Pathogenic determinants of the SIV envelope transmembrane cytoplasmic domain

描述（由申请人提供）：HIV-1是一种持续性感染，其特征是在强烈的宿主免疫应答、CD 4细胞耗竭和最终AIDS的情况下持续进行病毒复制。对于艾滋病的SIV模型，减毒的SIV为理解发病机制提供了强有力的工具，因为它们是可以被宿主控制的病毒。这些模型还提供了最好的证据，表明宿主免疫反应可以保护动物免受感染和/或疾病时，与致病性分离株的挑战。然而，即使是最佳表征的减毒病毒，其减毒的机制和这种保护的免疫相关性也是未知的。此外，当获得保护时，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。我们的实验室已经表明，SIVmac 239 Env胞质尾中保守的GYxxX运输基序中的突变产生了深度减毒的表型，使这种高致病性病毒对宿主免疫控制敏感。我们还表明，受感染的动物被保护免受同源SIVmac 239攻击的感染，并且显著地，能够控制异源攻击病毒（SIVmac E660）。我们建议扩展我们在这个模型中的初步结果，以表征GYxxX突变对体外病毒的影响，并在恒河猴中进行严格的体内研究，以确定致病性降低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尾突变对病毒体结构、组成、感染性和适应性的影响，并将探索我们在初步结果中注意到的这些病毒的中和敏感性增加的基础。目的#2将全面评价粘膜和其他淋巴部位的早期和晚期感染事件，以确定感染的细胞、发生的细胞病变效应、引发的炎症反应以及在该模型中观察到的减毒是否影响GYxxX突变体经粘膜传播的能力。目的#3将评估对GYxxX突变体的细胞和体液免疫应答，以确定与SIVmac 239相比在广度、特异性和幅度上存在什么差异。将在用致病性异源病毒攻击之前和之后评估应答，以确定保护的免疫相关性。这些目标还将利用已经发展出明显的补偿突变的病毒，因为它们将是未来研究的有力工具，以解决这种减毒和宿主免疫控制模型的基础细胞机制。总的来说，我们的工作表明，可以利用Env运输信号中合理设计的突变来破坏病毒/宿主平衡和感染结果。这一模型对于致病机理和疫苗研究都具有很高的参考价值。 开发有效的HIV疫苗的三个主要障碍是：1）缺乏可重复地显示出对遗传多样性挑战病毒的保护的动物模型; 2）对预防或控制HIV感染所需的免疫应答的组分的理解不足;以及3）如何开发将引发这些免疫应答的疫苗。我们的申请描述了一种减毒活SIV的新模型，其中病毒包膜基因中的合理设计的突变产生了可以完全由宿主免疫系统控制的病毒，同时赋予预防或控制遗传上不同于疫苗株的致病性病毒感染的能力。该提案描述了用最先进的技术评估这种新模型的病理学、免疫学和病毒学方面的计划，并解决了艾滋病毒疫苗科学的核心问题。

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.