homeRNA self-blood collection by exposed close contacts enables high-frequency temporal profiling of the pre-symptomatic host immune kinetics to respiratory viral infection

Background Host immunity is critical in determining outcomes of acute respiratory viral infections (ARVIs). However, detailed kinetics of host immune responses following natural exposures are poorly understood. Investigating the host response during the pre-symptomatic phase of viral infection is challenging, and prior work has largely relied on human challenge studies. In this prospective longitudinal study, we utilized a self-blood collection tool ( hom eRNA) to profile the host response during pre-symptomatic ARVIs in recently exposed adults and present a study framework for the conduct of large-scale longitudinal mechanistic studies. Methods We prospectively recruited non-symptomatic adults with recent exposure to ARVIs who subsequently tested negative (exposed uninfected) and positive for respiratory pathogens. Study participants performed self-collection of blood and nasal swabs across a 4-week observation window. Daily monitoring of symptoms, viral load, and blood transcriptional responses was performed for the first week followed by weekly monitoring of blood transcriptional responses and symptoms. Nasal swabs were assayed for respiratory pathogens including SARS- CoV-2. Immune kinetics from 132 longitudinal blood samples (8 SARS-CoV-2 infected and 4 exposed uninfected) were profiled at high temporal resolution for 773 host response genes. Findings 68 participants across 26 U.S. states completed the study between June 2021 – April 2022, with 97.6% of scheduled longitudinal blood collections (n=691), 97.9% of nasal swabs (n=466) and 97.2% of symptom surveys (n=688) returned. SARS-CoV-2 infection was confirmed in 25% of the participants (n=17) Expression of host immediate early genes (IEGs) involved in AP-1 transcriptional complex and prostaglandin biosynthesis along with genes encoding the early T-cell activation antigen ( CD69 ), pyrogenic cytokines (IL-6, MIP-1β, and IFN-γ), cytotoxic cell receptors and granule proteins, and interferon-induced GTPases were detected in the periphery prior to onset of viral shedding in the nasal passage. Upon onset of viral shedding, robust induction of interferon stimulated genes (ISGs) were observed. We also observed elevated expression of the host defense peptides DEFA4 , LCN2 , LTF , BPI (HDPs) in exposed uninfected individuals. Interpretation Signatures of T-cell responses prior to nasal viral shedding followed by robust induction of innate ISGs upon onset of viral shedding suggests that T-cell derived immune memory may play a role in pathogen control during early phases of the infection. Elevated levels of HDPs in exposed uninfected individuals suggest a potential role for neutrophil-mediated immunity in host defense during pathogen exposure. Finally, we demonstrated that unsupervised self-collection and stabilization of blood using home RNA can be used to study early host immune kinetics to natural ARVIs at a temporal resolution comparable to that of human challenge studies. ### Competing Interest Statement FYL, EB, and ABT filed patent 17/361,322 (Publication Number: US20210402406A1) through the University of Washington on homeRNA. ABT reports filing multiple patents through the University of Washington and receiving a gift to support research outside the submitted work from Ionis Pharmaceuticals. EB has ownership in Salus Discovery, LLC, and Tasso, Inc. that develops blood collection systems used in this manuscript, and is employed by Tasso, Inc. Technologies from Salus Discovery, LLC are not included in this publication. He is an inventor on multiple patents filed by Tasso, Inc., the University of Washington, and the University of Wisconsin. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research. AW reports receiving clinical trial support to their institution from Pfizer, Ansun Biopharma, Allovir, and GlaxoSmithKline/Vir; receiving personal fees from Kyorin Pharmaceuticals; and receiving grants from Amazon outside the submitted work. MB has clinical research support from Ansun Biopharma, Amazon, GSK, Vir Biotechnology, and Merck and receives personal fees from Allovir, Moderna, and Merck. JTS has clinical trial support from Aicuris and receives personal fees from Glaxo Smith Kline and Pfizer. ### Funding Statement R35GM128648 (ABT, for in-lab developments of homeRNA) Packard Research Fellowship from the David and Lucile Packard Foundation (ABT) R01AI153087 (AW) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was conducted at the University of Washington, Seattle WA and approved by the UW IRB under protocol STUDY00012546. All participants provided informed consent. All sample collections were performed remotely by study participants and online surveys were administered through REDCap. Participants were recruited to the study between June 2021- April 2022. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data are available in the main text or the supplementary materials. nCounter gene expression data available as normalized counts in data file S1. All statistical analyses were conducted using R and Rosalind Bio. Packages used to perform analyses are specified in the statistical section. R scripts developed to perform analyses and visualizations are available upon request.