Creation of Stable Cleaved Trimers of the HIV-1 Envelope Protein

描述（由申请人提供）：HIV-1流行病在2004年导致约500万新感染，共有4000万艾滋病毒/艾滋病感染者。临床试验表明，通过免疫天然存在的病毒包膜（Env）蛋白亚基无法预防HIV-1感染。然而，从实验研究中可以清楚地看出，Env蛋白的中和抗体可以保护灵长类动物免受感染。现在需要更好地暴露中和表位的修饰的Env蛋白来加速疫苗开发。对HIV-1和SIV Env糖蛋白的详细结构研究表明，该分子在与CD 4结合时发生了实质性变化，这是病毒-细胞相互作用的初始步骤。HIV疫苗研究的一个主要目标是创建基于三聚体Env分子的无配体形式的稳定结构。目前正在研究类似于病毒体表面上成熟包膜三聚体的稳定形式并且更好地暴露现有中和表位的分子。然而，由循环HIV-1携带的Env结构显然不会在感染个体中诱导广泛中和抗体。因此，无论使用三聚体Env复合物作为免疫原用于疫苗接种的益处如何，Env蛋白仍然必须被修饰以改善保守的中和表位的免疫原性。我们建议使用定向分子进化来实现两个主要目标。在这个I期SBIR提案中，我们将首先使用体外多基因DNA重组来创建新型嵌合HIV env基因的文库，这些基因将被筛选以鉴定许多稳定的、切割的、三聚体形式的HIV-1 Env胞外域。其次，在达到本工作的目标后，我们将在随后的II期SBIR提案中评估这些蛋白质的免疫原性。这项工作将对三聚体Env分子作为免疫原的价值进行严格评估，并将为进一步开发候选疫苗提供先导分子。我们最终的目标是研制一种对预防艾滋病毒/艾滋病有价值的商业疫苗产品。第一阶段SBIR提案的具体目标总结如下。它们旨在评估产生稳定和可溶形式的HIV-1 Env三聚体的可行性。- 具体目标#1：产生编码gp 140胞外域的重组env基因的文库; -特异性目标#2：鉴定和表征至少10种稳定形式的完全裂解的Env三聚体; -特异性目标#3：从每种稳定的裂解的Env三聚体制备单体gp 120。

DESCRIPTION (provided by applicant): The HIV-1 epidemic has resulted in ~5 million new infections in 2004 for a total of 40 million people living with HIV/AIDS. Clinical trials have shown that HIV-1 infection cannot be prevented by immunization with subunits of naturally occurring viral envelope (Env) proteins. However, it is clear from experimental studies that neutralizing antibodies to the Env protein can protect primates from infection. Modified Env proteins that better expose the neutralizing epitopes are now required to accelerate vaccine development. Detailed structural studies of the HIV-1 and SIV Env glycoproteins have revealed that this molecule undergoes substantial changes when it is bound by CD4, which is the initial step of virus-cell interaction. A major objective of HIV vaccine research is to create stable structures that are based on the unliganded form of the trimeric Env molecule. Molecules that resemble a stabilized version of the mature envelope trimer on the virion surface and that better expose existing neutralizing epitopes are currently being investigated. However, the Env structure carried by circulating HIV-1 clearly does not induce broadly neutralizing antibodies in infected individuals. Therefore, whatever the benefits of using a trimeric Env complex as an immunogen for vaccination, the Env protein must still be modified to improve the immunogenicity of conserved neutralizing epitopes. We propose to use directed molecular evolution to accomplish 2 main objectives. In this Phase I SBIR proposal, we will first use in vitro multigene DNA recombination to create libraries of novel chimeric HIV env genes that will be screened to identify a number of stable, cleaved, trimeric forms of the HIV-1 Env ectodomain. Second, upon reaching the goals of the present work, we will evaluate the immunogenicity of these proteins in a subsequent Phase II SBIR proposal. This work will provide a rigorous assessment of the value of trimeric Env molecules as immunogens and will provide lead molecules for further development as vaccine candidates. We are ultimately targeting a commercial vaccine product that will be of value in the prevention of HIV/AIDS. The Specific Aims of this Phase I SBIR proposal are summarized below. They are designed to evaluate the feasibility of creating stable and soluble forms of the HIV-1 Env trimer. -Specific Aim #1: Create libraries of recombined env genes encoding the gp140 ectodomain; -Specific Aim #2: Identify and characterize at least 10 stable forms of fully cleaved Env trimers; -Specific Aim #3: Prepare monomeric gp120 from each of the stable, cleaved Env trimers.