Development of gene transfer approach for prevention of vaginal HIV transmission

描述(申请人提供)：超过一半的新的HIV-1感染是由妇女通过阴道接触感染的。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。艾滋病毒-1跨越这一障碍的运输对艾滋病毒-1的定居和病毒传播至关重要。其长期目标是通过局部表达抗HIV-1中和抗体来阻止上皮细胞附着和病毒侵入，从而增强粘膜表面的抗HIV-1体液免疫。其他以AAV为基础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。在我担任研究员期间，我将研究将稳定的腺相关病毒(AAV)中和人类抗体基因转移到宫颈内、宫颈外和阴道上皮细胞是否可以提供针对HIV-1的持久保护。具体地说，我将1)确定为这些细胞提供最佳基因转移的AAV血清型(1-9)；2)从270亿人的噬菌体展示文库中分离新的中和人抗Syndecan 1和抗Syndecan 2抗体，并测试它们阻断HIV-1与原代人宫颈阴道上皮细胞附着的能力；以及构建病毒抑制肽(VIRIP)-Fc融合蛋白，并测试其通过阻断病毒融合来抑制感染的能力。抗Syndecan抗体和VIRIP-Fc蛋白将在体外测试对病毒内化和跨宫颈阴道上皮细胞的跨细胞作用的抑制，以及跨细胞病毒颗粒的感染性。3)确定经阴道持续分泌人抗Syndecan抗体和VIRIP-Fc融合蛋白的小鼠下生殖道能否实现稳定的AAV介导的基因转移。最后，在人类造血干细胞移植小鼠模型中，阴道内AAV-抗Syndecan和AAV-VIRIP-Fc基因转移研究以及随后的阴道内HIV-1攻击研究将为这一新假说提供实验检验。这一新方法的成功可以推广到非人类灵长类动物的研究，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。 公共卫生相关性：艾滋病毒-1感染最常通过性接触获得，超过一半的新感染是由妇女通过阴道感染艾滋病毒获得的。我建议开发一种基因疫苗，当它被注射到宫颈和阴道的粘膜表面时，允许衬里细胞稳定地产生人类抗体，阻止HIV-1的附着和感染。向女性下生殖道提供保护性基因疫苗可以极大地减缓艾滋病毒/艾滋病的传播。

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.