Impairment of B cell Responses by Pathogenic Chikungunya Viruses

致病性基孔肯雅病毒对 B 细胞反应的损害

• 批准号: 9753471
• 负责人: Michael S Diamond
• 金额: $ 60.17万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-25 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753471
• 关键词: Acute; Alphavirus; Amino Acid Sequence; Amino Acids; Anabolism; Antibodies; Antibody Response; Antigens; Arginine; Attenuated; Avidity; B-Lymphocytes; Biological Assay; Cells; Chikungunya virus; Chronic; Chronic Disease; Culicidae; Data; Defect; Development; Diamond; Dichloromethylene Diphosphonate; Enzymes; Epidemic; Epitopes; Failure; Fever; Flow Cytometry; Genetic; Genetic study; Glycine; Glycoproteins; Glycosaminoglycans; Goals; Human; IFNAR1 gene; Immunity; Impairment; Infection; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Interferons; Joints; Knowledge; Laboratories; Liposomes; LoxP-flanked allele; Lymphoid Tissue; Mediating; Modeling; Mouse Strains; Mus; Myeloid Cells; Nitrogen; Oxygen; Pathogenicity; Phagocytes; Polyarthralgias; Positioning Attribute; Process; Production; RNA; Reporter; Role; Signal Transduction; Sinus; Site; Structure of germinal center of lymph node; Swelling; Tenosynovitis; Tissues; Vaccines; Viral; Virus; Virus Diseases; Work; adaptive immune response; chronic infection; confocal imaging; diphtheria toxin receptor; improved; insight; joint stiffness; lymph nodes; macrophage; monocyte; mouse model; neutralizing antibody; novel therapeutic intervention; recruit; response; reverse genetics; therapy development; transcriptome sequencing; vaccine development

PROJECT SUMMARY B cell responses are essential for clearance of viral infections. The mechanisms by which chronic viruses subvert B cell immunity are poorly understood. Chikungunya virus (CHIKV) is a mosquito-transmitted RNA alphavirus that causes explosive epidemics of debilitating polyarthralgia. Joint swelling, joint stiffness, and tenosynovitis can last for months to years and are associated with persistent CHIKV infection. The mechanistic basis for how CHIKV evades B cell immunity to establish chronic infection is unknown. We found that an attenuated CHIKV strain, but not the parental CHIKV strain (which differs by 5 amino acids), is cleared from joints of WT mice. Persistent infection of the attenuated strain was restored in mice unable to produce virus-specific antibody (Ab). Mapping studies revealed that a single arginine or glycine residue at position 82 in the CHIKV E2 glycoprotein dictates viral clearance or persistence, respectively. In comparison with acutely cleared CHIKV, persistent CHIKV infection was associated with altered B cell responses, a failure to develop germinal centers in the draining lymph node (DLN), poorer quality virus-specific neutralizing Abs, and distinct viral localization and inflammatory responses in lymphoid tissue. Remarkably, depletion of inflammatory myeloid cells improved B cell responses to pathogenic CHIKV. The goal of this highly interactive project between the Morrison and Diamond laboratories is to define mechanistically how pathogenic CHIKV strains evade B cell immunity and the contribution of inflammatory monocytes to this process. In Specific Aim 1, the mechanisms by which a specific CHIKV E2-82 residue dictates inflammatory responses in the DLN will be determined. Viruses differing only at E2-82, and new mice with genetic deficiencies in glycosaminoglycans (GAGs), will be used to determine how E2-GAG interactions dictate CHIKV localization in the DLN. LN macrophage depletion and single-cell RNAseq will define the role of LN myeloid cells in inflammatory and B cell responses to persistent and acutely cleared CHIKV. In Specific Aim 2, how type I IFN signaling modulates B cell responses during acutely cleared and persistent CHIKV infection will be defined. Type I IFN reporter mice, flow cytometry, and IHC will be used to define the cells that produce type I IFN in lymphoid tissue. Ab-mediated blockade of IFNAR1 or distinct type I IFN subtypes (pan-α versus β), and mice with B cell-specific defects in type I IFN signaling will be used to determine the effects of type I IFN on B cell, Th, and Tfh cell responses. In Specific Aim 3, how inflammatory myeloid cells antagonize B cell responses during persistent CHIKV infection will be elucidated. The impact of inflammatory myeloid cells on the avidity, neutralization capacity, and epitope repertoire of the polyclonal anti- CHIKV Ab response will be defined. Using new Nos2F/F or Nox2F/F mice, and a panel of Cre driver strains, we will determine mechanisms by which specific subsets of myeloid cells inhibit optimal B cell responses. This work will elucidate new mechanisms by which viruses subvert B cell immunity and establish persistence. The knowledge gained may aid the development of vaccines or therapies against CHIKV or other chronic viral infections.

项目概要 B 细胞反应对于清除病毒感染至关重要，这是慢性病毒破坏的机制。 人们对 B 细胞免疫知之甚少。基孔肯雅病毒 (CHIKV) 是一种由蚊子传播的 RNA 甲病毒。 导致关节肿胀、关节僵硬和腱鞘炎的爆发性流行。 可以持续数月至数年，并与持续性 CHIKV 感染相关。 CHIKV 逃避 B 细胞免疫以建立慢性感染尚不清楚。我们发现减毒的 CHIKV。 从 WT 小鼠的关节中清除了 CHIKV 毒株，但不包括亲本 CHIKV 毒株（相差 5 个氨基酸）。 在无法产生病毒特异性抗体（Ab）的小鼠中，减毒株的持续感染得以恢复。 绘图研究表明，CHIKV E2 糖蛋白第 82 位有一个精氨酸或甘氨酸残基 分别决定病毒清除或持久性 与急性清除的 CHIKV 相比，持久性。 CHIKV 感染与 B 细胞反应改变有关，即 B 细胞无法发育生发中心。 引流淋巴结（DLN）、质量较差的病毒特异性中和抗体以及独特的病毒定位和 淋巴组织中的炎症反应 值得注意的是，炎症性骨髓细胞的消耗改善了 B 细胞。 Morrison 和 Diamond 之间这个高度互动的项目的目标。 实验室的目标是从机制上定义致病性 CHIKV 菌株如何逃避 B 细胞免疫以及 在特定目标 1 中，炎症单核细胞对此过程的贡献。 CHIKV E2-82 规定 DLN 中的炎症反应仅在以下情况下确定。 E2-82 和具有糖胺聚糖 (GAG) 遗传缺陷的新小鼠将用于确定如何 E2-GAG 相互作用决定了 CHIKV 在 DLN 中的定位和单细胞 RNAseq。 将定义 LN 骨髓细胞在炎症和 B 细胞对持续和急性清除反应中的作用 CHIKV。在具体目标 2 中，I 型 IFN 信号如何在急性清除和清除过程中调节 B 细胞反应。 将使用流式细胞术和 IHC 来定义持续性 CHIKV 感染。 定义了在淋巴组织中产生 I 型 IFN 的细胞，该细胞介导的 IFNAR1 或不同的 I 型干扰素阻断。 IFN 亚型（泛 α 与 β）以及 I 型 IFN 信号传导中具有 B 细胞特异性缺陷的小鼠将用于 确定 I 型 IFN 对 B 细胞、Th 和 Tfh 细胞反应的影响。在具体目标 3 中，炎症如何发生。 骨髓细胞在持续 CHIKV 感染期间拮抗 B 细胞反应的影响将得到阐明。 炎症性骨髓细胞对多克隆抗抗体的亲合力、中和能力和表位库的影响 我们将使用新的 Nos2F/F 或 Nox2F/F 小鼠以及一组 Cre 驱动菌株来定义 CHIKV Ab 反应。 这项工作将确定骨髓细胞特定亚群抑制最佳 B 细胞反应的机制。 阐明病毒破坏 B 细胞免疫并建立持久性的新机制。 获得的成果可能有助于开发针对 CHIKV 或其他慢性病毒感染的疫苗或疗法。