Development of a Pan-HIV Proteomic Chip

描述（由申请人提供）：艾滋病毒-1是当今世界上单一传染性病原体造成死亡人数最多的病毒。尽管在治疗HIV-1感染及其引起的艾滋病方面取得了相当大的进展，但迫切需要更好的预防和治疗HIV-1感染的方法。因此，研制一种安全有效的疫苗以预防艾滋病毒-1感染或随后发展为艾滋病是极为重要的。然而，由于缺乏对保护性免疫相关关系的了解，以及缺乏测量有效的抗hiv -1免疫反应所需的工具，这一努力受到了阻碍。我们开发了一种Multi-Clade HIV-1蛋白质组学芯片（MC-HIV-1芯片），可以作为快速筛选体内感染或疫苗接种引起的HIV-1抗体反应的工具。目前的MC-HIV-1芯片可以从抗原发现公司（ADI）买到，它表达了100多种HIV-1蛋白、蛋白片段和表位，这些蛋白片段和表位来自A1、A2、B、C和D进化支，它们共占全球HIV-1感染的74% （Stephens 2012）。我们的初步研究表明，MC-HIV-1芯片可用于鉴定HIV-1抗体的特异性，跟踪感染期间对HIV-1的体液免疫反应的变化，也可能能够鉴定HIV-1感染的进化支。在本申请中，我们提出通过扩大其对HIV-1亚型、循环重组形式（CRF）、二硫结合抗原、糖基化抗原、表位和HIV-2组A和B的覆盖范围来改进MC-HIV-1芯片，从而创建泛hiv蛋白质组学芯片。我们进一步提出用多种血清和抗体对MC-HIV芯片和Pan HIV芯片进行验证。我们假设改进的泛HIV芯片将允许识别HIV感染的类型、组和亚型，将促进对几乎所有HIV感染和疫苗接种方案的体液免疫反应的快速表征，将识别与一些广泛中和表位的反应性，并将区分自然免疫和疫苗诱导的体液免疫。我们的目标是：1 -我们将MC-HIV-1芯片扩展到包括HIV-1亚型G， CRF01 AE和CRF01 AG以及HIV-2组A和B，从而创建一个泛hiv芯片。这将使全球艾滋病毒1型感染和几乎所有艾滋病毒2型感染的覆盖范围扩大到90%。2 -我们将改进Pan-HIV芯片，在外部蛋白gp120和gp41及其片段（包括gp120的可变环）中加入二硫桥和糖基。我们还将包括区分进化支的关键表位的共识序列，以及那些广泛中和（BN）抗体的目标。这将提高与BN和进化特异性抗体以及识别构象和糖基表位的抗体的反应性。我们将检测当前MC-HIV-1芯片以及改进的Pan-HIV芯片对进展者、非进展者、感染早期和晚期、感染不同类型、群体和进化的HIV患者以及接种疫苗后的患者血清的反应能力。我们还将用当前和改进的HIV芯片检测所有类别的广泛中和单克隆抗体的反应性。这一目标将展示当前MC-HIV-1芯片和改进的Pan-HIV芯片在疫苗研究和表征HIV感染的体液免疫反应中的效用。未来的方向将包括筛选疫苗接种者血清和优化Pan HIV芯片，以区分疫苗接种的体液免疫反应和自然感染的反应。

DESCRIPTION (provided by applicant): HIV-1 causes the largest number of deaths from a single infectious agent in the world today. Despite considerable advances in treatment of HIV-1 infection and AIDS, which it causes, there is an urgent need for better methods of prevention and treatment of HIV-1 infection. The development of a safe and effective vaccine to prevent HIV-1 infection or the subsequent development of AIDS is therefore of the utmost importance. This effort, however, has been hampered by a lack of understanding of the correlates of protective immunity and a lack of the tools needed to measure effective anti-HIV-1 immune responses. We have developed a Multi-Clade HIV-1 Proteomic Chip (MC-HIV-1 chip) that can be used as a tool to rapidly screen antibody responses to HIV-1 elicited in vivo in response to infection or vaccination. The current version of the MC-HIV-1 chip which is available commercially from Antigen Discovery Incorporated (ADI) expresses over 100 HIV-1 proteins, protein fragments and epitopes from clades A1, A2, B, C and D which together comprise 74% of HIV-1 infections worldwide (Stephens 2012). Our preliminary studies show that the MC-HIV-1 chip can be used to identify the specificity of HIV-1 antibodies, track changes in the humoral immune response to HIV-1 during infection and may also be able to identify the clade of an HIV-1 infection. In this application we propose to improve the MC-HIV-1 chip by expanding it's coverage of HIV-1 subtypes, circulating recombinant forms (CRF), disulfide bound antigens, glycosylated antigens, epitopes and HIV-2 groups A and B thereby creating a Pan-HIV Proteomic Chip. We further propose to validate the MC-HIV chip and Pan HIV chip with a variety of sera and antibodies. We hypothesize that the improved Pan-HIV Chip will allow identification of the type, group and subtype of an HIV infection, will facilitate rapid characterization of humoral immune response to nearly all HIV infections and vaccination regimens, will identify reactivity with some broadly neutralizing epitopes and will differentiate natural from vaccine-induced humoral immunity. We have the following aims: 1 - We will expand the MC-HIV-1 chip to include HIV-1 subtype G, CRF01 AE and CRF01 AG as well as HIV-2 groups A and B, thereby creating a Pan-HIV chip. This will expand coverage to > 90% of HIV-1 infections and virtually all HIV-2 infections globally. 2 - We will improve the Pan-HIV chip by including disulfide bridges and glycosyl groups in the external proteins, gp120 and gp41, and in their fragments, including the variable loops of gp120. We will also include consensus sequences of key epitopes that differentiate clades as well as those that are the targets of braodly neutralizing (BN) antibodies. This will improve reactivity with BN and clade specific antibodies as well as antibodies that recognize conformational and glycosyl epitopes.3 - We will assay the ability of the current MC-HIV-1 chip as well as the improved Pan-HIV chip to react with patient sera from progressors, non-progressors, from early vs. late in infection, from patients infected with different types, groups and clades of HIV and following vaccination. We will also assay reactivity of all classes of broadly neutralizing monoclonal antibodies with the current and improved HIV chips. This aim will demonstrate the utility of the current MC-HIV-1 chip and improved Pan-HIV chip in vaccine research and in characterizing the humoral immune response to HIV infection. Future directions will include screening vaccinee sera and optimizing the Pan HIV chip for differentiating humoral immune responses to vaccinination from responses to natural infection.