Researching COVID to enhance recovery (RECOVER) adult study protocol: Rationale, objectives and design (preprint)

Importance: SARS-CoV-2 infection can result in ongoing, relapsing, or new symptoms or other health effects after the acute phase of infection; termed post-acute sequelae of SARS-CoV-2 infection (PASC), or long COVID. The characteristics, prevalence, trajectory and mechanisms of PASC are ill-defined. The objectives of the Researching COVID to Enhance Recovery (RECOVER) Multi-site Observational Study of PASC in Adults (RECOVER-Adult) are to: (1) characterize PASC prevalence; (2) characterize the symptoms, organ dysfunction, natural history, and distinct phenotypes of PASC; (3) identify demographic, social and clinical risk factors for PASC onset and recovery; and (4) define the biological mechanisms underlying PASC pathogenesis. Methods: RECOVER-Adult is a combined prospective/retrospective cohort currently planned to enroll 14,880 adults aged [≥]18 years. Eligible participants either must meet WHO criteria for suspected, probable, or confirmed infection; or must have evidence of no prior infection. Recruitment occurs at 86 sites in 33 U.S. states, Washington, DC and Puerto Rico, via facility- and community-based outreach. Participants complete quarterly questionnaires about symptoms, social determinants, vaccination status, and interim SARS-CoV-2 infections. In addition, participants contribute biospecimens and undergo physical and laboratory examinations at approximately 0, 90 and 180 days from infection or negative test date, and yearly thereafter. Some participants undergo additional testing based on specific criteria or random sampling. Patient representatives provide input on all study processes. The primary study outcome is onset of PASC, measured by signs and symptoms. A paradigm for identifying PASC cases will be defined and updated using supervised and unsupervised learning approaches with cross-validation. Logistic regression and proportional hazards regression will be conducted to investigate associations between risk factors, onset, and resolution of PASC symptoms. Discussion: RECOVER-Adult is the first national, prospective, longitudinal cohort of PASC among US adults. Results of this study are intended to inform public health, spur clinical trials, and expand treatment options.

Longitudinal analysis reveals elevation then sustained higher expression of autoantibodies for six months after SARS-CoV-2 infection (preprint)

High autoantibody levels are found in individuals hospitalized for COVID-19. The temporal trajectories and levels of these autoantibodies months into convalescence after SARS-CoV-2 infection are unclear. It is also unknown if the composite autoantibody signatures of convalescent SARS-CoV-2-infected individuals resemble those with diagnosed autoimmune diseases. We measured the circulating levels of 17 autoantibodies associated with autoimmune connective tissue diseases from SARS-CoV-2 hospitalized and outpatient subjects, as well as from individuals with scleroderma (SSc), systemic lupus erythematosus (SLE), and uninfected pre-pandemic controls. Seven of the 17 autoantibodies measured were higher in hospitalized and/or outpatient SARS-CoV-2 individuals an average of six months after symptom onset compared with controls, with multivariate analyses revealing links between SARS-CoV-2 infection and positivity of SSB-La, Sm, Proteinase 3, Myleoperoxidase, Jo-1, and Ku reactive IgG six months post-symptom onset. Autoantibody levels from SARS-CoV-2 infected individuals were followed over time from initial symptom onset for an average of six months, and different temporal autoantibody trajectories were classified. A 'negative, then positive' expression pattern was found for at least one autoantibody in 18% of the outpatient and 53% of the hospitalized subjects, indicating initiation and durable expression of self-reactive immune responses post-infection, particularly with severe acute illness. Analysis of individual subject autoantibody expression patterns revealed similar patterns between pre-pandemic and convalescent SARS-CoV-2 infected groups that are distinct from both the SSc and SLE subjects. As autoantibody positivity can occur years prior to autoimmune disease onset, the possibility that SARS-CoV-2-associated autoantibodies are a herald of future autoimmune disorders requires further investigation.

Distinct Autoimmune Antibody Signatures Between Hospitalized Acute COVID-19 Patients, SARS-CoV-2 Convalescent Individuals, and Unexposed Pre-Pandemic Controls (preprint)

Abstract Increasing evidence suggests that autoimmunity may play a role in the pathophysiology of SARS-CoV-2 infection during both the acute and "long COVID" phases of disease. However, an assessment of autoimmune antibodies in convalescent SARS-CoV-2 patients has not yet been reported. Methodology: We compared the levels of 18 different IgG autoantibodies (AABs) between four groups: (1) unexposed pre-pandemic subjects from the general population (n = 29); (2) individuals hospitalized with acute moderate to severe COVID-19 (n = 20); (3) convalescent SARS-COV-2-infected subjects with asymptomatic to mild viral symptoms during the acute phase with samples obtained between 1.8 and 7.3 months after infection (n = 9); and (4) unexposed pre-pandemic subjects with systemic lupus erythematous (SLE) (n = 6). Total IgG and IgA levels were also measured from subjects in groups 1-3 to assess non-specific pan-B cell activation. Results: As expected, in multivariate analysis, AABs were detected at much higher odds in SLE subjects (5 of 6, 83%) compared to non-SLE pre-pandemic controls (11 of 29, 38%) [odds ratio (OR) 19.4,95% CI, 2.0 - 557.0, p = 0.03]. AAB detection (percentage of subjects with one or more autoantibodies) was higher in SARS-CoV-2 infected convalescent subjects (7 of 9, 78%) [OR 17.4, 95% CI, 2.0 - 287.4, p = 0.02] and subjects with acute COVID-19 (12 of 20, 60%) compared with non-SLE pre-pandemic controls, but was not statistically significant among the latter [OR 1.8,95% CI, 0.6 - 8.1, p = 0.23]. Within the convalescent subject group, AABs were detected in 5/5 with reported persistent symptoms and 2/4 without continued symptoms (p = 0.17). The multivariate computational algorithm Partial Least Squares Determinant Analysis (PLSDA) was used to determine if distinct AAB signatures distinguish subject groups 1-3. Of the 18 autoantibodies measured, anti-Beta 2-Glycoprotein, anti-Proteinase 3-ANCA, anti-Mi-2 and anti-PM/Scl-100 defined the convalescent group; anti-Proteinase 3-ANCA, anti-Mi-2, anti-Jo-1 and anti-RNP/SM defined acute COVID-19 subjects; and anti-Proteinase 3-ANCA, anti-Mi-2, anti-Jo-1, anti-Beta 2-Glycoprotein distinguished unexposed controls. The AABs defining SARS-COV-2 infected from pre-pandemic subjects are widely associated with myopathies, vasculitis, and antiphospholipid syndromes, conditions with some similarities to COVID-19. Compared to pre-pandemic non-SLE controls, subjects with acute COVID-19 had higher total IgG concentration (p-value=0.006) but convalescent subjects did not (p-value=0.08); no differences in total IgA levels were found between groups. Conclusions: Our findings support existing studies suggesting induction of immune responses to self-epitopes during acute, severe COVID-19 with evidence of general B cell hyperactivation. Also, the preponderance of AAB positivity among convalescent individuals up to seven months after infection indicates potential initiation or proliferation, and then persistence of self-reactive immunity without severe initial disease. These results underscore the importance of further investigation of autoimmunity during SARS-CoV-2 infection and its role in the onset and persistence of post-acute sequelae of COVID-19.

An ELISA protocol with resolution at high sample concentration reveals reactive antibodies to SARS-CoV-2 in unexposed individuals (preprint)

The COVID-19 pandemic has significantly impacted work, economy, and way of life. The SARS-CoV-2 virus displays unique features including widely varying symptoms and outcomes between infected individuals. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into virus transmission dynamics, pre-existing cross-reactive immunity, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. This BU ELISA method exhibits very low signal from plasma or serum samples added to uncoated wells at as low as a 1:5 dilution. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (NP) reactive antibodies from blood samples drawn prior to May 2019. Of our pre-pandemic cohort, no SARS-CoV-2 RBD-reactive IgG antibodies were detected in subjects over 70 years of age, and SARS-CoV-2 NP-reactive antibodies were present at similar levels to infected subjects in some individuals and very low in others. Also, samples drawn in May 2020 from two individuals with no symptoms or no known virus exposure contained SARS-CoV-2 RBD-reactive antibodies at intermediate amounts compared with other subject groups (higher than pre-pandemic and lower than confirmed SARS-CoV-2 infected). The one asymptomatic SARS-CoV-2 convalescent subject in our study possessed comparable amounts of SARS-CoV-2 NP-specific IgM and IgG but drastically lower IgA than the symptomatic counterparts. Also, our assay detected positive signal from samples that gave negative results in a commercially available Lateral Flow Device (LFD) and the EUA approved Abbott IgG chemiluminescent microparticle immunoassay for SARS-CoV-2 antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and has promising implications for improved detection of all analytes measurable by this platform.