C5a as an Anti-HIV Microbicidal Candidate

描述(申请人提供)：我们鉴定了一个称为C5a的短肽SWLRDIWDWICEVLSDFK，它代表了一类新的杀微生物活性候选者。C5a在NM-M范围内中和HIV，对人类细胞没有明显的细胞毒性。C5a对应于丙型肝炎病毒非结构蛋白5A(NS5A)的一个小的(18个氨基酸)N末端区域(AA 3-20)(477个氨基酸)。C5a序列包含负责NS5A锚定到内质网膜上的区域。重要的是，与C5a(18aa)相比，全长NS5A(477aa)不能抑制HIV感染。我们证明了C5a破坏了HIV膜，但保留了细胞质膜的完整性。HIV膜破裂是特殊的，因为C5a即使在高剂量使用时也不会扰乱人类细胞质膜的完整性，也因为它不能抑制其他被包裹的病毒的感染，如流感病毒和水泡性口炎病毒。C5a具有多种诱人的杀微生物活性：i)阻断包括T细胞、巨噬细胞和树突状细胞在内的主要靶标的HIV感染；ii)对HIV初级分离株、多药耐药HIV分离株、SIV和SIV显示广泛的抗病毒活性；iii)阻断正在进行的T细胞感染；iv)防止艾滋病毒通过原代人类生殖器上皮细胞的迁移；v)阻断树突状细胞和朗格汉斯细胞的体外感染(皮肤组织)；vi)防止艾滋病毒从树突状细胞和朗格汉斯细胞向体外T细胞的转移；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加入细胞之前(至少1小时)和之后(至少1小时)都是有效的；ix)在低pH值下有效；x)在抑制浓度下溶于水；xi)对阴道中的共生乳杆菌没有毒性；xii)在体内的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)不影响细胞信号通路；XV)显然不允许病毒产生耐药性；xvi)在生殖液中稀释时有效地阻断HIV的传染性；最重要的是，在人源化的BLT小鼠艾滋病毒传播模型中，经阴道应用C5a可提供完全保护，免受阴道病毒的挑战。因此，C5a代表了新一代杀微生物剂的原型，可能有希望预防艾滋病毒。在这项申请中，我们希望通过充分探索C5a代表真正的杀微生物剂候选者的可能性来跟踪这些令人兴奋的数据。在这项应用的第一个目的中，我们建议进行一系列实验，旨在确定C5a与病毒膜结合的成分，因为C5a配体位于HIV膜上，是开发具有不寻常抗病毒作用机制的新型抗HIV药物的潜在靶点。有趣的是，我们获得了几条证据，证明被称为二氢鞘磷脂(DHSM)的鞘磷脂代表了HIV膜上C5a的主要靶点：i)DHSM被结合到HIV颗粒中，被C5a珠特异性地拉下；ii)C5a直接与吸附的DHSM结合；iii)C5a断裂由DHSM组成的脂质体；以及最重要的是iv)C5a与可溶性DHSM预先孵育可以防止C5a破裂并保持HIV的感染性。C5a的两亲性，DHSM作为HIV膜上C5a靶标的特性，以及含有DHSM的脂质体或HIV颗粒的特异性C5a断裂，为C5a的抗病毒作用机制提供了第一个线索：C5a通过与HIV膜内富含的DHSM结合，包含负责NS5A锚定到ER膜的N-末端区域，扰乱了病毒膜的完整性。在这一应用的第二个目标中，我们建议通过以亲本C5a多肽为原型创造第二代多肽来优化C5a的体外效力和体内安全性。所有新合成的多肽都将在生殖液中进行体外杀菌性能测试。新合成的多肽中最有效的化合物将被挑选出来。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生殖液中C5a的抗HIV活性。这是对鉴定具有增强的抗艾滋病毒活性的C5a衍生物的可行性的概念验证。在第三个目标中，将评估最有效的C5a衍生物在HIV阴道传播BLT小鼠模型中的安全性和有效性。将进行动力学管理研究，以确定在病毒挑战C5a阻止艾滋病毒传播之前和/或之后多久。在第四个目标中，安全性和有效性的初步研究将在另一个SIV/HIV阴道传播动物模型中进行：孕酮处理的猕猴模型。如果使用两种传播模型获得类似的保护结果，将进一步验证这些模型用于筛选杀微生物剂候选者的有效性。此外，保护性结果将为局部使用杀微生物剂，如C5a，预防生殖器艾滋病毒传播的有用性提供概念证明。

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.