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

描述（由申请人提供）：为了探索一种独特的方法，在非人灵长类动物模型中，在粘膜和全身淋巴组织中产生强大的长期免疫应答，我们建议利用我们实验室开发的减毒恒河巨细胞病毒（RhCMV）疫苗载体，口服递送一种类人猿免疫缺陷病毒（SIV）免疫原作为融合蛋白，包括全长编码序列或鞭毛蛋白的特定结构域。toll样受体（TLR）激动剂TLR5通过激活肠道和口腔粘膜相关淋巴组织（MALT）中的树突状细胞（DC）、滤泡相关上皮（FAE）和M细胞，是一种有效的粘膜免疫反应激活剂。因此，鞭毛蛋白已被证明是1)诱导血清和/或分泌抗体反应和2)诱导全身和粘膜组织中Th1和Th2反应的有效佐剂。这种口服疫苗方法结合了强效TLR佐剂与经口服递送/传播后可在整个宿主中传播的病毒载体（CMV）的优势，并在免疫反应存在的情况下建立终身持久性，从而限制宿主内多个部位的病毒致病性。此外，我们的减毒RhCMV载体通过删除RhCMV编码的病毒白细胞介素(IL)-10基因（RhCMV IL10）进行了修饰，该基因在体内导致原发感染部位的炎症增加，在体外消除了野生型CMV感染相关的DC成熟抑制，并且为了安全性减少了病毒复制。最后，数据显示先前对人类巨细胞病毒（HCMV）免疫并不排除再感染，这意味着基于巨细胞病毒的载体系统可以广泛应用于疫苗目标人群，而不管疫苗前对HCMV是否免疫。我们假设表达SIV疫苗免疫原的RhCMV IL10载体通过口服和全身给药可在多个粘膜部位诱导良好的疫苗抗原特异性细胞和体液免疫反应。此外，通过鞭毛蛋白序列与疫苗抗原融合，结合TLR5激动剂佐剂活性将进一步增强先天性和适应性粘膜免疫反应，并提供一种新的口服粘膜疫苗方法来保护HIV。因此，将构建与鞭毛蛋白全长和/或特定结构域融合的免疫原（SIVmac239 Capsid），以产生对粘膜组织具有最佳免疫原性的疫苗抗原，并将用于生成候选RhCMV IL10Capsid/鞭毛蛋白疫苗载体（Aim1）。接下来，候选RhCMV IL10Capid/鞭毛蛋白疫苗将在多种粘膜和全身组织中进行免疫原性（Aim 2）测试，并通过阴道SIVmac251攻击RhCMV血清阴性的雌性恒河猴进行有效性（Aim 3）测试。我们认为这些新的载体具有显著影响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.