禽流感病毒血凝素H5特异性单克隆抗体的制备及ELISA捕获法的建立

目的 制备和鉴定禽流感病毒(H5N1)血凝素(H5)特异性单克隆抗体(mAb),建立H5抗原的双抗体夹心ELISA捕获法.方法 以H5血凝素和携带H5全长基因的质粒免疫Balb/c小鼠制备mAb,利用血凝抑制(HI)实验筛选和鉴定,通过竞争抑制试验分析抗体识别表位,并采用抗体配对试验筛选捕获抗体和检测抗体,建立测定H5抗原的双抗体夹心ELISA捕获法.结果 获得16株特异性针对H5的单克隆抗体,与A型流感病毒H1、H3、H7、H9和B型流感病毒的血凝素无HI交叉反应,对H5血凝素的血凝抑制效价为1:100～1:51 200;通过配对实验,建立以单克隆抗体H5M9为捕获抗体,辣根过氧化物酶标记单克隆抗体H5M11为检测抗体的双抗体夹心ELISA;检测多株H5N1病毒和H5血凝素的最低检出值为1/32血凝单位,检测A型流感病毒H1N1、H3N2、B型流感病毒以及H7、H9血凝素均为阴性.结论 建立了一种灵敏度高、特异性强的测定H5抗原的ELISA捕获法,可应用于禽流感病毒H5N1感染的实验室早期诊断.     

表达HPV-16结构蛋白的重组痘苗病毒疫苗免疫效果研究

目的研究表达HPV—16结构蛋白L1和12的重组痘苗病毒（rVVL1L2）的免疫效果。方法以重组痘苗病毒rVVL1L2免疫C57BIJ6小鼠，用酶联免疫（ELISA）和酶联免疫斑点（ELISPOT）方法检测重组痘苗病毒诱发小鼠产生的体液免疫和细胞免疫应答水平；利用C3肿瘤细胞在C57BIJ6小鼠中的成瘤模型，观察重组痘苗病毒在抗肿瘤移植实验和肿瘤生长抑制实验中对小鼠的免疫保护效果。结果重组痘苗病毒rVVL1L2免疫的小鼠，可以检测到针对L1和12特异的抗体、L1165-175肽特异性的、分泌IFN-的T细胞；同时可以观察到免疫后的C57BI/6小鼠，可以有效预防HPV．16相关肿瘤细胞（1．5×10^5C3细胞）的攻击；对已产生的肿瘤，可以延缓肿瘤细胞的生长速度。结论重组痘苗病毒rVVL1L2可以有效诱发小鼠产生体液免疫和细胞免疫应答，为研究预防和治疗HPV-16感染的疫苗提供了实验资料。     

HIV-1中国流行株gag与hIL-2在重组痘苗病毒中的共表达及其与核酸疫苗的实验免疫研究

目的：将HIV-1中国流行株B亚型gag基因、gag和hIL-2基因在天坛株痘苗病毒中进行共表达，以期获得重组痘苗病毒，观察细胞因子的佐剂作用，与核酸疫苗混合免疫，评价免疫效果，为新型艾滋病疫苗研制开发打下基础。方法：将HIV-1中国流行株 gag基因、gag和hIL-2基因片段插入到 pJ38载体启动子下游，经同源重组和血凝素阴性空斑筛选重组痘苗病毒，SDS-PAGE、Western blot检测目的蛋白。以重组病毒和核酸疫苗免疫Balb/c小鼠，进行淋巴细胞转化实验、CTL、CD4 、CD8 T细胞数目以及血清抗体的细胞免疫和体液免疫指标检测。结果：获得了重组痘苗病毒 vJ38gag和 vJ38gag-IL-2。与 vJ38-gag相比，vJ38gag-IL-2，具有更好的免疫原性，重组痘苗病毒免疫3次的效果好于重组病毒免疫2次，以2rVV-DNA混合免疫效果最好。结论：重组痘苗病毒vJ38gag和vJ38gag-IL-2能够表达外源蛋白并诱导机体产生细胞免疫和体液免疫。细胞因子IL-2发挥了免疫佐剂的作用。     

去除痘苗病毒显性表位B8R对外源抗原免疫原性的增强

目的 去除B8R对外源抗原免疫原性的影响,为降低载体的免疫优势从而提高疫苗的有效性提供参考.方法 利用针对痘苗病毒的pSC11质粒,构建插入OVA的质粒pSC11-OVA,在CV-1细胞中与去除B8R的痘苗病毒重组,筛选纯化获得重组病毒.利用体外培养细胞,检测B8R缺失和外源抗原插入对病毒感染复制特性的影响;利用感染小鼠模型,检测重组病毒诱发的针对OVA的细胞免疫应答和免疫记忆效应;采用体征及神经行为学评分系统,检测重组病毒的毒力.结果 B8R缺失和OVA导入对痘苗病毒的生物学特性无明显影响;去除B8R后,外源性OVA成为显性表位,针对OVA的细胞免疫应答和免疫记忆效应明显增强;去除B8R还可显著降低痘苗病毒的毒力.结论 去除B8R可有效降低痘苗病毒的免疫优势效应,增加外源性抗原的免疫原性.去除痘苗病毒本身的显性表位,可为疫苗和基因治疗提供更为有效的载体.     

重组结核抗原痘苗病毒Ankara株的构建及其免疫原性研究

目的 构建5种不同类型的表达结核杆菌特异抗原的重组痘苗病毒,并研究其特异免疫原性.方法 运用同源重组技术将含结核分泌抗原Ag85A和ESAT-6的基因片段插入痘苗病毒表达质粒p18中.重组质粒导入痘苗病毒Ankara(MVA)后构建重组痘苗病毒,经筛选和Western blot鉴定,得到5个种类的带有结核抗原基因的重组病毒.用构建的5种重组病毒免疫小鼠,MTT法检测免疫后小鼠脾淋巴细胞对特异结核抗原的增殖反应;ELISA检测小鼠脾淋巴细胞培养上清液中IFN-γ的含量;结核菌素纯蛋白衍化物(PPD)皮内试验以检测重组病毒引发的针对结核抗原的特异细胞免疫应答.结果 构建的5种蘑组病毒介导的细胞表达产物经Western blot鉴定确认相对分子质量与结核抗原一致.免疫小鼠两次后,5种重组病毒免疫组脾淋巴细胞体外与Ag85A-ESAT-6融合蛋白共培养后表现出明显的增殖活性(P<0.01),培养上清液中IFN-γ的浓度均较同组细胞经生理盐水刺激明显增高(P<0.05);与空痘苗病毒或生理盐水免疫后小鼠相比,5种重组MVA免疫组脾淋巴细胞与AgB5A.ESAT-6融合蛋白共培养后同样表现出明显的增殖活性(P<0.01),与Ag85A-ESAT-6融合蛋白共培养的细胞上清液中IFN-γ的浓度均升高(P<0.01).与空痘苗病毒或生理盐水免疫后小鼠相比,5种重组MVA免疫组小鼠对PPD都表现出显著的迟发型超敏反应应答(P<0.05).结论 成功构建了5种不同类型的表达结核杆菌抗原的重组痘苗病毒疫苗,其免疫小鼠后可激发针对结核杆菌抗原的特异性细胞免疫.     

HIV-1CN54 DNA疫苗滴鼻免疫小鼠诱发系统和黏膜免疫应答

目的：探讨重组痘苗病毒rVVsyngp120或rVVmCN54gp120候选疫苗是否增强HIV-1CN54合成gp120基因(syngp120)DNA疫苗的免疫原性。方法：第0、7、14、21天用DNA疫苗滴鼻免疫小鼠,第28、35、42天再滴鼻接种rVVsyngp120或rVVmCN54gp120。体外测脾和肠系膜淋巴结(MLN)淋巴细胞增殖应答与CD8^ CTL应答。测血清和黏膜洗液特异的IgG和IgA,并测其是否中和实验室适应株HIV-1SF33。结果：单纯DNA免疫后,脾和MLN淋巴细胞在体外发生增殖应答和CTL应答,且测出血清特异的IgG和黏膜洗液特异的IgA。重组痘苗病毒末次免疫后第2周(第56天),发现rVVmCN54gp120增强MLN淋巴细胞增殖应答和CTL应答,脾CTL应答也增强。rVVsyngp120则增强MLN CTL应答。同时发现：2组重组痘苗病毒免疫的动物其血清中特异IgG抗体滴度均有所增高,但黏膜(粪便和阴道)洗液特异IgA抗体滴度却未增高,未测出血清特异IgA和黏膜洗液特异IgG。免疫血清可中和HIV-1SF33,而阴道洗液却不能。结论：单纯DNA疫苗滴鼻免疫可诱发较弱的系统和黏膜体液免疫与细胞免疫,但维持时间短。重组痘苗病毒主要增强局部黏膜的细胞免疫应答,且稍增强系统体液免疫应答,未增强黏膜的IgA应答。免疫血清有中和作用。     

重组结核抗原痘苗病毒免疫次数和免疫途径对免疫原性的影响

卡介苗(BCG)是目前用于预防结核病的惟一疫苗,但其对成年人结核病的预防效果不理想.本研究前期工作已构建了5种不同类型的表达结核杆菌特异抗原的重组痘苗病毒,其免疫小鼠2次后均可激发针对结核杆菌抗原的特异性细胞免疫.然而,重组安卡拉痘苗病毒(MVA)是一活的病毒载体[1],此重组病毒是否能多次免疫动物以及多次免疫动物后能否产生更有效的免疫反应尚不清楚.     

H5N1亚型禽流感病毒血凝素Th和B细胞表位预测及抗原性分析

目的:预测H5N1亚型禽流感病毒血凝素Th和B细胞相关抗原表位,并初步分析其抗原性.方法:依据近年H5N1亚型禽流感病毒流行趋势,下载得到相关HA蛋白氨基酸序列.进行生物信息学综合分析预测,获得Th和B细胞相关抗原表位,并比较其保守性和特异性.通过BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清,初步鉴定候选表位抗原性.结果:综合多项预测及空间构象模拟结果,我们获得了三条候选Th和B细胞表位,分别为HA141～155、HA206～223、HA302～316.候选表位处于H5N1亚型禽流感HA1 蛋白序列上相对保守的区域内,且与目前流行的H5N1亚型禽流感病毒HA相应区域具有较好的一致性.而不同候选表位在BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清反应中显示了不同抗体结合能力,预示了其成为功能表位的可能.结论:所筛选的表位具有成为H5N1亚型禽流感病毒HA Th和B细胞相关抗原表位的可能.本研究为深入揭示流感病毒感染与免疫机制,H5N1亚型禽流感功能表位认知及表位疫苗研究奠定了基础.     

H5N1型高致病性禽流感病毒血凝素蛋白及神经氨酸酶的B细胞抗原表位预测

目的预测H5N1亚型高致病性禽流感病毒HA蛋白和NA蛋白的B细胞表位,为基于B细胞表位的预防性疫苗设计提供依据。方法基于HA蛋白和NA蛋白的蛋白质序列,采用Kyte-Doolittle的亲水性方案,Emini方案,Karplus方案和Jameson-wolf抗原指数方案,并辅以MAGE蛋白的二级结构柔性区域分析,预测HA蛋白和NA蛋白的B细胞表位。结果分别预测出了6条血凝素蛋白(Hemagglutinin,HA)以及6条神经氨酸酶(Neuraminidase,NA)B细胞优势表位。结论这些B细胞表位可为禽流感疫苗的研制提供实验依据。     

人禽流感病毒H5N1多价重组痘苗病毒(天坛株)疫苗在小鼠中的免疫原性研究

Objective To develop an effective and broad immune protective H5N1 vaccine.Methods We first developed two recombinant vaccinia ( Tiantan strain) virus ( rTTV ) based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin(HA) and matrix protein 2(M2), or bicistron expressing the neuraminidase(NA) and matrix protein 1 (M1). The expression of H5N1 protein in rTTVs was confirmed. We immunized the BALB/c mice twice with two kind of dose ( 104 PFU, 107 PFU)using different combination. Subsequently, we assessed the humoral and cellular immune response in vaccinated mice. Results Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HA- and NAspecific antibody level and IFN-γ secreting form cell(SFC) with either single dose of 107 PFU or twice dose of 104 PFU or 107 PFU. We also detected significant neutralizing antibody and matrix-specific immune response. In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level of M2-specific antibody than that with single of rTTV-based H5N1 vaccine. Conclusion rTTVbased H5N1 vaccines in this study elicited board array of immunity and our study offers a promising alternative H5N1 vaccine candidates with favorable potential to prevent various H5N1 pandemic.     

禽流感重组禽痘病毒rFPV-HA-NA活载体疫苗的研究

目的：确定重组禽痘病毒rFPV-HA-NA疫苗株的最佳免疫剂量、免疫日龄、免疫产生时间及免疫持续期。方法：用重组禽痘病毒rFPV-HA-NA疫苗免疫SPF鸡，免疫后用HPAIVH5N1和H7N1AIV进行致死性攻击，观察疫苗免疫后的保护效果。结果：大约含100个PFU的重组病毒即能使机体获得对强毒攻击的100％保护；对1日龄SPF鸡进行免疫接种，4周后能100％抵抗HPAIV的致死性攻击；2周和3周龄的免疫鸡对免疫周1后的强毒攻击具有100％的保护力；重组病毒在免疫后10个月时，其诱导产生的血凝抑制（Haemagglutinin inhibition，HI）抗体仍保持在2-3log2水平，并可以提供100％抵抗病毒的致死性感染。结论：重组禽痘病毒rFPV-HA-NA疫苗是一种安全、高效的基因工程疫苗，它有望在不久的将来替代全病毒灭活疫苗用于高致病力禽流感的预防。     

Protective Efficacy of the Conserved NP,PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD₅₀) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD₅₀ and 10 LD₅₀ and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics.     

Experience with the clinical development of influenza vaccines for potential pandemics

During normal interpandemic influenza seasons, immune responses to vaccines are quite predictable and meet the licensing criteria of the European Union (EU) Committee for Proprietary Medicinal Products (CPMP). In a pandemic situation, large sections, if not all of the community will be immunologically na?ve and therefore new immunisation strategies will be needed. In 1976 and 1977 H1N1 vaccines were prepared and tested clinically. To stimulate 'protective' antibody responses, two doses of vaccine were needed in people below the age of 24 years (no previous experience of H1N1 virus), whereas one conventional dose was adequate in older people. In 1997, the highly pathogenic avian influenza H5N1 virus caused widespread concern when it infected man, with lethal effects. Due to safety concerns it was necessary to adopt new strategies for vaccine development and one such strategy was to produce vaccine from an avirulent H5N3 virus, A/Duck/Singapore-Q/F119-2/97. Clinical trials of a subunit vaccine prepared from A/Duck/Sing/97 virus revealed that even two doses of twice the normal vaccine concentration (i.e. 30 micro g haemagglutinin) were poorly immunogenic, whereas an H5N3 vaccine adjuvanted with microfluidised emulsion (MF) 59 stimulated antibody levels that complied with CPMP criteria after two half strength doses (i.e. 7.5 micro g haemagglutinin).     

Mucosal immunity induced by adenovirus-based H5N1 HPAI vaccine confers protection against a lethal H5N2 avian influenza virus challenge

Development of effective vaccines against highly pathogenic avian influenza (HPAI) H5N1 viruses is a global public health priority. Considering the difficulty in predicting HPAI H5N1 pandemic strains, one strategy used in their design includes the development of formulations with the capacity of eliciting broad cross-protective immunity against multiple viral antigens. To this end we constructed a replication-defective recombinant adenovirus-based avian influenza virus vaccine (rAdv-AI) expressing the codon-optimized M2eX-HA-hCD40L and the M1-M2 fusion genes from HPAI H5N1 human isolate. Although there were no significant differences in the systemic immune responses observed between the intramuscular prime-intramuscular boost regimen (IM/IM) and the intranasal prime-intramuscular boost regimen (IN/IM), IN/IM induced more potent CD8⁺ T cell and antibody responses at mucosal sites than the IM/IM vaccination, resulting in more effective protection against lethal H5N2 avian influenza (AI) virus challenge. These findings suggest that the strategies used to induce multi-antigen-targeted mucosal immunity, such as IN/IM delivery of rAdv-AI, may be a promising approach for developing broad protective vaccines that may be more effective against the new HPAI pandemic strains.     

An MDCK Cell Culture-Derived Formalin-Inactivated Influenza Virus Whole-Virion Vaccine from an Influenza Virus Library Confers Cross-Protective Immunity by Intranasal Administration in Mice

It is currently impossible to predict the next pandemic influenza virus strain. We have thus established a library of influenza viruses of all hemagglutinin and neuraminidase subtypes and their genes. In this article, we examine the applicability of a rapid production model for the preparation of vaccines against emerging pandemic influenza viruses. This procedure utilizes the influenza virus library, cell culture-based vaccine production, and intranasal administration to induce a cross-protective immune response. First, an influenza virus reassortant from the library, A/duck/Hokkaido/Vac-3/2007 (H5N1), was passaged 22 times (P22) in Madin-Darby canine kidney (MDCK) cells. The P22 virus had a titer of >2 ×10⁸ PFU/ml, which was 40 times that of the original strain, with 4 point mutations, which altered amino acids in the deduced protein sequences encoded by the PB2 and PA genes. We then produced a formalin-inactivated whole-virion vaccine from the MDCK cell-cultured A/duck/Hokkaido/Vac-3/2007 (H5N1) P22 virus. Intranasal immunization of mice with this vaccine protected them against challenges with lethal influenza viruses of homologous and heterologous subtypes. We further demonstrated that intranasal immunization with the vaccine induced cross-reactive neutralizing antibody responses against the homotypic H5N1 influenza virus and its antigenic variants and cross-reactive cell-mediated immune responses to the homologous virus, its variants within a subtype, and even an influenza virus of a different subtype. These results indicate that a rapid model for emergency vaccine production may be effective for producing the next generation of pandemic influenza virus vaccines.     

Antibody Breadth and Protective Efficacy Are Increased by Vaccination with Computationally Optimized Hemagglutinin but Not with Polyvalent Hemagglutinin-Based H5N1 Virus-Like Particle Vaccines

One of the challenges for developing an H5N1 influenza vaccine is the diversity of antigenically distinct isolates within this subtype. Previously, our group described a novel hemagglutinin (HA) derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA, when used as an immunogen, elicits a broad antibody response against H5N1 isolates from different clades. In this report, the immune responses elicited by the COBRA HA virus-like particle (VLP) vaccine were compared to responses elicited by a mixture of VLPs expressing representative HA molecules from clade 2.1, 2.2, and 2.3 primary H5N1 isolates (polyvalent). The COBRA HA VLP vaccine elicited higher-titer antibodies to a panel of H5N1 HA proteins than did the other VLPs. Both COBRA and polyvalent vaccines protected vaccinated mice and ferrets from experimental infection with highly lethal H5N1 influenza viruses, but COBRA-vaccinated animals had decreased viral replication, less inflammation in the lungs of mice, and reduced virus recovery in ferret nasal washes. Both vaccines had similar cellular responses postchallenge, indicating that higher-titer serum antibodies likely restrict the duration of viral replication. Furthermore, passively transferred immune serum from the COBRA HA VLP-vaccinated mice protected recipient animals more efficiently than immune serum from polyvalent-vaccinated mice. This is the first report comparing these two vaccine strategies. The single COBRA HA antigen elicited a broader antibody response and reduced morbidity and viral titers more effectively than a polyvalent mixture of primary H5N1 HA antigens.     

Evaluation of the antigenicity and reactogenicity of varying formulations of the rhesus rotavirus-based quadrivalent and the M37 rotavirus vaccine candidates

Three phase l trials of the rhesus rotavirus (RRV) based quardrivalent vaccine [composed of sero-type 3 (RRV), and serotypes 1 (D × RRV), 2 (DS1 × RRV), and 4 (ST3 × RRV) human rotavirus × RRV reassortants] and the M37 (nursery strain) rotavirus vaccine candidates were conducted in an attempt to find a safe and optimally antigenic formulation. Infants 10-20 weeks old received, in trial I, 1) the quadrivalent vaccine as two separate bivalent doses (1 × 10⁴PFUeachof D × RRV and RRV, followed 4 weeks later by 1 × 10⁴ PFU each of DS1 × RRV and ST3 × RRV) or 2) placebo; in trial II, 1) one dose of quadrivalent vaccine (10⁴ PFU of each component), or 2) two doses of quadrivalent vaccine, or 3) a 10⁴ PFU dose of M37 vaccine, or 4) M37 vaccine followed by the quadrivalent vaccine, or 5) placebo; in trial III, 1) a dose of a higher-titered quadrivalent vaccine (10⁵ PFU of each component), or 2) two doses of higher titered quadrivalent vaccine, or 3) a dose of higher titered M37 vaccine (10⁵ PFU) or 4) two doses of M37 vaccine (10⁵ PFU), or 5) M37 vaccine (10⁵ PFU) followed by the higher titered quadrivalent vaccine, or 6) placebo. A mild, transient fever during the first week post-vaccination was associated with the bivalent or quadrivalent vaccines but not with the M37 vaccine. Fourfold or greater serum IgA ELISA responses to rotavirus were observed in 48-92% of the infants receiving quadrivalent vaccine and in 32-50% of those receiving M37 vaccine. Neutralization seroresponses to human rotaviruses in infants receiving quadrivalent vaccine ranged from 9% (to serotype 3 in recipients of the first dose of bivalent vaccine in trial I) to 68% (to serotype 1 in recipients of two doses of higher titered quadrivalent vaccine in trial III). M37 vaccine induced homologous neutralization responses in 33-78% of the infants and responses to human strains sharing its VP7 (Wa) or VP4 (ST3) serotype in 13-35% of the infants. Overall, both vaccine candidates induced a greater number of antibody responses when two doses or a higher titered preparation was administered. © 1994 Wiley-Liss, Inc.     

Superior protection provided by a single dose of MF59-adjuvanted whole inactivated H5N1 influenza vaccine in type 1 diabetic mice

Vaccination is the preferred strategy for the prevention of influenza virus infection. Both H5N1 subunit and split vaccines have shown poor immunogenicity in clinical trials thus far. Therefore, it is urgent to develop more immunogenic and antigen-sparing H5N1 influenza vaccines as well as safe and effective adjuvants for humans, especially for immunocompromised people such as patients with diabetes mellitus. In this study, the protective effect of an MF59-adjuvanted inactivated whole-virion H5N1 vaccine was investigated in a type 1 diabetic mouse model. Mice (both healthy and diabetic) were immunized with a single dose of the inactivated vaccine, alone or adjuvanted with MF59 or Al(OH)₃. After four weeks, mice were challenged with a lethal dose of H5N1 virus. Antibody responses, survival rates, lung virus titers and body weight changes were tested. The results showed that addition of MF59 or Al(OH)₃ to the vaccine enhanced the antibody responses in both healthy mice and diabetic mice, but the MF59-adjuvanted groups showed higher antibody responses than the Al(OH)₃-adjuvanted groups. The addition of MF59 or Al(OH)₃ to the vaccine led to a conversion of the immune response from a Th1-biased response to an enhanced mixed Th1/Th2 profile. The MF59-adjuvanted inactivated whole-virion H5N1 vaccine provided superior protection in type 1 diabetic mice to either the vaccine alone or the vaccine adjuvanted with Al(OH)₃.     

Employing XIAP to Enhance the Duration of Antigen Expression and Immunity Against an Avian Influenza H5 DNA Vaccine

DNA vaccine represents a powerful approach for prevention of avian H5N1 influenza infection. Yet, DNA vaccine-induced immune responses might be limited by the short duration of antigen expression. As a strategy to enhance adaptive immune responses elicited by a hemagglutinin 5 (H5) DNA vaccine, we explored the effect of co-administration of a DNA encoding X-linked inhibitor of apoptosis protein (XIAP) as a modulator of apoptosis and a stimulator of inflammatory signaling. In cultured cells as early as 24?hours (h), we found that the DNA vaccine encoded H5 antigen was a potent stimulator of apoptosis, and the H5 pro-apoptotic activity was significantly suppressed by the co-expression of full-length XIAP or mutant XIAP (ΔRING). However, full-length XIAP showed a higher potency than mutant XIAP (ΔRING) in the inhibition of H5-induced apoptosis. We also compared the immunizing ability of transmembrane and secretory forms of H5. Mice vaccinated (twice with 3-week intervals) with the secretory form of H5 showed higher hemagglutination inhibition (HI) antibody titers than mice vaccinated with the transmembrane form of H5. Furthermore, co-administration of XIAP with the secretory form of H5 resulted into a stronger antibody response than the transmembrane form of H5. Our findings suggest that in the design of DNA vaccines for a given pro-apoptotic antigen, using an anti-apoptotic molecular adjuvant and the secretory form of antigen may be a greater stimulus to induce immune responses.     

Induction of cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza virus infection in mice by inoculation of apathogenic H5N1 influenza virus particles inactivated with formalin

We investigated whether a vaccine derived from an apathogenic reassortant type A H5N1 influenza strain could induce immune responses in vivo that mediated protection from highly pathogenic avian influenza virus infection in mice. After two subcutaneous immunizations with formalin-inactivated H5N1 whole virus particles (whole particle vaccine), significant killing specific for cells presenting a nucleoprotein peptide from the vaccine strain of the virus was observed. Similar vaccination with viruses treated with ether and formalin, which are commonly used for humans as ether-split vaccines, induced little or no cytotoxic T-cell response. Furthermore, whole particle vaccines of the apathogenic H5N1 strain were more effective than ether-split vaccines at inducing antibody production able to neutralize a highly pathogenic H5N1 strain. Finally, whole particle vaccines of H5N1 protected mice against infection by an H5N1 highly pathogenic avian influenza virus more effectively than did ether-split vaccines. These results suggest that formalin-inactivated virus particles of apathogenic strains are effective for induction of both cytotoxic T-lymphocyte and antibody responses against highly pathogenic avian influenza viruses in vivo, resulting in protection from infection by a highly pathogenic H5N1 virus.