ONRAB® oral rabies vaccine is shed from,but does not persist in,captive mammals

ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41 days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6 h to 3 days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies.     

Durability of humoral immune responses to rubella following MMR vaccination

BackgroundWhile administration of the measles-mumps-rubella (MMR-II®) vaccine has been effective at preventing rubella infection in the United States, the durability of humoral immunity to the rubella component of MMR vaccine has not been widely studied among older adolescents and adults.MethodsIn this longitudinal study, we sought to assess the durability of rubella virus (RV)-specific humoral immunity in a healthy population (n = 98) of adolescents and young adults at two timepoints: ~7 and ~17 years after two doses of MMR-II® vaccination. Levels of circulating antibodies specific to RV were measured by ELISA and an immune-colorimetric neutralization assay. RV-specific memory B cell responses were also measured by ELISpot.ResultsRubella-specific IgG antibody titers, neutralizing antibody titers, and memory B cell responses declined with increasing time since vaccination; however, these decreases were relatively moderate. Memory B cell responses exhibited a greater decline in men compared to women.ConclusionsCollectively, rubella-specific humoral immunity declines following vaccination, although subjects’ antibody titers remain well above the currently recognized threshold for protective immunity. Clinical correlates of protection based on neutralizing antibody titer and memory B cell ELISpot response should be defined.     

Inactivated influenza vaccine formulated with single-stranded RNA-based adjuvant confers mucosal immunity and cross-protection against influenza virus infection

Influenza vaccination is considered the most valuable means to prevent and control seasonal influenza infections, which causes various clinical symptoms, ranging from mild cough and fever to even death. Among various influenza vaccine types, the inactivated subunit type is known to provide improved safety with reduced reactogenicity. However, there are some drawbacks associated with inactivated subunit type vaccines, with the main ones being its low immunogenicity and the induction of Th2-biased immune responses. In this study, we investigated the role of a single-stranded RNA (ssRNA) derived from the intergenic region in the internal ribosome entry site of the Cricket paralysis virus as an adjuvant rather than the universal vaccine for a seasonal inactivated subunit influenza vaccine. The ssRNA adjuvant stimulated not only well-balanced cellular (indicated by IgG2a, IFN-γ, IL-2, and TNF-α) and humoral (indicated by IgG1 and IL-4) immune responses but also a mucosal immune response (indicated by IgA), a key protector against respiratory virus infections. It also increases the HI titer, the surrogate marker of influenza vaccine efficacy. Furthermore, ssRNA adjuvant confers cross-protective immune responses against heterologous influenza virus infection while promoting enhanced viral clearance. Moreover, ssRNA adjuvant increases the number of memory CD4⁺ and CD8⁺ T cells, which can be expected to induce long-term immune responses. Therefore, this ssRNA-adjuvanted seasonal inactivated subunit influenza vaccine might be the best influenza vaccine generating robust humoral and cellular immune responses and conferring cross-protective and long-term immunity.     

帕金森病相关蛋白α-共核蛋白的可溶性表达、纯化及抗体制备

目的 利用分子克隆接术在愿核细胞中表达α-共核蛋白（SNCP），并制备免多克隆抗体。方法 从人神母经细胞SH-SY5Y细胞中提取总RNA，逆转录成cDNA。利用PCR技术扩增获得SNCP的cDNA序列，测序正确后克隆至pQE-30-GST表达载体上，在大肠埃希菌中表达GST-SNCP融合蛋白。融合蛋白纯化后免疫新西兰大白兔，制备特异性抗血清，并对该抗体进行检测。结果 琼脂糖凝胶电泳显示获得SNCP cD-NA序列为420bp。SDS-PAGE和Western blot分析，表达的融合蛋白呈可溶性，相对分子质量约为45000。以其为抗原制备的SNCP抗血清的ELISA效价达到1：32000，并且该抗体可与BALB/c小鼠脑组织中内源性SNCP发生特异性反应。结论 人SNCP可在原核细胞中可溶性表达。用表达的蛋白制备获得特异性较好的SNCP抗血清，为进一步了解SCNP的结构和功能以及在中枢神经系统退行性疾病的作用提供了必要的实验基础。     

中国H3N2亚型流感病毒烷胺类药物耐药性研究

目的 通过监测我国自1989年以来H3N2亚型流感病毒对烷胺类药物产生耐药性的情况,为流感的临床治疗用药提供一定的指导.方法 从我国流感监测网络中所分离的H3N2亚型流感病毒中随机选择了584株病毒,通过对病毒与烷胺类药物耐药性相关的M2基因进行序列测定,同时在细胞水平上通过药物敏感试验分析病毒对药物的敏感性,从而分析病毒产生耐药的情况.结果 1989-1999年没有发现对烷胺类药物产生耐药性病毒株,但2003年对烷胺类药物产生耐药性病毒株的比例从2002年的3.4%升到了56.0%,2005年达到77.6%.结论 自2003年以后,流行的H3N2亚型流感病毒超过50%的病毒产生了对烷胺类药物的耐药性,并且耐药病毒株的比例呈逐年升高的趋势.     

HIV-1 B''亚型R5或R5/X4嗜性毒株在GHOST细胞的感染性分析

目的分析我国HIV-1B′亚型R5或R5／X4嗜性毒株在GHOST细胞的感染性。方法采用传统的共培养方法从HIV-1感染者PBMC中分离并培养病毒，用表达CD4和趋化因子受体CCR5或CXCR4的GHOST细胞系，测定毒株的辅助受体利用情况，使用相同病毒量即2mg的HIV-1 p24分别感染表达不同受体的GHOST细胞系，通过流式细胞仪检测分析绿色荧光蛋白（GFP）的表达反映病毒感染细胞的能力。结果35例B′亚型毒株利用CCR5受体，占22例（62．85％），双嗜性即CCR5／CXCR4均阳性占13例（37．15％）。GHOST-R5／X4细胞的感染性分析结果显示，R5／X4双嗜性毒株的感染性明显高于CD4〉200／μl的R5毒株的感染性（P〈0．05）；R5／X4毒株与CD4≤200／μl的R5毒株感染性比较差异无统计学意义（P〉0．05）；CD4〉200／μl的R5毒株与CD4≤200／μl的R5毒株感染性比较差异有统计学意义（P〈0．05）；GHOST-CCR5细胞感染性分析结果显示：R5／X4双嗜性毒株的感染性明显下降，与CD4〉200／μl的R5毒株的感染性比较差异无统计学意义（P〉0．05）。利用相同剂量的双嗜性毒株分别感染R5、X4或R5／X4的GHOST细胞系，显示双嗜性毒株可同时利用CCR5和CXCR4辅助受体，但69．23％的R5／X4毒株以CCR5受体为主，30．77％的R5／X4毒株以CXCR4受体为主。结论HIV-1B′亚型R5／X4病毒不仅有更广泛的宿主细胞嗜性，而且在GHOST-R5／X4细胞中感染性明显提高。持续使用CCR5受体的毒株在疾病进展的过程中虽然辅助受体的利用是一样的，但病毒感染细胞的能力增加。     

一种朊蛋白错误折叠循环扩增方法的建立

目的建立一种类似于PCR的蛋白质扩增方法-蛋白错误折叠循环扩增技术(PMCA),用于朊病毒病脑组织中PrPSc的检测。方法将不同浓度的羊瘙痒因子263K毒株原液与正常仓鼠脑组织匀浆混合,经反复孵育/超声,共10~15个循环。WesternBlot检测扩增产物中蛋白酶K抗性PrPSc信号。结果在本研究试验体系下,263K毒株可以利用仓鼠脑组织为基质在体外迅速复制。所建立的PrPSc-PMCA技术可检测到10-5稀释的毒株原液中的PrPSc。与常规的脑组织免疫印记方法相比,敏感度提高了105~106倍。研究还显示PrPSc还可利用小脑和脑干为基质进行体外扩增复制。结论成功建立了PrPSc-PMCA技术,为朊病毒病的早期诊断和朊病毒生物学特性的研究提供了一种新的手段。     

常见呼吸道病毒分子鉴别诊断技术的建立

目的 建立一种常见呼吸道感染病毒的快速检测方法,为尽早诊断、减少疾病的传播以及为临床提供良好的治疗依据.方法 采用液体芯片检测技术结合靶向多重RT-PCR技术建立可以同时检测13种呼吸道病原的检测技术.结果 该方法特异性方面检测13种常见呼吸道病毒没有交叉,标本的检出率为100%;灵敏度方面达到10e2-10e1（pfu/ml）.结论 该分子鉴别诊断可应用常见呼吸道病毒的检测,协助诊断病毒引起的病毒性呼吸道感染.