Viral immunomodulation and rational CMV vaccine design

描述（由申请方提供）：开发抗人巨细胞病毒（HCMV）的疫苗是一项主要的公共卫生优先事项。特别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，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有效性。然而，目前尚不清楚基于单一HCMV蛋白的疫苗是否能提供强大的长期保护，特别是对育龄妇女。HCMV疫苗的减毒活方法的一个潜在的显著优点是，此类疫苗可以引发模拟天然免疫的免疫应答，从而为宿主免疫应答的多种病毒编码靶标提供更广泛的保护。然而，这种疫苗具有建立潜伏期、在免疫抑制条件下重新激活或引起慢性HCMV相关疾病的理论风险。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。使用先天性CMV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。在上一个资助期，我们证明了重组GPCMV疫苗的有效性和安全性，该疫苗具有一个MHCI同源物块的基因组缺失。我们建议确定参与这种减毒的关键病毒基因，并验证我们的假设，即这种基因产物通过抑制NK细胞反应逃避宿主防御（目的1）。我们假设，通过细胞因子应答、CD 8+应答和抗体滴度评估，该病毒的免疫原性优于野生型病毒。一种缺失MHC I功能的重组病毒，？(MHC将在动物中评价减毒，并通过免疫测定和NK耗竭研究确认免疫机制。在前一个资助期，我们还确定了参与PKR失活的GPCMV基因。我们建议表征这种和其他PKR evasins（目的2），以产生一种不能抑制PKR反应的缺失病毒（PKR）。我们将联合收割机PKR和MHC突变结合在一个单一的减毒病毒构建体中，我们预测这将产生一种安全的、高度减毒的疫苗，具有改善的免疫原性。尽管有严格的先天免疫控制，但假设这种（MHC/（PKR）构建体引发强的、持久的保护性免疫。将通过评价和比较β MHC和β MHC/β PKR疫苗在GPCMV先天性感染模型中进一步测试该概念（目的3）。我们假设这些疫苗对野生型（对照）病毒感染的先天性感染和疾病具有同等或上级保护作用。通过在GPCMV模型中确定最佳的减毒疫苗策略，将出现具有强免疫原性和改善的安全性的HCMV基因设计疫苗的转化前景。 公共卫生相关性：感染性病原体可导致新生儿出生缺陷，在美国这些感染性疾病中最常见的是人巨细胞病毒（CMV）。在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。一种有吸引力的方法是设计一种减毒疫苗，这意味着疫苗与天然病毒本身非常相似，但无法引起疾病。然而，这种疫苗必须是安全的，因此需要获得更多关于如何修改病毒的知识，以便它非常安全，但仍然能够诱导强烈的免疫反应。这些研究使用豚鼠动物模型来产生这种新知识。这些研究将通过删除有助于其逃避免疫清除能力的基因来研究工程CMV病毒的方法，以设计一种对发育中的胎儿也具有高度保护性的安全疫苗。

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.