Analysis of Plasmodium vivax merozoite surface protein-1 gene sequences from resurgent Korean isolates

The merozoite surface protein-1 (MSP-1) of Plasmodium vivax exhibits great antigenic diversity among different isolates of this parasite. This antigen is a useful genetic marker for studying the polymorphism of natural P. vivax parasite populations. One or more of these populations has been responsible for resurgent malaria now occurring in Korea. This paper reports the analysis of a highly polymorphic region between interspecies conserved blocks 5 and 6 of the MSP-1 gene, using the polymerase chain reaction to amplify the DNA fragment encompassing these regions from 25 Korean isolates, followed by sequencing. Almost all amino acid sequences of Korean isolates were nearly identical to that of Thai isolates TD525A (96.6-99.7%) and TD424 (96.3-99.5%), and very similar to that of the France-Belem strain when compared with other isolates (Sal-1, Sri Lanka, and Colombia). Interallelic recombination was found in the poly-Q repeat and a Sal-1 type amino acid structure was observed in all isolates. This study shows that the MSP gene nucleotide sequence of resurgent P. vivax in Korea is most similar to that of Thai isolates; however, the Korean strains are phylogenetically unique.     

间日疟原虫环子孢子蛋白和裂殖子表面抗原的多态性

虽然恶性疟原虫疫苗抗原研究取得了较大进展，目前已制备出许多恶性疟原虫期特异性抗原［１］，但间日疟原虫的研究因不能体外培养而受限制。本文将主要描述至今已特征化了的两种间日疟原虫候选疫苗抗原：环子孢子蛋白和裂殖子表面抗原－１。为了资料的完整性和选择性，本...     

Cloning and characterization of the merozoite surface antigen 1 gene of Plasmodium berghei.

Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome.     

间日疟原虫不同MSP-1等位基因型形态学观察

目的　比较两种不同等位基因型间日疟原虫生物学形态特征。方法　现场采集间日疟病人血样并进行基因分型 ,镜下观察形态并测量大小。结果与结论　 72份现场分离株基因分型为Sal- 1型 4 0份 ,Belem型 2 5份 ,混合感染 7份 ,均查到典型的间日疟原虫 ,6例多重感染病例均发现在海南分离株 ,多重感染与基因型混合感染无关联。测量正常红细胞、寄生红细胞、环状体以及核大小差别有显著意义 ,Belem型比Sal- 1型大 ,两种不同基因型差异的分子机制需进一步探讨     

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.     

Limited polymorphism of the Plasmodium vivax merozoite surface protein 1 gene in isolates from Turkey

The 200-kD merozoite surface protein of Plasmodium vivax (PvMSP-1) is one of the leading vaccine candidates against P. vivax malaria. However, the gene encoding PvMSP-1 (pvmsp1) is highly polymorphic and is a major obstacle to effective vaccine development. To further understand polymorphism in pvmsp1, we obtained 30 full-length pvmsp1 sequences from southeastern Turkey. Comparative analysis of sequences from Turkey and other areas showed substantially limited polymorphism. Substitutions were found at 280 and 162 amino acid sites in samples from other regions and those from Turkey, respectively. Eight substitutions were unique to Turkey. In one of them, D/E at position 1706 in the C-terminal 19-kD region, the K/E change at 1709 was the only polymorphism previously known. Limited diversity was also observed in microsatellites. Data suggest a recent population bottleneck in Turkey that may have obscured a signature for balancing selection in the C-terminal 42-kD region, which was otherwise detectable in other areas.     

我国不同疟区间日疟原虫裂殖子表面蛋白1(PvMSP-1)基因多态性研究

目的　了解我国不同疟疾流行区间日疟原虫裂殖子表面蛋白 - 1基因多态性及其分布。方法　用套式PCR扩增间日疟原虫现场分离株MSP - 1第五多态区 (ICB5 -ICB6 )基因片段 ,部分样本进行序列测定、分析和比对。结果　 82份间日疟原虫现场分离株扩增出 4 70bp片段 5 0份 ,4 0 0bp片段 39份 ,其中 7份为两种片段的混合型。海南分离株混合型为 2 0 %(6 / 30 ) ,平均克隆数为 1 2 0 (36 / 30 ) ,安徽分离株混合型仅为 2 3% ,平均克隆数 1 0 2。对 33份样本进行序列测定 ,Sal- 1型17份 ,Belem型 2份 ,12份重组型 (Ⅲ型 )和朝鲜型 2份为我国新发现基因型。结论　我国间日疟原虫PvMSP - 1存在 4种不同的等位基因型 ,以Sal- 1型和重组型 (Ⅲ型 )占优势 ,南部疟区基因型比北部复杂。     

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。     

间日疟原虫和恶性疟原虫可溶性抗原免疫反应性比较

目的分析和比较间日疟原虫(Plasmodiumvivax,P.v)和恶性疟原虫(P.falciparum,P.f)可溶性抗原与间日疟感染者混合血清和单克隆抗体(McAb)M26-32免疫反应性的差别,以期寻找潜在的疟疾诊断抗原。方法取P.v感染者血样,用Plasmodipur滤器分离去除白细胞,经60%Percoll浓集其中的感染红细胞(i RBC)。分别制备P.v、P.f和正常红细胞(nRBC)可溶性抗原,应用P.v感染者、正常对照混合血清和M26-32单抗与相应的抗原进行免疫印迹分析。结果免疫印迹分析表明,P.v感染者能特异性识别26k、33、49、115kDaP.v抗原;100、102、110、150、175kDaP.f抗原;能够交叉识别的条带有:31、59、63、70、120kDa。M26-32单抗能识别P.f、P.v抗原中31kDa等抗原条带。结论P.v感染者能特异识别间日疟抗原组份,并能交叉识别P.f抗原,其中31kDa抗原组分具有较强的免疫原性,同时能被M26-32单抗识别,其作为P.v特异性诊断抗原的价值有待于进一步研究。     

Antigenic cross-reactivity between different alleles of the Plasmodium falciparum merozoite surface protein 2

The polymorphic domain of the gene encoding Plasmodium falciparum merozoite surface protein 2 (MSP2) was PCR amplified from blood of malaria patients, genotyped, and 19 distinct fragments were cloned and expressed in E. coli. The reactivity of naturally occurring antibodies against this panel of recombinant MSP2 antigens was tested using 67 homologous or heterologous sera from a serum bank of travel clinic patients. Sera from semi-immune individuals strongly recognized almost all recombinant antigens. Sera from primary infected patients either did not react at all (9 sera), or reacted weakly against varying numbers of antigens (39 sera). The antigens that showed reactions were mostly of the allelic family corresponding to the infecting clone, but in very few cases also of the alternative allelic family. Single clone infections and repeated samples from the same individual were analysed in greater detail. Thus, we were able to quantify cross-reactivity induced by a single P. falciparum infection. Within the two allelic families of MSP2, cross-reactivity was observed between some but not all alleles of the same family, whereas antibodies cross-reactive between variants belonging to different allelic families were detected in only a few cases.     

Longevity of naturally acquired antibody responses to the N- and C-terminal regions of Plasmodium vivax merozoite surface protein 1.

In an earlier study, we found that individuals with patent infection had significantly higher IgG antibody titers to the 19-kD C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP1) than individuals treated for malaria 1-4 months earlier. These results suggested that the antibody levels decreased rapidly following treatment. The present study was designed to determine the persistence of antibody response to the N- and C-terminal regions of PvMSP1 after infection with P. vivax in individuals from the city of Bélem in northern Brazil. Our results demonstrated that the vast majority of individuals had a significant decrease in antibody titers to the C-terminal region of PvMSP1 in a period of two months following treatment. Among responders to the C-terminal region, 44.4% became serologically negative and 44.4% had their antibody titers reduced by an average of 13-fold. Only 11.2% of the individuals had their antibody titers maintained or slightly increased during that period. A decrease in the antibody response to the recombinant protein representing the N-terminal region of PvMSP1 was also noted; however, it was not as dramatic. The rapid decrease in the antibody levels to the C-terminal region of PvMSP1 might contribute to the high risk of reinfection in these individuals.     

大湄公河次流域间日疟原虫裂殖子表面蛋白1(P vMSP142)的基因多态性研究

目的明确大湄公河次流域间日疟原虫红内期候选疫苗裂殖子表面蛋白1的C端42kDa的基因多态性特点。方法提取泰国24例和中缅边境地区26例间日疟患者血DNA,采用巢式PCR扩增Pvmsp142基因片段并测序。采用MEGA7.0,DnaSP6.10.1以及Network5.0软件对中缅边境,泰国以及基因库中柬埔寨流行株Pvmsp142基因序列进行多态性分析,中性检验,FST检验以及单倍体型分析。结果 3个地区Pvmsp142基因π值分别为0.014 16、0.024 05和0.023 06;中性检验显示三地区均处于平衡选择且泰国和柬埔寨株Pvmsp142基因的中间片段差异有统计学意义(P<0.05);中缅边境地区Pvmsp142基因与其他两个地区比较分化程度较高;三地区间单倍体型无明显的地理聚类。结论 Pvmsp142基因具有多态性并处于平衡选择;中间区域显著的平衡选择可能与人体免疫相关,可作为疫苗研制的靶点,也为Pvmsp142基因作为间日疟红内期疫苗候选研究提供相应的遗传信息。     

Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus

The organization and allelic recombination of the merozoite surface protein-1 gene of Plasmodium vivax (PvMsp-1), the most widely prevalent human malaria parasite, were evaluated in complete nucleotide sequences of 40 isolates from various geographic areas. Alignment of 31 distinct alleles revealed the mosaic organization of PvMsp-1, consisting of seven interallele conserved blocks flanked by six variable blocks. The variable blocks showed extensive variation in repeats and nonrepeat unique sequences. Numerous recombination sites were distributed throughout PvMsp-1, in both conserved blocks and variable block unique sequences, and the distribution was not uniform. Heterozygosity of PvMsp-1 alleles was higher in Asia (0.953 +/- 0.009) than in Brazil (0.813 +/- 0.047). No identical alleles were shared between Asia and Brazil, whereas all but one variable block nonrepeat sequence found in Brazil occurred in Asia. These observations suggest that P. vivax populations in Asia are ancestral to Brazilian populations, and that PvMsp-1 has heterogeneity in frequency of allelic recombination events. Recurrent origins of new PvMsp-1 alleles by repeated recombination events were supported by a rapid decline in linkage disequilibrium between pairs of synonymous sites with increasing nucleotide distance, with little linkage disequilibrium at a distance of over 3 kb in a P. vivax population from Thailand, evidence for an effectively high recombination rate of the parasite. Meanwhile, highly reduced nucleotide diversity was noted in a region encoding the 19-kDa C-terminal epidermal growth factor-like domain of merozoite surface protein-1, a vaccine candidate.     

Allelic dimorphism in the merozoite surface protein-3alpha in Korean isolates of Plasmodium vivax

To study the genetic diversity of re-emerging Plasmodium vivax in the Republic of Korea, nucleotide sequence variations at the merozoite surface protein-3alpha (PvMSP-3alpha) locus were analyzed using 24 re-emerging isolates and 4 isolates from imported cases. Compared with the well known Belem strain (Brazil), a large number of amino acid substitutions, deletions, and insertions were found at the locus of the isolates examined. The Korean isolates were divided into two allelic types; type I (15 isolates), similar to the Belem strain, and type II (9), similar to the Chess strain (New Guinea). Isolates from imported cases were classified into three types; type III (1 from Malaysia), similar to type B from western Thailand, type IV (1 each from Indonesia and India), and type V (1 from Pakistan), both being new types. Our results have shown that the MSP-3alpha locus of re-emerging Korean P. vivax is dimorphic with two allelic types coexisting in the endemic area.     

Polymorphism at the merozoite surface protein-3alpha locus of Plasmodium vivax: global and local diversity.

Allelic diversity at the Plasmodium vivax merozoite surface protein-3alpha (PvMsp-3alpha) locus was investigated using a combined polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) protocol. Symptomatic patient isolates from global geographic origins showed a high level of polymorphism at the nucleotide level. These samples were used to validate the sensitivity, specificity, and reproducibility of the PCR/RFLP method. It was then used to investigate PvMsp3alpha diversity in field samples from children living in a single village in a malaria-endemic region of Papua New Guinea, with the aim of assessing the usefulness of this locus as an epidemiologic marker of P. vivax infections. Eleven PvMsp-3alpha alleles were distinguishable in 16 samples with single infections, revealing extensive parasite polymorphism within this restricted area. Multiple infections were easily detected and accounted for 5 (23%) of 22 positive samples. Pairs of samples from individual children provided preliminary evidence for high turnover of P. vivax populations.     

Sequence heterogeneity of the merozoite surface protein-1 gene (MSP-1) of Plasmodium vivax wild isolates in southeastern Iran

The merozoite surface protein of Plasmodium vivax (PvMSP-1) has been considered as a vaccine candidate, which exhibits antigenic diversity among isolates. We investigated the extent of sequence variation in the polymorphic region 5 of PvMSP-1 in order to characterize the genetic structure and composition of P. vivax in clinical isolates from Iranshahr and Chahbahar districts of Sistan and Baluchistan province, Iran. The PvMSP-1 gene amplification revealed size variation among the isolates, ranging from 430 to 550 bp. Sequences were obtained for 15 Iranian and one Pakistani isolates and 14 different alleles were detected. Results also showed three distinct sequence types of the polymorphic region. Sequence analysis has shown several single nucleotide polymorphisms to occur in this block of PvMSP-1, creating different alleles in the progeny and also microheterogeneity in the region. Thus, this study provides preliminary evidence of sequence heterogeneity in the Iranian P. vivax population.     

Genetic polymorphism of Plasmodium vivax msp1p, a paralog of merozoite surface protein 1, from worldwide isolates

Plasmodium vivax msp1p, a paralog of the candidate vaccine antigen P. vivax merozoite surface protein 1, possesses a signal peptide at its N-terminus and two epidermal growth factor-like domains at its C-terminus with a glycosylphosphatidylinositol attachment site. The msp1p gene locus may have originated by a duplication of the msp1 gene locus in a common ancestor of the analyzed Plasmodium species and lost from P. yoelii, P. berghei, and P. falciparum during their evolutionary history. Full-length sequences of the msp1p gene were generally highly conserved; they had a few amino acid substitutions, one highly polymorphic E/Q-rich region, and a single-to-triple hepta-peptide repeat motif. Twenty-one distinguishable allelic types (A1-A21) of the E/Q-rich region were identified from worldwide isolates. Among them, four types were detected in isolates from South Korea. The length polymorphism of the E/Q-rich region might be useful as a genetic marker for population structure studies in malaria-endemic areas.     

Testing the efficacy of a recombinant merozoite surface protein (MSP-1(19) of Plasmodium vivax in Saimiri boliviensis monkeys.

Saimiri boliviensis monkeys were immunized with the yeast-expressed recombinant protein yP2P30Pv200(19). The antigen consisted of the C-terminus (amino acid Asn1622-Ser1729) of the merozoite surface protein 1 of the Plasmodium vivax Salvador I strain. Two universal T helper cell epitopes (P2 and P30) of tetanus toxin and six histidine residues for purification purposes were attached to the N- and C-termini, respectively. Four groups of five monkeys were given three immunizations at four-week intervals with either 250 microg of yP2P30Pv200(19) formulated with nonionic block copolymer P1005, 250 microg of antigen adsorbed to alum, 250 microg of antigen in phosphate-buffered saline (PBS), or PBS alone. Five weeks after the last immunization, each animal was inoculated with 100,000 parasitized erythrocytes of the Salvador I strain of P. vivax. Animals were splenectomized one week after challenge to increase parasite densities; after seven weeks of infection, animals were treated. Eighteen weeks later, the animals were rechallenged with the homologous parasite. Following the first challenge, three monkeys immunized with the antigen with P1005 were protected; no animals were protected from rechallenge. One monkey immunized with yP2P30Pv200(19) with alum was protected; no protection was seen after rechallenge. Two monkeys immunized with antigen alone were protected; none were protected from rechallenge. One control animal had a low parasite count following primary infection; none were protected against rechallenge. Adverse reactions were only observed with animals receiving P1005. It is proposed that splenectomy of the monkeys prevented adequate assessment of the efficacy of this antigen. Identification of a monkey host that supports high density parasitemia without splenectomy appears needed before further testing of blood-stage vaccines against P. vivax.     

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).