Safety and Immunogenicity of novel, live-attenuated V4020 vaccine for Venezuelan Equine Encephalitis (VEE) in healthy adults

摘要 委内瑞拉马脑炎病毒（VEEV）是一种威胁生命的NIH/NIAID类别B人类病原体 和潜在的生物恐怖主义威胁VEEV暴发发生在中美洲， 进入美国VEEV在美国重新出现的潜在破坏性影响要求 有效的疫苗，以保护免疫初治人群。目前使用的是TC-83减毒活疫苗 根据IND协议，为有风险的医务人员接种疫苗。疫苗会引起不良反应， 正在努力开发一种改进的VEEV疫苗。然而，由于疫苗的发展是一个 由于TC-83疫苗的供应有限，美国可能很快就会出现短缺。 VEEV疫苗。这可能使美国人口和处于危险中的人员得不到保护。更以 在没有疫苗的情况下，VEEV可能符合CDC对BSL 4选择剂的定义。 在以前的NIH支持的研究中，Medigen评估了一种新的平台技术， 开发针对VEEV和潜在的其他病毒性疾病的疫苗。V4020疫苗 包括减弱全长基因组RNA内衣壳和糖蛋白基因的重排。 减毒重排有效地防止了逆转，因为许多独立的突变将被逆转。 需要恢复到致病基因型。此外，疫苗是由重排的DNA拷贝制成的。 RNA基因组位于CMV启动子下游的iDNA®感染性克隆中。的关键特征 iDNA感染性克隆（与标准cDNA克隆的不同之处）是启动活减毒病毒 而不需要体外转录。使用遗传稳定的iDNA改善了 疫苗安全在初步研究中，V4020病毒含有重排的基因组RNA， 在BALB/c小鼠和小鼠中，iDNA克隆显示出安全性优势和对VEEV攻击的保护作用。 食蟹猴非人灵长类动物（NHP）模型。 具体目标1。V4020 VEEV减毒活疫苗在人类受试者中的安全性。Medigen合作 与美国陆军传染病医学研究所（USAMRIID）合作，进行1a期，首次 评价V4020 VEEV疫苗在健康成人中安全性的人体研究。我们提出一个双盲， 在39名18-50岁的健康成人中进行的安慰剂对照、单次剂量递增研究。梅迪根将 生产cGMP批次的V4020疫苗，并与FDA就临床试验设计进行沟通。组 的志愿者（13例受试者/组）将接受单次104、105或106 PFU剂量的V4020疫苗（10例/组）， 每组3名受试者随机接受安慰剂。首先给予低剂量， 另一批人士则须延迟最少十四天才可开始接受较高剂量的疫苗。的 本研究旨在评估V4020疫苗皮下（SC）给药的安全性。 具体目标2。评价接种V4020的人类受试者的免疫应答。为此，我们 将评价临床研究期间采集的临床样本（血浆、唾液、尿液），以确定给药方案 提供多方面免疫反应（中和抗体、T细胞反应、细胞因子， 等等）。将鉴定推定的早期特征，其可用于预测后期免疫原性并获得 深入了解V4020在人类受试者中的免疫原性机制。 我们的初步数据表明，合理的设计，减毒重排和iDNA技术可以 通过提高安全性、遗传稳定性，为V4020 VEEV疫苗提供安全有效的解决方案， 免疫原性此外，衰减重排可以容易地适用于以下的发展： 其他疫苗，包括制备WEEV、EEEV、其他甲病毒的减毒活疫苗，以及 其他RNA病毒如果成功，这项技术可能会改变减毒活疫苗领域 对于许多病毒性疾病。

ABSTRACT Venezuelan Equine Encephalitis virus (VEEV) is a life-threatening, NIH/NIAID category B human pathogen and a potential bioterrorism threat. Outbreaks of VEEV occur in Central America and have previously spread into the United States. The potentially devastating effects of VEEV reemergence in the U.S. demand an effective vaccine to protect immunologically naïve population. Currently, live attenuated TC-83 vaccine is used under IND protocol for vaccination of medical personnel at risk. The vaccine causes adverse effects, and efforts to develop an improved VEEV vaccine are underway. However, because vaccine development is a lengthy process and the supply of TC-83 vaccine is limited, the U.S. may soon experience a shortage of the VEEV vaccine. This can leave the U.S. population and at-risk personnel unprotected. Furthermore, in the absence of a vaccine, VEEV may fit the CDC definition of a BSL4 Select Agent. During previous NIH-supported research, Medigen evaluated a new platform technology for the development of vaccines against VEEV and, potentially, other viral diseases. The proposed V4020 vaccine includes attenuating rearrangement of the capsid and glycoprotein genes within the full-length genomic RNA. The attenuating rearrangement effectively prevents reversion because many independent mutations would be needed to revert to the pathogenic genotype. In addition, vaccine is made from a DNA copy of the rearranged RNA genome placed in the iDNA® infectious clone downstream from the CMV promoter. The key feature of iDNA infectious clone (and the difference from standard cDNA clone) is that live attenuated virus is launched from the plasmid, without the need for in vitro transcription. The use of genetically stable iDNA improves vaccine safety. In preliminary research, the V4020 virus containing rearranged genomic RNA and prepared from iDNA clone showed safety advantages and protection against VEEV challenges in BALB/c mice and in a cynomolgus macaque non-human primate (NHP) model. Specific Aim 1. Safety of V4020 live-attenuated VEEV vaccine in human subjects. Medigen in collaboration with U.S. Army Medical Research Institute for Infectious diseases (USAMRIID), to conduct Phase 1a, first-in- human study evaluating safety of V4020 VEEV vaccine in healthy adults. We propose a double blinded, placebo controlled, single-dose escalation study in 39 healthy adults, 18-50 years of age. Medigen will manufacture cGMP lot of V4020 vaccine and communicate with the FDA regarding clinical trial design. Groups of volunteers (13 subjects/group) will receive a single 104, 105, or 106 PFU dose of V4020 vaccine (10/group), with 3 subjects from each group randomized to receive placebo. The low dose will be administered first, and there will be at least 14 days delay before another group will start receiving the higher dose of vaccine. The study is designed to assess the safety of V4020 vaccine administered by subcutaneous (SC) administration. Specific Aim 2. Evaluate immune responses from human subjects vaccinated with V4020. In this aim, we will evaluate clinical samples (plasma, saliva, urine) to be collected during the clinical study to identify a dosing regimen that provides a multi-faceted immune response (neutralizing antibody, T-cell response, cytokines, etc). Putative early signatures will be identified that could be used to predict later immunogenicity and obtain insight into mechanisms of V4020 immunogenicity in human subjects. Our preliminary data suggest that the rational design, attenuating rearrangement, and iDNA technology can provide a safe and effective solution for V4020 VEEV vaccine by improving safety, genetic stability, and immunogenicity. Furthermore, the attenuating rearrangement can be easily adapted for the development of other vaccines including preparation of live attenuated vaccines for WEEV, EEEV, other alphaviruses, and other RNA viruses. If successful, this technology can potentially transform the field of live attenuated vaccines for many viral diseases.