A General Molecular Strategy for Attenuation of Human Pathogenic Arenaviruses

 描述(申请人提供)：几种禽流感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，东半球阿拉伯病毒(OWA)拉萨(LASV)每年感染西非数十万人，导致与高发病率和高死亡率相关的大量拉沙热(LF)病例。此外，有证据表明，世界各地分布的OWA LCMV是一种被忽视的临床重要人类病原体。此外，包括LASV和LCMV在内的几种禽类病毒构成了可信的生物防御威胁。现有的抗阿拉伯病毒疗法仅限于非标签使用利巴韦林，仅有部分有效，而且没有FDA许可的或目前处于临床试验中的阿拉伯病毒疫苗。然而，MOPV/LASV重组疫苗(ML29)是一种候选的LASV减毒活疫苗(L-阿特V)，在动物模型中显示出良好的效果。然而，与许多其他传统的L病毒一样，ML29的衰减机制仍不清楚，这引发了人们对ML29对额外突变的表型稳定性的担忧。这项应用的中心目标是检验这样一个假设，即我们可以通过将其L IGR替换为基因定义的合成S样IGR(SYN)来将致病的OWA转化为具有L-AttV特征的减毒形式。我们的假设得到了以下发现的支持：1)我们可以在S和S基因组片段中回收具有相同的S-IGR的重组LCMV(IGR/L-IGR)，并发现它在体内高度减毒。2)免疫小鼠可抵抗野生型(WT)LCMV的致死性攻击。3)我们获得的证据表明，S IGR内高度的序列可塑性与病毒活性是相容的，这支持了在L片段中设计合成S IGR(Ssyn)的重组ArenaVirus作为ArenaVirus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：目的1.鉴定重组人巨细胞病毒(IgR/S-Ssyn)：我们拯救了rLCMV(IgR/S-Ssyn)，其中合成的S Ig R(Ssyn)取代了L-Ig R。我们将在培养细胞中鉴定rLCMV(IGR/S-Syn)，并利用LCMV感染的小鼠模型检验rLCMV(IGR/S-Syn)显示出L-AttV的关键特征的假设。目的：制备并鉴定重组LASV(IGR/S-Ssyn)：我们将制备rLASV(IGR/S-Ssyn)，并在已建立的LASV感染的豚鼠模型上验证其在体内减毒但能够诱导保护性免疫的假说。目的3.检测重组LCMV(IGR/S-Ssyn)的稳定性：检验rLCMV(IGR/S-Ssyn)在不同生长条件下繁殖过程中遗传稳定的假说。这项应用的成功完成将揭示一种一般的禽流感病毒减毒的分子策略，可以促进一种新的策略来开发L-AttV来对抗人类致病性禽流感病毒。

 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.