Eliciting and isolating neutralizing antibodies against Powassan virus

摘要 鲍瓦桑病毒(POWV)是一种扁虱传播的黄病毒，可引起严重的脑炎、脑膜炎和 长期的神经损伤。虽然POWV感染相对罕见，但这种病毒分布广泛。 在常见的媒介中，如肩部硬蜱(鹿扁虱)和报告的病例数量 美国每年都在上升。目前还没有获得批准的POWV感染疫苗或治疗方法。这样做的目的是 R21提案有两个方面。在目标1中，我们将研究蛋白质纳米颗粒免疫原的潜力。 携带重组表达的POWV糖蛋白E结构域III诱导中和抗体的研究 对小鼠的反应。EDIII是黄病毒亚单位疫苗设计的一个有吸引力的目标，因为它相对 很小(~80个残基)，包含多种黄病毒保护性抗体的表位，如 登革病毒(DENV)、寨卡病毒(ZIKV)、西尼罗河病毒(WNV)和环状病毒(LIV)。然而，EDIII 作为一种单体，免疫原性很差，因为它缺乏使表面B细胞受体交联的能力 (BCRs)以刺激强大的抗体反应。这一限制可以通过在 多价形式作为蛋白质纳米颗粒的一部分。我们已经生成了一个原型POWV EDIII 使用SpyCatcher/SpyTag结合技术的纳米疫苗，初步研究表明 这种纳米颗粒可以在小鼠体内诱导中和抗体。我们将进一步优化这个和相关的POWV 纳米疫苗。在目标2中，我们将分离一大批人类单抗(Hu-mAbbs)。 通过单B细胞分选从一名POWV感染的活着的幸存者中分离出。HU-mAbs具有很强的应用潜力 免疫疗法，因为它们具有高度的特异性，而且它们的人体支架可将抗药性的风险降至最低 抗体反应。此外，分析Hu-mAbs提供了关于人类免疫的直接信息 然后可以用来指导疫苗设计的反应。我们将测试POWV Hu-mAbs的容量 结合POWV E并中和POWV报告病毒颗粒(RVP)。这项工作将提供新的见解 以及预防和治疗POWV感染的候选人。

SUMMARY Powassan virus (POWV) is a tick-disseminated flavivirus that causes severe encephalitis, meningitis, and long-term neurological damage. Although POWV infections are relatively rare, the virus is widely distributed among common vectors such as Ixodes scapularis (the deer tick) and the number of reported cases in the US is rising each year. There are no approved vaccines or treatments for POWV infection. The goals of this R21 proposal are two-fold. In Aim 1, we will investigate the potential of protein nanoparticle immunogens bearing recombinantly-expressed POWV glycoprotein E domain III (EDIII) to induce neutralizing antibody response in mice. EDIII is an attractive target for flavivirus subunit vaccine design because it is relatively small (~80 residues) and contains epitopes of protective antibodies against multiple flaviviruses such as Dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and Looping Ill virus (LIV). However, EDIII as a monomer is poorly immunogenic because it lacks the capacity to crosslink surface B-cell receptors (BCRs) to stimulate a robust antibody response. This limitation can be overcome by presenting EDIII in multivalent format as part of a protein nanoparticle. We have generated a prototypic POWV EDIII nanoparticle vaccine using Spycatcher/Spytag conjugation technology, and pilot studies have demonstrated this nanoparticle can elicit neutralizing antibodies in mice. We will further optimize this and related POWV nanoparticle vaccines. In Aim 2, we will isolate a large panel of human monoclonal antibodies (hu-mAbs) from a living survivor of POWV infection by single B cell sorting. Hu-mAbs have strong potential as immunotherapies because they are highly specific and their human scaffold minimizes the risk for anti-drug antibody responses. Furthermore, profiling hu-mAbs provides direct information about human immune response that can then be used to inform vaccine design. We will test the POWV hu-mAbs for their capacity to bind POWV E and neutralize POWV reporter virus particles (RVPs). This work will provide new insights and candidates for prevention and treatment of POWV infection.