Impairment of B cell Responses by Pathogenic Chikungunya Viruses

项目总结 B细胞的反应对于清除病毒感染是必不可少的。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 这会导致令人衰弱的多关节痛的爆炸性流行。关节肿胀、关节僵硬和腱鞘炎 可持续数月至数年，并与持续性CHIKV感染有关。如何实现的机制基础 CHIKV逃避B细胞免疫建立慢性感染尚不清楚。我们发现一辆减速的CHIKV 从WT小鼠关节中分离到CHIKV毒株，而不是亲本CHIKV毒株(相差5个氨基酸)。 在不能产生病毒特异性抗体(Ab)的小鼠身上，恢复了对减毒株的持续感染。 作图研究表明CHIKV E2糖蛋白第82位有一个精氨酸或甘氨酸残基 分别决定病毒的清除或持久性。与急性清除的CHIKV相比，持久 CHIKV感染与B细胞反应改变有关，B细胞反应改变是一种在 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，去除炎性髓系细胞可以改善B细胞 对致病性CHIKV的反应。这个莫里森和戴蒙德之间高度互动的项目的目标是 实验室将从机械上定义致病性CHIKV毒株如何逃避B细胞免疫，以及 炎性单核细胞在这一过程中的作用。在特定目标1中，特定目标的机制 CHIKV E2-82残基指示DLN中的炎症反应将被确定。不同的病毒只有 E2-82和新的糖胺多聚糖(GAG)遗传缺陷小鼠将被用来确定如何 E2-GAG相互作用决定CHIKV在DLN中的定位。巨噬细胞耗竭与单细胞RNA序列 将确定LN髓系细胞在炎症和B细胞对持续性和急性清除的反应中的作用 奇科夫。在特定的目标2中，I型干扰素信号如何在急性清除和 持续的CHIKV感染将被定义。I型干扰素报告小鼠、流式细胞仪和IHC将用于 定义淋巴组织中产生I型干扰素的细胞。AB介导的IFNAR1或不同类型I型的阻断 干扰素亚型(PAN-α与β)，以及I型干扰素信号中B细胞特异性缺陷的小鼠将被用于 确定I型干扰素对B细胞、Th和Tfh细胞反应的影响。在具体目标3中，煽动性有多大 髓系细胞在CHIKV持续感染期间拮抗B细胞的反应将被阐明。网络的影响 炎性髓系细胞对多克隆抗体亲和力、中和能力和表位的影响 将定义CHIKV抗体应答。使用新的Nos2F/F或Nox2F/F小鼠，以及一组Cre驱动株，我们将 确定特定的髓系细胞亚群抑制最佳B细胞反应的机制。这项工作将 阐明病毒破坏B细胞免疫并建立持久性的新机制。《知识》 GAIN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。

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.