In situ gene-modified DCs for an HIV-1 T cell vaccine

描述(申请人提供)：尽管已经针对许多病毒性传染病制定了保护性疫苗接种战略，但20年来，针对艾滋病毒-1的广泛活性的疫苗仍然难以获得。细胞免疫涉及(1)调节HIV-1复制，(2)延缓疾病进展，以及(3)预防感染，认为它们是成功免疫战略的重要组成部分。树突状细胞(DC)可通过MHC-I、II类途径递呈抗原，在细胞免疫应答中发挥重要作用。因此，基于DC的疫苗目前正被开发为诱导强烈的抗原特异性T细胞反应的试剂，但这些方案通常需要对DC进行体外基因修饰，然后重新输注回自体或MHC匹配的宿主，在那里它们成熟，迁移到淋巴结，并将候选抗原呈现给CD4和/或CD8T细胞以引发抗原特异性反应。虽然体外基因操作方法在建立实验环境中的概念验证方面是有用的，但开发用于该领域的预防性疫苗是不现实的。如果DC的转导能够在原位完成，DC靶向疫苗作为预防性药物的广泛使用将更加可行。然而，原位转导的一个关键组成部分是在不干扰DC函数的情况下将载体有效地靶向DC。为此，我们构建了一个通过CD40受体特异性靶向DC的腺病毒(Ad)载体系统，并证明了CD40靶向的Ad在体外有效地转导DC而不干扰DC的功能。原位转导的一个潜在限制是，疫苗接种部位必须有足够数量的DC细胞才能实现有效免疫。因此，我们建议在CD40靶向疫苗接种之前，通过一种新的技术，即趋化释放聚合物棒，将DC招募到皮下部位。在这项建议中，我们将评估CD40靶向的Ad载体在体外小鼠体内表达HIV-1抗原的效力，以及加入趋化释放聚合物的原位疫苗模型。首先，我们将优化原位DC募集的参数，并确定这对原位CD40靶向转导效率的影响程度。接下来，在存在或不存在DC募集的情况下，将比较非靶向和CD40靶向的Ad载体免疫后抗原特异性的CD4和CD8T细胞反应。我们的假设是，CD40靶向的Ad将在皮下募集到免疫场所后最佳地转导DC，从而诱导强大的T细胞免疫。能够实现DC的定向原位免疫，然后诱导强大的抗原特异性T细胞免疫，将是HIV-1疫苗开发的一个里程碑。这项研究的成功将推动未来在非人类灵长类动物模型中进行体内研究，以显示针对HIV-1感染高危人群的保护性免疫和人类临床试验。

DESCRIPTION (provided by applicant): Although protective vaccination strategies have been developed against many infectious diseases of viral origin, a vaccine with broad activity against HIV-1 has remained elusive for two decades. Cell-mediated immunity has been implicated in (i) modulation of HIV-1 replication, (ii) protraction of disease progression, and (iii) protection against infection, arguing their importance as components of a successful immunization strategy. Dendritic cells (DC) can present antigens via MHC class I and II pathways and are central to the development of cell-mediated immune responses. Thus, DC-based vaccines are currently being developed as agents to elicit strong, antigen-specific T cell responses, but these protocols usually require ex vivo genetic modification of DCs, followed by re-infusion back into an autologous or MHC-matched host where they mature, migrate to lymph nodes, and present the candidate antigen to CD4 and/or CD8 T cells to elicit antigen-specific responses. Although the ex vivo genetic manipulation approach has been useful in establishing proof-of-concept in an experimental setting, it is not practical for the development of prophylactic vaccines to be used in the field. If transduction of the DCs could be accomplished in situ, the broad use of DC-targeted vaccines as prophylactic agents would be more feasible. However, a critical component of in situ transduction is efficient targeting of the vector to DCs without perturbation of DC function. To this end, we have generated an adenovirus (Ad) vector system that specifically targets DCs via the CD40 receptor and have shown that the CD40-targeted Ad efficiently transduces DCs in vitro without interfering with DC function. A potential limitation of in situ transduction is that sufficient numbers of DC cells must be at the vaccination site to achieve effective immunization. Therefore, we propose to recruit DCs into subcutaneous locales prior to CD40-targeted vaccination by using a novel technology, chemoattractant-releasing polymer rods. In this proposal, we will evaluate the potency of CD40-targeted Ad vectors that express HIV-1 antigens in murine in vitro and in situ vaccination models that incorporate chemoattractant-releasing polymers. Initially, we will optimize the parameters of in situ DC recruitment and determine the degree to which this contributes to the efficiency of in situ CD40-targeted transduction. Next, antigen-specific CD4 and CD8 T cell responses will be compared following immunization with untargeted and CD40-targeted Ad vectors in the presence or absence of DC recruitment. Our hypothesis is that the CD40-targeted Ad will optimally transduce DCs following subcutaneous recruitment to the immunization locale, resulting in the induction of robust T cell immunity. The ability to achieve targeted in situ immunization of DCs followed by the induction of robust antigen-specific T cell immunity will be a milestone in HIV-1 vaccine development. The success of this study will be the impetus for future in vivo studies in non-human primate models to show protective immunity and human clinical trials for populations at high risk of HIV-1 infection.