Viral Immunomodulation and Rational CMV Vaccine Design

描述（由申请方提供）：由于迫切需要针对先天性人巨细胞病毒（HCMV）感染的疫苗在临床试验中取得进展，因此对保护新生儿的最佳疫苗接种策略仍不完全了解。基于免疫显性包膜糖蛋白糖蛋白B（gB）的蛋白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UL 83（pp 65）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，仍然存在对单独针对HCMV蛋白亚基的免疫应答是否能够赋予足够的免疫应答以保护免受先天性HCMV感染和疾病的担忧。减毒活HCMV疫苗是一种有吸引力的替代方案，因为此类疫苗提供了诱导对广泛病毒蛋白质的持久应答的可能性。活病毒疫苗的主要局限性是不能确保足够的安全性，防止潜伏期，再活化和经胎盘传播。基于活病毒疫苗可以通过删除病毒免疫调节基因来提高安全性的假设，该提案旨在研究三个病毒基因家族在活病毒疫苗介导的保护作用和先天性CMV感染的唯一小动物模型--豚鼠模型中的经胎盘病毒传播中的作用，具体而言，我们将研究一种合理的活病毒疫苗设计策略，该策略基于以下假设：与野生型病毒相比，病毒编码的免疫逃避基因的缺失将导致减毒表型，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。待评价的特定病毒基因为：1）病毒G蛋白偶联受体（GPCR）的同源物，UL 33和UL 78; 2）新型CC β趋化因子，GPCMV MIP-1; 3）GPCMV I类下调基因。将利用分子克隆技术，包括克隆为细菌人工染色体（BAC）的GPCMV基因组的诱变，对这些突变GPCMV减毒活疫苗候选物进行工程改造。 减毒活病毒的疫苗有效性研究将与发病机制分析同时进行，以评估候选疫苗（胎盘感染和损伤，经胎盘传播）与野生型GPCMV相比的安全性特征。 此外，将采用特征标记的诱变策略来产生用于体内实验的重组病毒，以阐明其他病毒基因在先天性传播和发病机制中的作用。这将是第一次应用的签名标签方法的研究先天性感染。这些调查的结果将扩大我们的知识免疫逃避策略所采用的CMV，将有助于了解的病毒因素负责的先天性CMV疾病的发病机制，并将使合理的活病毒HCMV疫苗的人类临床试验的发展。

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.