Development and In Vivo Characterization of Safety-Enhanced RhCMV/SIV Vectors

描述(申请人提供)：我们已经证明CMV/SIV载体可以1)再感染CMV+恒河猴(RM)，2)在再感染期间，以强烈的“效应记忆”(TEM)偏向诱导强大而持久的SIV特异性CD4+和CD8+T细胞反应，以及3)在限制剂量的直肠攻击高致病性SIVmac239病毒后，完全保护约50%的已接种的RM免受进行性感染。这些RM表现出的保护作用在突发性和程度上与以前的疫苗不同，受保护的RM在最初感染时在血浆中表现出不同大小的病毒爆发，然后立即控制到无法检测到的水平。尽管在受保护的RM中偶尔可以观察到血浆中的病毒斑点，但这些病毒斑点随着时间的推移而下降，一年后，保护不受CD8+或CD4+细胞耗尽的影响，用超灵敏的巢式PCR进行广泛的组织分析表明，只有罕见的单拷贝SIV核酸检测到，没有存活的SIV。保护与疫苗阶段产生的总的SIV特异性CD8+TEM值相关，并且发生时没有记忆反应。这些数据表明了一种新的保护模式，与非常早期的控制一致，可能发生在病毒进入部位和/或病毒复制和扩增的早期部位，并涉及组织驻留的CD8+透射电子显微镜。因此，CMV载体和“TEM型”疫苗概念为HIV/AIDS疫苗的开发提供了一种强有力的新途径。然而，由于CMV的致病潜力，完全复制的CMV载体不太可能推广到人类使用。因此，一个中心目标是开发保持免疫原性和有效性的CMV载体，但在人类身上使用是安全的。幸运的是，初步数据表明，复制/传播缺陷的CMV具有很强的免疫原性。在这里，我们将探索复制/传播缺陷的CMV载体在多大程度上可以在致病性和传播性方面减弱，同时保持免疫原性和有效性。1)设计、构建和体外鉴定复制/传播缺陷CMV/SIV载体，2)确定复制/传播缺陷CMV/SIV载体在胚胎RM中的致病性，3)确定复制/传播缺陷CMV/SIV载体在幼年RM中的免疫原性和脱落，并对最优的复制/传播缺陷载体设计进行有效性试验，以确定其对限制剂量直肠SIVmac239攻击的保护能力。 与公共卫生的相关性：在全球范围内，2007年发生了约250万新的艾滋病毒感染(南部非洲一些地区的流行率超过15%)，人们普遍认为，有效的预防性疫苗是控制艾滋病毒/艾滋病流行的唯一实际手段。我们已经证明，巨细胞病毒(CMV)载体可以利用组织驻留效应记忆T细胞在感染早期就对抗艾滋病病毒，并可以保护恒河猴在黏膜攻击后免受进行性感染。本申请中提出的工作将提高CMV载体的安全性，并为这一新疫苗方法的进一步临床开发提供关键信息。

DESCRIPTION (provided by applicant): We have demonstrated that CMV/SIV vectors can 1) re-infect CMV+ rhesus macaques (RM), 2) during re-infection, elicit potent and persistent SIV-specific CD4+ and CD8+ T cell responses with a strong "effector memory" (TEM) bias, and 3) completely protect ~50% of vaccinated RM from progressive infection after limiting dose rectal challenge with the highly pathogenic SIVmac239 virus. The protection manifested in these RM is distinct from previous vaccines in its abruptness and extent, with protected RM exhibiting a viral burst in plasma of varying size upon initial infection, followed by immediate control to undetectable levels. Although occasional viral blips in plasma are observed in protected RM, these decline with time, and after 1 year, protection is unaffected by CD8+ or CD4+ cell depletion, and extensive tissue analysis with ultrasensitive nested PCR has shown only rare detection of ~ single copy SIV nucleic acid and no viable SIV. Protection correlates with the total SIV-specific CD8+ TEM generated during the vaccine phase, and occurs without an anamnestic response. These data indicate a novel pattern of protection consistent with very early control, likely taking place at the site of viral entry and/or early sites of viral replication and amplification, and involving tissue-resident CD8+ TEM. Thus, CMV vectors and the "TEM" vaccine concept offer a powerful new approach to HIV/AIDS vaccine development. However, fully replicative CMV vectors are unlikely to be advanced to human use due to the pathogenic potential of CMV. A central goal is therefore to develop CMV vectors that maintain immunogenicity and efficacy, but are safe to use in humans. Fortunately, preliminary data indicates that replication/spread-deficient CMVs can be robustly immunogenic. Here, we will explore the extent to which replication/spread-deficient CMV vectors can be attenuated with respect to pathogenicity and transmissibility, while retaining immunogenicity and efficacy. Three specific aims are proposed: 1) to design, construct and in vitro characterize replication/spread-deficient CMV/SIV vectors, 2) to determine the pathogenicity of replication/spread-deficient CMV/SIV vectors in fetal RM, and 3) to determine the immunogenicity and shedding of replication/spread-deficient CMV/SIV vectors in juvenile RM, and to take the optimal replication/spread deficient vector design to an efficacy trial to determine its ability to protect against limiting dose rectal SIVmac239 challenge. PUBLIC HEALTH RELEVANCE: Worldwide, ~2.5 million new HIV infections occurred in 2007 (with prevalence rates in some areas of southern Africa exceeding 15%), and it is generally agreed that an effective prophylactic vaccine is the only practical means by which the HIV/AIDS epidemic can be controlled. We have demonstrated that cytomegalovirus (CMV) vectors can harness tissue-resident effector memory T cells to combat the AIDS virus very early in infection, and can protect rhesus monkeys from progressive infection after mucosal challenge. The work proposed in this application will enhance the safety of CMV vectors, and provide crucial information for the further clinical development of this novel vaccine approach.