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

项目摘要/摘要 生殖器单纯疱疹病毒2型(HSV-2)感染在美国影响着6000多万人，超过536人 全世界有一百万人。FDA批准的生殖器疱疹疫苗目前还不可用。在原发感染后 在阴道粘膜皮肤组织(VMC)中，病毒传播并在感觉神经元中建立潜伏期 区域背根节(DRG)。病毒从潜伏期开始零星地重新激活，并在生殖器中脱落。 肠道，在那里它可能导致严重的复发性损害。我们的长期目标是开发一种治疗性疫苗 预防复发性生殖器疱疹。在过去的5年里，我们在确定 HSV-2候选抗原和抗病毒CD4和CD8 T细胞的表型和功能特征 在血清阳性妇女和豚鼠复发性生殖器疱疹模型中具有保护作用：(A)我们 发现两个HSV-2被膜病毒蛋白(VP16和VP22)和两个核苷酸还原酶亚基 蛋白质(RR1和RR2)主要针对的是CD4和CD8 T细胞，使其不会受到自然的无症状保护 妇女(即使感染了，但从未发展为复发性生殖器疱疹的人)；(B)VP16、VP22、 RR1和RR2蛋白是组织驻留的CD4和CD8T细胞识别的主要HSV-2抗原 驻留在受保护的无症状豚鼠的背根节和VMC中；(C)表型和转录型RNA- 在受保护的豚鼠中，DRG和治愈的VMC驻留的CD4和CD8T细胞的SEQ图谱表明，它们 具有功能组织驻留的CXCR3 CD4和CXCR3 CD8 T细胞的所有特征；(D)治疗 RR2抗原免疫豚鼠对HSV-2感染的豚鼠有较强的保护作用，VP16、VP22和 RR1抗原提供了适度的保护；以及(E)用嗜神经药物治疗HSV-2感染的豚鼠 表达豚鼠CXCL11趋化因子(CXCR3配体)的腺相关病毒载体(AAV8)增强 感染的DRG和VMC中特异性的CD4和CD8T细胞的数量和增强的保护作用。基于 这些公布的和初步的结果，我们假设，促进强大和持久的抗病毒组织- DRG和VMC中驻留的CD4和CD8T细胞局部反应将产生更强健/持续的 防止HSV-2重新激活和脱落，减少复发性生殖器疱疹。为了检验这一假设， 我们提出了两个具体的目标：目的1.评价其在豚鼠生殖器中的安全性和保护效果 疱疹模型，一种创新的初级/拉动式治疗性疫苗方法，包括：(1)用 VP16、VP22、RR1和RR2抗原；以及(2)通过一种方法将准备好的T细胞“拉”入受感染的DRG和VMC组织。 使用嗜神经性AAV8局部传递吸引趋化因子CXCL9、CXCL10和/或CXCL11的T细胞 向量。目的2.确定增加抗病毒组织常驻细胞的数量和功能是否 CD8T细胞：(1)中枢神经免疫(DRG)；(2)外周上皮免疫(VMC) 对生殖器疱疹有保护作用。这项临床前研究的目标是将一种优质/拉动式疫苗推向临床。

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.