Understanding humoral responses to SARS-CoV-2 is critical for improving diagnostics, therapeutics, and vaccines. Deep serological profiling of 232 COVID-19 patients and 190 pre-COVID-19 era controls using VirScan revealed over 800 epitopes in the SARS-CoV-2 proteome, including 10 epitopes likely recognized by neutralizing antibodies. Pre-existing antibodies in controls recognized SARS-CoV-2 ORF1, while only COVID-19 patients primarily recognized spike and nucleoprotein. A machine learning model trained on VirScan data predicted SARS-CoV-2 exposure history with 99% sensitivity and 98% specificity; a rapid Luminex-based diagnostic was developed from the most discriminatory SARS-CoV-2 peptides. Individuals with more severe COVID-19 exhibited stronger and broader SARS-CoV-2 responses, weaker antibody responses to prior infections, and higher incidence of CMV and HSV-1, possibly influenced by demographic covariates. Among hospitalized patients, males make greater SARS-CoV-2 antibody responses than females.

The human virome plays important roles in health and immunity. However, current methods for detecting viral infections and antiviral responses have limited throughput and coverage. Here, we present VirScan, a high-throughput method to comprehensively analyze antiviral antibodies using immunoprecipitation and massively parallel DNA sequencing of a bacteriophage library displaying proteome-wide peptides from all human viruses. We assayed over 108 antibody-peptide interactions in 569 humans across four continents, nearly doubling the number of previously established viral epitopes. We detected antibodies to an average of 10 viral species per person and 84 species in at least two individuals. Although rates of specific virus exposure were heterogeneous across populations, antibody responses targeted strikingly conserved “public epitopes” for each virus, suggesting that they may elicit highly similar antibodies. VirScan is a powerful approach for studying interactions between the virome and the immune system.

Developing effective strategies to prevent or treat COVID-19 requires understanding the natural immune response to SARS-CoV-2. We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8 + T cells of COVID-19 patients. In total, we identified 3–8 epitopes for each of the six most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8 + T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8 + T cell immunity to SARS-CoV-2.

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.

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Measles virus is directly responsible for more than 100,000 deaths yearly. Epidemiological studies have associated measles with increased morbidity and mortality for years after infection, but the reasons why are poorly understood. Measles virus infects immune cells, causing acute immune suppression. To identify and quantify long-term effects of measles on the immune system, we used VirScan, an assay that tracks antibodies to thousands of pathogen epitopes in blood. We studied 77 unvaccinated children before and 2 months after natural measles virus infection. Measles caused elimination of 11 to 73% of the antibody repertoire across individuals. Recovery of antibodies was detected after natural reexposure to pathogens. Notably, these immune system effects were not observed in infants vaccinated against MMR (measles, mumps, and rubella), but were confirmed in measles-infected macaques. The reduction in humoral immune memory after measles infection generates potential vulnerability to future infections, underscoring the need for widespread vaccination.

Life-threatening pulmonary influenza can be caused by inborn errors of type I and III IFN immunity. We report a 5-yr-old child with severe pulmonary influenza at 2 yr. She is homozygous for a loss-of-function allele. Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-α2b. The transcriptome induced by IFN-α2b in the patient's cells is much narrower than that of control cells; however, induction of a subset of IFN-stimulated gene transcripts remains detectable. In vitro, the patient's cells do not control three respiratory viruses, influenza A virus (IAV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). These phenotypes are rescued by wild-type IRF9, whereas silencing IRF9 expression in control cells increases viral replication. However, the child has controlled various common viruses in vivo, including respiratory viruses other than IAV. Our findings show that human IRF9- and ISGF3-dependent type I and III IFN responsive pathways are essential for controlling IAV.

Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.