Anti-HIV Microbicidal Peptides

说明(申请人提供)：局部杀微生物剂被定义为阴道使用的产品，可防止艾滋病毒在男性与女性之间或女性与男性之间传播。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。目前对所有类别的HIV抑制剂，如在O群病毒中发现的非核苷类RT和蛋白酶(PR)抑制剂的泛耐药性，提出了一个重要的问题，即针对RT或PR的杀菌剂的开发。这些担忧可能会扩展到艾滋病毒进入的抑制剂，因为在选择性压力下，包膜(Env)突变迅速出现。因此，迫切需要确定新的抗艾滋病毒化合物，这些化合物针对PR、RT和Env以外的病毒成分，并可能被开发为安全有效的杀微生物剂。我们确定了一种短线性多肽，它在NM范围内中和HIV。这种多肽以不依赖于环境的方式阻止艾滋病毒进入细胞。有几条证据表明，这种多肽破坏了HIV膜的稳定，释放了Env糖蛋白。多肽介导的HIV膜不稳定是特异的，因为i)该肽不干扰其他被包裹的病毒(即HSV和VSV)的膜；ii)即使在每天两次给细胞施药时，它也不会使人宫颈细胞膜不稳定，其浓度是阻断HIV感染的浓度的20-200倍；iii)该肽不会伤害静脉注射的小鼠。每天两次，使用高浓度的多肽(0.5毫克)，持续三天；以及iv)多肽显然不会对人源化小鼠的阴道上皮造成损害，因为多肽不是促进传播，而是完全阻断阴道内传播。多肽代表了一个有吸引力的杀菌剂候选方案，原因有几个：i)多肽抑制各种主要人类细胞类型的初级分离株的感染；ii)它干扰涉及艾滋病毒传播的三种机制：生殖器上皮迁移、树突状细胞介导的传播和粘膜靶细胞的感染；iii)多肽非常有效，因为接触不到15分钟就足以中和HIV；Iv)在将艾滋病毒加入细胞之前和之后的两个小时内有效，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有效；v)该肽在低pH值下保持其抗病毒特性；vi)它对小鼠无毒或免疫原性，即使在重复静脉注射后也是如此。7)最重要的是，该多肽能完全阻断HIV在人源化小鼠体内的阴道传播。在这项应用中，我们建议充分表征多肽及其衍生物在体外的抗HIV效果以及它们在体内(R21期)的毒性和免疫原性。如果这些研究明确地证明这些肽代表了有吸引力的抗HIV杀微生物剂候选者，那么它们将作为体内局部杀微生物剂进行测试(R33阶段)。该项目的目标是进行体外和体内研究，旨在支持短肽作为新型抗HIV杀菌剂的临床前开发。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使用承担个人责任。开发一种安全、有效、可接受的局部杀微生物剂，以防止艾滋病毒-1的性传播，可在全世界减少每天14,000多例新的艾滋病毒-1感染方面发挥重要作用，并有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.