Optimizing Immunity and Maternal Host Defense Against Congenital Cytomegalovirus Infection

摘要 女性巨细胞病毒（HCMV）有效预防疫苗的研制 生育年龄是一个主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相关性。减毒活疫苗可能会引起更广泛的 免疫应答的所有谱，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， 检验关于预防先天性感染的安全有效疫苗的最佳设计的假设。我们最近 发现包含由GPCMV gH、gL和GP 129、131组成的五聚体复合物（PC）， 133（HCMV PC的同源物），使得能够产生活的减毒疫苗，该减毒疫苗针对 先天性GPCMV传播，但疫苗的毒性不可接受。在平行研究中，我们观察到 具有孕前免疫力的妊娠母鼠对原型GPCMV毒株22122的病毒攻击 与异源的“临床分离株”，CIDMTR菌株，导致母体再感染和先天性 新挑战菌株的传播。这些发现，以及在HCMV感染的临床观察， 妇女，建议疫苗可能需要比“自然免疫”更好地发挥作用，以防止 再感染我们假设，一个合理设计的，PC完整，但禁用感染性单周期（DISC） 与亚单位gB相比，疫苗将提供上级保护性免疫，包括防止再感染， “天然”免疫力在目标1中，我们通过比较经以下方法减毒的PC阳性疫苗来检验这一假设： GPCV编码的MHC I同源物和病毒蛋白激酶R（PKR）evasin的缺失，与MF 59- 佐剂化的重组gB。我们将研究在原发性母体感染后， 和再次感染具有孕前免疫力的母鼠， 怀孕我们将通过开发DISC疫苗，使用PC完整的 具有与基本病毒蛋白融合的不稳定结构域的病毒。与临床试验中的HCMV疫苗相比， 我们假设DISC疫苗中的病毒复制被阻断在DNA复制的下游， 进展到晚期基因表达并产生更广泛的免疫原性蛋白质。目标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 设计我们将检验GPCMV编码第二个PKR拮抗剂的假设，消除该拮抗剂将导致 有望产生安全有效的疫苗。此外，我们将在疫苗中过表达dsRNA， 目的是提高安全性和免疫原性。将评价疫苗的减毒情况， 免疫原性和优化的疫苗将用于鉴定保护的免疫相关性，包括非免疫相关性。 IgG的中和功能。我们的研究将探讨一个总体假设，即由一种 巨细胞病毒疫苗可能上级“自然”孕前免疫，这是巨细胞病毒疫苗的一个高度相关问题。

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.