Development of a novel vaccine against congenital cytomegalovirus infection

描述（由申请人提供）：在美国，每年约有40，000名婴儿发生人巨细胞病毒（HCMV）先天性感染，是导致出生缺陷的主要病毒原因。这些问题与HCMV在实体器官和造血干细胞移植受者中引起的严重疾病一起为有效疫苗的开发提供了强大的动力。然而，与被宿主清除的病毒不同，HCMV即使在高水平的宿主免疫力存在下也能持续存在的能力表明需要新的方法来开发疫苗。由于小鼠CMV（MCMV）模型的结果表明，对免疫显性抗原特异性的CD 8 + T细胞在限制病毒复制方面无效，因此我们推测，使用这些抗原进行疫苗接种最多可能引起的免疫力并不显著高于自然感染期间产生的免疫力：由于病毒持续存在并可能导致复发性感染和先天性传播，因此保护最终不完全。我们假设CMV中高度保守的必需病毒蛋白可能代表一类新的T细胞靶点。其基本原理是这些基因必须被表达用于病毒复制，并且维持蛋白质活性所需的高氨基酸保守性限制了突变引起的免疫逃逸。由于这些抗原在HCMV或MCMV感染过程中大多数是亚显性的，因此我们假设T细胞对这些抗原的致敏可能导致比自然感染更强的保护性免疫。为此，我们最近已经表明，DNA免疫接种使用两个保守的，必要的基因MCMV（DNA聚合酶M54和解旋酶M105）保护小鼠免受随后的亚致死性的挑战与强毒病毒。我们还开发了一种DNA初免-完整、灭活病毒加强策略，其激发CD 8 + T细胞应答以及强中和抗体，并且我们证明了这种初免-加强疫苗接种赋予针对全身性病毒攻击的完全保护和针对粘膜病毒攻击的显著保护。然而，由于MCMV在子宫内不引起感染，因此预防先天性CMV感染的研究使用了豚鼠CMV（GPCMV）模型。虽然已经在豚鼠模型中测试的GPCMV疫苗在妊娠结局方面提供了统计学上显著的改善，但没有疫苗能够完全保护免于死亡或感染。在本提案中，我们计划使用先天性CMV感染的GPCMV模型来证明“原理证明”，即用GPCMV的保守的必需基因进行初免-加强免疫，然后用化学方法杀死GPCMV，可以保护患者免受先天性GPCMV感染和疾病的侵害，并为进行更全面的免疫和保护临床前研究提供依据。该提案的具体目标是：1。在先天性感染和疾病的豚鼠模型中，确定用三价DNA（由DNA聚合酶、解旋酶和糖蛋白B基因组成）进行初免-加强疫苗接种，然后灭活GPCMV的保护效力，以及2. DNA聚合酶和解旋酶DNA、糖蛋白B DNA和灭活病毒对免疫和保护的相对贡献的表征。我们提出的疫苗的新方面是在引发步骤中选择用于产生T淋巴细胞应答的高度保守的必需病毒基因以及在加强步骤中使用完整的灭活病毒，它们一起可以完全保护免受系统性CMV攻击。公共卫生相关性：在美国，每年约有40，000名婴儿发生人巨细胞病毒（HCMV）先天性感染，是导致出生缺陷的主要病毒原因。这些问题与HCMV在实体器官和造血干细胞移植受者中引起的严重疾病一起为有效疫苗的开发提供了强大的动力。然而，与被宿主清除的病毒不同，HCMV即使在高水平的宿主免疫力存在下也能持续存在的能力表明，需要新的方法来开发针对HCMV和其他持久性病毒的疫苗。

DESCRIPTION (provided by applicant): Congenital infection with human cytomegalovirus (HCMV) occurs in approximately 40,000 infants a year in the United States annually and is the leading viral cause of birth defects. These problems together with the severe disease that HCMV causes in solid organ and hematopoietic stem cell transplant recipients provides a strong impetus for the development of an effective vaccine. However, unlike viruses that are cleared by the host, the ability of HCMV to persist even in the presence of high levels of host immunity suggests that novel approaches are needed for the development of a vaccine. Because results in the murine CMV (MCMV) model have shown that CD8+ T cells specific for an immunodominant antigen are ineffective at limiting viral replication, we speculated that vaccination using these antigens may at best elicit immunity that is not significantly greater than that generated during the natural infection: protection that is ultimately incomplete since the virus persists and can cause recurrent infection and congenital transmission. We hypothesized that the essential viral proteins that are highly conserved among the CMVs may represent a novel class of T cell targets. The rationale is that these genes must be expressed for viral replication, and the high amino acid conservation needed for maintaining protein activity limits immune escape by mutation. Because these antigens have been found to be mostly subdominant during infection with HCMV or MCMV, we hypothesized that priming of T cells against these antigens may result in qualitatively greater protective immunity than the natural infection. To this end, we have recently shown that DNA immunization using either of two conserved, essential genes of MCMV (the DNA polymerase M54 and the helicase M105) protects mice against a subsequent sublethal challenge with virulent virus. We have also developed a DNA prime - whole, inactivated virus boost strategy that elicits both CD8+ T cell responses as well as strong neutralizing antibodies, and we demonstrated that this prime-boost vaccination conferred complete protection against systemic viral challenge and significant protection against mucosal viral challenge. However, since MCMV does not cause infection in utero, studies of the prevention of congenital CMV infection have utilized the guinea pig CMV (GPCMV) model. While the GPCMV vaccines that have been tested in the guinea pig model have provided statistically significant improvements in pregnancy outcomes, no vaccine has conferred complete protection against mortality or infection. In this proposal, we plan to use the GPCMV model of congenital CMV infection to demonstrate "proof-of-principle" that prime-boost immunization with the conserved, essential genes of GPCMV followed by chemically killed GPCMV is protective against congenital GPCMV infection and disease and to provide the justification for pursuing more comprehensive preclinical studies of immunity and protection. The Specific Aims of this proposal are: 1. Determination of the protective efficacy of prime-boost vaccination with a trivalent DNA (consisting of DNA polymerase, helicase, and glycoprotein B genes) followed by inactivated GPCMV in the guinea pig model of congenital infection and disease, and 2. Characterization of the relative contributions to immunity and protection provided by the DNA polymerase and helicase DNAs, the glycoprotein B DNA, and the inactivated virus. The novel aspects of our proposed vaccine are both the choice of the highly conserved essential viral genes for generating T lymphocyte responses in the priming step as well as the use of a whole, killed virus in the boosting step that together can completely protect against systemic CMV challenge. PUBLIC HEALTH RELEVANCE: Congenital infection with human cytomegalovirus (HCMV) occurs in approximately 40,000 infants a year in the United States annually and is the leading viral cause of birth defects. These problems together with the severe disease that HCMV causes in solid organ and hematopoietic stem cell transplant recipients provides a strong impetus for the development of an effective vaccine. However, unlike viruses that are cleared by the host, the ability of HCMV to persist even in the presence of high levels of host immunity suggests that novel approaches are needed for the development of a vaccine against HCMV and other persistent viruses.