Antigen presentation by epithelial stem cells to promote life long immunity

描述（由申请人提供）： SIV疫苗研究产生的大量数据表明，艾滋病毒疫苗是可以实现的。在病毒传播的初始阶段，粘膜组织中的体液和细胞免疫应答阻断或抑制复制的能力可能对接种疫苗的宿主抵抗感染的能力产生深远影响。开发一种有效的疫苗，限制病毒在粘膜入口复制可能是我们控制HIV感染的最大希望。我们认为，成功的疫苗有两个必要的特征：1）用病毒抗原终身刺激免疫系统; 2）在HIV初次复制部位产生靶向免疫应答。同时解决这两个问题的疫苗方法将有可能实现稳固的长期保护。为了满足这些要求，我们提出了一种替代方法，成功地将疫苗递送到粘膜部位并引发保护性粘膜免疫应答。我们建议使用上皮干细胞作为病毒抗原的永久来源，并将其分化的后代作为抗原产生呈递细胞。使用单循环SIV（SIVsc）的方法，这已被证明是一个非常安全的战略相比，传统的减毒疫苗，我们建议克隆SIVsc基因组的控制下的外皮蛋白启动子，一个终末分化的角质形成细胞特异性启动子。然后将该病毒施用到来自不同组织（表皮、阴道、直肠）的靶向上皮干细胞。基底层细胞将分裂和分化，从而触发SIV抗原表达以及直接和交叉引发。在此，我们提议：1）通过皮肤、阴道和直肠途径免疫恒河猴，诱导并优化SIV抗原在终末分化角质形成细胞特异性启动子中的表达; 2）研究不同接种方案诱导的免疫应答的性质;和，3）如果这些动物与减毒SIV感染的动物相比能够产生令人满意的免疫应答，我们将尝试证明用同源SIV毒株对多次低剂量阴道和直肠攻击的保护作用。HIV病毒的性质已经通过导致慢性病毒复制的适应性免疫的体液和细胞臂对有效的免疫控制产生了几个障碍。在SIV/猕猴模型中测试的疫苗方法中，用活减毒慢病毒接种始终产生针对致病性异源SIV毒株的最有效和持久的保护。然而，安全性问题排除了在人类中使用活的减毒慢病毒。正如许多作者所强调的那样，这些结果得出的结论是，只有疫苗对免疫系统的终身刺激才足以实现长期保护。另一个关键问题是主要通过生殖器或直肠粘膜表面传播。在病毒感染的初始阶段，粘膜组织中的体液和细胞免疫应答阻断或抑制HIV复制的能力可能对接种疫苗的宿主抵抗感染的能力产生深远影响。开发一种有效的疫苗，限制病毒在粘膜入口复制，可能是我们控制艾滋病毒大流行的最大希望。开发有效的艾滋病疫苗的障碍之一是我们不能在粘膜入口处长时间递送抗原。为了解决这些问题，疫苗必须使用持久的疫苗载体证明抗原的长期表达，并被递送到上皮屏障以产生粘膜归巢细胞应答。为此，我们建议使用表皮干细胞作为抗原的永久来源，并将其分化的后代作为抗原产生细胞。所有复层鳞状上皮均由上皮层组成。增殖基底细胞层的细胞在分化和向上移动时上调蛋白质（如外皮蛋白）的转录。在这些启动子中，外皮蛋白启动子已被广泛研究，并仅限于表皮和粘膜中的终末分化上皮细胞。我们的概念是用慢病毒载体靶向上皮干细胞，在外皮蛋白启动子的控制下引入抗原构建体。这些细胞将携带构建体而不被免疫系统攻击，因为它们不表达抗原。随着子代细胞分化，启动子将被激活，并且子细胞在某种意义上将成为抗原呈递细胞，导致通过直接引发和交叉引发诱导体液和细胞免疫应答。

DESCRIPTION (provided by applicant): The significant amount of data generated by SIV vaccine studies has led to the suggestion that an HIV vaccine is achievable. The capacity of humoral and cellular immune responses in mucosal tissues to block or contain replication at the initial stage of virus transmission may have a profound impact on the ability of a vaccinated host to resist infection. The development of an effective vaccine that restricts viral replication at the mucosal portal of entry may be our best hope for controlling HIV infection. We believe there are two necessary features for a successful vaccine: 1) life-long stimulation of the immune system with viral antigens; and 2) a targeted immune response at the site of primary replication of HIV. A vaccine approach that simultaneously addresses these two issues would have the potential to achieve solid, long-term protection. To fulfill these requirements, we propose an alternative approach to successfully deliver a vaccine to mucosal sites and elicit protective mucosal immune responses. We propose to use the epithelial stem cell as a permanent source of viral antigen and their differentiated offspring as antigen producing presenting cells. Using a single cycle SIV (SIVsc) approach, which has been shown to be a very safe strategy compared to traditional attenuated vaccines, we propose to clone the SIVsc genome under the control of the involucrin promoter, a terminally differentiated keratinocyte specific promoter. This virus will be then administered to target epithelial stem cells from different tissues (epidermal, vaginal, rectal). Basal layer cells will divide and differentiate thus triggering SIV antigen expression and both direct and cross priming. Herein, we propose: 1) To elicit and optimize the SIV antigen expression from a terminally differentiated keratinocyte-specific promoter in rhesus macaques vaccinated by dermal, vaginal and rectal routes; 2) To investigate the nature of immune responses induced by the different inoculation protocols; and, 3) if these animals are able to mount a satisfactory immune response compared to attenuated SIV infected animals, we will attempt to demonstrate protection against multiple low-dose vaginal and rectal challenges with a homologous SIV strain. The nature of the HIV virus has created several barriers to effective immune control by the humoral and cellular arms of adaptive immunity leading to chronic viral replication. Of the vaccine approaches tested in the SIV/macaque model, vaccination with live attenuated lentiviruses has consistently yielded the most effective and durable protection against pathogenic heterologous SIV strains. However, safety issues preclude the use of live attenuated lentiviruses in humans. As highlighted by many Authors, these results lead to the conclusion that only a life-long stimulation of the immune system by the vaccine will be sufficient to achieve long-term protection. Another key issue is transmission which occurs predominantly across genital or rectal mucosal surfaces. The capacity of humoral and cellular immune responses in mucosal tissues to either block or contain HIV replication at the initial stages of virus infection may have a profound impact on the ability of a vaccinated host to resist infection. The development of an effective vaccine that restricts viral replication at the mucosal portal of entry may be our best hope for controlling the HIV pandemic. One of the obstacles to the development of an effective AIDS vaccine has been our inability to deliver antigen for a prolonged period at the mucosal portal of entry. To address these issues a vaccine will have to demonstrate long-term expression of antigens using a persistent vaccine vector and be delivered to the epithelial barrier to generate mucosal homing cellular responses. To that end, we propose to use epidermal stem cell as a permanent source of antigen and their differentiated offspring as antigen producing cells. All stratified squamous epithelia are composed of epithelial layers. Cells at the proliferative basal cell layer upregulate transcription of proteins (such as involucrin) as they differentiate and move upward. Among these promoters, the involucrin promoter has extensively been studied and is restricted to terminally differentiated epithelial cells in epidermis and mucosa. Our concept is to target epithelial stem cells with a lentivirus vector to introduce an antigen construct under the control of the involucrin promoter. These cells will carry the construct without being attacked by the immune system because they do not express the antigens. As the progeny cells differentiate, the promoter will become activated and the daughter cells will become, in a sense, antigen presenting cells leading to induction of humoral and cellular immune response via direct priming and cross-priming.