Geographical patterns of allelic diversity in the Plasmodium falciparum malaria-vaccine candidate, merozoite surface protein-2

The polymorphic merozoite surface protein-2 (MSP-2) of Plasmodium falciparum is a major malaria-vaccine candidate. In the present study, PCR and hybridization with allelic-specific probes were used to type the Msp-2 gene from isolates from hypo-endemic Brazil (N = 113), meso-endemic Vietnam (N = 208) and holo-endemic Tanzania (N = 67). The typing methods were designed to group isolates into the dimorphic allelic families FC27 and IC1 and to detect possible between-family recombination events. The analysis was complemented by a comparison of 156 Msp-2 sequences from the GenBank database with 12 additional sequences obtained during the present study. Statistically significant differences were detected in pair-wise comparisons of the distribution of Msp-2 allelic types in Brazil and Vietnam, and in Brazil and Tanzania, but not in Vietnam and Tanzania. The extent of allelic diversity in the Msp-2 gene, as estimated by the total number of different alleles found in a given parasite population and the mean multiplicity of infections, clearly paralleled the levels of malaria endemicity in the study areas. However, no correlation between age and multiplicity of infections was found in the subjects. The patterns of Msp-2 diversity in Brazil appeared to be temporally stable, since no significant difference was observed in the distribution of Msp-2 allelic types among isolates collected, 10--13 years apart, in the same area of Rond?nia. Despite the extensive sequence diversity found in Msp-2 alleles, especially in the central repetitive region of the molecule, several instances of identical or nearly identical alleles were found among isolates from different countries and regions, possibly as a result of extensive homoplasy. No recombinant allele was detected by molecular typing in any of the study sites, and the GenBank database included only 12 recombinant sequences (representing 7% of all reported Msp-2 sequences), all of them with an IC1-type 5' end and an FC27-type 3' end. A single, putative, crossover site was characterised for all recombinant alleles. Most of the allelic diversity observed was therefore attributable to variation in the repetitive region of the gene, instead of recombination between alleles of dimorphic families (as commonly found, for example, in the Msp-1 gene). The implications of these findings for studies on the genetic and antigenic diversity of malarial parasites are discussed.     

Structural diversity in the Plasmodium falciparum merozoite surface antigen 2.

Antigens associated with the surface of merozoites of the malaria parasite Plasmodium falciparum are directly accessible to immune attack and therefore are prime vaccine candidates. We have previously shown that one of the two known merozoite surface antigens (merozoite surface antigen 2; MSA-2) exhibits considerable sequence and antigenic diversity in different isolates. The sequences of MSA-2 from three isolates revealed a central domain composed of repeats that vary in number, length, and sequence, flanked in turn by nonrepetitive variable sequences and by conserved N- and C-terminal domains. We report here the sequences of a further four MSA-2 alleles, containing repetitive sequences that are related but not identical to each other. The seven alleles of MSA-2 can be divided into two distinct allele families on the basis of nonrepetitive sequences. Hybridization studies with repeat probes indicated that all of the 44 P. falciparum isolates examined contained repeat regions similar to those defined in known MSA-2 sequences.     

Genetic diversity in the merozoite surface protein 1 gene of Plasmodium falciparum in different malaria-endemic localities

A number of stage-specific antigens have been characterized for vaccine development in Plasmodium falciparum malaria. The polymorphic merozoite surface protein 1 (MSP-1) of Plasmodium falciparum is a major asexual blood stage malaria vaccine candidate antigen. In the present study, we analyzed the impact of hyperendemic malaria transmission, mesoendemic malaria transmission, and multiple infection on allelic diversity. We have used a simple strategy of polymerase chain reaction amplification and slot-blot hybridization to analyze variable regions of block-2, block-4 and blocks 6-10 of the MSP-1 gene. The allelic types of isolates collected from regions of hyperendemic malaria transmission (RHEMT) and mesoendemic malaria transmission (RMEMT) were compared. In RHEMT, 20 of 24 possible gene types were found among 163 isolates and more than one allelic type was found in 82 (50.3%) of the isolates. Thirteen of 24 possible gene types were found among 125 isolates in RMEMT and 27 (21.6%) of them contained more than one allele type. Our results suggest for the first time that the allelic distribution or allelic diversity and chances of finding multi-strain parasites in isolates in an area vary with the rate of transmission. Analyses of isolates containing more than one strain of parasite suggest that allelic types are randomly distributed, no specific type of alleles predominately show multi-strain infection, and neither strain of the parasite affect the process of infection and development of another.     

Antibody responses to Plasmodium falciparum merozoite surface protein-1 and efficacy of amodiaquine in Gabonese children with P. falciparum malaria

The relationship between the efficacy of amodiaquine for the treatment of uncomplicated Plasmodium falciparum malaria and preexisting antibodies against merozoite surface protein (MSP)-1, a blood-stage P. falciparum antigen, was investigated. The immunoglobulin G antibody response to different MSP-1 recombinant proteins was evaluated in plasma samples from Gabonese children with uncomplicated malaria who were treated with amodiaquine. The prevalence of anti-MSP-1 antibodies was similar among patients with either parasitological and clinical cure after treatment (n=102) or treatment failure (n=51) by day 28 (83% in both groups). However, associations between antibody responses to K1 and MAD20 allelic families and therapeutic success were found (P< .001 and P= .034, respectively). A high proportion of plasma samples recognizing several antigens was found in the cured group. This association was significant even when data were stratified by age, particularly for the K1 family antigens (P= .029). These results suggest that humoral immune responses play a supportive role in the efficacy of amodiaquine treatment.     

Genetic diversity of Plasmodium falciparum isolates from naturally infected children in north-central Nigeria using the merozoite surface protein-2 as molecular marker

ObjectiveTo characterize the genetic diversity of Plasmodium falciparum (P. falciparum) field isolates in children from Lafia, North-central Nigeria, using the highly polymorphic P. falciparum merozoite surface protein 2 (MSP-2) gene as molecular marker.MethodsThree hundred and twenty children were enrolled into the study between 2005 and 2006. These included 140 children who presented with uncomplicated malaria at the Dalhatu Araf Specialist Hospital, Lafia and another 180 children from the study area with asymptomatic infection. DNA was extracted from blood spot on filter paper and MSP-2 genes were genotyped using allele-specific nested PCR in order to analyze the genetic diversity of parasite isolates.ResultsA total of 31 and 34 distinct MSP-2 alleles were identified in the asymptomatic and uncomplicated malaria groups respectively. No difference was found between the multiplicity of infection in the asymptomatic group and that of the uncomplicated malaria group (P>0.05). However, isolates of the FC27 allele type were dominant in the asymptomatic group whereas isolates of the 3D7 allele type were dominant in the uncomplicated malaria group.ConclusionsThis study showed a high genetic diversity of P. falciparum isolates in North-central Nigeria and is comparable to reports from similar areas with high malaria transmission intensity.     

Genetic diversity in the merozoite surface protein 1 and 2 genes of Plasmodium falciparum from the Artibonite Valley of Haiti

Describing genetic diversity of the Plasmodium falciparum parasite provides important information about the local epidemiology of malaria. In this study, we examined the genetic diversity of P. falciparum isolates from the Artibonite Valley in Haiti using the allelic families of merozoite surface protein 1 and 2 genes (msp-1 and msp-2). The majority of study subjects infected with P. falciparum had a single parasite genotype (56% for msp-1 and 69% for msp-2: n=79); 9 distinct msp-1 genotypes were identified by size differences on agarose gels. K1 was the most polymorphic allelic family with 5 genotypes (amplicons from 100 to 300 base pairs [bp]); RO33 was the least polymorphic, with a single genotype (120-bp). Although both msp-2 alleles (3D7/IC1, FC27) had similar number of genotypes (n=4), 3D7/IC1 was more frequent (85% vs. 26%). All samples were screened for the presence of the K76T mutation on the P. falciparum chloroquine resistance transporter (pfcrt) gene with 10 of 79 samples positive. Of the 2 (out of 10) samples from individuals follow-up for 21 days, P. falciparum parasites were present through day 7 after treatment with chloroquine. No parasites were found on day 21. Our results suggest that the level of genetic diversity is low in this area of Haiti, which is consistent with an area of low transmission.     

恶性疟原虫主要裂殖子表面蛋白1C末端19ku片段(MSP1-19)的表达及免疫效应检测

目的 探讨恶性疟原虫主要裂殖子表面蛋白1C末端19ku片段（MSP1-19）重组蛋白的免疫活性。方法 利用毕赤酵母高效表达系统分泌表达MSP1-19，表达产物纯化后免疫新西兰兔，3次免疫后ELISA检测其血清中IgG滴度的变化，观测重组MSP“。免疫效应。结果 MSP1-19在毕赤酵母高效表达；重组MSP1-19免疫后，兔血清中IgG滴度的变化与对照组有显著差异。结论 MSP1-19重组蛋白具有较好的免疫原性。     

IgG antibody profile to c-terminal region of Plasmodium vivax merozoite surface protein-1 in Thai individuals exposed to malaria

Naturally acquired immune response to C-terminal region of Plasmodium vivax merozoite surface protein1 (PvMSP1) in 200 individuals with recent clinical episodes of malaria from malaria endemic areas along Thai-Myanmar border in the west and Thai-Cambodia border in the east of Thailand was evaluated by enzyme-linked immunosorbent assay (ELISA). The anti-PvMSP1-IgG antibody was observed in 110 individuals (55%). Among IgG responders, IgG1 coexpressed with IgG3 were the predominant subclasses. The levels of anti-PvMSP1 total IgG, IgG1 and IgG3 antibody response seem to be increased with age although no detectable significant correlation was found (r = 0.004, p = 0.484 for total IgG; r = 0.035, p = 0.386 for IgG1; r = -0.600, p = 0.142 for IgG2; r = 0.077, p = 0.227 for IgG3; r = 0.664, p = 0.051 for IgG4). However, the mean level of specific total IgG was highest in the age group of >40 years. These levels of either specific total IgG or each IgG isotype did not vary among individuals with different malaria episodes. A higher level of specific total IgG, IgG1 and IgG3 antibody response related with the lower of parasitemia density was observed although no significant correlation was found. Our data indicate that individuals exposed to vivax malaria in Thailand developed antibodies to the potential candidate vaccine antigen, PvMSP1 (C-terminal).     

Genetic diversity of Plasmodium falciparum and its relationship to parasite density in an area with different malaria endemicities in West Uganda

Field populations of Plasmodium falciparum can be effectively genotyped by PCR-amplification of selected fragments of the Merozoite Surface Proteins 1 and 2 (MSP1 and MSP2). Genetic diversity of P. falciparum populations in areas with different transmission levels (holo- vs. mesoendemic) was investigated in Kabarole District, West Uganda. 225 samples positive for P. falciparum were analysed by amplification of polymorphic regions and classified according to prevalence of allelic families. A large number of alleles was detected for each locus: 22 for MSP1 block 2 and 24 for MSP2 and, 175 (78%) of MSP1 alleles and 143 (64%) of MSP2 showed multiple infections within a range of 2-8 clones. Significant differences between holoendemic and mesoendemic areas in regards of population structure and number of multiclonal infections of P. falciparum were not apparent. However, a significant correlation between parasite density, selected MSP2 loci and differences between parasite density in monoclonal vs. multiclonal infections occurred. Multiplicity of infection was age-dependent.     

海南省消除疟疾后期间日疟原虫裂殖子表面蛋白1基因（MSP-1）多态性检测

目的 比对海南省在消除疟疾前期间日疟原虫裂殖子表面蛋白1的基因型。方法 采用PCR扩增特异性目的片段,基因测序,序列比对及进化树构建。结果 从27个本地感染间日疟样本分析来看,Sal-1型有24个,分别属于9个Sal-1亚型,为优势型。Belem 型有2个,同属于1个Belem型,重组型的有1个。在各亚型中大多数仍以个别氨基酸间的替换为主,但仍发现2例新亚型以连续的氨基酸(DKKLLKEYELNADEKTKINQN)叠加个别氨基酸替换,另发现1例新型的重组型亚型。Belem型以常见19个多聚谷氨酰胺型。进化树分析可以看出,间日疟Sal-1型与Belem型及重组型可以较好的区分开,属于不同进化簇。Sal-a和Sal-b与其他亚型差距较大,独成一类,而其余亚型相对聚集性一类。Belem型本次调查中虽有2例但同属于同一亚型,为常见20个谷氨酰胺的重复,从进化树得知,该型与其他亚型同属于常见Belem型。重组型仅有一个亚型,属于为II/Q/S型重组型,但该重组型在基因库中未发现同源序列,可能属于新型的重组亚型,有待于进一步研究。结论 海南消除疟疾后期(2009-2012)间日疟仍以Sal-1型为主,但仍存在该等位基因的多样性而非单一性。     

Genetic diversity in the C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium vivax (PvMSP-1(42)) among Indian isolates

Plasmodium vivax merozoite surface protein 1 (PvMSP-1) is a leading malaria vaccine candidate. This protein is processed to give rise to various sized fragments during merozoite maturation. Here, we describe the analysis of genetic diversity in the 42 kDa C-terminal part of this protein among 33 Indian P. vivax isolates. A total of 27 haplotypes with 72 mutations and 0.0212 ± 0.0005S.D. over all π nucleotide diversity were observed among the isolates. Twenty-six of 27 haplotypes reported here were new as they have not been reported so far from any other country. The difference between non-synonymous (dN) and synonymous (dS) mutations was found to be positive (0.0081 ± 0.0051) for the entire 42 kDa region. Further analysis revealed that 33 kDa (MSP-1₃₃) fragment of the MSP-1₄₂ was highly polymorphic with π nucleotide diversity 0.0290 ± 0.0007S.D. The dN-dS for this region of MSP-1 was also positive (0.0114 ± 0.0071S.E.). On the other hand, there was no non-synonymous mutation in the 19 kDa (MSP-1₁₉) fragment of the MSP-1₄₂ and thus it was highly conserved. In conclusion, MSP-1₃₃ fragment was highly polymorphic and appeared to be under diversifying selection whereas there was no selection at MSP-1₁₉ region among the isolates. Present study will be helpful for the development of PvMSP-1 based vaccine against P. vivax malaria.     

Molecular epidemiology of malaria in Cameroon. XVIII. Polymorphisms of the Plasmodium falciparum merozoite surface antigen-2 gene in isolates from symptomatic patients

Merozoite surface antigen-2 (MSA-2) is a polymorphic genetic marker that is highly discriminatory for characterizing Plasmodium falciparum field isolates. Genetic diversity of isolates obtained from symptomatic patients residing in Yaounde, Cameroon was analyzed by an allele-specific polymerase chain reaction and direct sequencing of amplification products. Of 137 isolates, 25 (18%) had only FC27-type alleles, 40 (29%) had only 3D7-type alleles, and 72 (53%) had multiple parasite populations with both alleles. Of 295 fragments, 145 (49.2%) and 150 (50.8%) belonged to FC27 and 3D7 alleles, respectively. There were 23 different MSA-2 alleles (10 FC27-type and 13 3D7-type that yielded 44 different combinations in multiple infections). DNA sequencing showed distinct individual sequences. Sequences belonging to the FC27 allelic family were relatively conserved, with most of the polymorphism arising from differences in the number of repeat units. In contrast, the sequences within the GSA-rich region in 3D7 allelic family were less conserved, but many of the sequences in Cameroonian isolates have been identified in other isolates from geographically distant origins. Our results show an extensive diversity of the central region of MSA-2 in size, allelic family, combinations of these two features in multiple infections, and sequence variations underlying the complex population structure of P. falciparum clinical isolates in Yaounde, Cameroon.     

表达恶性疟原虫裂殖子表面蛋白-1片段的减毒鼠伤寒杆菌的构建

目的 检测恶性疟原虫裂殖子表面蛋白１基因在减毒鼠伤寒直蓖Ｘ４０６４株中的表达。方法 将７个克隆有恶性疟原虫裂殖子表面蛋白１基因片段的表达质粒ｐＱＥＭ用氯化钙法转化到修饰系统正常、限制系统缺陷的鼠伤寒杆菌ＬＢ５０００中,再通过专一性噬菌体Ｐ２２转导入腺苷酸环化酶基因、环腺苷酸受体基因双重突变的减毒鼠伤寒杆菌Ｘ４０６４株中,用质粒酶切、Ｗｅｓｔｅｒｎ ｂｌｏｔ鉴定构建的重组菌株 组菌株的表达。结果 成     

Variant-specific antibodies to merozoite surface protein 2 and clinical expression of Plasmodium falciparum malaria in rural Amazonians

Naturally acquired antibodies to five variants of the merozoite surface protein 2 (MSP-2), a target of clinical immunity to Plasmodium falciparum malaria, were measured in a cohort of rural Amazonians. Local MSP-2 variants comprised both highly divergent families of alleles (FC27 and 3D7). Total IgG antibodies to two FC27-type antigens were found in 22-28% of subjects at baseline, with substantial cross-reactivity between variants and stable concentrations and specificities over time. The IgG antibodies to three 3D7-type antigens were less prevalent (6-7%), less cross-reactive, and short-lived; subsequent exposure to 3D7-type parasites rarely elicited homologous response. The clinical spectrum of 109 incident P. falciparum infections in our cohort ranged between asymptomatic infection and fully symptomatic but uncomplicated disease. Parasitemia at the time of diagnosis, rather than cumulative malaria exposure or acquired immunity (presence of variant-specific antibodies matching the MSP-2 type in infecting parasites), was a major predictor of perceived symptom severity.     

Limited allelic diversity of Plasmodium falciparum merozoite surface protein 1 gene from populations in the Solomon Islands

Meiotic recombination generates allelic diversity in the Plasmodium falciparum merozoite surface protein 1 (msp1) gene. In this study, we monitored recombination-based diversity of msp1 in Guadalcanal, the Solomon Islands, where malaria transmission is high. We identified 5' recombinant types, 3' sequence types, and msp1 haplotypes (unique associations of 5' recombinant types and 3' sequence types), and compared them with those from areas of low transmission in Thailand and Vanuatu. The mean number of 5' recombinant types per person (multiplicity) was lower in Guadalcanal than in Thailand. Guadalcanal populations had 6-8 msp1 haplotypes; the numbers are comparable to Vanuatu but much lower than in Thailand. There were marked geographic differences in distribution of msp1 haplo-types. Linkage disequilibrium in msp1 was stronger in Guadalcanal than in Thailand, suggesting limited recombination events in the Solomon Islands. We suggest that the frequency of recombination events in msp1 is determined not only by transmission intensity but by the number of msp1 alleles prevalent and multiplicity of infections.     

Genetic diversity of merozoite surface protein-1 gene of Plasmodium vivax isolates in mining villages of Venezuela (Bolivar State)

The merozoite surface protein-1 gene of Plasmodium vivax is highly polymorphic and so, currently used in epidemiological studies of P. vivax malaria. We sequenced the variable block 5 of the gene from 39 Venezuelan isolates, 18 of which were co-infected with Plasmodium falciparum. We observed a limited variability with 34 isolates belonging to the type Salvador I, none Belem type and only five recombinants. Among the recombinants, only two types of sequences were observed with, respectively, 18 and 21 poly-Q residues. Nucleotide substitutions explained the major differences of the 11 patterns observed. We could evidence neither specific MSP-1 genotype associated with co-infected samples, nor peculiar MSP-1 genotype distribution inside the investigated areas. In comparison with other low endemic regions in the world, our sampling has a lower genetic diversity, which could be mainly explained by the lack of Belem type. In fact, the variable repeats of poly-Q residues involved in the polymorphism of Belem type and recombinant isolates are responsible for a great part of variability observed in MSP-1 block 5.     

Temporal and spatial distribution of the variants of merozoite surface protein-1 (MSP-1) in Plasmodium falciparum populations in Brazil

The polymorphic, merozoite surface protein-1 (MSP-1) of Plasmodium falciparum, an antigen of the parasite's asexual blood-stages, is a major malaria-vaccine candidate. Nucleotide sequences of each variable domain or block of this antigen may be grouped into one of three possible allelic types (K1, MAD20 and RO33), and 24 major types of the msp-1 gene may be defined, as unique combinations of allelic types in these variable blocks. Isolates collected from the Brazilian Amazon, over a period of 14 years, have now been investigated, by PCR-based typing of the msp-1 gene. Thirteen of the 24 possible gene-types were identified, and 336 P. falciparum clones were fully typed among 239 isolates. Most parasites (87%) belonged to one of the seven most frequent gene-types. Marked temporal variation in the distribution of msp-1 variants was found when comparing parasites sampled in the same sites at intervals of at least 5 years. Spatial variations were also found when comparing parasites from both neighbouring and distant sites within the Amazon Basin. The between-population variance in the frequencies of msp-1 allelic types found in Brazil, as estimated by Wright's FST statistic, is of similar magnitude to that found in previous world-wide comparisons. The potential implications of these findings for the development of an MSP-1-based, multivalent malaria vaccine are discussed.     

Plasmodium falciparum subtilisin-like protease 2, a merozoite candidate for the merozoite surface protein 1-42 maturase.

The process of human erythrocyte invasion by Plasmodium falciparum parasites involves a calcium-dependent serine protease with properties consistent with a subtilisin-like activity. This enzyme achieves the last crucial maturation step of merozoite surface protein 1 (MSP1) necessary for parasite entry into the host erythrocyte. In eukaryotic cells, such processing steps are performed by subtilisin-like maturases, known as proprotein convertases. In an attempt to characterize the MSP1 maturase, we have identified a gene that encodes a P. falciparum subtilisin-like protease (PfSUB2) whose deduced active site sequence resembles more bacterial subtilisins. Therefore, we propose that PfSUB2 belongs to a subclass of eukaryotic subtilisins different from proprotein convertases. Pfsub2 is expressed during merozoite differentiation and encodes an integral membrane protein localized in the merozoite dense granules, a secretory organelle whose contents are believed to participate in a late step of the erythrocyte invasion. PfSUB2's subcellular localization, together with its predicted enzymatic properties, leads us to propose that PfSUB2 could be responsible for the late MSP1 maturation step and thus is an attractive target for the development of new antimalarial drugs.