Viral Immunomodulation and Rational CMV Vaccine Design

描述(由申请人提供)：随着针对先天性人类巨细胞病毒(HCMV)感染的迫切需要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存在不完全的理解。基于免疫优势的包膜糖蛋白糖蛋白B(GB)的蛋白亚单位疫苗是安全的，并能诱导出强大的中和抗体效价。针对主要细胞毒性T淋巴细胞靶标UL83(Pp65)蛋白的载体疫苗也在研究中。然而，鉴于临床分离株之间gB编码序列的多样性以及大量其他免疫原性CMV蛋白，人们仍然担心针对HCMV蛋白亚基的免疫反应是否能够单独产生足够的免疫反应，以预防先天性HCMV感染和疾病。减毒活的巨细胞病毒疫苗是一个有吸引力的选择，因为这种疫苗提供了对广泛的病毒蛋白诱导长期应答的可能性。活病毒疫苗的主要局限性是无法确保足够的安全性，防止潜伏期、再激活和经胎盘传播。基于这样的假设，即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，本建议旨在研究三个病毒基因家族在先天性CMV感染的唯一小动物模型-豚鼠模型中的活病毒疫苗介导的保护和经胎盘病毒传播中的作用。具体地说，我们将研究一种合理的活病毒疫苗设计策略，该策略基于这样一种假设，即与野生型病毒相比，缺失病毒编码的免疫逃避基因将导致表型减弱，经胎盘传播减少。我们进一步假设，这些候选疫苗将保持免疫原性，并提供针对先天性GPCMV传播和疾病的保护，同时具有更好的安全性。待评估的病毒特异性基因将是：1)病毒G蛋白偶联受体(GPCRs)的同源基因UL33和UL78；2)新的CC Beta趋化因子GPCMV MIP-1；3)GPCMV I类下调基因(S)。分子克隆技术，包括作为细菌人工染色体(BAC)克隆的GPCMV基因组的突变，将被用来设计这些突变的GPCMV减毒活疫苗候选。减毒活病毒的疫苗效力研究将与致病机理分析同时进行，以评估候选疫苗(胎盘感染和损伤、经胎盘传播)与野生型GPCMV的安全性。此外，还将使用签名标记突变策略来产生重组病毒，用于体内实验，以阐明其他病毒基因在先天性传播和发病机制中的作用。这将是签名标签方法在先天性感染研究中的首次应用。这些研究的结果将扩大我们对巨细胞病毒所采用的免疫逃避策略的了解，将有助于了解导致先天性巨细胞病毒病发病的病毒因素，并将有助于开发合理的活病毒巨细胞病毒疫苗用于人类临床试验。

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.