嵌合HAdV六邻体蛋白抗原表位的rFLU/HAdV/Hexon疫苗候选株构建及免疫效果研究

目的构建、拯救以PR8流感病毒为载体嵌合HAdV六邻体蛋白抗原表位的疫苗候选株,对其免疫原性及免疫保护性进行评价。方法应用反向遗传学技术,以流感病毒PR8为载体,将HAdV六邻体蛋白抗原表位的基因插入流感病毒非结构蛋白NS基因,构建重组质粒NS1-Hexon,将重组质粒与PR8流感病毒其他7个基因组质粒共转染COS-1/MDCK共培养细胞,成功拯救嵌合HAdV六邻体抗原表位重组疫苗候选株,命名为rFLU/HAdV/Hexon,通过HA、RT-PCR、IFA、Western blotting和电镜等方法对rFLU/HAdV/Hexon进行鉴定。rFLU/HAdV/Hexon经鸡胚培养、浓缩纯化后滴鼻免疫Balb/c小鼠,评价小鼠机体产生的体液免疫、细胞免疫和黏膜免疫应答,进一步采用PR8流感病毒和HAdV-3病毒攻击小鼠评价其免疫保护性。结果成功拯救出基于PR8流感病毒为载体嵌合HAdV六邻体蛋白抗原表位的rFLU/HAdV/Hexon疫苗候选株。rFLU/HAdV/Hexon疫苗株形态符合流感病毒典型特征,能够诱导小鼠机体产生高效价的针对PR8和HAdV-3的特异性抗体,肺鼻灌洗液可检测到高效价的sIgA抗体;攻毒实验结果显示,rFLU/HAdV/Hexon疫苗株免疫小鼠可明显减轻感染症状、有效降低肺鼻的病毒载量、显著改善肺组织的病理病变情况。结论研制的嵌合HAdV六邻体蛋白抗原表位的rFLU/HAdV/Hexon疫苗株是有发展前景的腺病毒候选疫苗株,为腺病毒疫苗的研究提供了新思路。     

重组嵌合表达G蛋白中和表位的流感病毒载体RSV疫苗构建和免疫保护效果

目的构建和拯救重组嵌合表达G蛋白表位的流感病毒载体RSV疫苗株,并通过动物实验评价其免疫原性及免疫保护性。方法利用流感病毒反向遗传学技术,以流感病毒为载体,将RSV保护性抗原G蛋白表位的基因插入到流感病毒非结构蛋白NS1基因,拯救重组嵌合表达G蛋白中和表位的流感病毒载体RSV疫苗株rFLU/RSV/G,进而对rFLU/RSV/G的特性进行全面鉴定。rFLU/RSV/G滴鼻免疫Balb/c小鼠,通过检测中和抗体效价、血清抗体(IgG、IgG1、IgG2a)和黏膜抗体sIgA效价以及细胞因子(IL-2、IL-4、IL-5、IFN-γ和TNF-α)的分泌量来评价机体产生的体液、细胞和黏膜免疫反应。攻毒实验评价rFLU/RSV/G的免疫保护性,观测小鼠的体质量变化、病毒载量、存活情况以及肺病理变化。结果成功拯救出重组嵌合表达G蛋白表位的流感病毒载体RSV疫苗株rFLU/RSV/G。重组病毒rFLU/RSV/G与流感病毒颗粒形态和大小分布相符,并且表达针对RSV的特异性蛋白NS1-G。动物实验表明rFLU/RSV/G诱导小鼠机体产生较高的中和抗体、RSV特异性的Th1型细胞免疫反应以及黏膜免疫反应。攻毒实验显示rFLU/RSV/G对RSV A2株和PR8流感病毒野毒株具有一定免疫保护效果,能够有效降低肺鼻的病毒载量,肺组织病变情况明显减轻,且无小鼠死亡。结论重组嵌合表达G蛋白表位的流感病毒载体RSV疫苗株rFLU/RSV/G是一个有前景的RSV候选疫苗株,具有较高的免疫原性和免疫保护性,值得在棉鼠和猴子进行进一步研究。     

汉坦病毒嵌合基因G2S0.7与CTL多表位重组腺病毒的构建及免疫学分析

目的:构建含有汉坦病毒M基因(编码糖蛋白G2)和部分S基因(编码核蛋白主要抗原区段)以及S基因的多个CTL表位的多种重组腺病毒表达载体,并对这些重组腺病毒的免疫学特性进行研究。方法:构建含目的基因的重组腺病毒载体,并在VeroE6细胞中进行表达,利用纯化后的重组腺病毒免疫BALB/c小鼠,通过ELISA、微量细胞中和试验、T淋巴细胞增殖实验及CTL杀伤实验等方法检测免疫反应。结果:成功表达出可被抗汉滩病毒核蛋白及糖蛋白G2的特异性单克隆抗体(mAb)所识别的融合蛋白;重组的腺病毒可以有效诱导针对汉坦病毒NP及GP的体液免疫应答和细胞免疫应答;同时我们发现含有CTL表位的重组腺病毒比不含CTL表位的重组腺病毒可以诱导小鼠产生更为有效的细胞免疫反应。结论:构建的含CTL多表位的重组腺病毒可以有效的提高嵌合基因刺激细胞免疫应答的能力。     

含HIV-1 gag基因的重组腺病毒5型与35型嵌合病毒免疫效果研究

目的探讨含HIV-1 gag基因的重组腺病毒5型与35型嵌合病毒(rAd5/F35)在BALB/c小鼠中的免疫效果。方法PCR,间接免疫荧光鉴定HIV-1gag基因在重组腺病毒(rAd5/F35-mod.gag)的正确插入和体外细胞水平的表达,用rAd5/F35-mod.gag以不同的方式免疫BALB/c小鼠,ELISA检测小鼠血清中的p24特异性抗体,细胞内细胞因子染色法检测小鼠的特异性细胞毒性T淋巴细胞(CTL)应答。结果重组腺病毒rAd5/F35-mod.gag在体外细胞水平可以很好的表达目的基因gag;在小鼠体内重组腺病毒rAd5/F35-mod.gag可诱导特异性的CTL应答和血清IgG抗体反应,用rAd5/F35单独免疫2次,所诱发的CTL和血清IgG反应最强。但Ad5初次感染后,产生的抗Ad5抗体可以抑制rAd5/F35疫苗的特异性免疫反应。结论重组腺病毒rAd5/F35-mod.gag单独免疫可在小鼠体内诱导特异性的CTL应答和血清IgG抗体反应。只改变Ad5纤突蛋白Fiber,不能避免抗Ad5抗体的抑制作用。     

流感疫苗株为载体的重组HAdV疫苗候选株构建及免疫效果

目的以冷适应、减毒流感活疫苗株为载体,构建并拯救喷鼻重组HAd V疫苗候选株,并评价其免疫效果,为腺病毒候选疫苗研制提供实验数据。方法应用反向遗传学技术,以冷适应、减毒流感活疫苗A/Ann Arbor/6/60ca(H2N2)株为骨架,与季节性流感病毒A/California/07/209疫苗株的血凝素(haemagglutination,HA)及插入HAd V六邻体蛋白抗优势原表位的2个重复序列经改造的神经氨酸酶(neuraminidase,NA)基因构建流感病毒八质粒系统,转染COS-1/MDCK细胞筛选疫苗候选株并鉴定;制备的候选疫苗株滴鼻免疫Balb/c小鼠,通过中和抗体、s Ig A黏膜抗体及细胞因子的测定评价其免疫原性,并用野生HAd V-7病毒株攻击评价免疫保护效果。结果成功拯救获得重组HAd V疫苗候选株,命名为rg Flu/HAd V-Ca,其形态符合流感病毒典型特征,可有效表达HAd V病毒Hexon优势表位,且具有冷适应、减毒表型。小鼠免疫后可产生针对HAd V-7及H1N1流感病毒的特异性中和抗体,肺鼻灌洗液中可检测到针对HAd V-7中和抗体s Ig A抗体,并可检测到脾淋巴细胞分泌HAd V-7特异性细胞因子IFN-γ、IL-4;攻毒实验显示,rg Flu/HAd V-Ca疫苗候选株可有效降低小鼠肺病毒载量。结论重组冷适应、减毒HAd V活疫苗株rg Flu/HAd V-Ca具有良好的免疫效果,为腺病毒候选疫苗的研制提供了新思路。     

结核分枝杆菌MPT64重组母牛分枝杆菌免疫学特性

目的:研究结核分枝杆菌MPT64重组母牛分枝杆菌疫苗免疫学特性.方法:用分泌表达MPT64的重组母牛分枝杆菌疫苗免疫BALB/c小鼠,ELISA法检测免疫小鼠的特异性抗体滴度和抗体亚类.分离免疫小鼠脾淋巴细胞,检测淋巴细胞增殖、IFN-γ和IL-12产生水平、CD4+细胞和CD8+细胞数、脾淋巴细胞特异性CTL杀伤效应.毒株攻击后对脾脏细菌负荷计数.结果:MPT64重组母牛分枝杆菌疫苗免疫可诱导小鼠高水平的体液免疫应答,免疫小鼠脾淋巴增殖明显,IFN-γ和IL-12含量增加,CD4+和CD8+细胞百分比明显增加,CTL杀伤效应明显,对MTB H37Rv攻击后有一定的保护作用.结论:MPT64重组母牛分枝杆菌疫苗可诱导小鼠有效的体液和细胞免疫应答,有可能作为新型TB疫苗候选.     

减毒鼠伤寒沙门氏菌运送CD8+T细胞表位的细胞免疫应答

目的： 探索减毒沙门氏菌运送CD8＋ T 细胞表位诱导机体产生特异性细胞免疫应答的规律性.方法： 通过构建融合表达OVA 257～264aa和LCMV NP 118～132aa CD8＋ T 细胞表位的原核表达质粒ptG2F, 以电穿孔法转化减毒鼠伤寒沙门氏菌SL7207, 筛选重组菌SL7207（ptG2F）.采用静脉注射免疫C57BL/6和BALB/c小鼠, 间隔2 wk, 分别于第2次和第3次免疫后, 取免疫小鼠脾细胞, 用ELISPOT法检测特异性IFN-γ分泌细胞和IL- 4分泌细胞.结果： 携带CD8＋ T细胞表位的重组菌SL7207（ptG2F）能诱导产生细胞免疫应答.在提呈OVA CD8＋ T细胞表位时, 2次免疫后, 诱导产生的细胞免疫应答趋向于Th1; 而在3次免疫后, 呈现Th1/Th2的平衡转换.在提呈LCMV NP CD8＋ T细胞表位过程中, Th2免疫应答水平高于Th1, 且有增强趋势.结论： 减毒沙门氏菌可以有效运送CD8＋ T细胞表位并诱导产生特异细胞免疫应答, 这为减毒细菌作为运送载体的研究提供了参考依据.     

EB病毒潜伏膜蛋白2多表位的原核表达及其抗原特性分析

目的 对EB病毒(EBV)潜伏膜蛋白2(LMP2)中富含T、B细胞多表位肽段基因进行原核表达,并分析该多表位蛋白的抗原特性.方法 利用计算机在线软件预测EBV LMP2蛋白的CTL表位、Th表位.选取富含CTL表位和Th表位的肽段,兼顾其上下游已预测的B细胞表位,组成含多个T、B细胞表位的EBV LMP2多表位,该多表位基因序列经原核密码子优化后全序列合成,并克隆入原核表达载体pET32a(+)得到pET32a(+)/EBV-LMP2多表位重组质粒,经IPTG诱导在E.coli BL21(DE3)表达并纯化,经SDS-PAGE和Western blot分析鉴定;采用EBV膜蛋白家兔免疫血清和鼻咽癌患者血清进行Western blot分析其抗原特性;并利用EBV-LMP2多表位蛋白免疫BALB/c小鼠,分别采用LDH和ELISA方法检测小鼠脾细胞特异性CTL杀伤效应及血清特异性抗体IgG水平,以分析该表位蛋白的免疫原性.结果 LMP2(aa195-232)和LMP2(aa419-436)肽段富含CTL、Th和B细胞表位,将其串联后作为EBV LMP2多表位,该多表位基因在大肠杆菌中获得了表达.表达产物的相对分子质量(Mr)约27×103,与预期Mr相符;经Western blot证实EBV-LMP2多表位具有抗原特异性,可被EBV膜蛋白家兔免疫血清和鼻咽癌患者血清特异性抗体识别;小鼠免疫结果显示EBVLMP2多表位可诱导机体产生特异性的CTL杀伤效应,随着效靶比(1:5,1:10,1:25)增加,CTL杀伤活性逐渐增强(12.52%±2.59%,21.80%±1.08%,23.68%±3.74%),同时产生了特异性血清IgG抗体反应(A490=0.258±0.040),与对照组比较差异均具有统计学意义(P＜0.05).结论 本研究没计的EBV-LMP2多表位具有良好的抗原性和一定的免疫原性.     

丙型肝炎病毒多表位抗原重组体免疫原性分析

目的：研究丙型肝炎病毒(HCV)多表位抗原重组体在大肠杆菌中的非融合表达及其免疫原性分析。方法：利用DNA重组技术将构建的HCV多表位抗原重组体克隆入原核表达载体pBV220并在BL-21中表达，非融合表达蛋白经SDS-PAGE检测，排阻层析纯化，利用ELISA和免疫印迹分析其抗原反应性。免疫昆明鼠和猕猴，并检测其抗体和特异性CTL(Cyto-toxic T lymphocyte)，在抗体转阴后再次用HCV病人血清攻击以检测其免疫应答能力。结果：HCV多表位重组体在pBV220／BL-21中表达量占菌体总蛋白量的15％。Western-blot和ELISA分析表明HCV多表位抗原能与HCV病人血清特异性结合，抗体水平和特异性CTL检测结果显示该抗原肽能诱导小鼠和猕猴产生较好的抗体水平和特异性CTL效应。用病人血清再次攻击后受试动物的抗体迅速阳转。结论：表达的多表位重组体具有特异的抗原反应性和免疫原性。     

呼吸道合胞病毒样颗粒疫苗制备及免疫效果研究

目的利用昆虫表达系统表达以流感病毒基质蛋白M1为骨架制备的嵌合呼吸道合胞病毒(RSV)融合蛋白F和表面糖蛋白G蛋白的呼吸道合胞病毒样颗粒(RSV-F/G VLPs)疫苗候选株,并滴鼻免疫Balb/c小鼠,评价其免疫原性与免疫保护性。方法通过基因重组技术制备以流感基质蛋白M1为骨架,并嵌合RSV F/G蛋白的RSV-F/G VLPs,经超滤浓缩、蔗糖密度梯度离心纯化VLP,滴鼻免疫Balb/c小鼠,检测血清抗体IgG、IgG1、IgG2a,黏膜sIgA效价以及细胞因子IL-4、INF-γ等,评价候选疫苗的免疫原性;二免后2周RSV A2株攻毒,通过检测小鼠体质量变化,肺病毒载量及肺病理切片来评价候选疫苗的免疫保护性。结果成功制备了RSV-F VLPs、RSV-G VLPs疫苗抗原,动物实验结果证实,候选疫苗株RSV-F/G VLPs可刺激机体产生特异性Th1型细胞免疫及黏膜免疫;攻毒实验结果表明候选疫苗株RSV-F/G VLPs针对RSV A2株的攻击可以产生一定的保护效果。结论成功制备了RSV-F VLPs、RSV-G VLPs候选疫苗抗原,并通过动物实验证明RSV-F/G VLPs具有良好的免疫原性和免疫保护性,为发展新型RSV疫苗提供实验依据。     

Efficient vagina-to-lower respiratory tract immune trafficking in a murine model of influenza A virus infection

Effective vaccination strategies for infectious diseases take into account the induction, long-term maintenance and recall of memory T-cell populations. To understand the immunological cross-talk within the mucosal compartments, we compared intranasal to vaginal immunization and demonstrated that vaginal infection of BALB/c mice with influenza A virus provides protective mucosal immunity against both homosubtypic and heterosubtypic virus challenge in the respiratory tract. We found that, prior to the viral challenge, in vaginally primed mice, antigen-specific CD8+ T cells were not detected in the lung airways and levels of serum antibodies were lower than those observed in intranasally immunized mice. However, following pulmonary challenge, NP147-specific CD8+ T cells were recruited and amplified in vaginally primed mice to the same extent as those in intranasally primed mice. Thus, the long-term memory immune response elicited by vaginal immunization with influenza virus is efficiently recalled and offers reasonable protection against infection in the respiratory tract.     

Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. III. Delivery of recombinant nucleoprotein to the immune system using attenuated Salmonella typhimurium as a live carrier

A plasmid encoding the influenza nucleoprotein gene from A/NT/60/68 virus was transduced into the attenuated Salmonella typhimurium aroA- strain SL3261. The bacterial vector expressing the viral gene product was able to induce both humoral and cell-mediated immune responses to the nucleoprotein antigen. CD4+ virus-specific T cells capable of proliferation were readily induced and, in some circumstances, class II major histocompatibility complex (MHC)-restricted cytotoxicity was detected. However, virus-specific class I MHC-restricted cytotoxic T lymphocytes (CTL) were not detected after such immunization. Mice immunized orally with the nucleoprotein-expressing bacteria mounted a strong anti-viral antibody response and spleen cells from such mice proliferated specifically to virus challenge in vitro, producing interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Orally immunized mice showed significant protection from challenge infection with influenza virus if the mice were also boosted intranasally before infection.     

Enhancement of mucosal immune responses by chimeric influenza HA/SHIV virus-like particles

To enhance mucosal immune responses using simian-human immunodeficiency virus-like particles (SHIV VLPs) as a mucosal HIV vaccine, we have produced phenotypically mixed, chimeric influenza HA/SHIV 89.6 VLPs and used them to immunize C57B/6J mice intranasally. Systemic and mucosal antibody responses, as well as cytotoxic T cell (CTL) responses, were compared in groups immunized with SHIV 89.6 VLPs or HA/SHIV 89.6 VLPs. Intranasal immunizations were given using VLPs either with or without the addition of the mucosal adjuvant cholera toxin. Total serum IgG, IgG1 and IgG2a, and IgA in saliva, vaginal lavage, lung wash, and fecal extracts were evaluated by enzyme-linked immunosorbent assay (ELISA). The level of serum IgG production to HIV Env was highest in the group immunized with chimeric HA/SHIV 89.6 VLPs. Similarly, mucosal IgA production was also enhanced in the mucosal HA/SHIV 89.6 VLP-immunized group. Analysis of the IgG1/IgG2a ratio indicated that a Th1-oriented immune response resulted from these VLP immunizations. High levels of serum IgG and mucosal IgA against influenza virus were also detected in mice immunized with HA/SHIV VLPs. HA/SHIV 89.6 VLP-immunized mice also showed significantly higher CTL responses than those observed in SHIV 89.6 VLP-immunized mice. Furthermore, a Major Histocompatibility Complex (MHC)-class-I-restricted T cell activation ELISPOT assay showed elevated interferon-gamma, interleukin-2, and interleukin-12 production in HA/SHIV 89.6 VLP-immunized mice, indicating that phenotypically mixed HA/SHIV 89.6 VLPs can enhance both humoral and cellular immune responses at multiple mucosal sites. Therefore, chimeric HA-containing VLPs represent a potential approach for mucosal immunization for prevention of HIV infection.     

Protective and cross-protective mucosal immunization of mice by influenza virus type A with bacterial adjuvant.

Mucosal immunization by inactivated viruses often fails to evoke a sufficient immune response. Intensive efforts have been made to enhance the response by suitable adjuvants. We used the G+ nonpathogenic delipidated bacterium Bacillus firmus with pronounced immunostimulatory properties as an adjuvant for immunizing mice with inactivated influenza virus type A. BALB/c mice were immunized intratracheally with inactivated influenza A H1N1 and H3N2 viruses. The production of antibodies in sera and secretions was determined by the ELISA. The local situation in the lungs was assessed histologically and by testing the cytokine expression. The protective and cross-protective effect against infection was tested in in vivo experiments after infection with influenza virus A H1N1. B. firmus as adjuvant increased both systemic and mucosal antibody responses, improved protection against homologous virus and induced cross-protection against virus H1N1 after immunization with virus H3N2.     

乙型肝炎病毒多表位抗原DNA疫苗的免疫原性

目的 :探讨HBV复合多表位DNA疫苗诱导体液及细胞免疫的可行性。方法 :将HBV多表位抗原基因BPT克隆到真核表达载体pcDNA3.1中 ,构建重组真核表达载体pcDNA3.1/BPT。将其通过肌肉注射免疫BALB/c小鼠 ,用间接免疫ELISA法、CTL杀伤功能检测和淋巴细胞增殖试验 ,检测特异性体液免疫和细胞免疫的水平 ,并观察其对免疫小鼠的毒副作用。结果 :用重组质粒pcDNA3.1/BPT免疫BALB/c小鼠后 ,在效靶比为 10 0∶1时 ,可诱导显著地特异性CTL应答 (P <0 .0 5 )。ELISA检测免疫小鼠血清特异性IgG的水平明显升高 (P <0 .0 5 )。在BPT基因原核表达蛋白的刺激下 ,免疫小鼠脾淋巴细胞增殖显著 (P <0 .0 5 )。RT PCR分析表明 ,IL 12mRNA的水平亦明显升高。结论 :HBV多表位基因疫苗可诱发特异性免疫应答 ,为预防、治疗性HBV疫苗的研制提供了一定的实验依据     

HIV DNA疫苗与重组腺病毒伴随病毒联合免疫效果的研究

目的 构建含HIV-1B亚型中国株gagV3基因的DNA疫苗及重组腺病毒伴随病毒(rAAV)疫苗，并研究DNA疫苗和rAAV联合免疫的免疫效果。方法 将HIV-1B亚型中国株gagV3基因克隆入真核表达载体pCI-neo上，构建了含HIV-1 gagV3基因的DNA疫苗pCI-gagV3。采用电击法将pCI-gagV3质粒转染p815细胞，用G418压力筛选，得到转入重组质粒的细胞系p815-gagV3，用免疫酶法检测细胞系中HIV-1基因的表达。用该DNA疫苗进行小鼠免疫实验，检测免疫效果；用该DNA疫苗初次免疫，含同样gagV3基因的重组腺病毒伴随病毒rAAV-gagV3加强免疫，采用免疫酶法检测免疫小鼠血清中HIV-1特异性的抗体水平，用乳酸脱氢酶法检测免疫小鼠的HIV-1特异性CTL水平。结果 pCI-gagV3可以在p815细胞中表达HIV-1的基因，免疫BALB／c小鼠后可以在小鼠体内诱发HIV-1特异性的细胞和体液免疫反应。HIV-1特异性抗体滴度为1：20；效靶比为50：1时，CTL平均杀伤率为41．7％。pCI-gagV3与rAAV-gagV3联合免疫并不能明显提高抗体水平，但可以提高CTL反应，效靶比为50：1时，CTL平均杀伤率为61.3％，高于单独用DNA疫苗或重组AAV疫苗免疫后产生的CTL活性。结论 DNA疫苗与重组腺病毒伴随病毒联合免疫可以提高免疫小鼠产生的HIV-1特异性CTL反应。     

人偏肺病毒DNA疫苗的构建和小鼠免疫反应的研究

目的 构建人偏肺病毒(hMPV)DNA疫苗,小鼠免疫后评价其细胞和体液免疫水平.方法 利用PCR方法,从hMPV的cDNA中扩增融合蛋白FATM(缺失跨膜区)基因和基质蛋白M基因,构建DNA疫苗pcDNA3.1His-FATM和pcDNA3.1His-M,瞬时转染后用Western Blot和间接免疫荧光方法检测F、M蛋白表达.疫苗肌内注射免疫小鼠,ELISA和ELISPOT方法分别检测血清IgG抗体和小鼠脾细胞CTL水平.结果 Western Blot和间接免疫荧光(IFA)方法证明构建的疫苗可表达FATM和M蛋白.peDNA3.1His-FATM单独免疫小鼠,血清抗体滴度为1:44;与pcDNA3.1His-M联合免疫后,血清抗体滴度为1:64.ELISPOT检测证明,联合免疫组小鼠脾细胞产生IFN-γ的效应CD8+T细胞数为42±8.9,高于单独免疫组32±7.4的水平.结论 DNA疫苗peDNA3.1His-F△TM可以诱导产生特异性的体液和细胞免疫,与pcDNA3.1His-M联合免疫,可以提高免疫水平.     

Mucosal immunization of CD4+ T cell-deficient mice with an inactivated virus induces IgG and IgA responses in serum and mucosal secretions

Immunoglobulin (Ig) class switching can occur in the absence of alphabeta+ or gammadelta+ T cells when mice are infected with certain live viruses, although CD4 T helper cells are believed to be essential for induction of a high-affinity antibody response and for efficient isotype switching from IgM to IgG and IgA production. However, little information is available about the immune responses after mucosal immunization of CD4+ T cell-deficient mice with inactivated virus. In this study, we show that intranasal immunization with formalin-inactivated influenza A/PR8/34 virus induces IgG and IgA responses in serum and IgA responses in mucosal secretions in CD4+ T cell-deficient mice. All four subclasses of IgG were produced. IgG1/IgG2a ratios were found to be from 1 to 1.75, indicating that both Th1 and Th2 immune responses are induced by the inactivated influenza virus. The sera and mucosal secretions were found to have neutralizing activity against influenza virus in vitro. In addition, the mucosally immunized CD4+ T cell-deficient mice were protected completely from challenge with a lethal dose of live, pathogenic influenza virus. To our knowledge, this is the first demonstration that mucosal immunization with an inactivated virus induces immune responses in serum and mucosal secretions in CD4+ T cell-deficient mice.     

含HIV-1 gp120基因的重组腺相关病毒和重组腺病毒疫苗联合免疫的研究

目的 探讨含HIV-1 gp120基因的重组腺相关病毒(rAAV)和重组腺病毒(rAdV)疫苗在BALB／c小鼠中联合免疫的效果。方法 将密码子优化的HIV-1 gp120基因分别插入腺相关病毒(AAV)和腺病毒(AdV)载体质粒，构建含该基因的rAVV和rAdV载体疫苗。将两种疫苗以不同的联合方式免疫BALB／c小鼠，ELISA检测小鼠血清中的gp120特异性抗体，细胞内细胞因子染色法检测小鼠的特异性细胞毒性T淋巴细胞(CTL)应答。结果 两种重组病毒均可表达目的基因gp120；在小鼠体内两种重组病毒联合免疫可诱导特异性的CTL应答和血清1gG抗体反应，但用rAAV初免2次，再用rAdV加强3次所诱发的CTL和血清1gG反应最强。结论 rAAV和rAdV疫苗联合免疫可在小鼠体内诱导特异性的CTL应答和血清1gG抗体反应。     

携带EBV-LMP2基因的重组MVA痘苗病毒初步免疫效果

目的 观察携带EBV lmp2基因的重组痘苗病毒疫苗在小鼠体内诱导的LMP2特异性细胞免疫应答效果.方法 使用表达EBV lmp2基因的重组痘苗病毒MVA-Lac-LMP2,采用低（2＆#215;105 pfu/只）、中（2 ＆#215; 106pfu/只）、高（2 ＆#215; 107pfu/只）三个剂量组,分别于0、2周免疫BALB/c小鼠,末次免疫1周后应用IFN-γ ELISPOT方法检测小鼠脾淋巴细胞中EBV LMP2特异性CTL应答水平.结果 MVA-Lac-LMP2能诱导小鼠产生EBV LMP2特异性的CTL应答,其中,中、高剂量组小鼠CTL水平明显高于低剂量组（P≤0.01）,中、高剂量组小鼠诱导的CTL应答水平差异没有统计学意义.结论 MVA-Lac-LMP2能够在小鼠体内诱导EBV-LMP2特异性的细胞免疫,应答水平的高低与免疫剂量有关.