Viral exposure signatures may define individuals vulnerable for COVID-19

我们与NIAID的Luigi Notarangelo医学博士、Irini Sereti医学博士和Andrea Lisco医学博士合作，正在使用VirScan平台确定病毒暴露特征的景观，作为对covid -19阳性患者病毒感染史的体液免疫反应。我们正在通过全基因组测序将VirScan数据与其他COVID-19联盟项目的数据进行生物信息学整合，包括t细胞受体(TCR)/ b细胞受体（BCR）谱、细胞因子谱、SARS-CoV-2血清学谱和种系变异。这些分析旨在全面了解可预测COVID-19临床结果的宿主特异性遗传和免疫学特征，并有可能为免疫治疗策略提供信息，从而改善SARS-CoV-2感染个体的诊断、监测和治疗。具体目标1：我们正在利用VirScan技术分析来自意大利多个地区和其他国家的SARS-CoV-2感染患者血清学样本中的VES。我们将对2020年1 - 5月期间在意大利和美国医院住院的700名SARS-CoV-2患者的血清学样本进行病毒状态检测，步骤如下：步骤1)T7噬菌体文库扩增：T7噬菌体混合物一次显示来自206种和1000多种不同病毒株的肽，提供所有已知人类病毒的蛋白质组全覆盖。文库扩增将使用标准扩增协议进行。将产生大量病毒滴度为1010pfu的T7噬菌体文库，以最大限度地减少批次差异，并确保整个研究期间的稳定性。步骤2)血清学标本筛选：将对COVID-19患者[伦巴第（意大利北部）400例、普利亚（意大利南部）200例和美国（华盛顿特区）100例患者]以及来自同一地理区域的健康献血者作为对照和具有获得性或原发性免疫缺陷的受试者（n=300）采集的血清学标本进行抗病毒抗体筛选。因此，本研究的血清学样本总数为1000个，其中10%的样本重复进行（n=200），因此将分析1100个样本。每个人大约1微升的血液将用于进行VirScan检测。步骤3)噬菌体文库构建及噬菌体DNA平行测序：噬菌体免疫沉淀测序（PhIP-Seq）和DNA测序将用于分析病毒-宿主相互作用。我们将进行PCR，在每次免疫沉淀反应中回收的DNA上添加测序接子和索引，并对噬菌体DNA进行大规模平行测序，通过计算免疫沉淀前后每个肽的读取计数，定量特定抗病毒抗体在其相应噬菌体文库输入中的富集/丰度。生物信息学和统计分析将使用鲍蒂和标准统计分析进行。具体目标2：我们正在确定与各种临床特征严重程度相关的SARS-CoV-2感染个体的独特VES: a)无症状/轻度感染，需要通过鼻插管补充氧气低于5升/分钟（L/m）； b)严重感染，需要通过鼻插管或其他非侵入性通气方式补充氧气大于5升/分钟；c)严重感染，需要机械通气；d)死亡。步骤1)我们将评估意大利健康献血者（n=100）、意大利HIV-1感染者（n=100）、美国健康献血者、HIV-1感染者和特发性CD4淋巴细胞减少症患者（n=100）以及意大利（北部和南部地区）和美国的COVID-19患者（n=700） VES特征的差异。因此，我们将评估不同地理区域和临床背景下VES特征的异质性。step2)我们将分析具有上述4种临床结局的受试者在VES谱上的差异。step3) COVID-19的严重临床病程与淋巴细胞严重减少有关，有报道称继发性淋巴器官淋巴细胞凋亡。这些发现表明，除了转运和淋巴细胞亚群的重新分布外，对B细胞和T细胞存活的特异性影响可能导致协调不良的适应性免疫反应，最终导致更差的临床结果。IL-7是一种T细胞和B细胞的稳态细胞因子，可以逆转这种作用。将在COVID-19患者中纵向评估VES谱，以评估IL-7对SARS-CoV-2和所有其他病毒性病原体的谱抗体反应的影响。特异性目标3：我们正在整合VES谱与宿主遗传学、t细胞/ b细胞谱、细胞因子谱和对SARS-CoV-2感染的血清学反应，以完善VES谱，用于监测、诊断和治疗SARS-CoV-2感染患者。利用我们最近发表的各种生物信息学工具，我们将通过将cov -19相关的VES与种系变异、TCR/BCR谱、细胞因子谱和SARS-CoV-2基因型联系起来，进行数据整合，以完善VES。预期结果和潜在影响：我们的项目将使我们能够询问感染SARS-CoV-2患者的病毒特征。本研究将为有效划分COVID-19患者的临床病程和免疫特征并预测疾病严重程度设计策略奠定基础。这项研究的独特优势包括结合了一种综合方法，该方法可能会通过分析过去的病毒感染来检测宿主免疫与其对sars - cov -2诱导的疾病严重程度的影响之间的关联。由于我们有来自意大利和美国不同地区的多个队列，因此可以很容易地验证改进的VES，以最大限度地减少潜在的混杂因素，尽管预计分析人群中的这种地理和遗传变异性也可能代表我们的分析在定义强大的免疫学预测因子方面的优势。VirScan将使我们了解病毒与宿主免疫之间的相互作用及其与sars - cov -2相关疾病的关系。如果成功，我们的项目将使我们能够全面了解可预测COVID-19临床结果的宿主特异性遗传和免疫学特征，并为免疫治疗策略提供信息，从而改善对SARS-CoV-2感染个体的诊断、监测和治疗。

In collaboration with Luigi Notarangelo, MD, Irini Sereti, MD and Andrea Lisco, MD, PhD of NIAID, we are working to determine the landscape of viral exposure signature as the humoral immunological responses to the history of viral infection in COVID-19-positive patients using the VirScan platform. We are in the process of bioinformatically integrating VirScan data to data from other COVID-19 consortium projects that include T-Cell receptor (TCR)/B-Cell Receptor (BCR) repertoires, cytokine profiles, SARS-CoV-2 serological profiles and germline variants by whole genome sequencing. These analyses are aiming to provide a comprehensive view of the host-specific genetic and immunological characteristics predictive of COVID-19 clinical outcomes and have the potential to inform immunological treatment strategies, thereby improving diagnosis, surveillance and treatments of individuals with SARS-CoV-2 infection. Specific Aim 1: We are utilizing VirScan technology to profile VES present in serological samples of patients with SARS-CoV-2 infection from multiple regions of Italy and other countries. We will test the viral status in serological samples from 700 patients who were hospitalized at the hospitals in Italy and US between Jan-May, 2020 due to SARS-CoV-2 infection by the following steps: Step 1) T7 Phage Library Amplification: The T7 bacteriophage mixture displays peptides from 206 species and over 1000 different strains of virus at a time, providing proteome-wide coverage from all known human viruses. Library amplification will be performed using standard amplification protocols. A large stock of the T7 bacteriophage library with viral titer 1010 pfu will be generated to minimize batch variability and to ensure stability across the duration of this study. Step 2) Screening of Serological Specimens: Antiviral antibodies will be screened in serological specimens collected from COVID-19 patients [400 from Lombardy (North Italy), 200 patients from Puglia (South of Italy) and 100 patients from US (Washington DC)] along with healthy blood donors as controls from the same geographical regions and subjects with acquired or primary immunological defects (n=300). Thus, the total serological sample set for this study will be 1,000 with 10% of the specimens performed in duplicate (n=200), thus 1,100 samples will be assayed. Approximately 1 microliter of blood from each individual will be used to perform the VirScan assay. Step 3) Library construction and Parallel Sequencing on the Recovered Phage DNA: Phage immunoprecipitation sequencing (PhIP-Seq) and DNA sequencing will be used to analyze viral-host interactions. We will perform PCR to add sequencing adaptor and index to DNA recovered from each immunoprecipitation reaction and massively parallel sequencing on the phage DNA to quantify enrichment/abundance of a specific antiviral antibody to its corresponding phage library input by calculating the read count for each peptide before and after immunoprecipitation. Bioinformatics and statistical analysis will be performed using Bowtie and standard statistical analysis. Specific Aim 2: We are identifying unique VES in individuals who are infected with SARS-CoV-2 infection linked to the severity of various clinical features: a) asymptomatic/mild infection requiring oxygen supplementation less than 5 Liters/minute (L/m) by nasal cannula, b) severe infection requiring oxygen supplementation greater than 5 L/m by nasal cannula or other non-invasive modalities of ventilation, c) severe infection requiring mechanical ventilation, d) death. Step 1) We will assess differences in VES profile from Italian heathy blood donors (n=100), Italian HIV-1 infected individuals (n=100), US healthy blood donors, HIV1-infected and subjects with Idiopathic CD4 lymphopenia (n=100), and COVID-19 patients from Italy (Northern and Southern regions) and US (n=700). We will therefore evaluate the heterogeneity of VES profile per geographic area and clinical context. Step 2) We will analyze differences in VES profile between subjects with the above mentioned 4 clinical outcomes. Step 3) The severe clinical course of COVID-19 is associated with profound lymphopenia with reports on lymphocytes apoptosis in secondary lymphoid organs. Such findings suggest that besides trafficking and redistributions of lymphoid subsets, specific effect on B and T cell survival may result in a poorly orchestrated adaptive immune response that ultimately contributes to worse clinical outcomes. IL-7 is a T and B cells homeostatic cytokine which can reverse such effect. VES profile will be evaluated longitudinally in individuals with COVID-19 to evaluate the effect of IL-7 on the profile antibody responses to SARS-CoV-2 and all other viral pathogens. Specific Aim 3: We are integrating VES profiles with host genetics, T-cell/B-cell repertoires, cytokine profile, and serological responses to SARS-CoV-2 infection to refine VES useful for surveillance, diagnosis and treatment of patients infected with SARS-CoV-2. Using various bioinformatics tools described in our recent publications, we will perform data integration by linking COV-19-related VES to germline variants as well as TCR/BCR repertoires, cytokine profiles and SARS-CoV-2 genotypes to refine VES. Anticipated Outcomes and Potential Impact: Our project will allow us to interrogate viral signatures in patients infected with SARS-CoV-2. This study will set the stage to design strategies to effectively stratify clinical course and immunological signature of COVID-19 patients and predict disease severity. Unique advantages of this study include the incorporation of a comprehensive method that will likely detect associations between host immunity from profiling past viral infections and its impact in SARS-CoV-2-induced disease severity. Since we have multiple cohorts from different regions of Italy as well as US, a refined VES can easily be validated to minimize potential confounding factors, although it is anticipated that such geographic and genetic variability in the analyzed population may also represent a strength of our analysis in the definition of robust immunological predictors. VirScan would allow us to understand the interplay between the virome and the host immunity and its relation to SARS-CoV-2-associated disease. If successful, our project will allow us to develop a comprehensive view of the host-specific genetic and immunological characteristics predictive of COVID-19 clinical outcomes and to inform immunological treatment strategies, thereby improving diagnosis, surveillance and treatments of individuals with SARS-CoV-2 infection.