A General Molecular Strategy for Attenuation of Human Pathogenic Arenaviruses

 描述（由申请方提供）：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，旧世界沙粒病毒（OWA）拉沙（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。此外，有证据表明，世界范围内分布的OWA LCMV是一种被忽视的临床重要的人类病原体。此外，包括LASV和LCMV在内的几种沙粒病毒构成了可信的生物防御威胁。现有的抗沙粒病毒疗法仅限于利巴韦林的标签外使用，其仅部分有效，并且没有FDA许可的或目前处于临床试验中的沙粒病毒疫苗。然而，MOPV/LASV抑制剂（ML 29）是LASV的候选减毒活疫苗（L-AttV），其在动物模型中显示出有希望的结果。然而，与许多其他传统的L-AttV一样，ML 29减毒的机制仍然未知，这引起了对ML 29响应于额外突变的表型稳定性的担忧。本申请的中心目标是检验我们可以通过用遗传上定义的合成S样IGR（Ssyn）替换其L IGR将致病性OWA转化为具有L-AttV特征的减毒形式的假设。我们的假设得到了以下结果的支持：1）我们可以拯救S和L基因组片段具有相同S-IGR的rLCMV（IGR/S-S），并且发现它在体内是高度减毒的。2)用rLCMV（IGR/S-S）免疫的小鼠被保护免受野生型（WT）LCMV的致死攻击。3)我们已经获得证据表明，S IGR内的高度序列可塑性与病毒活力相容，这支持了在L区段中用合成的S IGR（Ssyn）工程化重组沙粒病毒作为沙粒病毒减毒的一般分子策略的可行性。为了验证我们的假设，我们建议完成以下具体目标：目标1。表征rLCMV（IGR/S-Ssyn）：我们已经拯救了rLCMV（IGR/S-Ssyn），其中合成的S IGR（Ssyn）取代了L-IGR。我们将在培养的细胞中表征rLCMV（IGR/S-Ssyn），并使用LCMV感染的小鼠模型测试rLCMV（IGR/S-Syn）显示L-AttV的关键特征的假设。目标二：生成和表征rLASV（IGR/S-Ssyn）：我们将生成rLASV（IGR/S-Ssyn）并检验以下假设：其在体内减毒，但能够在完善的LASV感染豚鼠模型中诱导针对WT LASV致死性攻毒的保护性免疫。目标3。检查rLCMV（IGR/S-Ssyn）的稳定性：我们将检验rLCMV（IGR/S-Ssyn）在不同生长条件下增殖期间遗传稳定的假设。该申请的成功完成将揭示沙粒病毒减毒的一般分子策略，其可以促进开发L-AttV以对抗人类致病性沙粒病毒的新策略。

 DESCRIPTION (provided by applicant): Several arenaviruses cause hemorrhagic fever (HF) disease in humans and pose a serious public health problem in their endemic regions. Thus, the Old World arenavirus (OWA) Lassa (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. Moreover, evidence indicates that the worldwide-distributed OWA LCMV is a neglected clinically important human pathogen. In addition, several arenaviruses including LASV and LCMV pose a credible biodefense threat. Existing anti-arenaviral therapy is limited to an off-label use of ribavirin that is only partially effective, and there are no FDA-licensed, or currently in clinical trials, arenavirus vaccines. However, the MOPV/LASV reassortant (ML29) is a candidate live-attenuated vaccine (L-AttV) for LASV that has shown promising results in animal models. Nevertheless, as with many other traditional L-AttV, the mechanism of ML29 attenuation remains unknown, which raises concerns about the phenotypic stability of ML29 in response to additional mutations. The central goal of this application is to test the hypothesis that we can convert a pathogenic OWA into an attenuated form with features of L-AttV via replacement of its L IGR by a genetically defined synthetic S-like IGR (Ssyn). Our hypothesis is supported by our recent following findings: 1) We could rescued rLCMV(IGR/S-S) with the same S-IGR in both S and L genome segments, and found that it was highly attenuated in vivo. 2) Mice immunized with rLCMV(IGR/S-S) were protected against a lethal challenge with wild type (WT) LCMV. 3) We have obtained evidence that a high degree of sequence plasticity within the S IGR is compatible with virus viability, which supports the feasibility of engineering recombinant arenaviruses with a synthetic S IGR (Ssyn) in the L segment as a general molecular strategy for arenavirus attenuation. To test our hypothesis we propose to complete the following specific aims: Aim 1. Characterize rLCMV (IGR/S-Ssyn): We have rescued rLCMV(IGR/S-Ssyn) where a synthetic S IGR (Ssyn) substituted for the L-IGR. We will characterize rLCMV(IGR/S-Ssyn) in cultured cells and using the mouse model of LCMV infection test the hypothesis that rLCMV(IGR/S-Syn) displays key features for L-AttV. Aim 2: Generate and characterize rLASV(IGR/S-Ssyn): We will generate rLASV(IGR/S-Ssyn) and test the hypothesis that it is attenuated in vivo but able to induce protective immunity against a letha challenge with WT LASV in a well-established guinea pig model of LASV infection. Aim 3. Examine the stability of rLCMV(IGR/S-Ssyn): We will test the hypothesis that rLCMV(IGR/S-Ssyn) is genetically stable during multiplication under different growth conditions. The successful completion of this application will uncover a general molecular strategy for arenavirus attenuation that can facilitate a novel strategy to develop L-AttV to combat human pathogenic arenaviruses.