间日疟原虫云南分离株传播阻断疫苗候选抗原Pvs 28基因多态性分析

目的分析我国疟疾混合流行区云南分离株间日疟原虫（P．v）传播阻断疫苗候选抗原Pvs28基因特点。方法收集云南省血样17份；提取疟原虫基因组DNA；PCR扩增Pvs28基因；基因测序；DnaSP version4．0软件进行基因多态性分析。结果成功扩增Pvs28全长基因17个序列。与标准株Sal—I比较，检测出7个错义突变，7个基因型和7种氨基酸型。云南P.v分离株核苷酸多态性n值为0．0044。若不考虑重复片段拷贝数的差异，云南P．v分离株和湖北P．v分离株Pvs 28蛋白主导氨基酸型完全一致，即V^14-L^52-L^98-E^105-L^115-S^14-I^122。与泰国P．v分离株比较。我国主导基因型突变位点完全包含在泰国P．v分离株突变位点中。结论以Sal—I Pvs28为基础建立的传播阻断疫苗能够克服云南P．v分离株Pvs28抗原多样性而发挥传播阻断作用，提示间日疟原虫传播阻断疫苗在我国具有良好的应用前景。     

间日疟原虫传播阻断疫苗候选抗原Pvs25中国分离株高度保守

目的　研究我国单纯间日疟流行区 3 1例患者 (湖北18例、浙江13例 )间日疟原虫分离株传播阻断疫苗候选抗原Pvs2 5基因多态性 ,并与 14例孟加拉间日疟原虫株进行比较分析。　方法　从干燥滤纸血膜提取疟原虫基因组DNA ,对Pvs25基因进行聚合酶链反应 (PCR)扩增、纯化和直接序列分析。　结果　与间日疟原虫标准株Sal-I相比 ,获得的 45个Pvs25全长序列中有 3处点突变 ,引起相应氨基酸的替换。并且核苷酸多态性 (π值 )检验结果显示 ,Pvs25在不同流行区或同一流行区不同分离株之间核苷酸及其相应的氨基酸序列高度保守。　结论　与红前期和红内期候选抗原相比 ,Pvs25具有有限的抗原多态性 ,提示以Pvs25为基础构建的传播阻断疫苗在我国流行区具有普遍应用的可能性     

我国云南分离株间日疟原虫传播阻断疫苗候选抗原pvs25基因多态性分析

目的阐明我国疟疾流行区问日疟原虫传播阻断疫苗候选抗原pvs25基因多态性特点。方法收集全年流行区云南省血样31份；提取疟原虫基因组DNA；聚合酶链反应(PCR)扩增pvs25基因；Sanger双脱氧链终止法测序；DnaSP version 4．0软件统计学分析。结果成功扩增pvs25全长基因31个序列。与标准株Sal I比较。检测出4个错义突变位点。5种基因型。4处氨基酸置换。C^103G^289C^389C^391。为主导基因型，L^35E^97T^130Q^131是相应主导氨基酸型。其突变与季节流行区的主导基因型和氨基酸型完全一致。核苷酸多态性π值为0．0014。组间多态性π值比较表明全年流行区明显高于季节流行区。结论我国分离株闻日疟原虫传播阻断疫苗候选抗原Pvs25只有1种主导基因型和1种主导氨基酸型。有限的基因突变再次提示Pvs25是理想的候选抗原，以Pvs25为基础构建的传播阻断疫苗在我国具有广泛应用的可行性。     

疟疾疫苗候选抗原Pvs25和Pvs28的抗体能完全阻断蚊感染间日疟原虫

间日疟是流行于亚洲和南美州的发病率很高并可复发的疾病。传播阻断疫苗是针对疟原虫复杂的生活史中的传播环节而设计的一种疫苗。本文作者最近在酿酒酵母中表达了动合子表面蛋白Pvs25和Pvs28基因,而进一步的实验证实Pvs25和Pvs28是很有潜力的传播阻断疫苗候选抗原。     

疫苗候选抗原Pvs48/45在中缅边境的遗传多样性特点

目的 了解间日疟原虫传播阻断疫苗候选抗原Pvs48/45在中缅边境地区的基因多态性和自然选择特点。方法 提取39例来自中缅边境主要流行区(Laiza)的间日疟原虫基因组DNA,采用巢式PCR(nest-PCR)扩增Pvs48/45基因开放阅读区(Open reading frame, ORF);通过MEGA 4.0、DnaSP v5.10和NETWORK 4.6对Pvs48/45基因多态性和自然选择特点进行分析。结果 研究发现了6个突变位点,均为非同义突变(E35K ,H211N ,K250N ,D335Y,A376T,K418R),导致了5种单倍型;Pvs48/45基因的核苷酸多态性水平极低(π=0.00064),CRDⅢ区最高(π=0.00163);多种中性检验均表明Pvs48/45基因处于平衡选择;种群进化分析和单倍型网络图显示Pvs48/45具有较强的地理分化,且H2型为中缅边境地区频率最高的单倍型。结论 Pvs48/45在中缅边境地区遗传多样性很低,且在进化过程中经历平衡选择,提示其可作为传播阻断疫苗候选靶点;Pvs48/45在全球具有较强的地理分化,应根据地区遗传特点开发更加有效的传播阻断疫苗。     

《中国寄生虫学与寄生虫病杂志》2004年第22卷文题索引

疟疾流行病学遥感地理信息系统技术在疟疾研究中的应用 119………………洪涝灾害遥感资料用于疟疾和流行性乙型脑炎疫情分析　　 14 4……………………………………………………………济南市基本消灭疟疾后疟疾疫情分析 190………………………商水县 2 0 0 1～ 2 0 0 3年疟疾暴发调查与控制 3 74…………………山东省基本消灭疟疾后 15年来的监测 3 78……………………病原枸橼酸钠抗凝剂对疟原虫生长活性影响的研究 3 43……………分子生物学间日疟原虫传播阻断疫苗候选抗原Pvs2 5中国分离株高度　　保守 16………………………………………     

间日疟疫苗主要候选抗原的研究进展

疟疾是当今世界人类最严重的三大传染性疾病之一。根据疟原虫复杂的生活史和不同发育时期表达特异性抗原分子的特点 ,确定有效特异性候选抗原成为疟疾疫苗研制开发的关键。多年来 ,研究者致力于子孢子疫苗、红内期疫苗和传播阻断疫苗的研究。在我国间日疟分布最广 ,普遍流行。目前 ,对间日疟疫苗候选抗原的研究取得了卓有成效的进展。疫苗已在小鼠、家兔和猴等开展试验 ,有些已进入人体临床实验阶段。该文选择间日疟 3个疫苗阶段有代表性的候选抗原 ,就分子结构 ,免疫原性和免疫效果作一综述。     

间日疟疫苗主要候选抗原的研究进展

疟疾是当今世界人类最严重的三大传染性疾病之一。根据疟原虫复杂的生活史和不同发育时期表达特异性抗原分子的特点，确定有效特异性候选抗原成为疟疾疫苗研制开发的关键。多年来，研究者致力于子孢子疫苗、红内期疫苗和传播阻断疫苗的研究。在我国间日疟分布最广，普遍流行。目前，对间日疟疫苗候选抗原的研究取得了卓有成效的进展。疫苗已在小鼠、家兔和猴等开展试验，有些已进入人体临床实验阶段。该文选择间日疟3个疫苗阶段有代表性的候选抗原，就分子结构，免疫原性和免疫效果作一综述。     

恶性疟原虫传播阻断抗原Pfs25和Pfs48/45基因编码序列的体外扩增

目的：体外扩增编码恶性疟原虫传播阻断抗原Pfs和Pfs48／45的基因序列,为进一步对其进行克隆和体外高效表达创造条件。方法：特定寡核苷酸引物的设计、合成与纯化;恶性疟原虫FCC1／HN株体外培养;利用碱裂解法从培养的恶性疟原虫FCC1／HN株提取染色体DNA;PCR扩增和琼脂糖凝胶电泳分析。结果：从恶性疟原虫FCC1／HN株基因组DNA中特异扩增出编码Pfs25和Pfs48／45基因序列,其片段大小分别为657bp和1,359bp。而用间日疟原虫基因组DNA为模板作对照,无扩增条带出现。结论：体外扩增编码恶性疟原虫传播阻断抗原Pfs25和Pfs48／45基因序列与预期长度相符合,从而,为该基因的克隆、测序和表达奠定基础。     

疟疾传播阻断疫苗的研究现状和应用前景

随着疟疾传播阻断疫苗研究的深入开展,有望将疟疾控制在孤立地域内,阻止疟原虫再次引入无疟区,阻断抗药性和突变虫株的蔓延,减少对疟原虫“毒力”株的暴露。近来已鉴定了系列有望的候选传播阻断疫苗分子。本文对疟疾传播阻断疫苗的研究现状和应用前景作一概述。     

Blocking of transmission to mosquitoes by antibody to Plasmodium vivax malaria vaccine candidates Pvs25 and Pvs28 despite antigenic polymorphism in field isolates

We have previously demonstrated that mouse antisera against yeast-produced recombinant forms of the ookinete surface proteins of Plasmodium vivax (Pvs25 and Pvs28) blocks transmission of the homologous P. vivax (Sal I strain). In this study, we developed mouse and rabbit antisera against Pvs25 and Pvs28 and evaluated the efficacy of these vaccine candidates against natural isolates of P. vivax in Thailand. Although both Pvs25 and Pvs28 genes are polymorphic, sera from mice immunized using alum adjuvant completely inhibited oocyst development for most human isolates, whereas sera from rabbits immunized with either alum or Freund's adjuvant were partially inhibitory. All inhibition occurred in an antibody dose dependent fashion. Data from this study clearly demonstrates that antibodies raised against Sal I-based vaccines overcome the genetic polymorphism of Pvs25 and Pvs28 present in natural isolates of P. vivax, suggesting the wide range applicability of Sal I based vaccines.     

Immunogenicity in rhesus of the Plasmodium vivax mosquito stage antigen Pvs25H with Alhydrogel and Montanide ISA 720

Pvs25 is an ookinete surface protein from Plasmodium vivax that is the target of transmission-blocking antibodies. Two immunogenicity trials in rhesus monkeys with a recombinant form of the protein, Pvs25H, were undertaken. Monkeys were vaccinated with Pvs25H adsorbed to Alhydrogel or emulsified in Montanide ISA 720 at 0, 4 and 27 weeks (study 1) or in Montanide ISA 720 at 0 and 18 weeks (study 2) with 1.5 or 15 microg Pvs25H in 0.1 or 0.5 mL of emulsion (four combinations). Immunogenicity was assessed by ELISA and by membrane-feeding experiments using P. vivax-infected blood from human volunteers (studies 1 and 2) or from chimpanzees (study 1). Both vaccine trials generated antibodies that blocked transmission of P. vivax to mosquitoes. Antibody titres and transmission blocking were higher with Montanide ISA 720 than with Alhydrogel in the first trial and with the 15 microg Pvs25H/0.5 mL ISA 720 combination in the second trial.     

我国分离株间日疟原虫与红细胞黏附关键区DBPⅡ区基因多态性分析

目的　了解我国季节流行区间日疟原虫红内期候选抗原DBP基因多态性特点。方法　疟原虫基因组DNA提取。PCR扩增DBPRⅡ区基因片段,序列直接测序分析。结果　成功扩增18个DBPRⅡ区基因片段,发现9个核苷酸突变,产生4种基因型,导致9个氨基酸置换。其中G115 1-A116 9-A12 70为主导基因型,G384 -H390 -I4 2 4是主导氨基酸型。结论　我国季节流行区分离株DBP基因突变位点完全含盖在以往报道的位点中,同时表明我国分离株DBP基因突变位点有限,提示我国分离株红内期候选抗原DBP蛋白功能相对保守。     

Global distribution of a variant of the circumsporozoite gene of Plasmodium vivax

The global distribution of a newly described variant of the Plasmodium vivax circumsporozoite (CS) gene was determined by genetic analysis of wild isolates. Whole blood specimens were collected on filter paper from patients infected with P. vivax in South America. West Africa, and the Indian subcontinent. P. vivax DNA was released from the filter paper samples, and the CS gene was amplified by polymerase chain reaction and analyzed for genetic variation. Amplified DNA was probed with oligonucleotide probes that hybridize with the predominant CS repeat region (PV210) and the variant CS repeat region (PV247) of P. vivax. The PV247 variant was found in all three geographically diverse areas. In addition, five of six consecutive patients studied had simultaneous infection with both the predominant and variant forms of P. vivax. These findings suggest that a single-epitope vaccine based on the predominant CS domain is unlikely to be protective on even a regional basis.     

Assessment of transmission-blocking activity of candidate Pvs25 vaccine using gametocytes from chimpanzees

Macaca mulatta monkeys were immunized with the candidate transmission-blocking vaccine against Plasmodium vivax, Pvs25, combined with alum or Montanide ISA 720. Efficacy was measured by combining post-immunization sera with gametocytes obtained from infections induced in chimpanzees using membrane-feeding techniques. The results indicate that immunization of M. mulatta monkeys with Pvs25 and Montanide ISA 720 was more effective than with alum in efficacy and resulted in the maintenance of a lasting transmission-blocking immunity to P. vivax. This was evident two weeks after the second immunization, and more strongly demonstrable 62 and 152 days after the second immunization. This transmission-blocking activity was strongly reinforced by a third immunization given 181 days after the primary immunization, as measured three weeks later by indirect membrane feeding. The use of gametocytes of P. vivax derived from infections induced in chimpanzees can contribute to the selection of appropriate constructs, formulations, and immunization regimens for the development of effective transmission-blocking vaccines.     

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。     

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.