Guinea pig cytomegalovirus as a model for vaccines that target endocytic entry

描述(申请人提供)：人类巨细胞病毒(HCMV)是美国出生缺陷的主要感染原因。开发预防巨细胞病毒相关出生缺陷的疫苗被认为是国家优先事项。两种候选疫苗正处于第二阶段试验--汤恩减毒活疫苗和GB/MF59蛋白亚单位疫苗。两者都被证明能诱导中和抗体，阻止HCMV进入成纤维细胞。然而，最近的研究表明，HCMV通过一条与感染成纤维细胞的途径截然不同的“内吞”途径进入内皮细胞、上皮细胞和树突状细胞。我们发现，与成纤维细胞相比，人免疫血清在中和HCMV进入上皮细胞方面的中和活性平均高出48倍，这表明基于成纤维细胞的检测只揭示了HCMV中和反应的一小部分。此外，Towne和GB/MF59疫苗在诱导中和HCMV进入上皮细胞的抗体方面都远远低于自然感染(分别是自然感染的28倍和15倍)(崔等人，疫苗，2008年)。三种巨细胞病毒蛋白UL128、UL130和UL131是胞内进入所必需的，令人信服的证据表明，这三种蛋白都含有重要的表位，可诱导抗体中和胞内进入。这些蛋白质目前正在研究中，作为潜在的免疫原，用于诱导中和抗体，阻止病毒进入上皮细胞或内皮细胞。然而，虽然可以在体外测量诱导HCMV中和抗体的能力，但评估疫苗在预防宫内传播或胎儿发病方面的有效性将需要动物模型。由于巨细胞病毒具有严格的物种特异性，动物模型必须利用每个物种特有的相关巨细胞病毒。豚鼠巨细胞病毒(GPCMV)提供了唯一可以评估疫苗预防宫内传播和/或胎儿发病能力的小型动物模型。该项目的目的是确定：(1)GPCMV是否可以进入上皮细胞或内皮细胞并在其中复制；(2)自然感染是否会引起中和抗体，特异性地阻断内/上皮细胞进入，以及内/上皮细胞进入是否通过不同于成纤维细胞进入的机制发生。这些问题的答案将有助于确定基于UL128、UL130或UL131的免疫策略是否可以在使用等效GPCMV蛋白作为免疫原的豚鼠先天性感染模型中进行评估。如果是这样的话，豚鼠模型可以提供重要的疗效数据，有助于在人体试验之前指导新疫苗的设计。公共卫生相关性：在美国，人类巨细胞病毒是导致出生缺陷的主要感染原因。我们最近发现，目前的两种候选疫苗未能诱导出高水平的抗体，阻止病毒进入上皮细胞。根据这些结果，正在研究新的疫苗策略。该项目旨在确定豚鼠巨细胞病毒是否可以用于评估新的疫苗策略，这些策略旨在诱导阻断上皮细胞进入的抗体。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is the major infectious cause of birth defects in the United States. Development of a vaccine to prevent HCMV-associated birth defects has been deemed a national priority. Two candidate vaccines are in Phase II trials - the Towne live attenuated vaccine and the gB/MF59 protein subunit vaccine. Both have been shown to elicit neutralizing antibodies that block HCMV entry into fibroblasts. However, recent work has revealed that HCMV enters endothelial, epithelial, and dendritic cells by an "endocytic" pathway that is quite distinct from the pathway used to infect fibroblasts. We found that the neutralizing activities of human immune sera are on average 48-fold more potent in neutralizing HCMV entry into epithelial cells compared to fibroblasts, suggesting that fibroblast-based assays reveal only a minor component of the HCMV-neutralizing response. Moreover, both Towne and gB/MF59 vaccines fell far short (28- and 15-fold, respectively) of natural infection in eliciting antibodies that neutralize HCMV entry into epithelial cells (Cui et al., Vaccine, 2008). Three HCMV proteins, UL128, UL130, and UL131 are essential for endocytic entry and compelling evidence suggests that all three proteins contain epitopes important for inducing antibodies that neutralize endocytic entry. These proteins are now under study as potential immunogens for induction of neutralizing antibodies that block viral entry into epithelial or endothelial cells. However, while the ability to elicit HCMV neutralizing antibodies can be measured in vitro, evaluating vaccine efficacy in preventing in utero transmission or fetal pathogenesis will require animal models. As cytomegaloviruses are strictly species-specific, animal models must utilize related cytomegaloviruses native to each species. Guinea pig cytomegalovirus (GPCMV) provides the only small animal model in which the ability of vaccines to protect against in utero transmission and/or fetal pathogenesis can be evaluated. The aims of this project are to determine: (1) if GPCMV can enter and replicate in epithelial or endothelial cells; and (2) if natural infection elicits neutralizing antibodies that specifically block endo/epithelial cell entry and if endo/epithelial entry occurs by a mechanism different from fibroblast entry. The answers to these questions will help determine if immunization strategies based on UL128, UL130, or UL131 can be evaluated in the guinea pig congenital infection model using the equivalent GPCMV proteins as immunogens. If so, the guinea pig model could provide important efficacy data that would help guide the design of new vaccines prior to human trials. PUBLIC HEALTH RELEVANCE: Human cytomegalovirus is the major infectious cause of birth defects in the United States. We recently found that two current candidate vaccines fail to elicit high levels of antibodies that block viral entry into epithelial cells. Based on these results, new vaccines strategies are under investigation. This project aims to determine if guinea pig cytomegalovirus can be used to evaluate new vaccine strategies that are designed to elicit antibodies that block epithelial cell entry.