Attenuation of Lassa Virus Via Codon Deoptimization

几种沙粒病毒在人类中引起出血热（HF）疾病，并在其流行地区造成严重的公共卫生问题。因此，拉沙病毒（LASV）每年在西非感染数十万个体，导致与高发病率和死亡率相关的大量拉沙热（LF）病例。值得注意的是，旅行的增加导致LF病例输入到地球仪周围的非流行性大都市地区。除了对公共卫生的影响外，包括LASV在内的几种沙粒病毒还具有使其成为可信的生物防御威胁的特征。没有FDA许可的疫苗来对抗沙粒病毒感染，目前的抗沙粒病毒疗法仅限于利巴韦林的标签外使用，这只是部分有效并与副作用有关。沙粒病毒（包括LASV）在人类健康和生物防御准备中的重要性，以及对抗其感染的有限的现有医疗设备，突出了开发有效疫苗以对抗沙粒病毒引起的人类疾病的重要性。本申请的中心目标是测试基于密码子去优化（CD）的策略可用于开发安全、免疫原性、稳定和保护性的减毒活疫苗（LAV）以对抗由LASV感染引起的HF疾病的新假设。此外，我们还假设表达密码子去优化（CD）糖蛋白（GP）的重组LASV，rLASV/GPCD，将证明研究者具有有效的LASV替代物，其可以安全地用于BSL 2设施中，以促进LASV-宿主细胞相互作用的研究以及抗LASV候选治疗剂的鉴定和表征，而不需要BSL 4遏制，目前需要研究这种重要的人类生物防御病原体的活形式。我们假设的基础是：1）现有证据表明CD可用于产生减毒病毒，包括沙粒病毒，其能够提供针对野生型（WT）病毒攻击的保护。2)CD LAV具有与WT病毒相同的免疫原性，但CD LAV基因组内的大量（> 100 s）沉默突变使其不可能回复到强毒株。3)CD LAV，包括沙粒病毒，能够在FDA批准用于疫苗生产的Vero细胞中生长至与疫苗生产相容的高滴度。4)CD重组LASV的产生可以使用我们最先进的反向遗传学技术快速实现。为了检验我们的假设，我们将首先使用原型LCMV系统，该系统为我们提供了一种可安全用于BSL 2设施的优良参考病毒，以使用充分表征的LCMV感染小鼠模型来检测CD LASV GP是否可将强毒株转化为减毒（Aim 1）和稳定（Aim 2）形式，该形式仍能诱导保护性免疫（Aim 3）。我们将使用这些知识来产生rLASV/GPCD，其作为LAV的潜力将使用完善的LF疾病豚鼠模型进行评价（目的4）。

Several arenaviruses cause hemorrhagic fever (HF) disease in humans and pose a serious public health problem in their endemic regions. Thus, Lassa virus (LASV) infects several hundred thousand individuals yearly in West Africa resulting in a high number of Lassa fever (LF) cases associated with high morbidity and mortality. Notably, increased traveling has led to the importation of LF cases into non-endemic metropolitan areas around the globe. Besides their impact in public health, several arenaviruses, including LASV, have features that make them a credible biodefense threat. There are no FDA-licensed vaccines to combat arenavirus infections and current anti-arenaviral therapy is limited to the off-label use of ribavirin, which is only partially effective and associated with side effects. The significance of arenaviruses, including LASV, in human health and biodefense readiness, together with the limited existing armamentarium to combat their infections, highlight the importance of developing effective vaccines to combat arenavirus induced disease in humans. The central goal of this application is to test the novel hypothesis that a codon deoptimized (CD)-based strategy can be used for the development of a safe, immunogenic, stable and protective live-attenuated vaccines (LAV) to combat HF disease caused by LASV infection. Moreover, we also hypothesize that a recombinant LASV expressing a codon deoptimized (CD) glycoprotein (GP), rLASV/GPCD, will prove investigators with a valid LASV surrogate that could be safely used in BSL2 facilities to facilitate the investigation of LASV-host cell interactions as well as the identification and characterization of anti- LASV candidate therapeutics without the need of BSL4 containment, currently needed to work with live forms of this important human biodefense pathogen. The bases for our hypothesis are: 1) Existing evidence that CD can be used to generate attenuated viruses, including arenaviruses, that are able to provide protection against a challenge with wild-type (WT) virus. 2) CD LAV have identical immunogenic properties than WT virus, but the large number (>100s) of silent mutations within the genome of CD LAV makes impossible its reversion to a virulent strain. 3) CD LAV, including arenaviruses, are able to grow to high titers, compatible with vaccine production, in FDA-approved Vero cells for vaccine production. 4) Generation of CD recombinant LASV can be rapidly achieved using our state-of-the-art reverse genetics technologies. To test our hypothesis, we will first use the prototypic LCMV system that provides us with an excellent reference virus that can be safely used in BSL2 facilities to test whether a CD LASV GP can convert a virulent strain into an attenuated (Aim 1) and stable (Aim 2) form still able to induce protective immunity (Aim 3) using the well characterized mouse model of LCMV infection. We will use this knowledge to generate a rLASV/GPCD whose potential as LAV will be evaluated using the well-established guinea pig model of LF disease (Aim 4).