Anti-HIV Microbicidal Peptides

描述（由申请方提供）：局部杀微生物剂定义为阴道应用的产品，可预防男性对女性或女性对男性的HIV传播。HIV逆转录酶（RT）的高错误率推动了HIV耐药性和基因组多样性的发展。目前泛耐药性的所有类别的HIV抑制剂，如在O组病毒中发现的非核苷RT和蛋白酶（PR）抑制剂，提出了一个重要的问题，关于发展的杀微生物剂靶向RT或PR。这些问题可能会扩展到抑制剂的HIV进入由于快速出现的突变的信封（Env）在选择压力下。因此，迫切需要鉴定新的抗HIV化合物，其靶向除PR、RT和Env之外的病毒组分，并且其可以被开发为安全有效的杀微生物剂。我们鉴定了一种短的线性肽，其在nM范围内中和HIV。该肽以不依赖Env的方式阻断HIV进入细胞。一些证据表明，这种肽使HIV膜不稳定，释放出Env糖蛋白。肽介导的HIV膜去稳定化是特异性的，因为i）肽不干扰其他包膜病毒的膜（即，HSV和VSV）; ii）即使当以上级阻断HIV感染的浓度20-200倍的浓度每天两次应用于细胞时，它也不会使人宫颈细胞的膜不稳定;（0.5mg）持续三天;和iv）该肽显然不会在人源化小鼠的阴道上皮中产生损伤，因为该肽，而不是促进传递，完全阻断阴道内传播该肽代表了一种有吸引力的杀微生物剂候选物，原因如下：i）该肽抑制各种原代人细胞类型中的广泛的原代分离物的感染; ii）它干扰HIV传播中涉及的三种机制：生殖器上皮细胞迁移、树突细胞介导的传播和粘膜靶细胞的感染; iv）在向细胞中加入HIV之前和之后，它在两个小时内都是有效的，这表明它在性交之前和之后都应该在可接受的时间长度内是有活性的; v）所述肽在低pH下保持其抗病毒特性; vi）即使在重复静脉内给予高剂量的肽后，它在小鼠中也没有毒性或免疫原性;和vii）最重要的是，所述肽完全阻断HIV在人源化小鼠中的阴道内传播。在本申请中，我们建议充分表征肽和衍生物的体外抗HIV功效以及它们的体内毒性和免疫原性（R21阶段）。如果这些研究明确地证明这些肽代表有吸引力的抗HIV杀微生物剂候选物，则将它们作为局部杀微生物剂在体内进行测试（R33阶段）。该项目的目标是进行体外和体内研究，旨在支持临床前开发的短肽作为新型抗HIV杀微生物剂。迫切需要开发安全、有效的局部杀微生物剂，以防止艾滋病毒的性传播，并允许女性伴侣对使用和应用承担个人责任。开发一种安全、有效、可接受的局部杀微生物剂来预防HIV-1的性传播，可以在全世界每天减少14，000多例新的HIV-1感染方面发挥重要作用，并可能挽救数百万人的生命。

DESCRIPTION (provided by applicant): Topical microbicides are defined as vaginally applied products that prevent male-to-female or female-to-male HIV transmission. The high error rate of the HIV reverse transcriptase (RT) drives the development of resistance and genomic diversity in HIV. The current pan-resistance to all classes of HIV inhibitors such as that found in O group virus for non-nucleoside RT and protease (PR) inhibitors, raises an important question with regard to the development of microbicides targeting RT or PR. These concerns may be extended to inhibitors of HIV entry due to rapid emergence of mutations in the envelope (Env) under selective pressure. There is thus an urgent need to identify new anti-HIV compounds, which target viral components other than PR, RT and Env, and which may be developed as safe and effective microbicides. We identified a short linear peptide, which neutralizes HIV at an nM range. The peptide blocks HIV entry into cells in an Env-independent manner. Several lines of evidence suggest that the peptide destabilizes the HIV membrane, liberating the Env glycoprotein. The peptide-mediated HIV membrane destabilization is specific because i) the peptide does not disturb the membrane of other enveloped viruses (i.e., HSV and VSV); ii) it does not destabilize the membrane of human cervical cells even when applied twice daily to cells at a concentration 20-200-fold superior to that which blocks HIV infection; iii) the peptide does not harm mice injected i.v. twice daily with high peptide concentrations (0.5 mg) for a period of three days; and iv) the peptide apparently does not create lesions in the vaginal epithelium of humanized mice since the peptide, rather than promoting transmission, completely blocks intravaginal transmission The peptide represents an attractive microbicide candidate for several reasons: i) the peptide inhibits infection of a broad range of primary isolates in various primary human cell types; ii) it interferes with the three mechanisms involved in HIV transmission: genital epithelial transmigration, dendritic cell-mediated transmission, and infection of mucosal target cells; iii) the peptide is extremely efficacious since less than 15 minutes of exposure suffices to neutralize HIV; iv) it is potent for two hours both prior to and after addition of HIV to cells, suggesting that it should be active for an acceptable length of time both prior to and after sexual intercourse; v) the peptide retains its antiviral properties at a low pH; vi) it is not toxic or immunogenic in mice, even after repeated i.v. administrations of high peptide doses; and vii) most importantly, the peptide totally blocks the intravaginal transmission of HIV in humanized mice. In this application, we propose to fully characterize the anti-HIV efficacy of the peptide and derivatives in vitro as well as their toxicity and immunogenicity in vivo (R21 phase). If these studies unambiguously demonstrate that the peptides represent attractive anti-HIV microbicide candidates, they will then be tested as topical microbicides in vivo (R33 phase). The goal of this project is to conduct in vitro and in vivo studies designed to support the preclinical development of short peptides as novel anti-HIV microbicides. There is an urgent need for the development of safe, effective topical microbicides to prevent the sexual transmission of HIV and to allow the female partner to take personal responsibility for use and application. The development of a safe, effective acceptable topical microbicide to prevent the sexual transmission of HIV-1 could play a major role in worldwide reduction of the over 14,000 new HIV-1 infections per day, and potentially save millions of lives.