C5a as an Anti-HIV Microbicidal Candidate

描述（由申请人提供）：我们鉴定了称为C5 A的短肽SWLRDIWDWICEVLSDFK，其代表一类新的杀微生物候选物。C5 A在nM-M范围内中和HIV，对人细胞没有明显的细胞毒性。C5 A对应于丙型肝炎病毒（HCV）非结构蛋白5A（NS 5A）（477个氨基酸）的小（18个氨基酸）N-末端区域（aa 3-20）。C5 A的序列包括负责将NS 5A锚定到ER膜中的区域。重要的是，与C5 A（18 aa）相反，全长NS 5A（477 aa）不抑制HIV感染。我们证明了C5 A破坏了HIV膜，但保留了细胞质膜的完整性。HIV膜破裂是特异性的，因为C5 A即使在高剂量使用时也不会干扰人细胞质膜的完整性，并且因为它不会抑制其他包膜病毒如流感病毒和水泡性口炎病毒的感染。C5 A具有多种有吸引力的杀微生物特性：其i）阻断HIV对包括T细胞、巨噬细胞和树突细胞的主要靶标的感染; ii）对主要HIV分离株、多药耐药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HIV通过主要人生殖器上皮细胞的迁移; v）离体阻断树突细胞和朗格汉斯细胞的感染（皮肤组织）; vi）防止HIV从树突细胞和朗格汉斯细胞离体转移到T细胞; vii）非常有效，因为少于15分钟的暴露足以使C5 A中和HIV;（viii）在相当长的时间内都是有效的，（至少1小时）及之后（至少1小时）向细胞中加入HIV; ix）在低pH下是有效的; x）在抑制浓度下可溶于水; Xi）对存在于阴道中的乳酸杆菌无毒; 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）无免疫原性; 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）当稀释在生殖器液中时，有效地阻断HIV感染性;并且最重要的是xvii）C5 A的阴道应用在人源化BLT小鼠HIV传播模型中提供针对阴道病毒攻击的完全保护。因此，C5 A代表了新一代杀微生物剂的原型，可能有希望用于艾滋病毒预防。在本申请中，我们希望通过充分探索C5 A代表真正杀微生物候选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的中，我们建议进行一系列实验，旨在鉴定C5 A结合的病毒膜组分，因为驻留在HIV膜中的C5 A配体代表了开发具有不寻常的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，即称为二氢鞘磷脂（DHSM）的鞘脂代表了HIV膜中C5 A的主要靶标：i）掺入HIV颗粒中的DHSM被C5 A珠特异性地拉下; ii）C5 A直接结合吸附的DHSM; iii）C5 A破坏由DHSM构成的脂质体;最重要的是iv）C5 A与可溶性DHSM的预温育防止了HIV被C5 A破坏并保持了HIV感染性。C5 A的两亲性、DHSM作为HIV膜中C5 A靶标的特性以及含有DHSM的脂质体或HIV颗粒的特异性C5 A破裂，为C5 A作用的抗病毒机制提供了第一个提示：C5 A，其包含负责通过结合HIV膜内富集的DHSM将NS 5A锚定到ER膜中的N-末端区域，由于其两亲性而干扰病毒膜的完整性。在本申请的第二个目的中，我们提出通过使用亲本C5 A肽作为原型产生第二代肽来优化C5 A的体外效力和体内安全性。所有新合成的肽将在生殖液中测试其体外杀微生物特性。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C5 A的乙酰化、酰胺化和糖基化大大增强了C5 A在生殖器液中的抗HIV活性。这是鉴定具有增强的抗HIV活性的C5 A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C5 A衍生物在HIV阴道传播BLT小鼠模型中的安全性和有效性。将进行动态给药研究，以确定病毒攻毒前和/或后C5 A阻止HIV传播的时间。在第四个目标中，将在另一种SIV/HIV阴道传播动物模型中进行安全性和有效性初步研究：炔雌醇治疗的猕猴模型。如果使用两种传播模型获得相似的保护结果，则将进一步验证这些模型用于筛选杀微生物剂候选物的用途。此外，保护性结果将为局部应用杀微生物剂（如C5 A）预防生殖器艾滋病毒传播的有效性提供概念验证。

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.