Single cycle reporter assay for quantifying HIV-1 Nab

描述(申请人提供)：免疫反应的体液臂被认为是各种病毒疫苗引起的主要保护机制。诱导有效的中和抗体反应是HIV疫苗开发的主要目标之一。然而，很难确定中和抗体在遏制艾滋病毒初次感染、疾病自然病史或疫苗接种方面可能起到什么作用。我们认为，这些困难是由多种环境造成的，有些是生物的，有些是技术的。为了克服技术限制，我们开发了一种新的HIV-1进入试验来测定中和抗体(NAB)。该检测基于对HIV-1初级分离株敏感的报告细胞系(JC53-BL)，直接分析HIV进入步骤，并准确地量化单个感染周期中的进入。此外，该检测方法相对简单、可靠。最终，商业可行性将取决于这种进入试验产生的结果是否具有生物学、免疫学和临床相关性。也就是说，NAB体外对HIV-1感染的抑制作用，对JC53-BL细胞的抑制作用，是否与体内对相关靶细胞的抑制作用相似。我们感到鼓舞的是，使用这项检测的结果表明：(A)接受融合抑制剂(T-20)单一治疗的患者中出现了耐药的HIV-1，以及(B)在体内，NAB施加了足够的病毒抑制压力，导致中和敏感病毒被耐药病毒取代。我们的假设是-我们的进入试验在商业上是可行的，并且具有临床意义，因为它复制了大多数HIV-1毒株在自然靶细胞上的重要进入机制。为了解决这一假设，我们提出：(1)分析进入试验的报告细胞系对不同HIV-1毒株感染的敏感性；(2)确定基于JC53-BL的进入试验检测异源NAB的敏感性。

DESCRIPTION (provided by applicant): The humoral arm of the immune response is considered to be the major protective mechanism elicited by a variety of viral vaccines. The induction of a potent neutralizing antibody response is one of the principal goals in HIV vaccine development. However, it has been remarkably difficult to ascertain what affect neutralizing antibodies may play in the containment of HIV primary infection, the natural history of disease or vaccination. We suggest that these difficulties stem from a combination of circumstances, some biologic and some technical. To overcome the technical limitations, we have developed a novel HIV-1 entry assay for measuring neutralizing antibodies (Nab). This assay is based on a reporter cell line (JC53-BL) that is sensitive to primary HIV-1 isolates, directly analyzes HIV entry steps and accurately quantifies entry in a single cycle of infection. Moreover, the assay is relatively simple and robust. Ultimately, the commercial viability will depend on whether the results produced from this entry assay have biologic, immunologic and clinical relevance. That is - whether the inhibition of HIV-1 infection by Nab in vitro, on JC53-BL cells, mimics inhibition on relevant target cells in vivo. We are encouraged by results obtained using this assay that have show (a) the emergence of resistant HIV-1 in patients receiving fusion inhibitor (T-20) monotherapy, and (b) Nab exert sufficient virus inhibitory pressure, in vivo, to result in the replacement of neutralization sensitive virus by resistant virus. Our hypothesis is - our entry assay is commercially viable and clinically relevant because it replicates important entry mechanisms of most HIV-1 strains on natural target cells. To address this hypothesis we propose: (1) To analyze the susceptibility of the entry assay's reporter cell line to infection by diverse strains of HIV-1 and (2) To determine the sensitivity of the JC53-BL-based entry assay for detecting heterologous Nab.