间日疟原虫重组蛋白Pvs25和Pvs28免疫小鼠应答机制观察

目的观察间日疟传播阻断疫苗候选抗原Pvs25和Pvs28的免疫应答机制。方法分别用与弗氏佐剂乳化后的Pvs25和Pvs28重组蛋白皮下免疫DBA/2小鼠;采用ELISA分别对各组小鼠脾细胞培养上清的IFN-γ、IL-4、IL-10动态检测;以ELISPOT方法检测小鼠脾脏IFN-γ+和IL-4+细胞数量。结果经重组蛋白免疫的两组小鼠脾细胞培养上清IFN-γ的含量于初次免疫后第14d升高,与对照组相比差异显著(P<0.01),但随后降至对照组水平;IL-4和IL-10均在初次免疫后第28d出现有意义的升高(P<0.01),后升高更为明显,分别直至免疫后期70d、56d出现下降,但直至112d仍明显高于免疫前的水平(P<0.01)。初次免疫后第70d?98d小鼠脾脏IFN-γ+和IL-4+细胞数量,与对照组相比IFN-γ+无明显变化,而IL-4+则明显增多(P<0.01)。结论Pvs25和Pvs28重组蛋白可诱导小鼠产生以Th2反应为主的免疫应答。     

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。     

我国间日疟原虫传播阻断疫苗候选抗原Pvs48基因特点分析

目的明确我国疟疾流行区间日疟原虫传播阻断疫苗候选抗原Pvs48基因特点。方法收集疟疾单纯流行区浙江、湖北两省间日疟患者血样,制备血样干滤纸片;提取疟原虫基因组DNA;聚合酶链反应(PCR)扩增Pvs48基因;Sanger双脱氧链终止法测序并分析。结果成功扩增12例间日疟原虫分离株Pvs48全长基因。与间日疟原虫标准株SalⅠ比较,检测出6个错义突变位点,导致6处氨基酸置换和6种基因型。结论我国间日疟原虫传播阻断疫苗候选抗原Pvs48具有保守性。     

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。     

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。     

疟原虫蚊阶段虫体表面蛋白P25和P21/P28与传播阻断疫苗

疟疾是由媒介昆虫蚊传播、在热带和亚热带地区广泛流行的以发热、贫血和脾肿大为主要症状的感染性疾病。近年来 ,由于耐药性原虫和耐杀虫剂性蚊的出现 ,疟疾患者人数急剧增加。全世界每年由恶性疟原虫造成的死亡人数可达15 0～ 2 70万〔1〕。在中国 ,疟疾的流行范围分布于全国     

疟原虫传播阻断免疫的研究进展

疟疾疫苗的研究已经深入到分子水平，基因工程疫苗、合成肽疫苗、亚单位疫苗以及复合多价疫苗的研究进展迅速。本文概述了对疟原虫传播阻断靶抗原研究的进展，并对传播阻断疫苗的研制及佐剂的应用进行了探讨。     

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。     

Sequence polymorphisms of Plasmodium vivax ookinete surface proteins (Pvs25 and Pvs28) from clinical isolates in Korea

The Ookinete surface proteins of Plasmodium vivax (P. vivax), Pvs25 and Pvs28, were candidates for the transmission blocking vaccine (TBV), which exhibited great antigenic diversities among various isolates. Polymorphisms of these genes in the isolates from Republic of Korea (ROK) were analysed, which provided valuable baseline data for the field trials of TBV‐based vaccines. A total of 98 isolates were collected over 11 years from 1996 to 2007. pvs25 and pvs28 genes from the above isolates were amplified, sequenced and compared against Sal‐1 strain. Sequencing analysis of PCR products from P. vivax pvs25 revealed two allelic types, Q97T130 and E97/T130 alleles with the frequencies of 54.5% and 45.5%, respectively, in comparison with Sal I type sequence (E97/I130). From pvs28 gene, polymorphisms at M52L and T140S in the first and third EGF‐like domains in comparison to Sal‐1 strain were detected, respectively. Six GSGGE tandem repeats followed by GSGGDT or SSGGDT were identified at the end of the fourth EGF‐like domain in all Korean isolates. Interestingly, different tandem repeats of amino acid substitutions were observed from isolates collected after 2006 in comparison with preceding years. The ROK isolates revealed limited sequence polymorphisms in pvs25 and tandem repeats in pvs28 in comparison with reported isolates from other nations. Current observations suggested the rapid progresses of genetic changes among Korean isolates.     

Genetic diversity of transmission blocking vaccine candidate (Pvs25 and Pvs28) antigen in Plasmodium vivax clinical isolates from Iran

The leading candidates for a Transmission Blocking Vaccine (TBV) in Plasmodium vivax parasite are the ookinete surface protein 25 (Pvs25) and Pvs28, which their phase I clinical trial is ongoing. Therefore, we carried out survey of polymorphisms of the pvs25 and pvs28 genes in P. vivax populations that are circulating in the two malaria areas of contrasting endemicity in Iran, before field application of the TBV. To characterize the polymorphisms of pvs25 and pvs28 genes, 50 isolates were analyzed by sequencing method and their gene structure was compared with parasite populations from India, Bangladesh, Indonesia, Thailand, Mexico and Brazil. Three mutations were detected in pvs25 and pvs28 including Q87K, E97Q, I130T and M52L, T65K, T140S with two and four distinct haplotypes, in comparison with the Sal I sequence type, respectively. Both haplotypes of Pvs25 were found among northern and southern P. vivax isolates; however, only two and three of the Pvs28 variants were observed among the northern and southern isolates, respectively. In conclusion, the present results show the limited sequence polymorphism of the pvs25 and pvs28 genes among field P. vivax population in Iran. These results highly encourage with respect to applicability of Pvs25 and Pvs28-based vaccine against P. vivax infection in the region, where these parasites are prevalent, whether these occur in the temperate or tropical zones.     

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.     

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.     

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.