Frese Michael, Lee Eva, Larena Maximilian, Lim Pek Siew, Rao Sudha, Matthaei Klaus I, Khromykh Alexander, Ramshaw Ian, Lobigs Mario
John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.
J Virol. 2014 Feb;88(4):2056-70. doi: 10.1128/JVI.03051-13. Epub 2013 Dec 4.
Infectious clone technologies allow the rational design of live attenuated viral vaccines with the possibility of vaccine-driven coexpression of immunomodulatory molecules for additional vaccine safety and efficacy. The latter could lead to novel strategies for vaccine protection against infectious diseases where traditional approaches have failed. Here we show for the flavivirus Murray Valley encephalitis virus (MVEV) that incorporation of the internal ribosome entry site (IRES) of Encephalomyocarditis virus between the capsid and prM genes strongly attenuated virulence and that the resulting bicistronic virus was both genetically stable and potently immunogenic. Furthermore, the novel bicistronic genome organization facilitated the generation of a recombinant virus carrying an beta interferon (IFN-β) gene. Given the importance of IFNs in limiting virus dissemination and in efficient induction of memory B and T cell antiviral immunity, we hypothesized that coexpression of the cytokine with the live vaccine might further increase virulence attenuation without loss of immunogenicity. We found that bicistronic mouse IFN-β coexpressing MVEV yielded high virus and IFN titers in cultured cells that do not respond to the coexpressed IFN. However, in IFN response-sufficient cell cultures and mice, the virus produced a self-limiting infection. Nevertheless, the attenuated virus triggered robust innate and adaptive immune responses evidenced by the induced expression of Mx proteins (used as a sensitive biomarker for measuring the type I IFN response) and the generation of neutralizing antibodies, respectively. IMPORTANCE The family Flaviviridae includes a number of important human pathogens, such as Dengue virus, Yellow fever virus, Japanese encephalitis virus, West Nile virus, and Hepatitis C virus. Flaviviruses infect large numbers of individuals on all continents. For example, as many as 100 million people are infected annually with Dengue virus, and 150 million people suffer a chronic infection with Hepatitis C virus. However, protective vaccines against dengue and hepatitis C are still missing, and improved vaccines against other flaviviral diseases are needed. The present study investigated the effects of a redesigned flaviviral genome and the coexpression of an antiviral protein (interferon) on virus replication, pathogenicity, and immunogenicity. Our findings may aid in the rational design of a new class of well-tolerated and safe vaccines.
感染性克隆技术允许合理设计减毒活病毒疫苗，并有可能通过疫苗驱动共表达免疫调节分子来提高疫苗的安全性和有效性。后者可能会带来针对传统方法失效的传染病的新型疫苗保护策略。在此，我们针对黄病毒墨累谷脑炎病毒（MVEV）表明，在衣壳蛋白和prM基因之间插入脑心肌炎病毒的内部核糖体进入位点（IRES）可显著减弱毒力，并且由此产生的双顺反子病毒在基因上是稳定的且具有强大的免疫原性。此外，这种新型的双顺反子基因组结构有助于产生携带β干扰素（IFN-β）基因的重组病毒。鉴于IFN在限制病毒传播以及有效诱导记忆B细胞和T细胞抗病毒免疫方面的重要性，我们推测该细胞因子与活疫苗的共表达可能会进一步增强毒力减弱效果而不丧失免疫原性。我们发现，共表达小鼠IFN-β的双顺反子MVEV在对共表达的IFN无反应的培养细胞中产生了高病毒滴度和IFN滴度。然而，在对IFN有足够反应的细胞培养物和小鼠中，该病毒产生了自限性感染。尽管如此，减毒病毒分别通过诱导Mx蛋白表达（用作测量I型IFN反应的敏感生物标志物）和产生中和抗体，引发了强大的先天性和适应性免疫反应。重要性黄病毒科包括许多重要的人类病原体，如登革病毒、黄热病毒、日本脑炎病毒、西尼罗河病毒和丙型肝炎病毒。黄病毒在各大洲感染大量人群。例如，每年有多达1亿人感染登革病毒，1.5亿人患有丙型肝炎病毒慢性感染。然而，针对登革热和丙型肝炎的保护性疫苗仍然缺失，并且需要改进针对其他黄病毒疾病的疫苗。本研究调查了重新设计的黄病毒基因组以及抗病毒蛋白（干扰素）的共表达对病毒复制、致病性和免疫原性的影响。我们的发现可能有助于合理设计一类耐受性良好且安全的新型疫苗。
