Viral immunomodulation and rational CMV vaccine design

描述(由申请人提供)：人类巨细胞病毒(HCMV)疫苗的开发是一项主要的公共卫生优先事项。尤其迫切的是需要保护新生儿免受先天性巨细胞病毒感染的破坏性后果，包括智力迟缓、脑性瘫痪和耳聋。最近，一种基于重组人巨细胞病毒糖蛋白B(GB)的亚单位疫苗在临床试验中显示出效果。然而，目前还不清楚基于单一HCMV蛋白的疫苗是否能提供强有力的长期保护，特别是对育龄妇女。活的、减毒的巨细胞病毒疫苗的一个潜在的显著优势是，这种疫苗可以激发模拟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目标提供更广泛的保护。然而，这种疫苗在理论上存在建立潜伏期、在免疫抑制条件下重新激活或导致慢性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对感染的防御机制的病毒基因。利用先天性巨细胞病毒感染的豚鼠模型，我们建议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evasins)和蛋白激酶R(PKR)evasins。在之前的资助期间，我们展示了一种重组GPCMV疫苗的有效性和安全性，该疫苗具有一段MHC I同源物的基因组缺失。我们建议确定与这种衰减有关的关键病毒基因，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(目标1)。我们推测，通过细胞因子反应、CD8+反应和抗体滴度的评估，该病毒将表现出比野生型病毒更好的免疫原性。一种缺失MHC I功能的重组病毒(MHC)将在动物身上进行减毒试验，免疫机制将通过免疫试验和NK耗竭研究得到证实。在之前的资助期间，我们还发现了一个与PKR失活有关的GPCMV基因。我们建议表征这个和其他PKR evasins(目标2)，以达到产生一种不能抑制PKR反应((PKR))的缺失病毒的目标。我们将在一个单一的减毒病毒结构中结合PKR和MHC突变，我们预测将产生一种安全的、高度减毒的疫苗，具有更好的免疫原性。尽管有严格的先天性免疫控制，但这种(MHC/(PKR)结构被认为可以诱导强大的、持久的保护性免疫。这一概念将通过评估和比较(MHC和(MHC/(PKR)疫苗(目标3))在GPCMV先天性感染模型中得到进一步测试。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保护作用，以防止先天性感染和疾病。通过在GPCMV模型中确定最优的减毒疫苗策略，从基因上设计一种免疫原性强、安全性提高的巨细胞病毒疫苗的前景将会出现。 公共卫生相关性：感染源可导致新生儿出生缺陷，而在美国，这些传染病中最常见的是人类巨细胞病毒(CMV)。怀孕前给妇女接种疫苗可能会防止CMV对胎儿和新生儿造成伤害。一个有吸引力的方法是设计一种减毒疫苗，这意味着疫苗与自然病毒本身非常相似，但不会导致疾病。然而，这样的疫苗必须是安全的，所以需要获得更多关于如何修改病毒的知识，使其非常安全，但仍能诱导强烈的免疫反应。这些研究使用了豚鼠的动物模型来产生这种新的知识。这些研究将研究改造CMV病毒的方法，通过删除有助于其逃避免疫清除的基因，以设计一种安全的疫苗，同时也对发育中的胎儿具有高度保护作用。

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.