Optimizing Immunity and Maternal Host Defense Against Congenital Cytomegalovirus Infection

摘要 人巨细胞病毒(HCMV)预防疫苗的研制 生育年龄是一个重要的公共卫生优先事项。亚单位疫苗正在进行临床试验，但存在不确定性 关于最佳平台和保护的关联性。减毒活疫苗可能会导致更广泛的 一系列免疫反应，但在安全性和免疫原性之间取得平衡是具有挑战性的。 这项正在进行的合作的长期目标是使用豚鼠巨细胞病毒(GPCMV)模型来 关于优化设计安全有效的预防先天性感染疫苗的假设测试。我们最近 发现包含由GPCMV Gh，Gl和GP129,131组成的五聚体复合体(PC) 133(人巨细胞病毒PC的同源物)，能够产生具有保护作用的减毒活疫苗 GPCMV是先天性传播的，但疫苗的毒力令人无法接受。在平行研究中，我们观察到 对典型的巨细胞病毒22122株有先入性免疫的怀孕母鸡的病毒挑战 CIDMTR株是一种异源的临床分离株，导致母体再感染和先天性 新挑战毒株的传播。这些发现以及巨细胞病毒感染的临床观察 女性建议，疫苗的效果可能需要比自然免疫力更好，以防止感染 再感染。我们假设，一个设计合理、PC完好无损但禁用的感染性单周期(盘) 疫苗将提供更好的保护性免疫，包括防止再次感染，与亚单位gb和 ‘自然’豁免权。在目标1中，我们通过比较PC阳性疫苗，减毒后的疫苗来检验这一假设 GPCMV编码的MHC I同源物和病毒蛋白激酶R(PKR)evasin的缺失，与MF59- 佐剂重组GB。我们将检查两种主要母体感染后的保护程度。 在与异源毒株攻击后，具有先天免疫的水坝再次感染 怀孕了。我们将在AIM 2中进一步优化CMV疫苗，使用完整的PC开发圆盘疫苗 具有不稳定结构域的病毒与基本病毒蛋白融合。与临床试验中的巨细胞病毒疫苗相比， 我们假设病毒复制受阻于DNA复制下游的圆盘疫苗将 晚期基因表达的进展，并产生更广泛的免疫原性蛋白。AIM 3将评估 删除宿主细胞防御的病毒拮抗剂，如蛋白激酶R(PKR)是否有助于疫苗 设计。我们将测试GPCMV编码第二个PKR拮抗剂的假设，消除该拮抗剂将 有望产生一种安全有效的疫苗。此外，我们将在疫苗中过度表达dsrna。 建造的目的是提高安全性和免疫原性。将评估疫苗的减毒效果和 将使用免疫原性和优化疫苗来确定保护的免疫相关因素，包括非 免疫球蛋白的中和功能。我们的研究将探索一个重要的假设，即 巨细胞病毒疫苗可能优于与巨细胞病毒疫苗高度相关的“自然”先验免疫。

ABSTRACT Development of an effective preventative vaccine against human cytomegalovirus (HCMV) for women of childbearing age is a major public health priority. Subunit vaccines are in clinical trials, but uncertainty exists about optimal platforms and correlates of protection. Live, attenuated vaccines may induce a broader repertoire of immune responses, but striking the balance between safety and immunogenicity is challenging. The long-term goal of this ongoing collaboration is to use the guinea pig cytomegalovirus (GPCMV) model to test hypotheses about optimal design of safe and effective vaccines against congenital infection. We recently discovered that inclusion of a pentameric complex (PC), consisting of the GPCMV gH, gL and GP129, 131 and 133 (homologs of the HCMV PC), enabled generation of a live, attenuated vaccine that was protective against congenital GPCMV transmission, but the vaccine was unacceptably virulent. In parallel studies we observed that viral challenge of pregnant dams with preconception immunity to the prototypical GPCMV strain 22122 with a heterologous `clinical isolate', the CIDMTR strain, resulted in both maternal re-infection and congenital transmission of the new challenge strain. These findings, as well as clinical observations in HCMV-infected women, suggest that a vaccine may need to perform better than `natural immunity' in order to protect against reinfection. We hypothesize that a rationally designed, PC-intact, but disabled infectious single cycle (DISC) vaccine will provide superior protective immunity, including against re-infection, compared to subunit gB and to `natural' immunity. In Aim 1, we test this hypothesis by comparing a PC-positive vaccine, attenuated by deletions of GPCMV-encoded MHC I homologs and a viral protein kinase R (PKR) evasin, versus MF59- adjuvanted recombinant gB. We will examine the magnitude of protection after both primary maternal infection and re-infection of dams with preconception immunity following challenge with a heterologous strain during pregnancy. We will further optimize CMV vaccines in Aim 2 by developing DISC vaccines, using a PC-intact virus with destabilizing domains fused to essential viral proteins. In contrast to HCMV vaccines in clinical trials, we hypothesize that DISC vaccines in which viral replication is blocked downstream of DNA replication will progress to late gene expression and produce a broader array of immunogenic proteins. Aim 3 will evaluate whether deletion of viral antagonists of host cell defenses such as protein kinase R (PKR) can aid in vaccine design. We will test the hypothesis that GPCMV encodes a second PKR antagonist, elimination of which would be expected to generate a safe and effective vaccine. In addition, we will overexpress dsRNA in the vaccine construct with the aim of improving safety and immunogenicity. Vaccines will be evaluated for attenuation and immunogenicity and optimized vaccines will be used to identify immune correlates of protection, including non- neutralizing functions of IgG. Our studies will explore the overarching hypothesis that immunity conferred by a CMV vaccine can be superior to `natural' preconception immunity, a highly relevant issue for HCMV vaccines.