铜绿假单胞菌注射液对NCI-N87胃癌细胞株增殖能力的影响及其机制研究

目的 探讨铜绿假单胞菌注射液（Pseudomonas aeruginosa mannose-sensitive hemagglutinin,PA-MSHA）对NCI-N87胃癌细胞增殖能力的影响及其分子作用机制。方法 MTT法检测PA-MSHA对NCI-N87胃癌细胞增殖的影响,流式细胞术检测细胞凋亡率,划痕实验和侵袭实验检测细胞迁移和侵袭能力的变化,Western Blot检测PTEN蛋白和p-AKT蛋白的表达变化,转染PTEN siRNA敲低PTEN表达后,再次行MTT法检测PA-MSHA对细胞增殖的影响。结果 PA-MSHA抑制NCI-N87胃癌细胞的增殖具有剂量和时间依赖性。与对照组比较,NCI-N87细胞经PA-MSHA处理后,凋亡率显著增加（P<0.05）,迁移和侵袭能力显著下降（P<0.05）。与对照组比较,PA-MSHA显著提高PTEN蛋白表达,降低p-AKT蛋白表达（P<0.05）。通过siRNA转染敲低PTEN表达后,PA-MSHA抑制NCI-N87细胞增殖的能力较对照组显著下降（P<0.05）。结论 PA-MSHA对NCI-N87胃癌细胞具有抑制增殖、促进凋亡、抑制侵袭转移的作用,其机制与PTEN/AKT信号通路有关。     

Advanced oxidation technology for the development of a next-generation inactivated West Nile virus vaccine

West Nile virus (WNV) is the most frequent mosquito-borne disease reported in the continental United States and although an effective veterinary vaccine exists for horses, there is still no commercial vaccine approved for human use. We have previously tested a 3% hydrogen peroxide (H₂O₂)-based WNV inactivation approach termed, HydroVax, in Phase I clinical trials and the vaccine was found to be safe and modestly immunogenic. Here, we describe an advanced, next-generation oxidation approach (HydroVax-II) for the development of inactivated vaccines that utilizes reduced concentrations of H₂O₂ in combination with copper (cupric ions, Cu²⁺) complexed with the antiviral compound, methisazone (MZ). Further enhancement of this oxidative approach included the addition of a low percentage of formaldehyde, a cross-linking reagent with a different mechanism of action that, together with H₂O₂/Cu/MZ, provides a robust two-pronged approach to virus inactivation. Together, this new approach results in rapid virus inactivation while greatly improving the maintenance of WNV-specific neutralizing epitopes mapped across the three structural domains of the WNV envelope protein. In combination with more refined manufacturing techniques, this inactivation technology resulted in vaccine-mediated WNV-specific neutralizing antibody responses that were 130-fold higher than that observed using the first generation, H₂O₂-only vaccine approach and provided 100% protection against lethal WNV infection. This new approach to vaccine development represents an important area for future investigation with the potential not only for improving vaccines against WNV, but other clinically relevant viruses as well.     

A novel combination of intramuscular vaccine adjuvants,nanoemulsion and CpG produces an effective immune response against influenza A virus

BackgroundVaccination is the most effective approach to prevent infection with highly pathogenic avian influenza (HPAI). Adjuvants are often used to induce effective immune responses and overcome the immunological weakness of recombinant HPAI antigens. Given the logistical challenges of immunization to HPAI during pandemic situations, vaccines administered via the intramuscular (I.M.) route would be of value.MethodsA new formulation of nanoemulsion adjuvant (NE02) suitable for I.M. vaccination was developed. This NE02 was evaluated alone and in combination with CpG to develop H5 immune responses in mouse and ferret models. Measures of recombinant H5 (rH5) specific immunity evaluated included serum IgG and IgG subclasses, bronchoalveolar lavage fluid IgA, and cytokines. The activation of NF-kB was also analyzed. The efficacy of the vaccine was assessed by performing hemagglutination inhibition (HAI), virus neutralization (VN) assays, and viral challenges in ferrets.ResultsI.M. vaccination with rH5-NE02 significantly increased rH5-specific IgG and protected ferrets in the viral challenge model providing complete protection and sterile immunity in all animals tested. Combining NE02 and CpG produced accelerated antibody responses and this was accompanied by an elevation of IFN-γ and IL-17 responses and the downregulation of IL-5. The combination also caused a synergistic effect on NF-kB activation. In immunized ferrets after viral challenge, the rH5-NE02 + CpG vaccine via I.M. achieved at least 75% and 88% seroconversion of HAI and VN antibody responses, respectively, and improved body temperature stabilization and weight loss over NE02 alone.ConclusionsThe I.M. injection of NE02 adjuvanted rH5 elicits strong and broad immune responses against H5 antigens and effectively protects animals from lethal H5 challenge. Combining this adjuvant with CpG enhanced immune responses and provided improvements in outcomes to viral challenge in ferrets. The results suggest that combinations of adjuvants may be useful to enhance H5 immune responses and improve protection against influenza infection.     

江苏省2018年健康人群百日咳毒素和丝状血凝素IgG抗体水平调查

目的了解江苏省2018年健康人群百日咳毒素(Pertussis toxin,PT)和丝状血凝素(Filamentous hemagglutinin,FHA)IgG抗体水平。方法采用分层抽样在江苏省两个市选择7个年龄组健康人群,采用间接酶联免疫吸附试验检测血清PT IgG抗体(IgG antibody against PT,Anti-PT IgG)和FHA IgG抗体(IgG antibody against FHA,Anti-FHA IgG),分析抗体阳性(抗体浓度≥20IU/mL)率和几何平均浓度(Geometricmeanconcentration,GMC)。结果共纳入受试者752名,Anti-PT IgG、Anti-FHA IgG阳性率分别为14.76%、28.72%,GMC分别为12.37IU/mL、17.13IU/mL。各年龄组Anti-PT IgG阳性率在8.62%(3-5岁)-23.15%(10-14岁)之间(χ^2=15.59,P<0.05),Anti-FHAIgG阳性率在10.38%(0-1.4岁)-53.27%(10-14岁)之间(χ^2=57.50,P<0.05)。在<3岁儿童中,无细胞百白破联合疫苗0-2剂次、3剂次、4剂次免疫儿童Anti-PT IgG阳性率分别为6.67%、10.75%、13.25%(χ^2=1.30,P>0.05),Anti-FHA IgG阳性率分别为6.67%、17.39%、30.12%(χ^2=6.74,P<0.05)。结论江苏省2018年健康人群Anti-PTIgG和Anti-FHAIgG水平较低;建议对特定人群进行百日咳组分疫苗选择性免疫或补充免疫。     

2018-2019北京市门头沟区甲型H3N2流感疫情病毒HA基因分析

目的分析2018-2019北京市门头沟区流感疫情中甲型H3N2流感病毒的HA基因特性,了解其与WHO推荐的疫苗株和同时期北京其他地区的毒株匹配情况。方法选取门头沟区2018-11/2019-01多起流感疫情中的13株甲型H3N2流感病毒,进行HA基因扩增和测序,并从全球共享禽流感数据库(GISAID)上下载北京其他地区同时间段H3N2流感病毒HA基因序列,运用MEGA-X软件分析HA的分子特征,并构建系统进化树。结果2018-11/2019-01北京市门头沟区和北京市其他地区的数据显示,H3N2流行株以3C.2a.1b和3C.3a两个亚分支为主。系统进化树分析显示3C.2a.1b亚分支同WHO2018-2019推荐疫苗株3C.2a.1进化距离比较近,但与3C.3a进化距离较远。分子特征显示,没有发现氨基酸序列的丢失与插入。在抗原决定簇区域,3C.2a.1b亚分支和3C.2a.1比对有3处氨基酸位点发生变异;3C.3a亚分支和3C.2a.1比对有10处氨基酸位点发生变异。结论引起流感疫情的毒株3C.2a.1b与2018-2019 WHO推荐疫苗株匹配,3C.3a与2019-2020WHO推荐疫苗株匹配。应加强开展流感分子生物学监测力度。分析病毒基因特性和构建进化树,对流感的防控有重要意义。     

抗H9亚型禽流感病毒血凝素单克隆抗体的制备及初步鉴定

目的 :制备抗禽流感病毒 (AIV)H9亚型血凝素蛋白的单克隆抗体 (mAb)。方法 :以AIVH9亚型油乳剂灭活疫苗作为免疫原 ,免疫 8wk龄BALB/c小鼠。采用淋巴细胞杂交瘤技术制备抗AIVH9亚型血凝素蛋白的mAb ;采用ELISA和血凝抑制试验(HI)检测腹水mAb的效价 ;采用ELISA、HI、免疫荧光染色 (IF)及Westernblot鉴定mAb的特异性。结果 :获得 3株可稳定分泌特异性mAb的杂交瘤细胞株 2A3、2H1和 1C8,其腹水mAb的ELISA效价依次为 1× 10 7、1× 10 5和 5× 10 6,血凝抑制效价为 1× 2 8～ 1× 2 13 ;3株mAb的Ig亚类均为IgG1。以mAb 2H1进行Westernblot的结果显示 ,该mAb能与AIV的Mr 为 75 0 0 0的蛋白条带起反应 ,表明其是针对AIVH9亚型血凝素蛋白的mAb。与 32株AIVH9亚型国内分离株进行血凝抑制试验表明 ,mAb 2H1具有良好的广谱性。结论 :成功地制备了抗AIVH9亚型血凝素蛋白的mAb ,为AIV的抗原性分析、血清学诊断、疫苗质量的监测及流行病学调查等奠定了基础     

抗禽流感病毒H7亚型血凝素特异性单克隆抗体的研制

目的:研制禽流感病毒H7亚型血凝素特异性单克隆抗体(mAb)。方法:以H7亚型禽流感诊断抗原为免疫原免疫6～8周雌性BALB/c小鼠,末次加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14进行融合。通过HA和HI试验筛选阳性克隆。应用HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得4株分泌抗AIVH7亚型HAmAbs的杂交瘤细胞株,分别命名为2E2、2A4、5F5、7G5。这些mAb的腹水HI效价在5×27～5×211之间,其中2E2属于IgM亚类,2A4属于IgG1亚类,5F5、7G5属于IgG2a亚类。Western blot分析结果显示,4株AIVH7亚型HAmAb能与AIVH7蛋白在Mr75000处反应,但不与新城疫病毒(NDV)蛋白发生反应,表明这些mAb能特异性识别AIVH7亚型HA。mAbHI反应性测定结果表明:4株mAb中,2E2、5F5、7G5只与H7亚型AIV发生特异性HI反应,而不与其他亚型AIV以及NDV、传染性支气管炎病毒(IBV)反应,显示出良好的特异性;而2A4除了与H7亚型AIV反应外,还与H15N8标准株发生低水平交叉反应。结论:这些mAb不仅为H7亚型AIV的HA结构分析提供了工具,而且为建立快速廉价的H7亚型禽流感诊断方法提供了核心试剂。     

Antigenic and genetic variation in the hemagglutinins of H1N1 and H3N2 human influenza a viruses in the Shanghai area from 2005 to 2008

Continued rapid evolution of the influenza A virus is responsible for annual epidemics and occasional pandemics in the Shanghai area. In the present study, the representative strains of A/H1N1 and A/H3N2 influenza viruses isolated in the Shanghai area from 2005 to 2008 were antigenically and genetically characterized. The antigenic cartography method was carried out to visualize the hemagglutination-inhibition data. Antigenic differences were detected between circulating A/H1N1 strains isolated from 2005 to 2006 and the epidemic A/H1N1 strains isolated in 2008, which were found to be associated with the amino acid substitution K140E in HA1. The present vaccine strain A/Brisbane/59/2007 is considered to be capable of providing sufficient immunity against most of the circulating A/H1N1 viruses isolated in 2008 from the Shanghai population. The study showed that there were significant antigenic differences between the epidemic A/H3N2 strains isolated in 2007 and 2008, suggesting that antigenic drift had occurred in the A/H3N2 strains isolated in 2008. The P194L mutation was thought to be responsible for the antigenic evolution of influenza A/H3N2 viruses isolated from Shanghai in 2008. Evidence of antigenic drift suggests that the influenza A/H3N2 vaccine component needs to be updated.     

Genetic characterization of wild type measles virus isolated in Croatia during the 2003-2004 outbreak

Viral epidemiology is determined by the movement of infected people within and between geographical areas. The genetic characterization of wild-type isolates combined with standard epidemiological methods may enable the identification of the source and transmission pathways and permit differentiation between indigenous and imported viruses. We investigated the genetic characteristics of the wild-type measles virus isolated in Croatia during a 2003-2004 outbreak. The results of this study indicate the presence of the D4 measles virus genotype in Europe. The isolated virus is closely related to virus isolates from the India-like subgroup of the D4 measles virus genotype. The virus responsible for this outbreak differs in the hemagglutinin gene sequence from other virus strains belonging to the D4 genotype. The hemagglutinin gene sequence also differs when compared to viruses from other genotypes that are known to circulate in Europe and from vaccine strains.