Attenuated RhCMV Delta 10 SIV Oral Vaccine Vectors Encoding TLR5 Ligand Sequences

描述（申请人提供）：为了探索一种独特的方法，在非人灵长类动物人类免疫缺陷病毒（HIV）模型中，在粘膜和全身淋巴组织中产生稳健的长期免疫应答，我们建议利用我们实验室开发的减毒恒河猴巨细胞病毒（RhCMV）疫苗载体，用于猴免疫缺陷病毒（SIV）的口服给药免疫原被工程化为融合蛋白，以包括鞭毛蛋白的全长编码序列或特异性结构域，鞭毛蛋白是toll样受体（TLR）5的激动剂。TLR 5是通过激活包括在肠和口腔粘膜相关淋巴组织（MALT）内的树突状细胞（DC）、滤泡相关上皮（FAE）和M细胞的粘膜免疫应答的有效激活剂。因此，鞭毛蛋白已被证明是i）诱导血清和/或分泌性抗体应答和ii）诱导全身和粘膜组织中的Th 1和Th 2应答的有效佐剂。这种口服疫苗方法结合了有效TLR佐剂与病毒载体（CMV）的优点，所述病毒载体显示在口服递送/传播后在整个宿主中传播，并且在存在免疫应答的情况下建立终身持久性，所述免疫应答限制宿主内多个位点的病毒致病性。此外，我们的减毒RhCMV载体已经通过缺失RhCMV编码的病毒白细胞介素（IL）-10基因（RhCMV IL 10）进行了修饰，这导致体内原发感染部位的炎症增加，消除了与体外野生型CMV感染相关的DC成熟抑制，并为了安全性而减弱了病毒复制。最后，数据显示，对人CMV（HCMV）的先前免疫力并不排除再感染，这意味着基于CMV的载体系统可以广泛应用于疫苗靶群体，而不管疫苗接种前对HCMV的免疫力如何。我们假设表达SIV疫苗免疫原的RhCMV IL 10载体经口服和全身给药将在多个粘膜部位诱导上级疫苗抗原特异性细胞和体液免疫应答。此外，通过鞭毛蛋白序列与疫苗抗原的融合来掺入TLR 5激动剂佐剂活性将进一步增强先天性和适应性粘膜免疫应答，并提供一种用于保护免受HIV的新的口腔粘膜疫苗方法。因此，将构建与鞭毛蛋白的全长和/或特异性结构域融合的免疫原（SIVmac 239衣壳）以产生对粘膜组织具有最佳免疫原性的疫苗抗原，并将其用于产生候选RhCMV IL 10衣壳/鞭毛蛋白疫苗载体（Aim 1）。接下来将通过阴道SIVmac 251攻击测试候选RhCMV IL 10 Capid/鞭毛蛋白疫苗在多个粘膜和全身组织中的免疫原性（目的2）以及在RhCMV血清阴性雌性恒河猴中的功效（目的3）。我们认为这些新型载体具有显著影响HIV疫苗设计的潜力。

DESCRIPTION (provided by applicant): To explore a unique approach for generating robust long term immune responses at both mucosal and systemic lymphoid tissues in the nonhuman primate model for human immunodeficiency virus (HIV), we propose to utilize an attenuated rhesus cytomegalovirus (RhCMV) vaccine vector developed in our laboratories for oral delivery of a simian immunodeficiency virus (SIV) immunogen engineered as a fusion protein to include either full length coding sequence or specific domains of flagellin, the agonist for toll-like receptor (TLR)5. TLR5 is a potent activator of mucosal immune responses through activation of dendritic cells (DC), follicle-associated epithelium (FAE) and M cells included within gut and oral cavity mucosal- associated lymphoid tissue (MALT). As such, flagellin has proven to be a potent adjuvant for i) induction of serum and/or secretory antibody responses and for ii) induction of Th1 and Th2 responses in both systemic and mucosal tissues. This oral vaccine approach combines the advantages of a potent TLR adjuvant with a viral vector (CMV) shown to disseminate throughout the host after oral delivery/transmission and to establish a lifelong persistence in the presence of immune responses that limit viral pathogenicity at multiple sites within the host. Furthermore, our attenuated RhCMV vector has been modified by deletion of the RhCMV-encoded viral interleukin (IL)-10 gene (RhCMV IL10) that results in increased inflammation at the site of primary infection in vivo, an abrogation of the inhibition of DC maturation associated with wild type CMV infection in vitro, and attenuated viral replication for safety. Lastly, data has shown that prior immunity to human CMV (HCMV) does not preclude re-infection, implying that a CMV-based vector system may be broadly applied to a vaccine target population irrespective of pre-vaccine immunity to HCMV. We hypothesize that RhCMV IL10 vectors expressing SIV vaccine immunogens delivered orally and systemically will induce superior vaccine antigen-specific cellular and humoral immune responses in multiple mucosal sites. Also, incorporation of TLR5 agonist adjuvant activity by fusion of flagellin sequences with vaccine antigen will further enhance both innate and adaptive mucosal immune responses and provide a novel oral mucosal vaccine approach for protection against HIV. Accordingly, immunogens (SIVmac239 Capsid) fused with full length and/or specific domains of flagellin will be constructed to generate a vaccine antigen with optimal immunogenicity for mucosal tissues and will be used for generation of candidate RhCMV IL10Capsid/flagellin vaccine vectors (Aim1). Candidate RhCMV IL10Capid/flagellin vaccines will next be tested for immunogenicity (Aim 2) in multiple mucosal and systemic tissues and for efficacy (Aim 3) in RhCMV-seronegative female rhesus macaques by vaginal SIVmac251 challenge. We propose that these novel vectors carry the potential to significantly impact HIV vaccine design.