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.