A Novel Prime/Pull Therapeutic Vaccine Strategy to Prevent Recurrent Genital Herpes

项目概要/摘要 生殖器单纯疱疹病毒2型（HSV-2）感染影响美国超过6000万人， 全球百万。目前还没有获得FDA批准的生殖器疱疹疫苗。原发性感染后， 阴道粘膜皮肤组织（VMC），病毒传播并在感觉神经元中建立潜伏期， 区域背根神经节（DRG）。病毒从潜伏期偶尔重新激活，并在生殖器中脱落 道，在那里它可以导致严重的复发性病变。我们的长期目标是开发治疗性疫苗， 预防生殖器疱疹复发。在过去的五年里，我们在识别 候选HSV-2抗原，并表征抗病毒CD 4+和CD 8 + T细胞的表型和功能， 与血清阳性妇女和豚鼠复发性生殖器疱疹模型中的保护相关：（A）我们 发现两个HSV-2被膜病毒蛋白（VP 16和VP 22）和两个核苷酸还原酶亚单位 蛋白（RR 1和RR 2）主要由来自“天然”保护的无症状的CD 4+和CD 8 + T细胞靶向 女性（那些尽管被感染但从未复发生殖器疱疹的人）;（B）类似地，VP 16，VP 22， RR 1和RR 2蛋白是HSV-2的主要抗原，被组织驻留的CD 4+和CD 8 + T细胞识别 （C）表型和转录组RNA- 在受保护的豚鼠中DRG-和治愈的VMC-驻留的CD 4+和CD 8 + T细胞的Seq谱显示， 具有功能性组织驻留CXCR 3 + CD 4+和CXCR 3 + CD 8 + T细胞的所有标志;（D）虽然治疗性 在HSV-2感染的豚鼠中，用RR 2抗原接种产生强保护，VP 16、VP 22和 RR 1抗原提供适度的保护;和（E）用神经营养素治疗HSV-2感染的豚鼠 表达豚鼠CXCL 11趋化因子（CXCR 3配体）的腺相关病毒载体（AAV 8）加强了 在感染的DRG和VMC中特异性的CD 4+和CD 8 + T细胞的数量和改善的保护。基于 根据这些已发表的初步结果，我们假设，增强强大而持久的抗病毒组织- DRG和VMC中局部存在的CD 4+和CD 8 + T细胞应答将产生更强/持续的 防止HSV-2再活化和脱落，减少复发性生殖器疱疹。为了验证这个假设， 我们提出了两个具体目标：目标1。评价其安全性和对豚鼠生殖器的保护作用 本发明涉及一种创新的引发/拉动治疗性疫苗方法的疱疹模型，其包括：（1）用以下物质引发T细胞： VP 16、VP 22、RR 1和RR 2抗原;和（2）通过免疫组织化学方法将致敏的T细胞“拉入”感染的DRG和VMC组织。 使用亲神经性AAV 8局部递送T细胞吸引趋化因子CXCL 9、CXCL 10和/或CXCL 11 vector.目标2.为了确定是否增加抗病毒组织驻留的CD 4+和CD 4+的数量和功能， CD 8 + T细胞：（1）DRG（中枢神经元免疫）;和（2）VMC（外周上皮免疫）相关 预防生殖器疱疹本临床前研究的目标是将初免/拉免疫苗推向临床。

PROJECT SUMMARY/ABSTRACT Genital herpes simplex type virus-2 (HSV-2) infection affects over 60 million people in the U.S. and over 536 million worldwide. An FDA-approved genital herpes vaccine is currently unavailable. After primary infection of the vaginal mucocutaneous tissue (VMC), the virus spreads and establishes latency in sensory neurons of regional dorsal root ganglia (DRG). The virus reactivates sporadically from latency and sheds back in the genital tract, where it can cause severe recurrent lesions. Our long-term goal is to develop a therapeutic vaccine to prevent recurrent genital herpes. Over the last 5 years, we have made significant progress in identifying candidate HSV-2 antigens and characterizing the phenotype and function of antiviral CD4+ and CD8+ T cells that associate with protection in seropositive women and in the guinea pig recurrent genital herpes model: (A) We found that two HSV-2 tegument virion proteins (VP16 and VP22) and two ribonucleotide reductase subunit proteins, (RR1 and RR2) are mainly targeted by CD4+ and CD8+ T cells from “naturally” protected asymptomatic women (those who, despite being infected, never develop recurrent genital herpes); (B) Similarly, VP16, VP22, RR1, and RR2 proteins were the main HSV-2 antigens recognized by tissue-resident CD4+ and CD8+ T cells that reside in DRG and VMC of protected asymptomatic guinea pigs; (C) Phenotypic and transcriptomic RNA- Seq profiling of DRG- and healed VMC-resident CD4+ and CD8+ T cells in protected guinea pigs show that they bear all the hallmarks of functional tissue-resident CXCR3+CD4+ and CXCR3+CD8+ T cells; (D) While therapeutic vaccination with RR2 antigen produced strong protection in HSV-2 infected guinea pigs, the VP16, VP22 and RR1 antigens provided modest protection; and (E) Treatment of HSV-2 infected guinea pigs with a neurotropic adeno-associated virus vector (AAV8) expressing the guinea pig CXCL11 chemokine (a CXCR3 ligand) boosted the number of CD4+ and CD8+ T cells specifically in infected DRG and VMC and improved protection. Based on these published and preliminary results, we hypothesize that boosting strong and long-lasting antiviral tissue- resident CD4+ and CD8+ T cell responses locally in DRG and VMC would produce a more robust/sustained protection against HSV-2 reactivation and shedding and reduce recurrent genital herpes. To test this hypothesis, we propose two Specific Aims: Aim 1. To evaluate the safety and protective efficacy, in the guinea pig genital herpes model, of an innovative prime/pull therapeutic vaccine approach that consists of: (1) Priming T cells with VP16, VP22, RR1, and RR2 antigens; and (2) “Pulling” primed T cells into infected DRG and VMC tissues by a local delivery of T-cell attracting chemokines, CXCL9, CXCL10 and/or CXCL11, using a neurotropic AAV8 vector. Aim 2. To determine whether increasing the number and function of antiviral tissue-resident CD4+ and CD8+ T cells within: (1) DRG (central neuronal immunity); and (2) VMC (peripheral epithelial immunity) correlates with protection against genital herpes. The goal of this pre-clinical study is to bring a prime/pull vaccine to clinic.