Comparison of immunogenicity,efficacy and transcriptome changes of inactivated rabies virus vaccine with different adjuvants

Adjuvants have been proven to be very effective in enhancement of immune response of many antigens. However, few studies involved head-to-head comparison of their potentials in inactive rabies virus vaccine. In this study, we investigated two types of aluminum adjuvants and five other adjuvants (BLPs, AS02, AS03, MF59 and Poly I:C) on their capacity in enhancing the efficacy of rabies vaccine. The differences in immunogenicity and potency of rabies vaccines with different adjuvants were evaluated by immunizations in ICR mice. Compared with other adjuvants, nano-sized aluminum induced earlier and more vigorous production of rabies virus neutralizing antibodies and facilitated a more effective protection in the challenge test. Based on these results, to comprehensively and systematically explore the role of adjuvants in rabies vaccine immunization, blood samples from four groups were chosen to perform mRNA sequencing. The differentially expressed genes (DEGs) of groups were identified, both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted for these DEGs. The results showed that there were significant differences in mRNA expression between the mice after immunization with different adjuvants, but the two aluminum adjuvant vaccines induced similar gene expression. Moreover, the data of enrichment analysis indicated that adjuvants were more advantageous in activating the pathways associated with antigen processing, presentation and initial immunity. These results revealed that adjuvants can be used as an enhancer in rabies vaccination, and nano-sized aluminum may be a candidate adjuvant for the development of more effective rabies vaccines. And these data also provide a basis for understanding the mechanisms underlying adjuvants enhancement of the immune response.     

浙江省不同宿主来源狂犬病病毒N基因分子特征分析

目的 测定浙江省不同宿主（人、鼬獾、犬）来源的狂犬病病毒街毒株N基因序列, 分析病毒遗传变异特征及其与流行的关系。方法 采用直接免疫荧光试验和反转录聚合酶链式反应检测狂犬病病毒阳性标本N基因核苷酸序列, 利用生物信息学软件分析基因序列和编码蛋白。结果 共获得浙江省2 个人源、5 个鼬獾源和11 个犬源狂犬病病毒街毒株N基因核苷酸序列, 18 个核苷酸和氨基酸序列同源性在89.7%～100.0%和98.4%～100.0%, N蛋白一级结构上绝大部分为稳定区域, 编码基因的核苷酸变异多为无义突变, 系统发育分析显示18 个街毒株均属于传统的基因1 型。结论 浙江省不同宿主来源狂犬病病毒的流行具有地域性特征, 同类宿主动物病毒株或来自同一县域/相邻县域的毒株在地理位置上最为近缘, 但人源株病毒更具有复杂性。浙江省狂犬病病毒街毒株的流行具有通过犬向鼬獾和人传播, 并在犬、鼬獾中跨区域循环传播的特点。     

狂犬病病毒浙江街毒株糖蛋白基因序列分析

目的 测定狂犬病病毒浙江株(鼬獾源和犬源)糖蛋白(GP)基因组全序列,从分子水平比较狂犬病病毒浙江株与其他地区代表性街毒株和疫苗株GP之间的差异.方法 乳鼠接种分离狂犬病病毒,RT-PCR反应测定狂犬病病毒浙江株GP基因核苷酸序列,并进行序列和编码蛋白的比较分析.结果测序获得浙江5株鼬獾源狂犬病病毒和9株犬源狂犬病病毒GP基因序列,全长1575个核苷酸,编码524个氨基酸.浙江病毒株与其他地区街毒株、疫苗株核苷酸和氨基酸序列相似性在82.3%～99.9%和85.1%～99.8%之间,种系分析显示浙汀株均为基因1型,GP编码蛋白无重组,主要抗原位点未出现较大的变异.结论 对GP基因一级结构综合分析表明,在某些区域存在优势抗原表位的可能性较大,可能出现潜在的蛋白质抗原决定簇,浙江株GP基因变异较小,与国内其他地区流行的代表性街毒株相似,但高于其他疫苗株,在基因结构及种系进化关系上存在一定的距离.     

Competitive ELISA using a rabies glycoprotein-transformed cell line to semi-quantify rabies neutralizing-related antibodies in dogs

Dogs are the principal vectors for rabies virus transmission to humans in many parts of the world. The current “gold standard” World Health Organization- and Office International des Epizooties-recommended tests for measuring anti-rabies virus antibodies in dogs, such as the fluorescent antibody virus neutralization (FAVN) test, requires high containment facilities and several days for results. Here, we describe a new competitive ELISA (c-ELISA) that utilizes a cell line stably expressing the rabies virus glycoprotein as the coating antigen and two horseradish peroxidase-labeled monoclonal rabies-neutralizing antibodies as indicator, which can be carried out in any basic laboratory. We demonstrate that the c-ELISA has high specificity and sensitivity. Application to 4350 dog serum samples showed a parallel relationship with the FAVN at neutralizing antibody levels up to 8.00 IU/ml. Since it can assay 40 or more samples in 1.5 h, the c-ELISA is suitable for the surveillance of mass vaccination in dogs where rabies causes public concern.     

The immune response to rabies virus infection and vaccination

Infection with rabies virus causes encephalitis in humans that has a case fatality rate of almost 100%. This inability to resolve infection is surprising since both pre-exposure vaccination and, if given promptly, post-exposure vaccination is highly effective at preventing encephalitic disease. The principal immunological correlate of protection produced by vaccination is neutralizing antibody. T-helper cells contribute to the development of immunity whereas cytotoxic T cells do not appear to play a role in protection and may actually be detrimental to the host. One reason for a failure to protect in humans may be the poor immunological response the virus provokes, despite the period between exposure to virus and the development of disease being measured in months. Few individuals have measurable neutralizing antibody on presentation with disease, although in many cases this develops as symptoms become more severe. Furthermore, when antibody is detected in serum it rarely appears in cerebrospinal fluid suggesting limited penetration into the CNS, the site where it is most needed. The role of the modest mononuclear cell infiltrate into the brain parenchyma is unclear. Some studies suggest the virus can suppress cell-mediated immunity early during the infection although there is little mechanistic evidence to support this beyond suppression of intracellular interferon production by the viral phosphoprotein. In contrast, levels of antibody in the CNS correlate to the peak virus production within the CNS. Here we review the current understanding of immune responses to rabies infection and vaccination against this disease. This article identifies a need to understand how rabies antigens are initially presented and how this can influence the subsequent development of antibody responses. This could help identify ways in which the response to prophylactic vaccination can be enhanced and how the natural immune response to infection can be boosted to combat neuroinvasion.     

抗狂犬病毒核蛋白单克隆抗体在狂犬病街毒检测中的应用

目的　测试抗狂犬病毒核蛋白单克隆抗体用于间接免疫荧光法检测已确认的狂犬病阳性及狂犬病阴性的动物脑组织标本的灵敏度和特异性。方法　将巴斯德研究所狂犬病参考中心保存的来自不同国家的 6 2份狂犬病街毒动物脑组织标本以及 2 71份法国境内收集的正常动物脑组织标本 ,用上述间接免疫荧光检测 ,并以巴斯德研究所狂犬病参考中心提供的狂犬病毒酶联免疫吸附法、组织细胞分离法及直接免疫荧光法等三个试验作确认试验 ,进行比较。结果　间接免疫荧光法可检测涵盖狂犬病毒 7个基因型在内的所有 6 2份街毒标本 ,对确认为狂犬病阴性的动物脑组织标本检测均为阴性 ,其特异性和灵敏度均达到 10 0 %。结论　抗狂犬病毒核蛋白单抗作为间接免疫荧光法检测狂犬病毒具有良好的应用前景。     

中国不同地区狂犬病病毒种群分布的差异

目的 明确我国不同地区流行的狂犬病病毒种群类别及其流行特征。方法 汇总GenBank数据库所有中国狂犬病流行株的N、G和全基因组序列以及国家狂犬病实验室新测定毒株序列,分别构建N基因和G基因种系发生树,明确每个毒株的种群归属。统计各地区流行株的种群类别及不同种群的毒株数量。结果 全国共流行6个毒株群（ChinaⅠ~Ⅵ）,云南和湖南是我国大陆种群最丰富省份,均有多达4个群流行;河南、福建等6省份均有3个毒株群流行;上海、江西等8省份皆流行2个病毒种群;北京、天津等14省份目前只监测到1个毒株群流行。优势毒株群ChinaⅠ已蔓延至我国东北部和西部地区,共计覆盖25个省份;China Ⅲ群近年在内蒙古、新疆地区的野生动物中流行且溢出至家畜中;China Ⅳ是青海、西藏地区的流行种群,同时流行于内蒙古、黑龙江地区的野生动物中。结论 我国不同地区狂犬病病毒种群类别和数量差异明显。     

狂犬病病毒G蛋白基因重组研究

目的探讨狂犬病病毒G蛋白基因重组及其对病毒属性的影响。方法收集GenBank狂犬病病毒G蛋白基因全序列,经过MEGA4.1对齐、去除空位处理后,运用RDP 2.0软件进行筛查,然后利用SimPlot 3.5.1软件进行相似性分析和BOOTSCAN分析,对基因重组加以验证,并构建最大似然进化树,观察重组片断拓扑结构的改变。结果利用RDP 2.0软件共检测到3个重组序列,分别是DQ849069、AF334789与AY009100。相似性分析和BOOTSCAN分析确认重组序列DQ849069重组特征明显,重组片段为746～1 566nt,重组片断最大似然进化树的拓扑结构也发生了相应改变。而AF334789与AY009100未发现有意义重组信号;地缘分析表明DQ849069是基因重组的产物。结论狂犬病病毒G蛋白在进化过程中存在重组,重组事件对未来病毒毒力的影响有待于深入研究。     

Rabies virus pathogenesis in relationship to intervention with inactivated and attenuated rabies vaccines

Despite progress in vaccine development in the past century the mechanisms behind immune responses elicited by rabies biologics or via natural infection remain largely unknown. In this study, we compared protection elicited by standard, early, or delayed prophylaxis with a reduced number of vaccine doses using inactivated and live-attenuated vaccines. Two-month-old Syrian hamsters, 4-week-old ICR mice or adult rhesus macaques were inoculated with canine rabies virus variants. Thereafter, prophylaxis was initiated 6 h, 1, 2, 3, 4, 5, 6 or 7 days post-exposure (p.e.). One or several doses of inactivated (HDCV), or reverse genetically attenuated (live), or gamma-irradiated (inactivated)-ERAG333 vaccines were administered intramuscularly. The dynamics of virus spread were measured over time in the rodent models. Rabies virus reached the spinal cord at day 4 and brain at day 6 p.e. All hamsters succumbed in groups in which live ERAG333 was delayed until days 5 and 6 p.e. However, 78%, 44%, 56% and 22% of hamsters survived when one dose of live ERAG333 was administered 6 h, 1, 2, 3, and 4 days p.e., respectively. Similarly, 67% survived when inactivated ERAG333 was administered at 24 h p.e. All hamsters succumbed when standard prophylaxis (the Essen regimen) was delayed until days 3–6, but 67% and 33% of hamsters survived when PEP began 1 or 2 days p.e., respectively. Macaques were protected by one dose of attenuated ERAG333 at 24 h p.e. The highly attenuated (live) and inactivated ERAG333 vaccines elicited potent protective immune responses, even when prophylaxis initiation was delayed. When 2–5 doses of commercial vaccine and HRIG were administered according to the Essen scheme, 89–100% of the animals survived. Reduced vaccine schedules provided efficacious intervention, regardless of the total number of vaccine doses administered.     

A single center,open label study of intradermal administration of an inactivated purified chick embryo cell culture rabies virus vaccine in adults

In the USA, rabies vaccines (RVs) are licensed for intramuscular (IM) use only, although RVs are licensed for use by the intradermal (ID) route in many other countries. Recent limitations in supplies of RV in the USA reopened discussions on the more efficient use of available biologics, including utilization of more stringent risk assessments, and potential ID RV administration. A clinical trial was designed to compare the immunogenic and adverse effects of a purified chicken embryo cell (PCEC) RV administered ID or IM. Enrollment was designed in four arms, ID Pre-Exposure Prophylaxis (Pre-EP), IM Pre-EP, ID Booster, and IM Booster vaccination. Enrollment included 130 adult volunteers. The arms with IM administration received vaccine according to the current ACIP recommendations: Pre-EP, three 1 mL (2.5 I.U.) RV doses, each on day 0, 7, and 21; or a routine Booster, one 1 ml dose. The ID groups received the same schedule, but doses administered were in a volume of 0.1 mL (0.25 I.U.). The rate of increase in rabies virus neutralizing antibody titers 14–21 days after vaccination were similar in the ID and correspondent IM groups. The GMT values for ID vaccination were slightly lower than those for IM vaccination, for both naïve and booster groups, and these differences were statistically significant by t-test. Fourteen days after completing vaccination, all individuals developed RV neutralizing antibody titers over the minimum arbitrary value obtained with the rapid fluorescent focus inhibition test (RFFIT). Antibodies were over the set threshold until the end of the trial, 160 days after completed vaccination. No serious adverse reactions were reported. Most frequent adverse reactions were erythema, induration and tenderness, localized at the site of injection. Multi use of 1 mL rabies vaccine vials for ID doses of 0.1 was demonstrated to be both safe and inmunogenic.     

广西狂犬病病毒与狂犬病疫苗株N基因序列分析

目的了解广西狂犬病病毒（RABV）街毒株与疫苗株N基因序列的差异,为疫苗使用提供理论依据。方法采用直接免疫荧光法（DFA）和巢式RT—PCR法检测,测定病毒N基因序列全长,同时根据N基因序列同源性及系统进化树比较,分析广西近年流行的RABV与国内外10株疫苗代表株N基因序列之间的差异。结果DFA法阳性率为8．84％（350／3961）,RT—PCR法检测阳性率1．29％（51／3961）,获得N基因全序15份;15份N基因核苷酸与氨基酸序列同源性为89．40％。100％和98．40％~99．80％;广西15份（RABV）与国内外10株疫苗代表株N基因核苷酸和氨基酸序列同源性分别为85．70％～99．70％和94．90％～99．80％,与中国CTN疫苗株N基因的核苷酸和氨基酸同源性最高,分别为89．60％,99．70％和98．40％。99．80％。N基因系统发生树分为两大分支,15份（RABV）与中国人用CTN株构成第一分支,其余的疫苗毒株构成第二分支。结论广西犬间流行的RABV与中国人用CTN疫苗株N基因序列同源性最高、亲缘关系最近,在广西地区使用CTN疫苗株效果可能较好。     

广西狂犬病病毒分子流行病学研究

目的分析广西狂犬病毒的分布和来源,从病原学角度分析广西狂犬病疫情高发的原因。方法2005年9月~2006年4月在广西玉林、贵港、柳州、来宾、南宁和河池等6市共收集健康犬脑标本1 352份,用直接免疫荧光法(DFA)和RT-PCR法检测狂犬病毒,其中22份标本完成狂犬病毒N基因编码区下游720bp核苷酸序列的测定。结果用DFA法检测出阳性标本160份,阳性率为11.83%,RT-PCR检测出阳性标本26份,阳性率1.9%,对其中22份标本完成N基因编码区下游720bp核苷酸序列的测定,核苷酸序列同源性为87.8%~100%,推导氨基酸序列的同源性为97.5%~99.6%,表明广西病毒标本N基因核苷酸序列的变异主要是同义突变。种系发生分析显示,广西病毒标本分为A、B、C群,各群分布范围不同,具有明显地域性特征;中国存在的3群狂犬病毒(CHINA-1、CHINA-2和CHINA-3群)目前在广西均有分布。广西流行的3群病毒来源各不相同:A群可能来源于与广西相邻的湖南、贵州;B群可能由广西北部省区传入;C群是在广西境内循环的毒株。结论广西境内普遍存在狂犬病毒感染犬只,并有外省狂犬病毒的传入,可能是近年广西狂犬病疫情上升的重要原因。     

中国狂犬病毒感染分布状况调查

目的 了解中国狂犬病不同流行区狂犬病毒的感染分布情况.方法 采集中国狂犬病高、中、低发病区动物脑组织以及疑似病例标本,利用直接免疫荧光法(DFA)和RT-PCR法检测狂犬病毒感染.结果 DFA法检测3007份犬脑组织标本,狂犬病毒阳性率为8.4%(254/3007),再经RT-PCR法验证,狂犬病毒阳性率为30.7%(78/254).DFA法检测93份伤人犬和猫的脑组织标本,狂犬病毒阳性率为67.7%(63/93),再经RT-PCR检测全部为阳性.此外,调查区域的黑线姬鼠、鼬獾和疑似病例标本中也检测到狂犬病毒.不同省份以及同一省份不同发病地犬狂犬病毒感染率差异无统计学意义.结论 中国狂犬病流行区家养犬(猫)中存在较高的狂犬病毒感染率,野生动物中也有狂犬病毒感染.     

广西家犬携带狂犬病毒的实验研究

目的：调查研究广西地区外观健康的家犬是否携带狂犬病毒。方法：从广西13个县（市收集了外观健康的家犬犬脑标本140份，采用小鼠颅内接种试验、间接免疫荧光技术、夹心ELISA试验、小鼠中和试验以及逆转录聚合酶链反应（RT-PCR）方法检测脑标本中的狂犬病毒及共相应的抗原。结果：除RT-PCR的检出率稍低（2.86％，4/140）外，其余4种方法均获得3.57％（5/140）的检出率。而且，不同县（市）的检出率亦有所不同。结论：以实验室方法首次证实了广西外观健康的家犬确实携带有狂犬病毒，从而为过去的流行病学调查资料提供了科学的实验室依据，对进一步做好狂犬病的预防和控制有重要的意义。     

登革2型病毒B株E基因部分序列原核蛋白表达及单克隆抗体制备

目的 通过构建登革2型病毒B株E基因区1～476 bp的原核表达载体,进行原核表达,并制备单克隆抗体,为研制登革病毒诊断试剂奠定基础.方法 将登革2型病毒B株E基因序列克隆入原核表达载体pET28a(+),构建原核表达重组体pET28a(+)-E,将重组表达载体转化BL21(DE3)菌株进行原核表达.纯化后的蛋白免疫B...     

Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus

Both rabies virus (RABV) and canine parvovirus (CPV) cause lethal diseases in dogs. In this study, both high egg passage Flury (HEP-Flury) strains of RABV and recombinant RABV carrying double RABV glycoprotein (G) gene were used to express the CPV virion protein 2 (VP2) gene, and were designated rHEP-VP2 and, rHEP-dG-VP2 respectively. The two recombinant RABVs maintained optimal virus titration according to their viral growth kinetics assay compared with the parental strain HEP-Flury. Western blotting indicated that G protein and VP2 were expressed in vitro. The expression of VP2 in Crandell feline kidney cells post-infection by rHEP-VP2 and rHEP-dG-VP2 was confirmed by indirect immunofluorescence assay with antibody against VP2. Immunogenicity of recombinant rabies viruses was tested in Kunming mice. Both rHEP-VP2 and rHEP-dG-VP2 induced high levels of rabies antibody compared with HEP-Flury. Mice immunized with rHEP-VP2 and rHEP-dG-VP2 both had a high level of antibodies against VP2, which can protect against CPV infection. A challenge experiment indicated that more than 80% mice immunized with recombinant RABVs survived after infection of challenge virus standard 24 (CVS-24). Together, this study showed that recombinant RABVs expressing VP2 induced protective immune responses to RABV and CPV. Therefore, rHEP-VP2 and rHEP-dG-VP2 might be potential combined vaccines for RABV and CPV.     

浙江省慈溪市犬类狂犬病病毒携带状况调查及变异研究

目的研究犬类狂犬病病毒的携带状况及变异,为正确有效控制狂犬病疫情提供依据。方法根据狂犬病疫情分布,用分层采样法,采集相关地区犬脑51只,用免疫荧光法、夹心酶联吸附法、小鼠颅内接种试验法及RT-PCR进行检测。分离狂犬病病毒并进行N基因测序。结果在流浪犬只中发现狂犬病毒株(命名CXs)。流浪犬带毒率7.69%,CXs与疫苗株和固定毒株的NJ进化树比较,发现与WZO(H)株100%同源,与狂犬病毒固定毒株相比更接近CTN株(该地区使用的疫苗株)。结论当前使用的狂犬疫苗用于预防狂犬病是可靠的。经狂犬病流行特征和暴露后免疫预防处理分析,加强犬只的管理、免疫,暴露后采用规范清创,正确使用狂犬疫苗,合理使用狂犬免疫球蛋白(或血清)仍是当前防治狂犬病需遵循的原则。     

Supplementation of omega 3 fatty acids improves oxidative stress in activated BV2 microglial cell line

Abstract

Many reports have shown promising beneficial effects of long-chain polyunsaturated fatty acids (L-PUFAs) of the omega 3 series in several brain diseases. In the present study, we tested the hypothesis that omega 3 fatty acids supplement reduced pro-inflammatory functions in vitro and in vivo. We demonstrated that a supplement rich in PUFAs (SRP) increased cell viability in a dose-dependent manner suggesting its protective role against lipopolysaccharide (LPS)-induced cell death in BV2 microglial cell line. In the same cultures, the supplement rich in PUFAs reduced the reactive oxygen species (ROS) and nitric oxide (NO) production. A most prominent target for ROS management is the family of peroxisome proliferator-activated receptors (PPARs). The co-treatment with SRP and LPS increased significantly the nuclear immunoreactivity of PPAR-γwhen compared the LPS treatment alone. Moreover, the chronic administration of the SRP in rats, increased the immunoreactivity of the PPAR-γ1 protein confirming its potential neuroprotective effect.     

乙型肝炎病毒体外感染HepG2细胞的初步研究

目的建立HBV阳性血清体外感染HepG2细胞的实验模式。方法在六孔板上培养HepG2细胞,用HBV阳性血清体外感染HepG2细胞,设立感染组和对照组。用HBV阳性血清和感染组细胞共孵育24 h,0.01 mol/L PBS清洗细胞后,每孔板中加入4 ml DMEM细胞培养液,每隔12 h收集细胞上清液至PBS洗后84 h。收集完最后一次细胞上清液,收集细胞培养板中的细胞。ELSIA检测细胞培养上清中HBsAg;PCR方法检测细胞培养上清和细胞中HBV DNA;免疫荧光定量PCR检测细胞培养上清中HBV DNA的含量。结果HBV感染组细胞培养上清,在PBS洗后第12 h,ELISA即可检测到HBsAg并持续到84 h,PCR检测感染组细胞培养上清和感染组细胞的HBV DNA均阳性。荧光定量PCR检测感染组细胞培养上清中HBV DNA的结果:感染组PBS洗后(0 h)的HBV定量为阴性,而在PBS洗后36 h8、4 h细胞培养上清中HBV的量达到5×105,3×106copies/ml)。结论用HBV阳性血清体外感染HepG2细胞的实验是可行的。     

贵州及周边省市狂犬病病毒的分群和进化研究

目的 全面掌握贵州及周边省市狂犬病病毒(RABV)的遗传进化特征和分群特点,了解狂犬病疫苗株与流行株的分子遗传差异,为贵州狂犬病疫情的预防和控制提供指导依据.方法 搜索GenBank数据库中贵州及周边5个省市具有明确时间、地区及宿主信息的流行毒株,对其糖蛋白基因(G)全序列进行汇总,并结合本实验室获得的贵州毒株和国内现役疫苗株G序列,利用MEGA和BEAST软件分别进行系统进化和时间进化分析.结果 贵州及周边省市79株RABV均属于基因1型,9个类群的RABV流行株具有明显的地域特征,11株疫苗株分化为2个类群;RABV流行株与疫苗株G基因核苷酸和氨基酸同源性分别为0.827～0.999和0.713～0.998;贵州及周边省市RABV流行毒株的最近共同祖先(TMRCA)可追溯至150年前.结论 贵州及周边省市流行的RABV优势毒株群仍属于基因1型,但毒株经过多年流行已经出现了分化,有必要对流行毒株与疫苗株的遗传变异一致性进行深入研究.