NASAL DNA/PROTEIN VACCINE FOR ANTI-HIV ANTIBODY AND CTL

描述（由申请方提供）：本项目将检验以下假设：如果同时通过皮内和鼻内途径给药，补充重组HIV包膜蛋白加佐剂的DNA疫苗将在粘膜和全身区室中诱导强烈的抗病毒体液和细胞免疫应答。 具体目的I是确认胃肠外佐剂QS-21也可用于辅助鼻内给药DNA疫苗或可溶性蛋白的免疫应答，并确定这些疫苗制剂的有效鼻内QS-21剂量。雌性恒河猴组经鼻接种10或25 pg QS-21加rgp 41-血凝素融合蛋白（gp 41 HA）或编码非感染性SHIV 89.6P颗粒的DNA（SHIV DNA）。将通过ELISA定量测定血清和分泌物中诱导的HIV和SIV特异性抗体。通过使用[3 H]胸苷摄取试验、ELISPOT试验和流式细胞术检测分离的单核细胞的抗原特异性淋巴细胞增殖、IFN-β分泌和细胞内IFN-γ产生，评价外周、直肠粘膜和宫颈/阴道粘膜中的T辅助细胞（Th）和细胞毒性T淋巴细胞（CTL）应答。发现产生最大免疫应答的QS-21剂量将用于随后的鼻疫苗接种。 特定目的II是在非人灵长类动物中使用QS-21、SHIV DNA和gp 41 HA，以确定最佳、实用的DNA/蛋白质疫苗接种方案，用于诱导全身腔室、直肠粘膜和生殖道粘膜中的体液和细胞免疫应答。我们将首先确认SHIV DNA加gp 41 HA和QS-21可以作为一种制剂给药而不丧失免疫原性。最后，将测试包括同时鼻/皮内施用SHIV DNA、gp 41 HA和QS-21，然后同时鼻/皮内加强表达重组修饰的安卡拉牛痘病毒的SHIV抗原的疫苗接种策略的免疫原性和直肠SHIV 89.6P攻击后的保护效力。在所有这些研究中，上文在AIM I中描述的方法将用于分析抗体和T细胞应答，包括粘膜中的那些。 具体目标III是完善和优化HIV/上皮细胞结合试验，该试验可用于测试疫苗接受者的分泌物中阻断HIV附着于上皮细胞的抗体。将在本试验中分析从上述接种动物中采集的分泌物，以确定gp 41 HA疫苗抗原是否能够诱导粘膜抗体，从而可能阻止HIV进入宿主。 这项研究预计将显着进一步我们的知识，佐剂的DNA疫苗，以及鼻接种途径，潜在的HIM疫苗亚单位成分的免疫原性，可用于评估的方法，用于预防传播粘膜抗体的分泌物，和设计的疫苗接种方案，是最有可能诱导保护性免疫艾滋病毒。

DESCRIPTION (provided by applicant): This project will test the hypothesis that supplementation of a DNA vaccine with recombinant HIV envelope protein plus adjuvant will induce strong antiviral humoral as well as cellular immune responses in both mucosal and systemic compartments if simultaneously administered by both the intradermal and nasal routes. Specific Aim I is to confirm that the parenteral adjuvant QS-21 can also be used to adjuvant immune responses to nasally administered DNA vaccines or soluble protein, and to establish an effective nasal QS-21 dosage for these vaccine preparations. Groups of female rhesus macaques will be nasally vaccinated with 10 or 25 pg QS-21 plus rgp41-hemagglutinin fusion protein (gp41HA) or DNA encoding noninfectious SHIV89.6P particles (SHIV DNA). HIV- and SIV-specific antibodies induced in sera and secretions will be quantitated by ELISA. T helper cell (Th) and cytotoxic T lymphocyte (CTL) responses in the periphery, rectal mucosa, and the cervical/vaginal mucosa will be evaluated by testing isolated mononuclear cells for antigen-specific lymphoproliferation, IFN-( secretion, and intracellular IFN-gamma production using the [3H] thymidine uptake assay, ELISPOT assay, and flow cytometry. The QS-21 dose found to produce greatest immune responses would be used for subsequent nasal vaccinations. Specific AIM II is to use QS-21, SHIV DNA, and gp41HA in nonhuman primates to identify an optimal, practical DNA/protein vaccination protocol for induction of humoral and cellular immune responses in the systemic compartment, rectal mucosa, and genital tract mucosa. We will first confirm that SHIV DNA plus gp41HA and QS-21 can be administered as one formulation without loss of immunogenicity. Ultimately, a vaccination strategy involving simultaneous nasal/intradermal administration of SHIV DNA, gp41HA, and QS-21 followed by simultaneous nasal/intradermal boosting with recombinant modified vaccinia ankara virus-expressing SHIV antigens will be tested for immunogenicity, and protective efficacy after rectal SHIV89.6P challenge. In all of these studies, the methods described above in AIM I will be used to analyze antibody and T cell responses, including those in the mucosa. Specific Aim III is to refine and optimize an HIV/epithelial cell-binding assay that could be used to test secretions of vaccine recipients for antibodies that block HIV attachment to epithelial cells. Secretions collected from animals vaccinated above will be analyzed in this assay to determine if the gp41HA vaccine antigen is capable of inducing mucosal antibodies that could potentially prevent entry of HIV into the host. This study is expected to significantly further our knowledge concerning adjuvants for DNA vaccines as well as the nasal vaccination route, the immunogenicity of potential HIM vaccine subunit components, methods that could be used for evaluating secretions for transmission-preventing mucosal antibodies, and the design of vaccination protocols that are most likely to induce protective immunity to HIV.