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导致了艾滋病毒疫苗是可以实现的。在病毒传播的初始阶段，粘膜组织中体液和细胞免疫反应阻止或遏制复制的能力可能对接种疫苗的宿主抵抗感染的能力有深远的影响。开发一种有效的疫苗，限制病毒在进入粘膜的入口复制，可能是我们控制艾滋病毒感染的最大希望。我们认为，一种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)在艾滋病毒的初级复制部位进行有针对性的免疫反应。同时解决这两个问题的疫苗方法将有可能实现坚实的长期保护。为了满足这些要求，我们提出了一种替代方法，成功地将疫苗输送到粘膜部位，并引发保护性粘膜免疫反应。我们建议使用上皮干细胞作为病毒抗原的永久来源，并将其分化的后代用作产生抗原的呈递细胞。使用单循环SIV(SIVsc)方法，与传统的减毒疫苗相比，这是一种非常安全的策略，我们建议在终末分化的角质形成细胞特异性启动子--总蛋白启动子的控制下克隆SIVsc基因组。然后，该病毒将被注射到来自不同组织(表皮、阴道、直肠)的靶上皮干细胞。基底层细胞会分裂和分化，从而触发SIV抗原的表达，以及直接和交叉启动。在此，我们建议：1)诱导和优化SIV抗原在恒河猴皮肤、阴道和直肠途径免疫的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反应的性质；3)如果这些动物能够与减毒的SIV感染动物相比产生令人满意的免疫反应，我们将尝试展示对同源SIV毒株的多次低剂量阴道和直肠攻击的保护作用。艾滋病毒的性质对适应性免疫的体液和细胞手臂的有效免疫控制造成了几个障碍，导致慢性病毒复制。在SIV/猕猴模型中测试的疫苗方法中，用减毒慢病毒活疫苗始终产生了对致病的异种SIV毒株最有效和最持久的保护。然而，安全问题排除了在人类身上使用减毒活慢病毒的可能性。正如许多作者强调的那样，这些结果导致了这样的结论，即只有疫苗对免疫系统的终身刺激才足以实现长期保护。另一个关键问题是传播，主要发生在生殖器或直肠粘膜表面。在病毒感染的初始阶段，粘膜组织中体液和细胞免疫反应阻止或遏制艾滋病毒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的影响。开发一种有效的疫苗，限制病毒在黏膜入口处的复制，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无法在进入粘膜的入口处长期递送抗原。为了解决这些问题，疫苗必须使用持久性疫苗载体证明抗原的长期表达，并将其输送到上皮屏障以产生粘膜归巢细胞反应。为此，我们建议使用表皮干细胞作为永久的抗原来源，并将其分化的后代用作抗原产生细胞。所有复层鳞状上皮均由上皮层组成。在增殖的基底细胞层的细胞在分化和向上移动的过程中上调蛋白质(如总环蛋白)的转录。在这些启动子中，总蛋白启动子已经得到了广泛的研究，并且仅限于表皮和粘膜中的终末分化的上皮细胞。我们的概念是用慢病毒载体靶向上皮干细胞，在总蛋白启动子的控制下引入抗原结构。这些细胞将携带这种结构，而不会受到免疫系统的攻击，因为它们不表达抗原。随着子代细胞的分化，启动子将被激活，在某种意义上，子代细胞将成为抗原提呈细胞，通过直接启动和交叉启动诱导体液和细胞免疫反应。

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.