High-resolution profiling of pathways of escape for SARS-CoV-2 spike-binding antibodies.

Defining long-term protective immunity to SARS-CoV-2 is one of the most pressing questions of our time and will require a detailed understanding of potential ways this virus can evolve to escape immune protection. Immune protection will most likely be mediated by antibodies that bind to the viral entry protein, spike (S). Here, we used Phage-DMS, an approach that comprehensively interrogates the effect of all possible mutations on binding to a protein of interest, to define the profile of antibody escape to the SARS-CoV-2 S protein using coronavirus disease 2019 (COVID-19) convalescent plasma. Antibody binding was common in two regions, the fusion peptide and the linker region upstream of the heptad repeat region 2. However, escape mutations were variable within these immunodominant regions. There was also individual variation in less commonly targeted epitopes. This study provides a granular view of potential antibody escape pathways and suggests there will be individual variation in antibody-mediated virus evolution.

Distinct Early Serological Signatures Track with SARS-CoV-2 Survival.

As SARS-CoV-2 infections and death counts continue to rise, it remains unclear why some individuals recover from infection, whereas others rapidly progress and die. Although the immunological mechanisms that underlie different clinical trajectories remain poorly defined, pathogen-specific antibodies often point to immunological mechanisms of protection. Here, we profiled SARS-CoV-2-specific humoral responses in a cohort of 22 hospitalized individuals. Despite inter-individual heterogeneity, distinct antibody signatures resolved individuals with different outcomes. Although no differences in SARS-CoV-2-specific IgG levels were observed, spike-specific humoral responses were enriched among convalescent individuals, whereas functional antibody responses to the nucleocapsid were elevated in deceased individuals. Furthermore, this enriched immunodominant spike-specific antibody profile in convalescents was confirmed in a larger validation cohort. These results demonstrate that early antigen-specific and qualitative features of SARS-CoV-2-specific antibodies point to differences in disease trajectory, highlighting the potential importance of functional antigen-specific humoral immunity to guide patient care and vaccine development.

Risk of Subsequent Respiratory Virus Detection After Primary Virus Detection in a Community Household Study-King County, Washington, 2019-2021.

BACKGROUND: The epidemiology of respiratory viral infections is complex. How infection with one respiratory virus affects risk of subsequent infection with the same or another respiratory virus is not well described. METHODS: From October 2019 to June 2021, enrolled households completed active surveillance for acute respiratory illness (ARI), and participants with ARI self-collected nasal swab specimens; after April 2020, participants with ARI or laboratory-confirmed severe acute respiratory syndrome coronavirus 2 and their household members self-collected nasal swab specimens. Specimens were tested using multiplex reverse-transcription polymerase chain reaction for respiratory viruses. A Cox regression model with a time-dependent covariate examined risk of subsequent detections following a specific primary viral detection. RESULTS: Rhinovirus was the most frequently detected pathogen in study specimens (406 [9.5%]). Among 51 participants with multiple viral detections, rhinovirus to seasonal coronavirus (8 [14.8%]) was the most common viral detection pairing. Relative to no primary detection, there was a 1.03-2.06-fold increase in risk of subsequent virus detection in the 90 days after primary detection; risk varied by primary virus: human parainfluenza virus, rhinovirus, and respiratory syncytial virus were statistically significant. CONCLUSIONS: Primary virus detection was associated with higher risk of subsequent virus detection within the first 90 days after primary detection.

Comparative diagnostic utility of SARS-CoV-2 rapid antigen and molecular testing in a community setting.

BACKGROUND: SARS-CoV-2 antigen-detection rapid diagnostic tests (Ag-RDTs) have become widely utilized but longitudinal characterization of their community-based performance remains incompletely understood. METHODS: This prospective longitudinal study at a large public university in Seattle, WA utilized remote enrollment, online surveys, and self-collected nasal swab specimens to evaluate Ag-RDT performance against real-time reverse transcription polymerase chain reaction (rRT-PCR) in the context of SARS-CoV-2 Omicron. Ag-RDT sensitivity and specificity within 1 day of rRT-PCR were evaluated by symptom status throughout the illness episode and Orf1b cycle threshold (Ct). RESULTS: From February to December 2022, 5,757 participants reported 17,572 Ag-RDT results and completed 12,674 rRT-PCR tests, of which 995 (7.9%) were rRT-PCR-positive. Overall sensitivity and specificity were 53.0% (95% CI: 49.6-56.4%) and 98.8% (98.5-99.0%), respectively. Sensitivity was comparatively higher for Ag-RDTs used 1 day after rRT-PCR (69.0%), 4 to 7 days post-symptom onset (70.1%), and Orf1b Ct ≤20 (82.7%). Serial Ag-RDT sensitivity increased with repeat testing ≥2 (68.5%) and ≥4 (75.8%) days after an initial Ag-RDT-negative result. CONCLUSION: Ag-RDT performance varied by clinical characteristics and temporal testing patterns. Our findings support recommendations for serial testing following an initial Ag-RDT-negative result, especially among recently symptomatic persons or those at high-risk for SARS-CoV-2 infection.

Incidence of SARS-CoV-2 infection and associated risk factors among staff and residents at homeless shelters in King County, Washington: an active surveillance study.

Homeless shelter residents and staff may be at higher risk of SARS-CoV-2 infection. However, SARS-CoV-2 infection estimates in this population have been reliant on cross-sectional or outbreak investigation data. We conducted routine surveillance and outbreak testing in 23 homeless shelters in King County, Washington, to estimate the occurrence of laboratory-confirmed SARS-CoV-2 infection and risk factors during 1 January 2020-31 May 2021. Symptom surveys and nasal swabs were collected for SARS-CoV-2 testing by RT-PCR for residents aged ≥3 months and staff. We collected 12,915 specimens from 2,930 unique participants. We identified 4.74 (95% CI 4.00-5.58) SARS-CoV-2 infections per 100 individuals (residents: 4.96, 95% CI 4.12-5.91; staff: 3.86, 95% CI 2.43-5.79). Most infections were asymptomatic at the time of detection (74%) and detected during routine surveillance (73%). Outbreak testing yielded higher test positivity than routine surveillance (2.7% versus 0.9%). Among those infected, residents were less likely to report symptoms than staff. Participants who were vaccinated against seasonal influenza and were current smokers had lower odds of having an infection detected. Active surveillance that includes SARS-CoV-2 testing of all persons is essential in ascertaining the true burden of SARS-CoV-2 infections among residents and staff of congregate settings.

Nausea, Vomiting, and Diarrhea Are Common in Community-Acquired Acute Viral Respiratory Illness.

BACKGROUND: Gastrointestinal (GI) symptoms are recognized sequelae of acute respiratory illness (ARI), but their prevalence is not well documented. Our study aim was to assess the incidence of GI symptoms in community ARI cases for persons of all ages and their association with clinical outcomes. METHODS: We collected mid-nasal swabs, clinical, and symptom data from Seattle-area individuals during the 2018-2019 winter season as part of a large-scale prospective community surveillance study. Swabs were tested by polymerase chain reaction (PCR) for 26 respiratory pathogens. Likelihood of GI symptoms given demographic, clinical, and microbiological covariates were analyzed with Fisher's exact, Wilcoxon-rank-sum, and t-tests and multivariable logistic regression. RESULTS: In 3183 ARI episodes, 29.4% had GI symptoms (n = 937). GI symptoms were significantly associated with pathogen detection, illness interfering with daily life, seeking care for the illness, and greater symptom burden (all p < 0.05). Controlling for age, > 3 symptoms, and month, influenza (p < 0.001), human metapneumovirus (p = 0.004), and enterovirus D68 (p = 0.05) were significantly more likely to be associated with GI symptoms than episodes with no pathogen detected. Seasonal coronaviruses (p = 0.005) and rhinovirus (p = 0.04) were significantly less likely to be associated with GI symptoms. CONCLUSION: In this community-surveillance study of ARI, GI symptoms were common and associated with illness severity and respiratory pathogen detection. GI symptoms did not track with known GI tropism, suggesting GI symptoms may be nonspecific rather than pathogen-mediated. Patients presenting with GI and respiratory symptoms should have respiratory virus testing, even if the respiratory symptom is not the primary concern.

The Clinical and Genomic Epidemiology of Rhinovirus in Homeless Shelters-King County, Washington.

BACKGROUND: Rhinovirus (RV) is a common cause of respiratory illness in all people, including those experiencing homelessness. RV epidemiology in homeless shelters is unknown. METHODS: We analyzed data from a cross-sectional homeless shelter study in King County, Washington, October 2019-May 2021. Shelter residents or guardians aged ≥3 months reporting acute respiratory illness completed questionnaires and submitted nasal swabs. After 1 April 2020, enrollment expanded to residents and staff regardless of symptoms. Samples were tested by multiplex RT-PCR for respiratory viruses. A subset of RV-positive samples was sequenced. RESULTS: There were 1066 RV-positive samples with RV present every month of the study period. RV was the most common virus before and during the coronavirus disease 2019 (COVID-19) pandemic (43% and 77% of virus-positive samples, respectively). Participants from family shelters had the highest prevalence of RV. Among 131 sequenced samples, 33 RV serotypes were identified with each serotype detected for ≤4 months. CONCLUSIONS: RV infections persisted through community mitigation measures and were most prevalent in shelters housing families. Sequencing showed a diversity of circulating RV serotypes, each detected over short periods of time. Community-based surveillance in congregate settings is important to characterize respiratory viral infections during and after the COVID-19 pandemic. CLINICAL TRIALS REGISTRATION: NCT04141917.

Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Most individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop neutralizing antibodies that target the viral spike protein. In this study, we quantified how levels of these antibodies change in the months after SARS-CoV-2 infection by examining longitudinal samples collected approximately 30-152 days after symptom onset from a prospective cohort of 32 recovered individuals with asymptomatic, mild, or moderate-severe disease. Neutralizing antibody titers declined an average of about 4-fold from 1 to 4 months after symptom onset. This decline in neutralizing antibody titers was accompanied by a decline in total antibodies capable of binding the viral spike protein or its receptor-binding domain. Importantly, our data are consistent with the expected early immune response to viral infection, where an initial peak in antibody levels is followed by a decline to a lower plateau. Additional studies of long-lived B cells and antibody titers over longer time frames are necessary to determine the durability of immunity to SARS-CoV-2.

Incidence of Medically Attended Acute Respiratory Illnesses Due to Respiratory Viruses Across the Life Course During the 2018/19 Influenza Season.

BACKGROUND: Although multiple respiratory viruses circulate in humans, few studies have compared the incidence of different viruses across the life course. We estimated the incidence of outpatient illness due to 12 different viruses during November 2018 through April 2019 in a fully enumerated population. METHODS: We conducted active surveillance for ambulatory care visits for acute respiratory illness (ARI) among members of Kaiser Permanente Washington (KPWA). Enrolled patients provided respiratory swab specimens which were tested for 12 respiratory viruses using reverse transcription polymerase chain reaction (RT-PCR). We estimated the cumulative incidence of infection due to each virus overall and by age group. RESULTS: The KPWA population under surveillance included 202 562 individuals, of whom 2767 (1.4%) were enrolled in the study. Influenza A(H3N2) was the most commonly detected virus, with an overall incidence of 21 medically attended illnesses per 1000 population; the next most common viruses were influenza A(H1N1) (18 per 1000), coronaviruses (13 per 1000), respiratory syncytial virus (RSV, 13 per 1000), and rhinovirus (9 per 1000). RSV was the most common cause of medically attended ARI among children aged 1-4 years; coronaviruses were the most common among adults aged ≥65 years. CONCLUSIONS: Consistent with other studies focused on single viruses, we found that influenza and RSV were major causes of acute respiratory illness in persons of all ages. In comparison, coronaviruses and rhinovirus were also important pathogens. Prior to the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coronaviruses were the second-most common cause of medically attended ARI during the 2018/19 influenza season.

Remote Household Observation for Noninfluenza Respiratory Viral Illness.

BACKGROUND: Noninfluenza respiratory viruses are responsible for a substantial burden of disease in the United States. Household transmission is thought to contribute significantly to subsequent transmission through the broader community. In the context of the coronavirus disease 2019 (COVID-19) pandemic, contactless surveillance methods are of particular importance. METHODS: From November 2019 to April 2020, 303 households in the Seattle area were remotely monitored in a prospective longitudinal study for symptoms of respiratory viral illness. Enrolled participants reported weekly symptoms and submitted respiratory samples by mail in the event of an acute respiratory illness (ARI). Specimens were tested for 14 viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using reverse-transcription polymerase chain reaction. Participants completed all study procedures at home without physical contact with research staff. RESULTS: In total, 1171 unique participants in 303 households were monitored for ARI. Of participating households, 128 (42%) included a child aged <5 years and 202 (67%) included a child aged 5-12 years. Of the 678 swabs collected during the surveillance period, 237 (35%) tested positive for 1 or more noninfluenza respiratory viruses. Rhinovirus, common human coronaviruses, and respiratory syncytial virus were the most common. Four cases of SARS-CoV-2 were detected in 3 households. CONCLUSIONS: This study highlights the circulation of respiratory viruses within households during the winter months during the emergence of the SARS-CoV-2 pandemic. Contactless methods of recruitment, enrollment, and sample collection were utilized throughout this study and demonstrate the feasibility of home-based, remote monitoring for respiratory infections.

SwabExpress: An End-to-End Protocol for Extraction-Free COVID-19 Testing.

BACKGROUND: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. METHODS: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. CONCLUSION: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

Isotopic and genetic methods reveal the role of the gut microbiome in mammalian host essential amino acid metabolism.

Intestinal microbiota perform many functions for their host, but among the most important is their role in metabolism, especially the conversion of recalcitrant biomass that the host is unable to digest into bioavailable compounds. Most studies have focused on the assistance gut microbiota provide in the metabolism of carbohydrates, however, their role in host amino acid metabolism is poorly understood. We conducted an experiment on Mus musculus using 16S rRNA gene sequencing and carbon isotope analysis of essential amino acids (AAESS) to quantify the community composition of gut microbiota and the contribution of carbohydrate carbon used by the gut microbiome to synthesize AAESS that are assimilated by mice to build skeletal muscle tissue. The relative abundances of Firmicutes and Bacteroidetes inversely varied as a function of dietary macromolecular content, with Firmicutes dominating when mice were fed low-protein diets that contained the highest proportions of simple carbohydrates (sucrose). Mixing models estimated that the microbial contribution of AAESS to mouse muscle varied from less than 5% (threonine, lysine, and phenylalanine) to approximately 60% (valine) across diet treatments, with the Firmicute-dominated microbiome associated with the greatest contribution. Our results show that intestinal microbes can provide a significant source of the AAESS their host uses to synthesize structural tissues. The role that gut microbiota play in the amino acid metabolism of animals that consume protein-deficient diets is likely a significant but under-recognized aspect of foraging ecology and physiology.

Evaluation of a novel university-based testing platform to increase access to SARS-CoV-2 testing during the COVID-19 pandemic in a cohort study.

OBJECTIVE: We aimed to evaluate the feasibility and utility of an unsupervised testing mechanism, in which participants pick up a swab kit, self-test (unsupervised) and return the kit to an on-campus drop box, as compared with supervised self-testing at staffed locations. DESIGN: University SARS-CoV-2 testing cohort. SETTING: Husky Coronavirus Testing provided voluntary SARS-CoV-2 testing at a university in Seattle, USA. OUTCOME MEASURES: We computed descriptive statistics to describe the characteristics of the study sample. Adjusted logistic regression implemented via generalised estimating equations was used to estimate the odds of a self-swab being conducted through unsupervised versus supervised testing mechanisms by participant characteristics, including year of study enrolment, pre-Omicron versus post-Omicron time period, age, sex, race, ethnicity, affiliation and symptom status. RESULTS: From September 2021 to July 2022, we received 92 499 supervised and 26 800 unsupervised self-swabs. Among swabs received by the laboratory, the overall error rate for supervised versus unsupervised swabs was 0.3% vs 4%, although this declined to 2% for unsupervised swabs by the spring of the academic year. Results were returned for 92 407 supervised (5% positive) and 25 836 unsupervised (4%) swabs from 26 359 participants. The majority were students (79%), 61% were female and most identified as white (49%) or Asian (34%). The use of unsupervised testing increased during the Omicron wave when testing demand was high and stayed constant in spring 2022 even when testing demand fell. We estimated the odds of using unsupervised versus supervised testing to be significantly greater among those <25 years of age (p<0.001), for Hispanic versus non-Hispanic individuals (OR 1.2, 95% CI 1.0 to 1.3, p=0.01) and lower among individuals symptomatic versus asymptomatic or presymptomatic (0.9, 95% CI 0.8 to 0.9, p<0.001). CONCLUSIONS: Unsupervised swab collection permitted increased testing when demand was high, allowed for access to a broader proportion of the university community and was not associated with a substantial increase in testing errors.

Utilizing a university testing program to estimate relative effectiveness of monovalent COVID-19 mRNA booster vaccine versus two-dose primary series against symptomatic SARS-CoV-2 infection.

Vaccine effectiveness (VE) studies utilizing the test-negative design are typically conducted in clinical settings, rather than community populations, leading to bias in VE estimates against mild disease and limited information on VE in healthy young adults. In a community-based university population, we utilized data from a large SARS-CoV-2 testing program to estimate relative VE of COVID-19 mRNA vaccine primary series and monovalent booster dose versus primary series only against symptomatic SARS-CoV-2 infection from September 2021 to July 2022. We used the test-negative design and logistic regression implemented via generalized estimating equations adjusted for age, calendar time, prior SARS-CoV-2 infection, and testing frequency (proxy for test-seeking behavior) to estimate relative VE. Analyses included 2,218 test-positive cases (59 % received monovalent booster dose) and 9,615 test-negative controls (62 %) from 9,066 individuals, with median age of 21 years, mostly students (71 %), White (56 %) or Asian (28 %), and with few comorbidities (3 %). More cases (23 %) than controls (6 %) had COVID-19-like illness. Estimated adjusted relative VE of primary series and monovalent booster dose versus primary series only against symptomatic SARS-CoV-2 infection was 40 % (95 % CI: 33-47 %) during the overall analysis period and 46 % (39-52 %) during the period of Omicron circulation. Relative VE was greater for those without versus those with prior SARS-CoV-2 infection (41 %, 34-48 % versus 33 %, 9 %-52 %, P < 0.001). Relative VE was also greater in the six months after receiving a booster dose (41 %, 33-47 %) compared to more than six months (27 %, 8-42 %), but this difference was not statistically significant (P = 0.06). In this relatively young and healthy adult population, an mRNA monovalent booster dose provided increased protection against symptomatic SARS-CoV-2 infection, overall and with the Omicron variant. University testing programs may be utilized for estimating VE in healthy young adults, a population that is not well-represented by routine VE studies.

Care-seeking correlates of acute respiratory illness among sheltered adults experiencing homelessness in Seattle, WA, 2019: a community-based cross-sectional study.

Objective: Multifarious barriers to accessing healthcare services among people experiencing homelessness (PEH) lead to delays in seeking care for acute infections, including those caused by respiratory viruses. PEH are at high risk of acute respiratory illness (ARI)-related complications, especially in shelter settings that may facilitate virus spread, yet data characterizing healthcare utilization for ARI episodes among sheltered PEH remained limited. Methods: We conducted a cross-sectional study of viral respiratory infection among adult residents at two homeless shelters in Seattle, Washington between January and May 2019. We assessed factors associated with seeking medical care for ARI via self-report. We collected illness questionnaires and nasal swabs were tested for respiratory viruses by reverse transcription quantitative real-time PCR (RT-qPCR). Results: We observed 825 encounters from 649 unique participants; 241 (29.2%) encounters reported seeking healthcare for their ARI episode. Seasonal influenza vaccine receipt (adjusted prevalence ratio [aPR] 1.39, 95% CI 1.02-1.88), having health insurance (aPR 2.77, 95% CI 1.27-6.02), chronic lung conditions (aPR 1.55, 95% CI 1.12-2.15), and experiencing influenza-like-illness symptoms (aPR 1.63, 95% CI 1.20 - 2.20) were associated with increased likelihood of seeking care. Smoking (aPR 0.65, 95% CI 0.45-0.92) was associated with decreased likelihood of seeking care. Discussion: Findings suggest that care seeking for viral respiratory illness among PEH may be supported by prior engagement with primary healthcare services. Strategies to increase healthcare utilization may lead to earlier detection of respiratory viruses.

Results from a test-and-treat study for influenza among residents of homeless shelters in King County, WA: A stepped-wedge cluster-randomized trial.

BACKGROUND: Persons experiencing homelessness face increased risk of influenza as overcrowding in congregate shelters can facilitate influenza virus spread. Data regarding on-site influenza testing and antiviral treatment within homeless shelters remain limited. METHODS: We conducted a cluster-randomized stepped-wedge trial of point-of-care molecular influenza testing coupled with antiviral treatment with baloxavir or oseltamivir in residents of 14 homeless shelters in Seattle, WA, USA. Residents ≥3 months with cough or ≥2 acute respiratory illness (ARI) symptoms and onset <7 days were eligible. In control periods, mid-nasal swabs were tested for influenza by reverse transcription polymerase chain reaction (RT-PCR). The intervention period included on-site rapid molecular influenza testing and antiviral treatment for influenza-positives if symptom onset was <48 h. The primary endpoint was monthly influenza virus infections in the control versus intervention periods. Influenza whole genome sequencing was performed to assess transmission and antiviral resistance. RESULTS: During 11/15/2019-4/30/2020 and 11/2/2020-4/30/2021, 1283 ARI encounters from 668 participants were observed. Influenza virus was detected in 51 (4%) specimens using RT-PCR (A = 14; B = 37); 21 influenza virus infections were detected from 269 (8%) intervention-eligible encounters by rapid molecular testing and received antiviral treatment. Thirty-seven percent of ARI-participant encounters reported symptom onset < 48 h. The intervention had no effect on influenza virus transmission (adjusted relative risk 1.73, 95% confidence interval [CI] 0.50-6.00). Of 23 influenza genomes, 86% of A(H1N1)pdm09 and 81% of B/Victoria sequences were closely related. CONCLUSION: Our findings suggest feasibility of influenza test-and-treat strategies in shelters. Additional studies would help discern an intervention effect during periods of increased influenza activity.

Respiratory syncytial virus and other respiratory virus infections in residents of homeless shelters - King County, Washington, 2019-2021.

Respiratory syncytial virus (RSV) causes disproportionate morbidity and mortality in vulnerable populations. We tested residents of homeless shelters in Seattle, Washington for RSV in a repeated cross-sectional study as part of community surveillance for respiratory viruses. Of 15 364 specimens tested, 35 had RSV detected, compared to 77 with influenza. The most common symptoms for both RSV and influenza were cough and rhinorrhea. Many individuals with RSV (39%) and influenza (58%) reported that their illness significantly impacted their ability to perform their regular activities. RSV and influenza demonstrated similar clinical presentations and burden of illness in vulnerable populations living in congregate settings.

CASCADIA: a prospective community-based study protocol for assessing SARS-CoV-2 vaccine effectiveness in children and adults using a remote nasal swab collection and web-based survey design.

INTRODUCTION: Although SARS-CoV-2 vaccines were first approved under Emergency Use Authorization by the Food and Drug Administration in late 2020 for adults, authorisation for young children 6 months to <5 years of age did not occur until 2022. These authorisations were based on clinical trials, understanding real-world vaccine effectiveness (VE) in the setting of emerging variants is critical. The primary goal of this study is to evaluate SARS-CoV-2 VE against infection among children aged >6 months and adults aged <50 years. METHODS: CASCADIA is a 4-year community-based prospective study of SARS-CoV-2 VE among 3500 adults and paediatric populations aged 6 months to 49 years in Oregon and Washington, USA. At enrolment and regular intervals, participants complete a sociodemographic questionnaire. Individuals provide a blood sample at enrolment and annually thereafter, with optional blood draws every 6 months and after infection and vaccination. Participants complete weekly self-collection of anterior nasal swabs and symptom questionnaires. Swabs are tested for SARS-CoV-2 and other respiratory pathogens by reverse transcription-PCR, with results of selected pathogens returned to participants; nasal swabs with SARS-CoV-2 detected will undergo whole genome sequencing. Participants who test positive for SARS-CoV-2 undergo serial swab collection every 3 days for 21 days. Serum samples are tested for SARS-CoV-2 antibody by binding and neutralisation assays. ANALYSIS: The primary outcome is SARS-CoV-2 infection. Cox regression models will be used to estimate the incidence rate ratio associated with SARS-CoV-2 vaccination among the paediatric and adult population, controlling for demographic factors and other potential confounders. ETHICS AND DISSEMINATION: All study materials including the protocol, consent forms, data collection instruments, participant communication and recruitment materials, were approved by the Kaiser Permanente Interregional Institutional Review Board, the IRB of record for the study. Results will be disseminated through peer-reviewed publications, presentations, participant newsletters and appropriate general news media.

Comprehensive characterization of the antibody responses to SARS-CoV-2 Spike protein finds additional vaccine-induced epitopes beyond those for mild infection.

Background: Control of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal. Methods: The epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions, we determined potential sites of escape by comparing antibody binding of peptides containing wild-type residues versus peptides containing a mutant residue. Results: Individuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination. Conclusions: The finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests that protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level. Funding: This work was supported by NIH grants AI138709 (PI JMO) and AI146028 (PI FAM). JMO received support as the Endowed Chair for Graduate Education (FHCRC). The research of FAM was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.

When SARS-CoV-2 ­ the virus that causes COVID-19 ­ infects our bodies, our immune system reacts by producing small molecules called antibodies that stick to a part of the virus called the spike protein. Vaccines are thought to work by triggering the production of similar antibodies without causing disease. Some of the most effective antibodies against SARS-CoV-2 bind a specific area of the spike protein called the 'receptor binding domain' or RBD. When SARS-CoV-2 evolves it creates a challenge for our immune system: mutations, which are changes in the virus's genetic code, can alter the shape of its spike protein, meaning that existing antibodies may no longer bind to it as effectively. This lowers the protection offered by past infection or vaccination, which makes it harder to tackle the pandemic. As it stands, it is not clear which mutations to the virus's genetic code can affect antibody binding, especially to portions outside the RBD. To complicate things further, the antibodies people produce in response to mild infection, severe infection, and vaccination, while somewhat overlapping, exhibit some differences. Studying these differences could help minimize emergence of mutations that allow the virus to 'escape' the antibody response. A phage display library is a laboratory technique in which phages (viruses that infect bacteria) are used as a 'repository' for DNA fragments that code for a specific protein. The phages can then produce the protein (or fragments of it), and if the protein fragments bind to a target, it can be easily detected. Garrett, Galloway et al. exploited this technique to study how different portions of the SARS-CoV-2 spike protein were bound by antibodies. They made a phage library in which each phage encoded a portion of the spike protein with different mutations, and then exposed the different versions of the protein to antibodies from people who had experienced prior infection, vaccination, or both. The experiment showed that antibodies produced during severe infection or after vaccination bound to similar parts of the spike protein, while antibodies from people who had experienced mild infection targeted fewer areas. Garrett, Galloway et al. also found that mutations that affected the binding of antibodies produced after vaccination were more consistent than mutations that interfered with antibodies produced during infection. While these results show which mutations are most likely to help the virus escape existing antibodies, this does not mean that the virus will necessarily evolve in that direction. Indeed, some of the mutations may be impossible for the virus to acquire because they interfere with the virus's ability to spread. Further studies could focus on revealing which of the mutations detected by Garrett, Galloway et al. are most likely to occur, to guide vaccine development in that direction. To help with this, Garrett, Galloway et al. have made the data accessible to other scientists and the public using a web tool.