Development of a novel vaccine against congenital cytomegalovirus infection

描述（由申请人提供）：在美国，每年大约有40,000名婴儿先天性感染人类巨细胞病毒（HCMV），这是导致出生缺陷的主要病毒原因。这些问题加上HCMV在实体器官和造血干细胞移植受者中引起的严重疾病，为开发有效疫苗提供了强大的动力。然而，与被宿主清除的病毒不同，HCMV即使在宿主免疫水平很高的情况下也能持续存在，这表明开发疫苗需要新的方法。由于小鼠CMV （MCMV）模型的结果表明，针对免疫优势抗原的CD8+ T细胞在限制病毒复制方面是无效的，我们推测使用这些抗原的疫苗接种最多可能引起的免疫力并不比自然感染期间产生的免疫力大得多：由于病毒持续存在并可能导致复发性感染和先天性传播，这种保护最终是不完整的。我们假设在cmv中高度保守的必需病毒蛋白可能代表了一类新的T细胞靶标。其基本原理是，这些基因必须表达才能进行病毒复制，而维持蛋白质活性所需的高氨基酸守恒限制了突变导致的免疫逃逸。由于这些抗原在感染HCMV或MCMV时大多是亚显性的，我们假设T细胞对这些抗原的启动可能比自然感染产生更大的保护性免疫。为此，我们最近表明，使用MCMV的两个保守的必需基因（DNA聚合酶M54和解旋酶M105）中的任何一个进行DNA免疫，可以保护小鼠免受随后的强毒病毒的亚致死攻击。我们还开发了一种DNA启动-整体灭活病毒增强策略，可引起CD8+ T细胞反应和强中和抗体，我们证明了这种启动-增强疫苗可完全保护机体免受全身病毒攻击，并显著保护机体免受粘膜病毒攻击。然而，由于MCMV不会在子宫内引起感染，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防止死亡或感染。在本研究中，我们计划使用先天性巨细胞病毒感染的GPCMV模型来证明“原理证明”，即使用GPCMV保守的必需基因进行初始强化免疫，然后化学杀死GPCMV，可以预防先天性GPCMV感染和疾病，并为进行更全面的免疫和保护的临床前研究提供依据。本建议的具体目标是：1。在先天性感染和疾病的豚鼠模型中，用三价DNA（由DNA聚合酶、解旋酶和糖蛋白B基因组成）接种灭活GPCMV后，确定初级加强疫苗的保护效果；鉴定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。我们提出的疫苗的新颖之处在于在启动阶段选择高度保守的必需病毒基因来产生T淋巴细胞反应，以及在增强阶段使用一个完整的、被杀死的病毒，两者一起可以完全防止系统性巨细胞病毒的攻击。公共卫生相关性：美国每年约有40,000名婴儿先天性感染人类巨细胞病毒（HCMV），这是导致出生缺陷的主要病毒原因。这些问题加上HCMV在实体器官和造血干细胞移植受者中引起的严重疾病，为开发有效疫苗提供了强大的动力。然而，与被宿主清除的病毒不同，HCMV即使在宿主免疫水平很高的情况下也能持续存在，这表明开发针对HCMV和其他持久性病毒的疫苗需要新的方法。

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.