C5a as an Anti-HIV Microbicidal Candidate

描述（由申请人提供）：我们鉴定了一种称为 C5A 的短肽 SWLRDIWDWICEVLSDFK，它代表了一类新型的杀菌候选药物。 C5A 在 nM-M 范围内中和 HIV，对人体细胞没有明显的细胞毒性。 C5A 对应于丙型肝炎病毒 (HCV) 非结构蛋白 5A (NS5A)（477 个氨基酸）的一个小（18 个氨基酸）N 末端区域（aa 3-20）。 C5A 的序列包含负责将 NS5A 锚定到 ER 膜上的区域。重要的是，与 C5A（18 个氨基酸）相比，全长 NS5A（477 个氨基酸）不会抑制 HIV 感染。我们证明 C5A 会破坏 HIV 膜，但保留细胞质膜的完整性。 HIV膜破裂具有特异性，因为即使在高剂量使用时，C5A也不会破坏人体细胞质膜的完整性，并且它不会抑制其他包膜病毒（例如流感病毒和水泡性口炎病毒）的感染。 C5A 具有多种有吸引力的杀菌特性： i) 阻止 HIV 感染主要目标，包括 T 细胞、巨噬细胞和树突状细胞； ii) 对原发性 HIV 分离株、多重耐药 HIV 分离株、SIV 和 SHIV 表现出广泛的抗病毒活性； iii) 中断正在进行的 T 细胞感染； iv) 防止艾滋病毒通过人类原代生殖上皮细胞迁移； v) 阻断离体树突状细胞和朗格汉斯细胞（皮肤组织）的感染； vi) 阻止HIV从树突状细胞和朗格汉斯细胞离体转移至T细胞； vii) 非常有效，因为 C5A 暴露不到 15 分钟就足以中和 HIV； viii) 在将HIV添加到细胞之前（至少1小时）和之后（至少1小时）在相当长的时间内有效； ix) 在低pH值下有效； x) 在抑制浓度下可溶于水； xi) 对阴道内的共生乳杆菌没有毒性； xii) 在体内宫颈阴道组织中表现出最小的不良变化、炎症和毒性； xiii) 不具有免疫原性； xiv) 不影响细胞信号传导途径； xv) 显然不允许病毒产生抗性； xvi) 在生殖器液中稀释时可有效阻止 HIV 感染；最重要的是 xvii) C5A 的阴道应用在人源化 BLT 小鼠 HIV 传播模型中提供了针对阴道病毒攻击的完全保护。因此，C5A 代表了新一代杀菌剂的原型，可能有望预防艾滋病毒。在此应用中，我们希望通过充分探索 C5A 代表真正的杀菌候选药物的可能性来跟进这些令人兴奋的数据。在本申请的第一个目标中，我们建议进行一系列实验，旨在识别 C5A 结合的病毒膜成分，因为驻留在 HIV 膜上的 C5A 配体代表了开发一类具有不寻常抗病毒作用机制的新型抗 HIV 疗法的潜在靶标。有趣的是，我们获得了几条证据，表明称为二氢鞘磷脂 (DHSM) 的鞘脂代表了 HIV 膜中 C5A 的主要靶标： i) DHSM 掺入 HIV 颗粒中，被 C5A 珠特异性拉下来； ii) C5A 直接与吸附的 DHSM 结合； iii) C5A使由DHSM构成的脂质体破裂；最重要的是 iv) C5A 与可溶性 DHSM 预孵育可防止 HIV 被 C5A 破裂并保留 HIV 感染性。 C5A 的两亲特性、DHSM 作为 HIV 膜中 C5A 靶点的特性，以及含有 DHSM 的脂质体或 HIV 颗粒的特异性 C5A 破裂，为 C5A 作用的抗病毒机制提供了第一个线索：C5A 包含负责将 NS5A 锚定到内质网膜上的 N 末端区域，通过与 HIV 膜内富集的 DHSM 结合，扰乱了病毒膜的完整性，因为它具有两亲性。在本申请的第二个目标中，我们建议通过使用亲本 C5A 肽作为原型创建第二代肽来优化 C5A 的体外效力和体内安全性。所有新合成的肽都将在生殖器液中测试其体外杀菌特性。将选择新合成的肽中最有效的化合物。值得注意的是，我们发现 C5A 的乙酰化、酰胺化和糖基化大大增强了 C5A 在生殖器液中的抗 HIV 活性。这是鉴定具有增强抗 HIV 活性的 C5A 衍生物的可行性的概念验证。第三个目标是评估最有效的 C5A 衍生物在 HIV 阴道传播 BLT 小鼠模型中的安全性和有效性。将进行动力学给药研究，以确定 C5A 病毒攻击之前和/或之后多久可以阻止 HIV 传播。第四个目标是在另一种 SIV/HIV 阴道传播动物模型：黄体酮治疗的猕猴模型中进行安全性和有效性试点研究。如果使用两种传播模型获得类似的保护结果，则将进一步验证这些模型在筛选杀菌候选药物中的用途。此外，保护性结果将提供局部使用杀微生物剂（例如 C5A）在预防生殖器 HIV 传播方面的有效性的概念证明。 公共健康相关性：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、有效的局部杀菌剂，以防止艾滋病毒的性传播，并让女性伴侣承担个人使用和应用的责任。开发一种安全、有效、可接受的局部杀菌剂来预防 HIV 性传播，可以在全球范围内减少每天 14,000 多例新的 HIV 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): We identified a short peptide SWLRDIWDWICEVLSDFK called C5A, which represents a novel class of microbicidal candidates. C5A neutralizes HIV at an nM-M range without apparent cytotoxicity to human cells. C5A corresponds to a small (18 amino acids) N-terminal region (aa 3-20) of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) (477 amino acids). The sequence of C5A encompasses the region responsible for the anchoring of NS5A into the ER membrane. Importantly, in contrast to C5A (18 aa), full length NS5A (477 aa) does not inhibit HIV infection. We demonstrated that C5A disrupts the HIV membrane, but preserves the integrity of the cellular plasma membrane. The HIV membrane rupture is specific because C5A does not disturb the integrity of the plasma membrane of human cells even when used at high doses and because it does not inhibit the infection of other enveloped viruses such as influenza and vesicular stomatitis viruses. C5A possesses multiple attractive microbicidal properties: it i) blocks HIV infection of primary targets including T cells, macrophages and dendritic cells; ii) exhibits a broad range of antiviral activity against primary HIV isolates, multi-drug resistant HIV isolates, SIV and SHIV; iii) interrupts an ongoing T cell infection; iv) prevents transmigration of HIV through primary human genital epithelial cells; v) blocks infection of dendritic and Langerhans cells ex vivo (skin tissues); vi) prevents HIV transfer from dendritic and Langerhans cells to T cells ex vivo; vii) is extremely efficacious since less than 15 min of exposure suffices for C5A to neutralize HIV; viii) is potent for a considerable length of time both prior to (at least 1 h) and after (at least 1 h) addition of HIV to cells; ix) is potent at a low pH; x) is soluble in water at inhibitory concentrations; xi) is not toxic to commensal Lactobacilli present in the vaginal tract; xii) exhibits minimal adverse changes, inflammation and toxicity in cervicovaginal tissue in vivo; xiii) is not immunogenic; xiv) does not affect cellular signaling pathways; xv) apparently does not allow viral development resistance; xvi) efficiently blocks HIV infectivity when diluted in genital fluids; and most importantly xvii) vaginal application of C5A offers complete protection against a vaginal viral challenge in the humanized BLT mouse HIV transmission model. Thus, C5A represents the prototype of a new generation of microbicidal agents that may have promise for HIV prevention. In this application, we would like to follow up on these exciting data by fully exploring the possibility that C5A represents a true microbicidal candidate. In the first aim of this application, we propose to conduct a series of experiments aimed at identifying the component of the viral membrane to which C5A binds because the C5A ligand, which resides in the membrane of HIV, represents a potential target for the development of a novel class of anti-HIV therapies with an unusual mechanism of antiviral action. Interestingly, we obtained several lines of evidence that the sphingolipid called dihydrosphingomyelin (DHSM) represents the main target of C5A in the HIV membrane: i) DHSM, incorporated into HIV particles, is specifically pulled down by C5A beads; ii) C5A binds directly to adsorbed DHSM; iii) C5A ruptures liposomes constituted with DHSM; and most importantly iv) pre-incubation of C5A with soluble DHSM prevents HIV rupture by C5A and preserves HIV infectivity. The amphipathic property of C5A, the identity of DHSM as the C5A target in the HIV membrane, and the specific C5A rupture of DHSM-containing liposomes or HIV particles, provide the first hint for the antiviral mechanism of C5A action: C5A, which encompasses the N-terminal region responsible for the anchoring of NS5A into the ER membrane, by binding to DHSM enriched within the HIV membrane, disturbs the integrity of the viral membrane due to its amphipathic nature. In the second aim of this application, we propose to optimize the in vitro potency and in vivo safety of C5A by creating a second generation of peptides using the parental C5A peptide as the archetype. All newly synthesized peptides will be tested in genital fluids for their in vitro microbicidal properties. The most potent compounds among the newly synthesized peptides will be selected. Remarkably, we found that acetylation, amidation and glycosylation of C5A greatly enhanced C5A anti-HIV activities in genital fluids. This is the proof-of-concept for the feasibility of identifying C5A derivates with enhanced anti-HIV activities. In the third aim, the most potent C5A derivates will be assessed for safety and efficacy in the HIV vaginal transmission BLT mouse model. Kinetic administration studies will be executed to determine how long before and/or after the viral challenge C5A precludes HIV transmission. In the fourth aim, safety and efficacy pilot studies will be conducted in another SIV/HIV vaginal transmission animal model: the progesterone-treated macaque model. If similar protective results were obtained using the two transmission models, it would further validate the use of these models for the screening of microbicidal candidates. In addition, protective results would provide proof-of-concept of the usefulness of topically applied microbicides, such as C5A, to prevent genital HIV transmission. PUBLIC HEALTH RELEVANCE: 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 could play a major role in worldwide reduction of the over 14,000 new HIV infections per day, and potentially save millions of lives.