Viral immunomodulation and rational CMV vaccine design

描述（由申请人提供）：人类巨细胞病毒（HCMV）疫苗的开发是一项重要的公共卫生优先事项。尤其紧迫的是需要保护新生儿免受先天性HCMV感染的破坏性后果，包括智力迟钝、脑瘫和耳聋。最近，一种基于重组HCMV糖蛋白B （gB）的亚单位疫苗在临床试验中显示出疗效。然而，目前尚不清楚基于单一HCMV蛋白的疫苗是否会提供强大的长期保护，特别是对育龄妇女。HCMV减毒活疫苗的一个潜在显著优势是，这种疫苗可以引发模仿自然免疫的免疫反应，从而为宿主免疫反应的各种病毒编码靶点提供更广泛的保护。然而，这类疫苗在理论上有建立潜伏期、在免疫抑制条件下重新激活或引起慢性hcmv相关疾病的风险。一种产生免疫原性但安全的活疫苗的方法是删除破坏宿主防御感染机制的病毒基因。利用先天性巨细胞病毒感染的豚鼠模型，我们提出通过靶向破坏两个关键宿主防御的病毒基因来测试这种方法：MHC I同源基因（NK细胞逃避蛋白）和蛋白激酶R （PKR）逃避蛋白。在之前的资助期内，我们证明了重组GPCMV疫苗的有效性和安全性，该疫苗具有MHC I同源物的基因组缺失块。我们建议确定参与这种衰减的关键病毒基因，并验证我们的假设，即该基因产物通过抑制NK细胞反应来逃避宿主防御（目的1）。我们假设，通过细胞因子反应、CD8+反应和抗体滴度评估，该病毒将表现出优于野生型病毒的免疫原性。重组病毒删除MHC I功能，？（MHC）将在动物中进行衰减评估，免疫机制将通过免疫测定和NK消耗研究来证实。在之前的资助期内，我们还发现了一个参与PKR失活的GPCMV基因。我们建议对这种和其他PKR规避进行表征（目的2），以产生一种不能抑制PKR反应的缺失病毒（PKR）。我们将把PKR和MHC突变结合在一个单一的减毒病毒结构中，我们预测这将产生一种安全的、高度减毒的疫苗，并提高免疫原性。尽管有严格的先天免疫控制，这种(MHC/(PKR)结构被认为可以引发强大的、持久的保护性免疫。通过评估和比较(MHC和(MHC/(PKR)疫苗（目标3），将在GPCMV先天性感染模型中进一步验证这一概念。我们假设这些疫苗将显示出与野生型（对照）病毒感染相同或更好的预防先天性感染和疾病的保护作用。通过在GPCMV模型中确定最佳减毒疫苗策略，基因设计具有强免疫原性和更高安全性的HCMV疫苗的转化前景将出现。

DESCRIPTION (provided by applicant): The development of a vaccine against human cytomegalovirus (HCMV) is a major public health priority. Of particular urgency is the need to protect newborn infants from the devastating consequences of congenital HCMV infection, including mental retardation, cerebral palsy, and deafness. Recently, a subunit vaccine based on recombinant HCMV glycoprotein B (gB) showed efficacy in a clinical trial. However, it is not clear that a vaccine based on a single HCMV protein will provide robust, long-term protection, particularly for women in their childbearing years. A potential significant advantage of the live, attenuated approach to HCMV vaccines is that such vaccines could elicit an immune response that mimics natural immunity and hence provide broader protection to a wide variety of virus-encoded targets of the host immune response. However, such vaccines carry the theoretical risk of establishing latency, reactivating under conditions of immune suppression, or causing chronic HCMV-associated diseases. An approach to generate an immunogenic, yet safe live vaccine is the deletion of viral genes that subvert host defense mechanisms to infection. Using the guinea pig model of congenital CMV infection, we propose to test this approach by targeting viral genes that subvert two key host defenses: MHC I homolog genes (NK cell evasins) and protein kinase R (PKR) evasins. In the previous funding period, we demonstrated efficacy and safety of a recombinant GPCMV vaccine which had a genomic deletion of a block of MHC I homologs. We propose to identify the key viral gene involved in this attenuation and to test our hypothesis that this gene product evades host defenses by inhibition of the NK cell response (aim 1). We hypothesize this virus will demonstrate superiority immunogenicity over wild-type virus, as assessed by cytokine response, CD8+ response, and antibody titer. A recombinant virus deleted of the MHC I function, ?(MHC, will be evaluated for attenuation in animals, and immune mechanisms will be confirmed by immune assays and NK depletion studies. In the previous funding period we have also identified a GPCMV gene involved in the inactivation of PKR. We propose to characterize this and other PKR evasins (aim 2) toward the goal of generating a deletion virus incapable of inhibiting the PKR response ((PKR). We will combine PKR and MHC mutations in a single attenuated virus construct that we predict will yield a safe, highly attenuated vaccine with improved immunogenicity. Despite tight innate immune control, this (MHC/(PKR construct is hypothesized to elicit strong, long-lasting protective immunity. This concept will be further tested in the GPCMV congenital infection model by evaluating and comparing the (MHC and (MHC/(PKR vaccines (aim 3). We hypothesize these vaccines will demonstrate equal or superior protection to infection with wild-type (control) virus against congenital infection and disease. By identifying optimal attenuated vaccine strategies in the GPCMV model, the translational prospect of genetically designing a vaccine for HCMV that has strong immunogenicity and improved safety will emerge. PUBLIC HEALTH RELEVANCE: Infectious agents can cause birth defects in newborn infants, and the most common of these infectious diseases in the United States is human cytomegalovirus (CMV). A vaccine given to a woman before pregnancy would likely prevent CMV from causing injury to the fetus and newborn infant. An attractive approach is to design a vaccine that is attenuated, meaning that the vaccine is very similar to the natural virus itself, but unable to cause disease. However, such a vaccine must be safe, so there needs to be more knowledge gained about how to modify the virus so that it is very safe, but still able to induce a strong immune response. These studies use an animal model in guinea pigs to generate this new knowledge. These studies will examine ways to engineer the CMV virus, by deleting genes that contribute to its ability to evade immune clearance, in order to design a safe vaccine that is also highly protective for the developing fetus.