A General Molecular Strategy for Attenuation of Human Pathogenic Arenaviruses

人类致病性沙粒病毒减毒的通用分子策略

• 批准号: 9217579
• 负责人: Juan C. de la Torre
• 金额: $ 24.06万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-05 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9217579
• 关键词: Adverse effects; Africa; Animal Model; Antigens; Area; Arenaviridae; Arenavirus; Attenuated; Attenuated Live Virus Vaccine; Cavia; Cell Line; Cells; Clinical; Clinical Trials; Competence; Consensus Sequence; Cultured Cells; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Engineering; Exhibits; Future; Genetic; Genome; Goals; Growth; Heterophile Antigens; Human; Immune response; Immunity; Immunize; Individual; Infection; Interferons; Knowledge; Label; Lassa Fever; Lassa virus; Lujo virus; Lymphocytic choriomeningitis virus; Mediating; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Old World Arenaviruses; Pathogenicity; Phenotype; Population; Production; Property; Protocols documentation; Public Health; Reassortant Viruses; Recombinants; Resources; Ribavirin; Serial Passage; South Africa; Symptoms; Testing; Travel; Vaccines; Viral; Viral Hemorrhagic Fevers; Virulence; Virus; Virus Diseases; attenuation; base; biodefense; clinically significant; combat; design; epidemiology study; genetic approach; in vivo; interest; metropolitan; mortality; mouse model; neglect; novel; novel strategies; pathogen; public health relevance; pyrosequencing; response; reverse genetics

 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.

 描述（由申请人提供）：几种沙粒病毒会引起人类出血热（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) 拉沙病毒 (LASV) 每年在西非感染数十万人，导致大量拉沙热 (LF) 病例，并伴有高发病率和死亡率。此外，有证据表明，全球分布的 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 在内的几种沙粒病毒构成了可靠的生物防御威胁。现有的抗沙粒病毒疗法仅限于利巴韦林的标签外使用，其效果仅部分有效，而且尚无获得 FDA 许可或目前正在进行临床试验的沙粒病毒疫苗。然而，MOPV/LASV 重配体 (ML29) 是 LASV 的候选减毒活疫苗 (L-AttV)，已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，ML29 减毒机制仍然未知，这引起了人们对 ML29 对额外突变的表型稳定性的担忧。该应用的中心目标是测试以下假设：我们可以通过用基因定义的合成 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。我们最近的以下发现支持了我们的假设：1）我们可以在S和L基因组片段中使用相同的S-IGR来拯救rLCMV（IGR/S-S），并发现它在体内高度减毒。 2)用rLCMV(IGR/S-S)免疫的小鼠免受野生型(WT) LCMV的致命攻击。 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段中合成S IGR（Ssyn）作为沙粒病毒减毒通用分子策略的工程重组沙粒病毒的可行性。为了检验我们的假设，我们建议完成以下具体目标： 目标 1. 表征 rLCMV (IGR/S-Ssyn)：我们拯救了 rLCMV(IGR/S-Ssyn)，其中合成的 S IGR (Ssyn) 取代了 L-IGR。我们将表征培养细胞中的 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 关键特征的假设。目标 2：生成并表征 rLASV(IGR/S-Ssyn)：我们将生成 rLASV(IGR/S-Ssyn) 并测试其在体内减弱但能够在完善的 LASV 感染豚鼠模型中诱导针对 WT LASV 致死攻击的保护性免疫的假设。目标 3. 检查 rLCMV(IGR/S-Ssyn) 的稳定性：我们将检验 rLCMV(IGR/S-Ssyn) 在不同生长条件下的增殖过程中遗传稳定的假设。该申请的成功完成将揭示沙粒病毒减毒的通用分子策略，该策略可以促进开发 L-AttV 来对抗人类致病性沙粒病毒的新策略。

项目成果

A Lassa Virus Live-Attenuated Vaccine Candidate Based on Rearrangement of the Intergenic Region

• DOI: 10.1128/mbio.00186-20
• 发表时间: 2020-03-01
• 期刊: MBIO
• 影响因子: 6.4
• 作者: Cai, Yingyun;Iwasaki, Masaharu;de la Torre, Juan Carlos
• 通讯作者: de la Torre, Juan Carlos

Molecular Engineering of a Mammarenavirus with Unbreachable Attenuation.

具有不可破坏的减毒能力的乳腺病毒的分子工程。

• DOI: 10.1128/jvi.01385-22
• 发表时间: 2023
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Sakabe,Saori;Cubitt,Beatrice;Martinez-Sobrido,Luis;delaTorre,JuanC
• 通讯作者: delaTorre,JuanC