Viral exposure signatures may define individuals vulnerable for COVID-19

病毒暴露特征可能会定义个体是否容易感染 COVID-19

• 批准号: 10702767
• 负责人: Xin Wei Wang
• 金额: $ 25.47万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702767
• 关键词: 2019-nCoV; Acute Respiratory Distress Syndrome; Affect; Animal Model; Antibody Response; B cell repertoire; B-Cell Antigen Receptor; B-Lymphocytes; Bacteriophage T7; Bacteriophages; Bioinformatics; Biological Assay; Biological Markers; Blood; Blood Coagulation Disorders; Blood donor; COVID-19; COVID-19 diagnosis; COVID-19 patient; COVID-19 susceptibility; Cannulas; Cell Survival; Cessation of life; Characteristics; Chronic Disease; Clinical; Clinical Trials; Collaborations; Complex; Country; DNA; DNA sequencing; Data; Defect; Diagnosis; Disease; Disease Progression; District of Columbia; Doctor of Philosophy; Double-Blind Method; Endothelium; Ensure; Genetic; Genotype; Geographic Locations; Geography; HIV-1; Health; Heterogeneity; Hospitals; Human; Hypoxemic Respiratory Failure; Hypoxia; IL7 gene; Immune response; Immunity; Immunologics; Immunoprecipitation; Individual; Infection; Inflammasome; Inflammatory Response; Interstitial Pneumonia; Italy; Libraries; Link; Lower respiratory tract structure; Lymphoid; Lymphopenia; Malignant Neoplasms; Massive Parallel Sequencing; Mechanical ventilation; Methods; Middle East Respiratory Syndrome Coronavirus; Modality; Modeling; Molecular; Multiple Organ Failure; National Institute of Allergy and Infectious Disease; Nose; Outcome; Oxygen; Pathogenesis; Pathogenicity; Patients; Peptides; Phage ImmunoPrecipitation Sequencing; Phase; Placebos; Population Analysis; Predisposition; Process; Proteome; Protocols documentation; Publications; RNA Viruses; Randomized Clinical Trials; Reaction; Recombinants; Recording of previous events; Reporting; Research Personnel; Research Project Grants; Resolution; Respiratory distress; SARS coronavirus; SARS-CoV-2 infection; Sampling; Serology; Severe Acute Respiratory Syndrome; Severities; Severity of illness; Shapes; Specimen; Statistical Data Interpretation; Supplementation; Symptoms; Syndrome; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Technology; Testing; Time; United States National Institutes of Health; Variant; Viral; Viral Antibodies; Virus; Virus Diseases; Virus Shedding; adaptive immune response; apoptosis in lymphocytes; betacoronavirus; bioinformatics tool; cohort; cytokine; data integration; design; genome sequencing; improved; indexing; older patient; pathogenic virus; post SARS-CoV-2 infection; response; screening; secondary lymphoid organ; trafficking; treatment strategy; ventilation; virome; whole genome

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.

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，利用 VirScan 平台确定病毒暴露特征作为对 COVID-19 阳性患者病毒感染史的体液免疫反应。我们正在将 VirScan 数据与其他 COVID-19 联盟项目的数据进行生物信息整合，其中包括 T 细胞受体 (TCR)/B 细胞受体 (BCR) 库、细胞因子谱、SARS-CoV-2 血清学谱和全基因组测序的种系变异。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 技术来分析来自意大利和其他国家多个地区的 SARS-CoV-2 感染患者的血清学样本中存在的 VES。我们将通过以下步骤测试 2020 年 1 月至 5 月期间在意大利和美国医院住院的 700 名患者的血清学样本中的病毒状态，这些患者因感染 SARS-CoV-2 而进行： 步骤 1) T7 噬菌体文库扩增：T7 噬菌体混合物一次展示来自 206 个物种和 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范围覆盖。将使用标准扩增方案进行文库扩增。将生成大量病毒滴度为 1010 pfu 的 T7 噬菌体文库，以最大限度地减少批次变异性并确保整个研究期间的稳定性。步骤 2) 血清学样本筛选：将从 COVID-19 患者 [400 名来自伦巴第（意大利北部）、200 名来自普利亚（意大利南部）的患者和 100 名来自美国（华盛顿特区）的患者] 以及来自相同地理区域的健康献血者（作为对照）和患有获得性或原发性免疫缺陷的受试者 (n=300) 收集的血清学样本中筛选抗病毒抗体。 因此，本研究的血清学样本总数为 1,000 个，其中 10% 的样本重复进行 (n=200)，因此将检测 1,100 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) 对回收的噬菌体 DNA 进行文库构建和并行测序：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，将测序接头和索引添加到从每个免疫沉淀反应中回收的 DNA 中，并对噬菌体 DNA 进行大规模并行测序，通过计算免疫沉淀前后每个肽的读取计数，来量化特定抗病毒抗体对其相应噬菌体库输入的富集/丰度。 将使用 Bowtie 和标准统计分析进行生物信息学和统计分析。具体目标 2：我们正在识别 SARS-CoV-2 感染者中与各种临床特征严重程度相关的独特 VES：a) 无症状/轻度感染，需要通过鼻插管补充氧气少于 5 升/分钟 (L/m)，b) 严重感染，需要通过鼻插管或其他无创通气方式补充氧气超过 5 L/m，c) 重度感染 需要机械通气的感染，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、HIV1 感染者和特发性 CD4 淋巴细胞减少症受试者 (n=100) 以及来自意大利（北部和南部地区）和美国的 COVID-19 患者 (n=700) 的 VES 特征差异。因此，我们将评估每个地理区域和临床背景的 VES 概况的异质性。步骤 2) 我们将分析具有上述 4 个临床结果的受试者之间 VES 概况的差异。步骤 3) COVID-19 的严重临床病程与严重的淋巴细胞减少有关，有报告称次级淋巴器官中淋巴细胞凋亡。这些发现表明，除了淋巴亚群的运输和重新分配之外，对 B 细胞和 T 细胞存活的特定影响可能会导致适应性免疫反应的协调不良，最终导致更糟糕的临床结果。 IL-7 是一种 T 细胞和 B 细胞稳态细胞因子，可以逆转这种效应。将在 COVID-19 个体中纵向评估 VES 谱，以评估 IL-7 对 SARS-CoV-2 和所有其他病毒病原体的谱抗体反应的影响。具体目标 3：我们正在将 VES 谱与宿主遗传学、T 细胞/B 细胞库、细胞因子谱和对 SARS-CoV-2 感染的血清学反应相结合，以完善 VES，用于监测、诊断和治疗 SARS-CoV-2 感染患者。使用我们最近出版物中描述的各种生物信息学工具，我们将通过将 COV-19 相关 VES 与种系变异以及 TCR/BCR 库、细胞因子谱和 SARS-CoV-2 基因型联系起来来进行数据集成，以完善 VES。预期结果和潜在影响：我们的项目将允许我们询问感染 SARS-CoV-2 的患者的病毒特征。这项研究将为设计有效分层 COVID-19 患者的临床病程和免疫学特征并预测疾病严重程度的策略奠定基础。这项研究的独特优势包括采用了一种综合方法，该方法可能会检测过去病毒感染的宿主免疫力与其对 SARS-CoV-2 引起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，因此可以轻松验证改进的 VES，以最大程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在稳健免疫学预测因子定义中的分析优势。 VirScan 将使我们能够了解病毒组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 感染者的诊断、监测和治疗。