Anti-HIV Microbicidal Peptides

描述（由申请人提供）：外用杀菌剂被定义为阴道使用的产品，防止男性对女性或女性对男性的艾滋病毒传播。HIV逆转录酶（RT）的高错误率推动了HIV耐药和基因组多样性的发展。目前对所有类型的HIV抑制剂（如O群病毒对非核苷RT和蛋白酶（PR）抑制剂的泛耐药）提出了一个重要的问题，即针对RT或PR的杀菌剂的开发。由于在选择压力下包膜（Env）中突变的快速出现，这些担忧可能扩展到HIV进入抑制剂。因此，迫切需要鉴定新的抗hiv化合物，这些化合物可以靶向PR、RT和Env以外的病毒成分，并可能开发为安全有效的杀微生物剂。我们发现了一种短的线性肽，它在nM范围内中和HIV。这种肽以不依赖于环境的方式阻止HIV进入细胞。一些证据表明，肽破坏了HIV膜的稳定性，释放了Env糖蛋白。肽介导的HIV膜失稳是特异性的，因为i)肽不会干扰其他包膜病毒（即HSV和VSV）的膜；ii)即使每天两次，浓度比阻断HIV感染的浓度高20-200倍，也不会破坏人宫颈细胞的膜；Iii)高肽浓度（0.5 mg）每日静脉注射两次对小鼠无伤害，持续三天；iv)该肽明显不会在人源化小鼠的阴道上皮中造成病变，因为该肽不是促进传播，而是完全阻断阴道内传播。由于以下几个原因，该肽代表了一个有吸引力的杀微生物剂候选物：i)该肽抑制各种原代人细胞类型中广泛的原代分离株的感染；ii)干扰HIV传播的三种机制：生殖器上皮转移、树突状细胞介导的传播和粘膜靶细胞的感染；iii)肽非常有效，因为接触不到15分钟就足以中和艾滋病毒；iv)在将HIV添加到细胞之前和之后的两个小时内，它都是有效的，这表明它在性交之前和之后都应该有一个可接受的时间长度的活性；v)肽在低pH下保持抗病毒特性；6)即使在多次静脉注射高剂量肽后，对小鼠也没有毒性或免疫原性；最重要的是，该肽完全阻断了HIV在人源化小鼠阴道内的传播。在这项应用中，我们建议在体外充分表征肽及其衍生物的抗hiv功效以及它们在体内的毒性和免疫原性（R21期）。如果这些研究明确地证明这些肽是有吸引力的抗hiv杀微生物剂候选物，那么它们将作为局部杀微生物剂在体内进行测试（R33期）。该项目的目标是进行体外和体内研究，旨在支持短肽作为新型抗hiv杀菌剂的临床前开发。目前迫切需要开发安全、有效的外用杀菌剂，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、可接受的局部杀菌剂来预防艾滋病毒-1的性传播，可以在全世界每天减少14,000多例新的艾滋病毒-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.