C5a as an Anti-HIV Microbicidal Candidate

C5a 作为抗 HIV 杀菌候选药物

• 批准号: 8073648
• 负责人: PHILIPPE ANDRE GALLAY
• 金额: $ 47万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8073648
• 关键词: AIDS prevention; Acetylation; Address; Affect; Amino Acids; Animal Model; Anti-HIV Therapy; Antiviral Agents; Binding; Bone Marrow; Cell membrane; Cells; Complement 5a; Data; Dendritic Cells; Development; Dissection; Dose; Epithelial Cells; Exhibits; Female; Generations; Genital system; Goals; Grant; HIV; HIV Infections; Hepatitis C virus; Human; In Vitro; Infection; Inflammation; Influenza; Inhibitory Concentration 50; Kinetics; Lactobacillus; Langerhans cell; Length; Ligands; Liposomes; Liquid substance; Liver; Local Microbicides; Macaca; Manuscripts; Membrane; Modeling; Multi-Drug Resistance; Mus; N-terminal; Nature; Nonstructural Protein; Peptides; Pilot Projects; Play; Preparation; Progesterone; Property; Publishing; Research Design; Resistance development; Role; Rupture; SIV; Safety; Screening procedure; Series; Sexual Transmission; Signal Pathway; Skin Tissue; Sphingolipids; Study Section; T-Lymphocyte; Testing; Thymus Gland; Time; Tissues; Titrations; Topical application; Toxic effect; Vagina; Vesicular stomatitis Indiana virus; Viral; Virus; Water; Work; amidation; anti-HIV microbicide; comparative efficacy; cytotoxicity; follow-up; glycosylation; immunogenic; improved; in vivo; in vivo Model; macrophage; microbicide; mouse model; mutant; novel; particle; pre-clinical; preclinical evaluation; prevent; prototype; public health relevance; research study; simian human immunodeficiency virus; tandem mass spectrometry; transmission process; vaginal transmission

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.

描述（由申请人提供）：我们鉴定了一种名为C5A的短肽swlrdiwdwwicevlsdfk，它代表了一类新的杀微生物候选物。C5A在nM-M范围内中和HIV，对人体细胞无明显的细胞毒性。C5A对应于丙型肝炎病毒（HCV）非结构蛋白5A (NS5A)（477个氨基酸）的一个小的（18个氨基酸）n端区（aa 3-20）。C5A序列包含负责将NS5A锚定到内质网膜的区域。重要的是，与C5A （18 aa）相比，全长NS5A （477 aa）不抑制HIV感染。我们证明C5A破坏HIV膜，但保留细胞质膜的完整性。HIV膜破裂是特异性的，因为C5A即使在高剂量使用时也不会破坏人类细胞质膜的完整性，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。C5A具有多种有吸引力的杀微生物特性：它i)阻断HIV感染的主要靶点，包括T细胞、巨噬细胞和树突状细胞；ii)对原发HIV分离株、耐多药HIV分离株、SIV和SHIV表现出广泛的抗病毒活性；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生殖器上皮细胞传播；v)体外阻断树突状细胞和朗格汉斯细胞（皮肤组织）的感染；vi)防止HIV在体外从树突状细胞和朗格汉斯细胞转移到T细胞；7)非常有效，因为C5A暴露不到15分钟就足以中和艾滋病毒；viii)在HIV加入细胞之前（至少1小时）和之后（至少1小时）都有相当长的有效时间；ix)在低pH值下有效；X)在抑制浓度下可溶于水；xi)对存在于阴道的共生乳酸菌没有毒性；Xii)在体内对宫颈阴道组织的不良改变、炎症和毒性最小；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；（十六）在生殖器液体中稀释后有效阻断艾滋病毒的传染性；最重要的是，在人源化BLT小鼠HIV传播模型中，阴道应用C5A对阴道病毒攻击提供了完全的保护。因此，C5A代表了可能有希望预防艾滋病毒的新一代杀微生物剂的原型。在这项应用中，我们希望通过充分探索C5A代表真正的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旨在确定C5A结合的病毒膜成分的实验，因为C5A配体存在于HIV膜中，代表了开发一类具有不同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，表明鞘磷脂二氢鞘磷脂（DHSM）代表了C5A在HIV膜中的主要靶点：i) DHSM被纳入HIV颗粒，被C5A珠特异性地拉下；ii) C5A直接与吸附的DHSM结合；iii) C5A破坏DHSM构成的脂质体；最重要的是，C5A与可溶性DHSM预孵育可防止C5A破坏HIV，保持HIV的传染性。C5A的两亲性，DHSM在HIV膜中作为C5A靶点的特性，以及含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锚定到内质膜中的n端区域，通过与HIV膜内富集的DHSM结合，由于其两亲性，破坏了病毒膜的完整性。在本应用的第二个目标中，我们建议通过以亲本C5A肽为原型创建第二代肽来优化C5A的体外效价和体内安全性。所有新合成的多肽将在生殖器液中测试其体外杀微生物性能。将从新合成的多肽中选择最有效的化合物。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证明。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。将进行动态给药研究，以确定在病毒攻击C5A之前和/或之后多久可以阻止HIV传播。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试点研究：黄体酮治疗的猕猴模型。如果使用这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候选杀微生物剂的有效性。此外，保护性结果将证明局部应用杀菌剂（如C5A）对预防生殖器艾滋病毒传播的有用性。