Development of gene transfer approach for prevention of vaginal HIV transmission

描述（由申请人提供）：超过一半的新HIV-1感染是由女性通过阴道内暴露获得的。宫颈阴道上皮细胞排列在女性下生殖道的粘膜表面，提供了对HIV-1感染的初步防御，但保护是不完整的。HIV-1通过这一屏障的运输对HIV-1定植和病毒传播至关重要。长期目标是通过局部表达抗hiv -1中和抗体来阻断上皮细胞附着和病毒进入，从而增强粘膜表面的抗hiv -1体液免疫。其他基于AAV的基因疗法的安全性、低免疫原性和进入人体临床试验的快速进展使其成为预防HIV-1的可行方法。在我的研究期间，我将研究稳定的腺相关病毒（AAV）中和人抗体基因转移到宫颈内膜、宫颈外和阴道上皮细胞是否能提供持久的抗HIV-1保护。具体来说，我将1)确定AAV血清型（1-9），提供最佳的基因转移到这些细胞没有毒性；2)从270亿个噬菌体展示文库中分离出新的中和性人抗syndecan 1和抗syndecan 2抗体，并测试其阻断HIV-1粘附人原代宫颈阴道上皮细胞的能力；构建病毒抑制肽(VIRIP)-Fc融合蛋白，并通过阻断病毒融合检测其抑制感染的能力。抗syndecan抗体和VIRIP-Fc蛋白将在体外测试病毒内化和跨宫颈阴道上皮细胞的胞吞作用以及胞吞病毒颗粒的感染性。3)通过持久的阴道内分泌人抗syndecan抗体和vilip - fc融合蛋白的中和滴度，确定AAV介导的基因转移能否在小鼠的下生殖道实现稳定。最后，在人类造血干细胞植入小鼠模型中，阴道内AAV-anti-syndecan和AAV-VIRIP-Fc基因转移研究以及阴道内HIV-1攻击将为这一新假设提供实验验证。这种新方法的成功可以推进到非人类灵长类动物研究，并支持预防性疫苗研究，有可能推进艾滋病预防性疫苗战略。

DESCRIPTION (provided by applicant): More than half of new HIV-1 infections are acquired by women through intravaginal exposure. The cervico-vaginal epithelial cells lining the mucosal surfaces of the female lower genital track provide an initial defense against HIV-1 infection, but protection is incomplete. Transport of HIV-1 across this barrier is critical for HIV-1 colonization and virus dissemination. The long-term goal is to enhance anti-HIV-1 humoral immunity at the mucosal surface by local expression of anti-HIV-1 neutralizing antibodies to block epithelial cell attachment and virus entry. The safety profile, low immunogenecity and rapid advancement of other AAV based gene therapies into human clinical trials makes it a feasible approach to HIV-1 prevention. During my fellowship, I will investigate whether stable adeno-associated virus (AAV)-neutralizing human antibody gene transfer to endocervical, ectocervical and vaginal epithelial cells can provide durable protection against HIV-1. Specifically, I will 1) Determine the AAV serotype (1-9) that provides optimal gene transfer to these cells without toxicity; 2) Isolate new neutralizing human anti-syndecan 1 and anti-syndecan 2 antibodies from a 27 billion member phage display library and test their abilities to block attachment of HIV-1 to human primary cervico-vaginal epithelial cells; and construct a Virus Inhibitory Peptide (VIRIP)-Fc fusion protein and test its ability to inhibit infection by blocking viral fusion. The anti-syndecan antibodies and VIRIP-Fc proteins will be tested for inhibition of virus internalization and transcytosis across the cervico- vaginal epithelial cells and infectivity of the transcytosed virus particles in vitro. 3) Determine if stable AAV- mediated gene transfer can be achieved in the lower genital track of mice with durable intravaginal secretion of neutralizing titers of human anti-syndecan antibodies and VIRIP-Fc fusion proteins. Finally, intravaginal AAV-anti-syndecan and AAV-VIRIP-Fc gene transfer studies followed by intravaginal HIV-1 challenge in a human hematopoietic stem cell-engrafted mice model will provide an experimental test of this novel hypothesis. The success of this novel approach could be advanced to non-human primate studies, and support prophylactic vaccine research with the potential to advance AIDS prophylactic vaccine strategies. PULBIC HEALTH RELEVANCE: HIV-1 infections are acquired most often through sexual contact and more than half of new infections are acquired by women through intravaginal HIV exposure. I propose to develop a genetic vaccine that when delivered to the mucosal surface of the cervix and vagina to allow the lining cells to stably produce human antibodies that block HIV-1 attachment and infection. A protective genetic vaccine delivered to the female lower genital track could dramatically slow the spread of HIV/AIDS.