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

描述（由申请人提供）：人巨细胞病毒（HCMV）是美国出生缺陷的主要感染原因。研制预防HCMV相关出生缺陷的疫苗已被视为国家优先事项。两种候选疫苗正在进行II期试验-Towne减毒活疫苗和gB/MF 59蛋白亚单位疫苗。两者都被证明可以引发中和抗体，阻断HCMV进入成纤维细胞。然而，最近的研究表明，HCMV进入内皮细胞，上皮细胞和树突状细胞的“内吞”途径，这是非常不同的途径，用于感染成纤维细胞。我们发现，人免疫血清的中和活性在中和HCMV进入上皮细胞方面平均比成纤维细胞强48倍，这表明基于成纤维细胞的测定仅揭示了HCMV中和反应的一小部分。此外，Towne和gB/MF 59疫苗在引发中和HCMV进入上皮细胞的抗体方面远远低于天然感染（分别为28倍和15倍）（Cui等人，Vaccine，2008）。三种HCMV蛋白，UL 128、UL 130和UL 131对于内吞进入是必需的，并且令人信服的证据表明，所有三种蛋白含有对于诱导中和内吞进入的抗体重要的表位。目前正在研究这些蛋白质作为潜在的免疫原，用于诱导中和抗体，阻断病毒进入上皮细胞或内皮细胞。然而，虽然可以在体外测量引发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功效将需要动物模型。由于巨细胞病毒具有严格的种属特异性，因此动物模型必须使用每个种属的相关巨细胞病毒。豚鼠巨细胞病毒（GPCMV）提供了唯一的小动物模型，其中疫苗的能力，以防止在子宫内传播和/或胎儿发病机制可以进行评估。本项目的目的是确定：（1）GPCMV是否可以进入上皮细胞或内皮细胞并在其中复制;（2）天然感染是否产生特异性阻断内皮/上皮细胞进入的中和抗体，以及内皮/上皮细胞进入是否通过与成纤维细胞进入不同的机制发生。这些问题的答案将有助于确定是否可以使用等效的GPCMV蛋白作为免疫原在豚鼠先天性感染模型中评价基于UL 128、UL 130或UL 131的免疫策略。如果是这样的话，豚鼠模型可以提供重要的有效性数据，这将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：人类巨细胞病毒是美国出生缺陷的主要传染性原因。我们最近发现，目前的两种候选疫苗未能引发高水平的抗体，阻止病毒进入上皮细胞。根据这些结果，正在研究新的疫苗战略。该项目旨在确定豚鼠巨细胞病毒是否可用于评估新的疫苗策略，这些策略旨在引发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.