Viral Immunomodulation and Rational CMV Vaccine Design

描述（由申请人提供）：由于迫切需要的预防先天性人类巨细胞病毒（HCMV）感染的疫苗在临床试验中取得进展，对保护新生儿的最佳疫苗接种策略仍有不完全的了解。基于免疫优势包膜糖蛋白，糖蛋白B （gB）的蛋白质亚单位疫苗是安全的，并诱导稳健的中和抗体滴度。针对主要细胞毒性t淋巴细胞靶点UL83 （pp65）蛋白的载体疫苗也在研究中。然而，考虑到临床分离株中gB编码序列的多样性和大量其他免疫原性CMV蛋白，人们仍然担心单独针对HCMV蛋白亚基的免疫反应是否能够提供足够的免疫反应，以防止先天性HCMV感染和疾病。减毒HCMV活疫苗是一种有吸引力的替代方案，因为这类疫苗提供了对多种病毒蛋白诱导持久反应的可能性。活病毒疫苗的主要限制是不能确保足够的安全性，以防止潜伏、再激活和经胎盘传播。基于活病毒疫苗可以通过缺失病毒免疫调节基因来提高安全性的假设，本研究旨在研究三个病毒基因家族在先天性巨细胞病毒感染的唯一小动物模型——豚鼠模型中，在活病毒疫苗介导的保护和经胎盘病毒传播中的作用。我们将研究一种合理的活病毒疫苗设计策略，该策略基于这样的假设：与野生型病毒相比，删除病毒编码的免疫逃避基因将导致表型衰减，经胎盘传播减少。我们进一步假设，这些候选疫苗将保留免疫原性，并赋予对先天性GPCMV传播和疾病的保护，同时具有更好的安全性。待评估的特定病毒基因将包括：1)病毒g蛋白偶联受体（gpcr）的同源基因UL33和UL78；2)一种新的CC β趋化因子GPCMV MIP-1；3) GPCMV I类下调基因(s)。分子克隆技术，包括克隆成细菌人工染色体（BAC）的GPCMV基因组诱变，将用于设计这些突变型GPCMV减毒活疫苗候选物。减毒活病毒的疫苗效力研究将与发病机制分析同时进行，以评估候选疫苗与野生型GPCMV相比的安全性（胎盘感染和损伤、经胎盘传播）。此外，将采用签名标记诱变策略生成重组病毒进行体内实验，以阐明其他病毒基因在先天性传播和发病机制中的作用。这将代表首次应用签名标签方法研究先天性感染。这些研究结果将扩大我们对巨细胞病毒所采用的免疫逃避策略的认识，将有助于了解先天性巨细胞病毒疾病发病机制的病毒因素，并将有助于开发用于人体临床试验的合理的活病毒HCMV疫苗。

DESCRIPTION (provided by applicant): As urgently needed vaccines against congenital human cytomegalovirus (HCMV) infection move forward in clinical trials, there remains an incomplete understanding of the optimal vaccination strategy for protection of the newborn. Protein subunit vaccines based on the immunodominant envelope glycoprotein, glycoprotein B (gB), are safe, and induce robust neutralizing antibody titers. Vectored vaccines directed against the major cytotoxic-T-lymphocyte target, the UL83 (pp65) protein, are also under study. However, concerns remain about whether immune responses against HCMV protein subunits alone would be capable of conferring adequate immune responses for protection against congenital HCMV infection and disease, given the diversity of gB coding sequences among clinical isolates and given the large number of other immunogenic CMV proteins. Live, attenuated HCMV vaccines are an attractive alternative, since such vaccines offer the possibility of inducing long-lasting responses to a broad range of viral proteins. The major limitation of live virus vaccines is the inability to ensure adequate safety against latency, reactivation, and transplacental transmission. Based on the hypothesis that live virus vaccines can be engineered for improved safety by deletion of viral immunomodulatory genes, this proposal aims to examine the role of three viral gene families in live virus vaccine-mediated protection and transplacental viral transmission in the only small animal model of congenital CMV infection, the guinea pig model Specifically, we will be investigating a rational live-virus vaccine design strategy based on the hypothesis that deletion of virally-encoded immunoevasive genes will result in an attenuation phenotype, with decreased transplacental transmission, compared to wild-type virus. We further hypothesize that such vaccine candidates will retain immunogenicity and confer protection against congenital GPCMV transmission and disease, while having a better safety profile. The specific viral genes to be evaluated will be: 1) homologs of the viral G-protein coupled receptors (GPCRs), UL33 and UL78; 2) a novel CC Beta chemokine, GPCMV MIP-1; 3) the GPCMV class I down-regulation gene(s). Molecular cloning techniques, including mutagenesis of the GPCMV genome cloned as a bacterial artificial chromosome (BAC), will be utilized to engineer these mutant GPCMV live attenuated vaccine candidates. Vaccine efficacy studies of the live, attenuated viruses will be performed in parallel with pathogenesis analyses, to assess the safety profile of the candidate vaccines (placental infection and injury, transplacental transmission) compared to wild-type GPCMV. Additionally, a signature-tagged mutagenesis strategy will be employed to generate recombinant viruses for in vivo experiments to elucidate the role of other viral genes in congenital transmission and pathogenesis. This will represent the first application of signature tag methodology to the study of congenital infection. The results of these investigations will expand our knowledge of immune evasion strategies employed by the CMVs, will aid understanding of the viral factors responsible for the pathogenesis of congenital CMV disease, and will enable development of rational live-virus HCMV vaccines for human clinical trials.