targeted to the surface of developing merozoites. In the sequential proteolytic processing of MSP7, N- and C-terminal 20 and 33kDa products of primary processing, MSP7(20) and MSP7(33) are formed and MSP7(33) remains bound to full length MSP1. Later in the mature schizont, MSP7(20) disappears from the merozoite surface and on merozoite release MSP7(33) undergoes a secondary cleavage yielding the 22kDa MSP7(22) associated with MSP1. In free merozoites, both MSP7(22) and a further cleaved product, MSP7(19) present only in some parasite lines, were detected; these two derivatives are shed as part of the protein complex with MSP1 fragments during erythrocyte invasion. Primary processing of MSP7 is brefeldin A-sensitive while secondary processing is resistant to both calcium chelators and serine protease inhibitors. Primary processing of MSP7 occurs prior to that of MSP1 in a post-Golgi compartment, whereas the secondary cleavage occurs on the surface of the developing merozoite, possibly at the time of MSP1 primary processing and well before the secondary processing of MSP1.

Merozoite surface protein-1 (MSP-1) is a major candidate in the development of a vaccine against malaria. Immunisation with a recombinant fusion protein containing the two Plasmodium yoelii MSP-1 C-terminal epidermal growth factor-like domains (MSP-1(19)) can protect mice against homologous but not heterologous challenge, and therefore, antigenic differences resulting from sequence diversity in MSP-1(19) may be crucial in determining the potential of this protein as a vaccine. Representative sequence variants from a number of distinct P. yoelii isolates were expressed in Escherichia coli and the resulting recombinant proteins were screened for binding to a panel of monoclonal antibodies (Mabs) capable of suppressing a P. yoelii YM challenge infection in passive immunisation experiments. The sequence polymorphisms affected the binding of the antibodies to the recombinant proteins. None of the Mabs recognised MSP-1(19) of P. yoelii yoelii 2CL or 33X or P. yoelii nigeriensis N67. The epitopes recognised by the Mabs were further distinguished by their reactivity with the other fusion proteins. The extent of sequence variation in MSP-1(19) among the isolates was extensive, with differences detected at 35 out of the 96 positions compared. Using the 3-dimensional structure of the Plasmodium falciparum MSP-1(19) as a model, the locations of the amino acid substitutions that may affect Mab binding were identified. The DNA sequence of MSP-1(19) from two Plasmodium vinckei isolates was also cloned and the deduced amino acid sequence compared with that in other species.

Merozoite surface protein-3 (MSP-3) is a secreted polymorphic antigen associated with erythrocytic schizonts and merozoites of Plasmodium falciparum asexual blood-stages. A prominent structural feature of MSP-3 is a domain composed of three blocks of tandemly-repeated heptads with the consensus sequence AXXAXXX. The three blocks of four alanine heptad-repeats are separated by short stretches of non-repetitive sequence unrelated to the heptad-repeat. C-terminal to the heptad-repeats, MSP-3 contains a glutamic acid-rich domain followed by another heptad-repeat similar to a leucine-zipper motif. An analysis of the msp-3 gene from four P. falciparum isolates shows that polymorphism in MSP-3 is predominantly due to sequence diversity in the N-terminal half of the predicted polypeptide within and flanking the heptad-repeats. Mutations in the region of the gene that encodes the alanine heptad-repeats appear to be of two types. Unique mutations in non-repetitive sequence have generated amino acid substitutions and deletions that result in unique sequences among MSP-3 variants. In contrast, mutations in the heptad-coding sequence are largely dimorphic and are clustered in one or two heptads in each of the three blocks of heptads. Despite the diversity within and flanking the heptad domain the AXXAXXX motif is highly conserved as are other features of the sequence that predict the formation of alpha-helical secondary structure. Recombinant proteins and a synthetic peptide were used to raise antisera to conserved and variable regions of MSP-3. Differential reactivity of these reagents with the parasite antigen identified the alanine heptad-repeat domain as a site of antigenic diversity among MSP-3 polypeptides.

Proteins located on the surface of the pathogenic malaria parasite Plasmodium falciparum are objects of intensive studies due to their important role in the invasion of human cells and the accessibility to host antibodies thus making these proteins attractive vaccine candidates. One of these proteins, merozoite surface protein 3 (MSP3) represents a leading component among vaccine candidates; however, little is known about its structure and function. Our biophysical studies suggest that the 40 residue C-terminal domain of MSP3 protein self-assembles into a four-stranded alpha-helical coiled coil structure where alpha-helices are packed "side-by-side". A bioinformatics analysis provides an extended list of known and putative proteins from different species of Plasmodium which have such MSP3-like C-terminal domains. This finding allowed us to extend some conclusions of our studies to a larger group of the malaria surface proteins. Possible structural and functional roles of these highly conserved oligomerization domains in the intact merozoite surface proteins are discussed.

Plasmodium vivax merozoite surface protein-9 (Pvmsp-9) is characterized here along with orthologues from the related simian malarias Plasmodium cynomolgi and Plasmodium knowlesi. We show that although the corresponding MSP-9 proteins do not have acidic-basic repeated amino acid (aa) motifs, they are related to the Plasmodium falciparum acidic-basic repeat antigen (ABRA) also known as p101. Recognition of this new interspecies Plasmodium MSP family stems from the prior identification of related MSP termed PvMSP-185, PcyMSP-150, and PkMSP-110 on the surface of P. vivax, P. cynomolgi and P. knowlesi merozoites. A clone containing the nearly complete P. knowlesi gene encoding PkMSP-110/MSP-9 provided a hybridization probe and initial sequence information for the design of primers to obtain the P. vivax and P. cynomolgi orthologues using polymerase chain reaction (PCR) amplification strategies. The P. vivax, P. cynomolgi and P. knowlesi msp-9 genes encode proteins that range in calculated molecular mass from 80 to 107 kDa, have typical eukaryotic signal peptides and diverse repeated motifs present immediately upstream of their termination codon. Another feature conserved among these proteins, including the P. falciparum ABRA protein, is the positions of four cysteine residues near the N-terminus, suggesting this conservation maintains structural and perhaps functional characteristics in the MSP-9 family. Rabbit polyclonal antisera raised against recombinantly expressed N-termini of P. knowlesi and P. vivax MSP-9 cross-react with the counterpart proteins in immunofluorescence and immunoblot assays. Comparative interspecies investigations of the potential role(s) of Plasmodium MSP-9 in merozoite invasion of erythrocytes and as a malaria vaccine candidate can now be pursued.

A gene encoding a 352 amino acid protein with a putative signal sequence, transmembrane domain and thrombospondin structural homology repeat was identified in the genome of the human malaria parasite, Plasmodium falciparum and the rodent malaria parasite, Plasmodium berghei. The protein localises in the apical organelles of P. falciparum and P. berghei merozoites within intraerythrocytic schizonts and has, therefore, been termed the Plasmodium thrombospondin-related apical merozoite protein (PTRAMP). PTRAMP co-localises with the Apical Merozoite Antigen-1 (AMA-1) in developing micronemes and subsequently relocates onto the merozoite surface. Although the gene appears to be specific to the Plasmodium genus, orthologues are present in the genomes of all malaria parasite species examined suggesting a conserved function in host-cell invasion. PTRAMP, therefore, has all the features to merit further evaluation as a malaria vaccine candidate.

Many surface antigens of the human malaria parasite Plasmodium falciparum show extraordinary diversity, with different alleles being so divergent as to be unalignable in some coding regions. To better understand the population history and modes of selection on such loci, we sequenced genomic regions flanking the highly polymorphic genes merozoite surface protein-1, merozoite surface protein-2, and circumsporozoite protein, from reference isolates of P. falciparum. Diversity was much lower in genomic flanking regions than in the coding sequences. Average pairwise nucleotide diversity for these regions was 0.00088, similar to other genomic regions not thought to be evolving under balancing selection, suggesting against balancing selection acting on promoter regions of these genes. Most observed polymorphisms were singletons. A higher ratio of SNPs to indels than previously reported for P. falciparum was observed. An 11 bp repeat upstream of msp2 showed an intriguing pattern of polymorphism possibly suggestive of purifying selection on total allele length.

The processing and localization of Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) products were examined using polyclonal and monoclonal antibodies raised to a recombinant protein containing residues 1-294 of RAP-1. Immunoblot and epitope mapping results with antibodies that selectively bound epitopes in the RAP-1 products Pr86, p82, and p67 showed that p82 and p67 are formed from Pr86 by progressive removal of epitopes from the amino-terminus of the RAP-1 coding sequence. The capacity of Pr86 to form complexes was revealed after size fractionation of parasite proteins radiolabeled in the presence of brefeldin A to prevent processing of Pr86. Fractions containing complexed Pr86 also contained the RAP-2 product p39 and the RAP-3 product p37, suggesting that Pr86, p39 and p37 may form complexes similar to complexes previously reported for p82 and p67 with p39 or p37. Immunofluorescence localization and immunoblot studies revealed that Pr86 is present in the rhoptries, but only transiently, and that it is not detected in segmenting schizonts or extracellular merozoites. p67 and p82, on the other hand, were shown to be major RAP-1 components in purified merozoites. Neither p67 nor p82 were relocalized from the intracellular rhoptries to the merozoite surface under conditions that promoted relocalization of the rhoptry protein PF83/apical membrane antigen 1. These results suggest that processing of Pr86 begins after Pr86 complexes are transported to the forming rhoptries and that two site-selective processing reactions occur in the rhoptries, a rapid cleavage of Pr86 to p82 and a delayed cleavage of p82 to p67. Since p67 is missing from ring-stage parasites (Howard et al., Am J Trop Med Hyg, 1984;33:1055 59), the present results indicate there is a narrow time during which p67 may play a role in merozoite invasion of erythrocytes.

Polymorphic regions of the genes encoding Plasmodium vivax apical membrane antigen 1 (PvAMA1) and P. vivax merozoite surface protein 1 (PvMSP1) were sequenced to examine population diversity both within and between geographical areas. Sequences were obtained for 219 isolates for PvAMA1 and for 175 isolates for PvMSP1 from Africa, China, India, Indonesia, Philippines, Papua New Guinea, Solomon Islands and Thailand. Over half of the isolates were obtained from different regions within the Philippines, and this was used to look at the diversity within a country. Sixty nine haplotypes and 22 polymorphic sites in a 414-bp region of PvAMA1 and 41 haplotypes and 34 polymorphic sites in a 249-bp fragment of PvMSP1 were detected. For both PvAMA1 and PvMSP1, four previously unreported polymorphic nucleotide positions were identified. Population analysis indicated that there were significant differences in allele frequencies between different regions but these differences were small compared to the diversity within populations (Fixation index, F(ST), of 0.126 and 0.078 for PvAMA1 and PvMSP1, respectively). PvAMA1 and PvMSP1 had similar nonsynonymous substitution frequencies but surprisingly, the synonymous substitution frequency for PvMSP1 was eight times the frequency for PvAMA1 suggesting that synonymous substitutions in at least PvAMA1 are not neutral.