Pathogenic determinants of the SIV envelope transmembrane cytoplasmic domain

描述(由申请人提供)：HIV-1建立了一种持续的感染，其特征是在面对强烈的宿主免疫反应、CD4细胞耗尽以及最终的艾滋病的情况下进行病毒复制。对于艾滋病的SIV模型，减毒SIV为理解发病机制提供了强大的工具，因为它们是可以由宿主控制的病毒。这些模型也提供了最好的证据，证明宿主免疫反应可以保护动物在受到致病菌株攻击时免受感染和/或疾病。然而，即使是特性最好的减毒病毒，其减毒机制(S)和这种保护的免疫相关性也是未知的。此外，当实现保护时，通常是针对同源病毒而不是异源病毒，这种情况与有效疫苗所需的情况相去甚远。我们的实验室已经证明，SIVmac239Env细胞质尾部保守的GYxxX运输基序的突变产生了一种严重减弱的表型，使这种高致病性病毒容易受到宿主免疫控制。我们还表明，感染的动物可以通过同源SIVmac239挑战保护免受感染，并且值得注意的是，能够控制异源挑战病毒(SIVmac E660)。我们建议在这个模型中扩展我们的初步结果，在体外表征GYxxX突变对病毒的影响，并在猕猴体内进行严格的研究，以确定降低致病性和宿主免疫控制的相关性。提出了三个目标。目的#1将评估TM尾部突变对病毒粒子结构、组成、传染性和适合性的影响，并将探索我们在初步结果中注意到的这些病毒中和敏感性增加的基础。目的#2将综合评估粘膜和其他淋巴组织部位感染的早期和晚期事件，以确定哪些细胞被感染，发生哪些细胞病变，引发哪些炎症反应，以及在该模型中观察到的衰减是否影响GYxxX突变体的粘膜传播能力。目的#3将评估GYxxX突变体的细胞和体液免疫反应，以确定与SIVmac239在广度、特异性和大小方面存在哪些差异。将对致病性异源病毒攻击前后的反应进行评估，以确定保护的免疫相关因素。这些目标还将利用已发展出明显代偿性突变的病毒，因为它们将成为未来研究的强大工具，以解决奠定这种衰减模型和宿主免疫控制基础的基本细胞机制。总之，我们的工作表明，环境运输信号中合理设计的突变可以被利用来破坏病毒/宿主的平衡和感染的结果。这个模型对于发病机制和疫苗研究都应该是一个信息量很大的模型。 开发有效的艾滋病毒疫苗的三个主要障碍是：1)缺乏可重复显示对遗传多样性挑战病毒的保护的动物模型；2)对免疫反应的哪些组成部分需要预防或控制艾滋病毒感染缺乏了解；3)如何开发能够引发这些免疫反应的疫苗。我们的应用描述了一种新的活体减毒SIV模型，在该模型中，病毒包膜基因的合理设计突变创建了一种可完全由宿主免疫系统控制的病毒，同时赋予了预防或控制不同于疫苗株的致病病毒感染的能力。该提案描述了利用最先进的技术评估这一新模式的病理学、免疫学和病毒学方面的计划，并解决了对艾滋病毒疫苗科学至关重要的问题。

DESCRIPTION (provided by applicant): HIV-1 establishes a persistent infection characterized by ongoing viral replication in the face of vigorous host immune responses, CD4 cell depletion, and ultimately AIDS. For SIV models of AIDS, attenuated SIVs have provided powerful tools to understand pathogenesis, as they are viruses that can be controlled by the host. These models have also provided the best evidence that host immune responses can protect animals from infection and/or disease when challenged with pathogenic isolates. However, for even the best characterized attenuated viruses, the mechanism(s) that underlie their attenuation and the immune correlates of this protection are unknown. Moreover, when protection is achieved, it is typically for homologous rather than heterologous viruses, a scenario far removed from what will be required for an effective vaccine. Our laboratory has shown that mutations in a conserved GYxxX trafficking motif in the SIVmac239 Env cytoplasmic tail produce a profoundly attenuated phenotype that renders this highly pathogenic virus susceptible to host immune control. We have also shown that infected animals are protected from infection with a homologous SIVmac239 challenge, and remarkably, are able to control a heterologous challenge virus (SIVmac E660). We propose to extend our preliminary results in this model to characterize the effects of GYxxX mutations on the virus in vitro and, in rigorous in vivo studies in rhesus macaques, to identify the correlates of reduced pathogenicity and host immune control. Three aims are proposed. Aim #1 will evaluate the effects of TM tail mutations on virion structure, composition, infectivity, and fitness, and will explore the basis for the increased neutralization sensitivity of these viruses that we noted in our preliminary results. Aim #2 will comprehensively evaluate early and late events of infection in mucosal and other lymphoid sites to determine what cells are infected, what cytopathic effects occur, what inflammatory responses are elicited, and whether the attenuation observed in this model affects the ability of GYxxX mutants to be transmitted mucosally. Aim #3 will evaluate cellular and humoral immune responses to GYxxX mutants to determine what differences exist compared to SIVmac239 in breadth, specificity and magnitude. Responses will be assessed before and after challenge with a pathogenic heterologous virus to determine what are the immune correlates of protection. These aims will also take advantage of viruses that have developed apparent compensatory mutations, as they will be powerful tools for future studies to address basic cellular mechanisms that underlie this model of attenuation and host immune control. Collectively, our work shows that rationally designed mutations in Env trafficking signals can be exploited to disrupt the viral/host balance and the outcome of infection. This model should be a highly informative for both pathogenesis and vaccine research.Project Narrative Three major obstacles in the development of an effective HIV vaccine have been 1) the lack of animal models that reproducibly show protection from genetically diverse challenge viruses; 2) a poor understanding of what components of the immune response are needed to prevent or control HIV infection; and 3) how a vaccine can be developed that will elicit these immune responses. Our application describes a new model of a live attenuated SIV in which a rationally designed mutation in the viral envelope gene creates a virus that can be completely controlled by the host immune system while conferring the ability to prevent or control infection by pathogenic viruses that are genetically different from the vaccine strain. The proposal describes plans to evaluate the pathological, immunological and virological aspects of this new model with state of the art technologies and addresses issues that are central to HIV vaccine science.