Immunobiology, molecular virology and countermeasures of highly pathogenic viruses

1.调查接种和挑战后的保护性免疫反应，开发针对新出现病毒的暴露前和暴露后疫苗 我们在FDA批准的埃博拉病毒(ERVEBO，VSV-EBOV)疫苗的基础上，开发了一种针对高致病性禽流感病毒H5N1的VSV疫苗。与亲本载体类似，该疫苗是快速有效的，并保护小鼠免受同源H5病毒以及其他H5分离株的攻击(Furuyama等人，NPJ疫苗2020)。重要的是，我们可以证明，与其他流感疫苗一样，保护主要由抗原特异性免疫球蛋白反应提供。我们对新冠肺炎使用了同样的方法(见另一份报告；Furuyama等人，正在筹备中)。 为了改进亲本载体，以指导抗原呈递和对第二抗原的免疫反应，我们从EBOV GP中删除了粘蛋白样结构域和糖帽，并可以证明该抗原确实具有较低的免疫原性(Marzi，未发表的数据)。我们将使用这种方法来优化基于VSV-EBOV的未来疫苗。 VSV-EBOV本身并不是一种有效的暴露后治疗方法，因此，我们在针对不同EBOV蛋白的载体中包括了microRNAs(MiRNA)。第一个小鼠实验结果是，在攻击后注射疫苗时，疫苗的miRNA表达带来的好处有限(ODonnell，未发表的数据)。我们正致力于加入小干扰(SiRNA)或单抗(MAb)序列。MAb的表达可以立即控制病毒复制，并为疫苗生效赢得所需的时间。 此外，我们更详细地研究了EBOV所描述的抗体依赖的感染增强。我们发现补体因子C1q介导EBOV的ADE(Furuyama等人，Plos NTD在出版社)。我们仍在分析这种只在体外描述的现象是否可能在EBOV感染和发病机制中发挥作用。这将影响未来丝状病毒的疫苗设计。 在与Kim Hasenkrug的合作中，我们测试了抗CD47抗体治疗是否对小鼠的EBOV感染有好处。不幸的是，情况并非如此(Marzi等人，未发表的数据)，然而，我们将继续测试这种治疗方法是否适用于其他新出现的病毒。 2.利用丝状病毒反向遗传学方法鉴定致病因子和未知病毒的特性 我们正在分析EBOV的可溶性糖蛋白(SGP)对病毒致病性的贡献。首先，我们建立了一种SGP捕获ELISA来检测由NIAID Chertow博士提供的动物血清样本和人类样本中的生物相关水平(Furuyama等人，正在准备中)。该检测方法有可能发展成为EBOV的诊断工具。接下来，我们从细胞中产生了SGP，并产生了一种敲除病毒(EBOV-SGP Ko)，使我们能够在细胞培养和小鼠体内的生物学相关水平上研究SGP的影响。我们发现，SGP确实在体外增强了病毒复制和斑块大小，并增加了小鼠肝脏中的病毒载量(Furuyama等人，正在准备中)。分析先天免疫途径的初步数据表明，MAP激酶途径可能在加入SGP后EBOV复制增加中发挥作用。 在塞拉利昂、中国等地的蝙蝠体内都发现了丝状病毒序列。我们正在利用我们的反向遗传学专业知识从质粒中恢复这些病毒，并在体外和体内鉴定这些病毒。 此外，在与耶鲁大学Anthony van den Pol的合作项目中，我们可以证明EBOV GP的粘蛋白样结构域增强了VSV-EBOV的溶瘤潜力(Zhang等人，J Virol 2020)。在这项研究中，我们使用了上述目标1中提到的VSV载体和VSV-EBOV(批准的疫苗)。 3.决定丝状病毒感染易感性的遗传宿主因素分析 最后，实验室感兴趣的是确定限制丝状病毒宿主范围的因素。与北海道大学的Ayato Takada合作，我们可以证明丝状病毒受体Nieman Pick-1的多态决定了不同种类的蝙蝠是否易受丝状病毒感染(Takadate等人，Cell Rep 2020)。 我们采用生物信息学的方法，与北卡罗来纳大学的拉尔夫·巴里克合作，使用协作杂交(CC)小鼠，鉴定出患有致命疾病或轻度/无疾病的CC菌株。我们培育了这两个毒株，并建立了这些小鼠的F2代，以确定EBOV的易感等位基因。有趣的是，我们发现TRIM基因与EBOV易感性有关(Schaefer，Marzi等人，未发表的数据)。我们目前正在培育TRIM KO小鼠，并进行EBOV感染研究，以确认这种宿主因素确实起到了作用。

1. Investigate protective immune responses after vaccination and challenge, and develop pre- and post-exposure vaccines for emerging viruses We have developed a VSV-based vaccine protective against highly pathogenic avian influenza virus H5N1 based on the FDA-approved Ebola virus (ERVEBO, VSV-EBOV) vaccine. Similar to the parental vector, the vaccine is fast-acting and protected mice against challenge with the homologous H5 virus, but also with other H5 isolates (Furuyama et al., npj vaccines 2020). Importantly, we could show that, as with other influenza vaccines, protection was mainly provided by the antigen-specific IgG response. We have used the same approach for COVID-19 (see separate report; Furuyama et al., in preparation). In order to improve the parental vector for directing antigen presentation and immune responses to the 2nd antigen, we deleted the mucin-like domain and glycan cap from the EBOV GP and could demonstrate that this antigen indeed is less immunogenic (Marzi, unpublished data). We will be using this approach to optimize future vaccines based on VSV-EBOV. The VSV-EBOV is not a potent post-exposure treatment by itself, therefore, we have included micro RNAs (miRNA) in the vectors targeting different EBOV proteins. A first mouse experiment resulted in limited benefit from the miRNA expression from the vaccine when administered after challenge (ODonnell, unpublished data). We are working towards incorporating small interfering (siRNA) or monoclonal antibody (mAb) sequences instead. Expression of the mAb may provide immediate control of the virus replication and buy the time needed for the vaccine to become effective. Furthermore, we investigated in more detail the antibody-dependent enhancement of infection described for EBOV. We found that the complement factor C1q is mediating ADE for EBOV (Furuyama et al., Plos NTD in press). We are still analyzing if this phenomenon, which has only been described in vitro, might play a role for EBOV infection and pathogenesis. This would impact future vaccine design for filoviruses. In collaboration with Kim Hasenkrug we tested if anti-CD47 antibody treatment results in a benefit against EBOV infection in mice. Unfortunately, this was not the case (Marzi et al., unpublished data), however, we will continue to test of this treatment is a viable option for other emerging viruses. 2. Identification of pathogenicity factors and characterization of unknown viruses using reverse genetics approaches for filoviruses We are in the process of analyzing the contributions of the soluble glycoprotein (sGP) of EBOV to viral pathogenicity. First, we developed a sGP capture ELISA to determine biologically relevant levels in animal serum samples and human specimen provided by Dr. Chertow, NIAID (Furuyama et al, in preparation). This assay could potentially be developed into a diagnostic tool for EBOV. Next, we produced sGP from cells and generated a knock-out virus (EBOV-sGP ko) allowing us to study the effects of sGP at a biologically relevant level in cell culture and in mice. We found that sGP indeed enhances virus replication and plaque size in vitro and increases virus loads in then liver in mice (Furuyama et al, in preparation). Preliminary data analyzing innate immune pathways indicate that the MAP kinase pathway might play a role in the increased EBOV replication after sGP addition. Filovirus sequences have been found in bats in Sierra Leone, China and other places. We are in the process of employing our reverse genetics expertise to recover these viruses from plasmid and characterize in vitro and in vivo. Furthermore, in a collaborative project with Anthony van den Pol, Yale we could demonstrate that the mucin-like domain of EBOV GP enhances the oncolytic potential of the VSV-EBOV (Zhang et al., J Virol 2020). For this study we utilized the VSV vector mentioned above in objective 1 and the VSV-EBOV (approved vaccine). 3. Analysis of genetic host factors determining the susceptibility of filovirus infections Lastly, the lab is interested in identifying factors restricting the host range of filoviruses. In collaboration with Ayato Takada, Hokkaido University we could demonstrate that polymorphisms in the filovirus receptor Nieman Pick-1 determine if different bat species are susceptible to filovirus infection (Takadate et al., Cell Rep 2020). We took a bioinformatics approach using the collaborative cross (CC) mice in collaboration with Ralph Baric, UNC and identified CC strains with fatal disease or mild/no disease. We bred these 2 strains and established a F2 generation of these mice to identify a susceptibility allele for EBOV. Interestingly, we found that the trim locus is involved in EBOV susceptibility (Schaefer, Marzi et al., unpublished data). We are currently breeding trim ko mice and performing EBOV-infection studies to confirm that this host factor plays indeed a role.