Plasma-based antigen persistence in the post-acute phase of SARS-CoV-2 infection (preprint)

BACKGROUND: Although RNA viruses like SARS-CoV-2 are generally thought to be transient, the persistence of viral components beyond the acute phase can be driven by a variety of virologic and immunologic factors. Recent studies have suggested that SARS-CoV-2 antigens may persist following COVID-19 but were limited by a lack of comparison to a large number of true negative control samples. METHODS: Using single molecule array (Simoa) assays for SARS-CoV-2 spike, S1, and nucleocapsid antigen in plasma from 171 pandemic-era individuals in the post-acute phase of SARS-CoV-2 infection and 250 pre-pandemic control samples, we compared prevalence of antigen detection. We used logistic regression models and prevalence ratios (PRs) to assess the relationship between demographic and disease factors and antigen persistence. RESULTS: Compared to the proportion of antigen positivity in the pre-pandemic controls (2%), detection of any SARS-CoV-2 antigen was more frequent across all post-acute COVID-19 time bins (3-6 months: 12.6%, p<0.001; 6-10 months, 10.7%, p=0.0002; 10-14 months, 7.5%, p=0.017). These differences were driven by spike protein for up to 14 months and nucleocapsid in the first 6 months after infection. The co-occurrence of multiple antigens at a single timepoint was uncommon. Hospitalization for acute COVID-19 (versus not hospitalized) and worse self-reported health during acute COVID-19 among those not hospitalized (versus more benign illness) were associated with higher prevalence of post-acute antigen detection (PR 1.86, p=0.03; PR 3.5, p=0.07, respectively) in the pandemic era. CONCLUSIONS: Our findings provide strong evidence that SARS-CoV-2 antigens can persist beyond the period of acute illness. The observation that more than 10% of plasma samples for over a year following initial SARS-CoV-2 infection contain detectable viral antigen, which are potentially immunogenic, has significant implications given the sheer number of people infected with SARS-CoV-2 to date. More work will be needed to determine whether these antigens have a causal role in post-acute sequelae of SARS-CoV-2 infection (PASC).

Multimodal Molecular Imaging Reveals Tissue-Based T Cell Activation and Viral RNA Persistence for Up to Two Years Following COVID-19 (preprint)

The etiologic mechanisms of post-acute medical morbidities and unexplained symptoms (Long COVID) following SARS-CoV-2 infection are incompletely understood. There is growing evidence that viral persistence and immune dysregulation may play a major role. We performed whole-body positron emission tomography (PET) imaging in a cohort of 24 participants at time points ranging from 27 to 910 days following acute SARS-CoV-2 infection using a novel radiopharmaceutical agent, [18F]F-AraG, a highly selective tracer that allows for anatomical quantitation of activated T lymphocytes. Tracer uptake in the post-acute COVID group, which included those with and without Long COVID symptoms, was significantly higher compared to pre-pandemic controls in many anatomical regions, including the brain stem, spinal cord, bone marrow, nasopharyngeal and hilar lymphoid tissue, cardiopulmonary tissues, and gut wall. Although T cell activation tended to be higher in participants imaged closer to the time of the acute illness, tracer uptake was increased in participants imaged up to 2.5 years following SARS-CoV-2 infection. We observed that T cell activation in spinal cord and gut wall was associated with the presence of Long COVID symptoms. In addition, tracer uptake in lung tissue was higher in those with persistent pulmonary symptoms. Notably, increased T cell activation in these tissues was also observed in many individuals without Long COVID. Given the high [18F]F-AraG uptake detected in the gut, we obtained colorectal tissue for in situ hybridization SARS-CoV-2 RNA and immunohistochemical studies in a subset of participants with Long COVID symptoms. We identified cellular SARS-CoV-2 RNA in rectosigmoid lamina propria tissue in all these participants, ranging from 158 to 676 days following initial COVID-19 illness, suggesting that tissue viral persistence could be associated with long-term immunological perturbations.

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.

Association of SARS-CoV-2 Infection and Cardiopulmonary Long COVID with Exercise Capacity and Chronotropic Incompetence among People with HIV (preprint)

Background: Long COVID has been associated with reduced exercise capacity, but whether SARS-CoV-2 infection or Long COVID is associated with reduced exercise capacity among people with HIV (PWH) has not been reported. We hypothesized that PWH with cardiopulmonary post-acute symptoms of COVID-19 (PASC) would have reduced exercise capacity due to chronotropic incompetence. Methods: We conducted cross-sectional cardiopulmonary exercise testing within a COVID recovery cohort that included PWH. We evaluated associations of HIV, prior SARS-CoV-2 infection, and cardiopulmonary PASC with exercise capacity (peak oxygen consumption, VO2) and adjusted heart rate reserve (AHRR, chronotropic measure) with adjustment for age, sex, and body mass index. Results: We included 83 participants (median age 54, 35% female). All 37 PWH were virally suppressed; 23 (62%) had prior SARS-CoV-2 infection, and 11 (30%) had PASC. Peak VO2 was reduced among PWH (80% predicted vs 99%; p=0.005), a difference of 5.5 ml/kg/min (95%CI 2.7-8.2, p<0.001). Chronotropic incompetence more prevalent among PWH (38% vs 11%; p=0.002), and AHRR was reduced among PWH (60% vs 83%, p<0.0001). Among PWH, exercise capacity did not vary by SARS-CoV-2 coinfection, but chronotropic incompetence was more common among PWH with PASC: 3/14 (21%) without SARS-CoV-2, 4/12 (25%) with SARS-CoV-2 without PASC, and 7/11 (64%) with PASC (p=0.04 PASC vs no PASC). Conclusions: Exercise capacity and chronotropy are lower among PWH compared to SARS-CoV-2 infected individuals without HIV. Among PWH, SARS-CoV-2 infection and PASC were not strongly associated with reduced exercise capacity. Chronotropic incompetence may be a mechanism limiting exercise capacity among PWH.

The Breadth of the Neutralizing Antibody Response to Original SARS-CoV-2 Infection is Linked to the Presence of Long COVID Symptoms (preprint)

Background: The associations between longitudinal dynamics and the breadth of SARS-CoV-2 neutralizing antibody response with various Long COVID (LC) phenotypes prior to vaccination are not known. The capacity of antibodies to cross neutralize a variety of viral variants may be associated with ongoing pathology and persistent symptoms. Methods: We measured longitudinal neutralizing and cross-neutralizing antibody responses to pre- and post-SARS-CoV-2 Omicron variants in participants infected during the early waves of the COVID-19 pandemic, prior to wide-spread rollout of SARS-CoV-2 vaccines. Cross sectional regression models adjusted for various clinical covariates and longitudinal mixed effects models were used to determine the impact of the breadth and rate of decay of neutralizing responses on the development of Long COVID symptoms in general, as well as LC phenotypes. Results: We identified several novel relationships between SARS-CoV-2 antibody neutralization and the presence of LC symptoms. Specifically, we show that, although neutralizing antibody responses to the original, infecting strain of SARS-CoV-2 were not associated with LC in cross-sectional analyses, cross-neutralization ID50 levels to the Omicron BA.5 variant approximately 4 months following acute infection was independently and significantly associated with greater odds of LC and with persistent gastrointestinal and neurological symptoms. Longitudinal modeling demonstrated significant associations in the overall levels and rates of decay of neutralization capacity with LC phenotypes. A higher proportion of participants had antibodies capable of neutralizing Omicron BA.5 compared with BA.1 or XBB.1.5 variants. Conclusions: Our findings suggest that relationships between various immune responses and LC are likely complex but may involve the breadth of antibody neutralization responses.

Association of Nirmatrelvir/Ritonavir Treatment with Long COVID Symptoms in an Online Cohort of Non-Hospitalized Individuals Experiencing Breakthrough SARS-CoV-2 Infection in the Omicron Era (preprint)

Background: Oral nirmatrelvir/ritonavir is a treatment for COVID-19, but whether treatment during the acute phase reduces the risk of developing Long COVID is unknown. Methods: Using the Covid Citizen Science (CCS) online cohort, we surveyed individuals who reported their first SARS-CoV-2 positive test between March and August 2022 regarding Long COVID symptoms. We excluded those who were pregnant, unvaccinated, hospitalized for COVID-19, or received other antiviral therapy. The primary exposure was oral nirmatrelvir/ritonavir. The primary outcome was the presence of any Long COVID symptoms reported on cross-sectional surveys in November and December 2022. We used propensity-score models and inverse probability of treatment weighting to adjust for differences in treatment propensity. Our secondary question was whether symptom or test positivity rebound were associated with Long COVID. Results: 4684 individuals met the eligibility criteria, of whom 988 (21.1%) were treated and 3696 (78.9%) were untreated; 353/988 (35.7%) treated and 1258/3696 (34.0%) untreated responded to the survey. Median age was 55 years and 66% were female. We did not identify an association between nirmatrelvir/ritonavir treatment and Long COVID symptoms (OR 1.15; 95%CI 0.80-1.64). Among n=666 treated with nirmatrelvir/ritonavir who responded who responded to questions about rebound, rebound symptoms or test positivity were not associated with Long COVID symptoms (OR 1.34; 95%CI 0.74-2.41; p=0.33). Conclusions: Within this cohort, treatment with nirmatrelvir/ritonavir among vaccinated, non-hospitalized individuals was not associated with lower prevalence of Long COVID symptoms or severity of Long COVID. Experiencing rebound symptoms or test positivity is not strongly associated with developing Long COVID.

Long COVID manifests with T cell dysregulation, inflammation, and an uncoordinated adaptive immune response to SARS-CoV-2 (preprint)

Long COVID (LC), a type of post-acute sequelae of SARS-CoV-2 infection (PASC), occurs after at least 10% of SARS-CoV-2 infections, yet its etiology remains poorly understood. Here, we used multiple omics assays (CyTOF, RNAseq, Olink) and serology to deeply characterize both global and SARS-CoV-2-specific immunity from blood of individuals with clear LC and non-LC clinical trajectories, 8 months following infection and prior to receipt of any SARS-CoV-2 vaccine. Our analysis focused on deep phenotyping of T cells, which play important roles in immunity against SARS-CoV-2 yet may also contribute to COVID-19 pathogenesis. Our findings demonstrate that individuals with LC exhibit systemic inflammation and immune dysregulation. This is evidenced by global differences in T cell subset distribution in ways that imply ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. Individuals with LC harbored increased frequencies of CD4+ T cells poised to migrate to inflamed tissues, and exhausted SARS-CoV-2-specific CD8+ T cells. They also harbored significantly higher levels of SARS-CoV-2 antibodies, and in contrast to non-LC individuals, exhibited a mis-coordination between their SARS-CoV-2-specific T and B cell responses. Collectively, our data suggest that proper crosstalk between the humoral and cellular arms of adaptive immunity has broken down in LC, and that this, perhaps in the context of persistent virus, leads to the immune dysregulation, inflammation, and clinical symptoms associated with this debilitating condition.

Autoantigen profiling reveals a shared post-COVID signature in fully recovered and Long COVID patients (preprint)

Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as Long COVID. The underlying pathophysiology of Long COVID remains unknown. Given that autoantibodies have been found to play a role in severity of COVID infection and certain other post-COVID sequelae, their potential role in Long COVID is important to investigate. Here we apply a well-established, unbiased, proteome-wide autoantibody detection technology (PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with Long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected which separates individuals with prior COVID infection from those never exposed to COVID, we did not detect patterns of autoreactivity that separate individuals with Long COVID relative to individuals fully recovered from SARS-CoV-2 infection. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and Long COVID was apparent by this assay.

Longitudinal and Quantitative Fecal Shedding Dynamics of SARS-CoV-2, Pepper Mild Mottle Virus and CrAssphage (preprint)

Wastewater-based epidemiology (WBE) emerged during the COVID-19 pandemic as a scalable and broadly applicable method for community-level monitoring of infectious disease burden, though the lack of high-quality, longitudinal fecal shedding data of SARS-CoV-2 and other viruses limits the interpretation and applicability of wastewater measurements. In this study, we present longitudinal, quantitative fecal shedding data for SARS-CoV-2 RNA, as well as the commonly used fecal indicators Pepper Mild Mottle Virus (PMMoV) RNA and crAss-like phage (crAssphage) DNA. The shedding trajectories from 48 SARS-CoV-2 infected individuals suggest a highly individualized, dynamic course of SARS-CoV-2 RNA fecal shedding, with individual measurements varying from below limit of detection to 2.79x10^6 gene copies/mg - dry mass of stool (gc/mg-dw). Of individuals that contributed at least 3 samples covering a range of at least 15 of the first 30 days after initial acute symptom onset, 77.4% had at least one positive SARS-CoV-2 RNA stool sample measurement. We detected PMMoV RNA in at least one sample from all individuals and in 96% (352/367) of samples overall; and measured crAssphage DNA above detection limits in 80% (38/48) of individuals and 48% (179/371) of samples. Median shedding values for PMMoV and crAssphage nucleic acids were 1x10^5 gc/mg-dw and 1.86x10^3 gc/mg-dw, respectively. These results can be used to inform and build mechanistic models to significantly broaden the potential of WBE modeling and to provide more accurate insight into SARS-CoV-2 prevalence estimates.

Factors Associated with Long Covid Symptoms in an Online Cohort Study (preprint)

Importance: Prolonged symptoms following SARS-CoV-2 infection, or Long COVID, is common, but few prospective studies of Long COVID risk factors have been conducted. Objective: To determine whether sociodemographic factors, lifestyle, or medical history preceding COVID-19 or characteristics of acute SARS-CoV-2 infection are associated with Long COVID. Design: Cohort study with longitudinal assessment of symptoms before, during, and after SARS-CoV-2 infection, and cross-sectional assessment of Long COVID symptoms using data from the COVID-19 Citizen Science (CCS) study. Setting: CCS is an online cohort study that began enrolling March 26, 2020. We included data collected between March 26, 2020, and May 18, 2022. Participants: Adult CCS participants who reported a positive SARS-CoV-2 test result (PCR, Antigen, or Antibody) more than 30 days prior to May 4, 2022, were surveyed. Exposures: Age, sex, race/ethnicity, education, employment, socioeconomic status/financial insecurity, self-reported medical history, vaccination status, time of infection (variant wave), number of acute symptoms, pre-COVID depression, anxiety, alcohol and drug use, sleep, exercise. Main Outcome: Presence of at least 1 Long COVID symptom greater than 1 month after acute infection. Sensitivity analyses were performed considering only symptoms beyond 3 months and only severe symptoms. Results: 13,305 participants reported a SARS-CoV-2 positive test more than 30 days prior, 1480 (11.1% of eligible) responded to a survey about Long COVID symptoms, and 476 (32.2% of respondents) reported Long COVID symptoms (median 360 days after infection). Respondents' mean age was 53 and 1017 (69%) were female. Common Long COVID symptoms included fatigue, reported by 230/476 (48.3%), shortness of breath (109, 22.9%), confusion/brain fog (108, 22.7%), headache (103, 21.6%), and altered taste or smell (98, 20.6%). In multivariable models, number of acute COVID-19 symptoms (OR 1.30 per symptom, 95%CI 1.20-1.40), lower socioeconomic status/financial insecurity (OR 1.62, 95%CI 1.02-2.63), pre-infection depression (OR 1.08, 95%CI 1.01-1.16), and earlier variants (OR 0.37 for Omicron compared to ancestral strain, 95%CI 0.15-0.90) were associated with Long COVID symptoms. Conclusions and Relevance: Variant wave, severity of acute infection, lower socioeconomic status and pre-existing depression are associated with Long COVID symptoms.

Reduced Exercise Capacity, Chronotropic Incompetence, Inflammation and Symptoms in Post-Acute COVID-19 (preprint)

BACKGROUND Mechanisms underlying persistent cardiopulmonary symptoms following SARS-CoV-2 infection (post-acute sequelae of COVID-19 "PASC" or "Long COVID") remain unclear. The purpose of this study was to elucidate the pathophysiology of cardiopulmonary PASC using multimodality cardiovascular imaging including cardiopulmonary exercise testing (CPET), cardiac magnetic resonance imaging (CMR) and ambulatory rhythm monitoring. METHODS In the Long-Term Impact of Infection with Novel Coronavirus (LIINC) Cohort, we performed CMR, CPET, and ambulatory rhythm monitoring among adults > 1 year after PCR-confirmed SARS-CoV-2 infection. We used logistic and linear regression to compare those with and without cardiopulmonary symptoms (dyspnea, chest pain, palpitations) adjusting for confounders. RESULTS One hundred twenty individuals were studied, among whom 46 participants (unselected for symptom status) had at least one advanced test performed at median 17 months (IQR 15-18). Median age was 52 (IQR 42-61), 18 (39%) were female, and 6 (13%) were hospitalized for severe acute infection. On CMR (n=39), smaller RV volume and stroke volume and higher extracellular volume were present among those with symptoms, but no evidence of late-gadolinium enhancement or differences in T1 or T2 mapping were demonstrated. We did not find arrhythmias on ambulatory monitoring. In contrast, on CPET (n=39), 13/15 (87%) participants with reduced exercise capacity (<85% predicted) reported cardiopulmonary symptoms or fatigue (p=0.008). Adjusted peak VO2 was 2.7 ml/kg/min lower among those with cardiopulmonary symptoms (95%CI -6.9 to 1.5; p=0.20) or -11% predicted (95%CI -27 to 5, p=0.17). Including fatigue along with cardiopulmonary symptoms, the adjusted difference in peak VO2 was -5.9 ml/kg/min (-9.6 to -2.3; p=0.002) or -21% predicted (-35 to -7; p=0.006). Chronotropic incompetence was the primary abnormality among 9/15 with reduced peak VO2. Adjusted heart rate reserve <80% was associated with reduced exercise capacity (OR 15.6, 95%CI 1.30-187; p=0.03). Those with chronotropic incompetence had higher hsCRP, lower heart rate recovery, and lower heart rate variability suggestive of autonomic dysfunction. CONCLUSIONS Reduced exercise capacity and reduced heart rate response to exercise, and hsCRP are associated with persistent cardiopulmonary symptoms more than 1 year following COVID-19. Chronic inflammation and autonomic dysfunction may underlie cardiopulmonary PASC.

Infectious viral shedding of SARS-CoV-2 Delta following vaccination: a longitudinal cohort study (preprint)

The impact of vaccination on SARS-CoV-2 infectiousness is not well understood. We compared longitudinal viral shedding dynamics in unvaccinated and fully vaccinated adults. SARS-CoV-2-infected adults were enrolled within 5 days of symptom onset and nasal specimens were self-collected daily for two weeks and intermittently for an additional two weeks. SARS-CoV-2 RNA load and infectious virus were analyzed relative to symptom onset stratified by vaccination status. We tested 1080 nasal specimens from 52 unvaccinated adults enrolled in the pre-Delta period and 32 fully vaccinated adults with predominantly Delta infections. While we observed no differences by vaccination status in maximum RNA levels, maximum infectious titers and the median duration of viral RNA shedding, the rate of decay from the maximum RNA load was faster among vaccinated; maximum infectious titers and maximum RNA levels were highly correlated. Furthermore, amongst participants with infectious virus, median duration of infectious virus detection was reduced from 7.5 days (IQR: 6.0-9.0) in unvaccinated participants to 6 days (IQR: 5.0-8.0) in those vaccinated (P=0.02). Accordingly, the odds of shedding infectious virus from days 6 to 12 post-onset were lower among vaccinated participants than unvaccinated participants (OR 0.42 95% CI 0.19-0.89). These results indicate that vaccination had reduced the probability of shedding infectious virus after 5 days from symptom onset.

Effect of oral nirmatrelvir on Long COVID symptoms: a case series (preprint)

As the SARS-CoV-2 pandemic continues to evolve, efforts to understand variability in COVID-19 recovery, as well as the impact of factors including viral variants, vaccine status, and COVID-19 treatment on the development and persistence of Long COVID symptoms have intensified. We report three cases that demonstrate that variability in the timing of nirmatrelvir therapy may be associated with different outcomes and underscores the need for systematic study of antiviral therapy for this disease condition.

POST-ACUTE SEQUELAE AND ADAPTIVE IMMUNE RESPONSES IN PEOPLE LIVING WITH HIV RECOVERING FROM SARS-COV-2 INFECTION (preprint)

Background: Limited data are available on the long-term clinical and immunologic consequences of SARS-CoV-2 infection in people with HIV (PWH). Methods: We measured SARS-CoV-2 specific humoral and cellular immune responses in people with and without HIV recovering from COVID-19 (n=39 and n=43, respectively) using binding antibody, surrogate virus neutralization, intracellular cytokine staining, and inflammatory marker assays. We identified individuals experiencing symptomatic post-acute sequelae of SARS-CoV-2 infection (PASC) and evaluated immunologic parameters. We used linear regression and generalized linear models to examine differences by HIV status in the magnitude of inflammatory and virus-specific antibody and T cell responses, as well as differences in the prevalence of PASC. Results: Among PWH, we found broadly similar SARS-CoV-2-specific antibody and T cell immune responses as compared with a well-matched group of HIV-negative individuals. PWH had 70% lower relative levels of SARS-CoV-2 specific memory CD8+ T cells (p=0.007) and 53% higher relative levels of PD-1+ SARS-CoV-2 specific CD4+ T cells (p=0.007). Higher CD4/CD8 ratio was associated with lower PD-1 expression on SARS-CoV-2 specific CD8+ T cells (0.34-fold effect, p=0.02). HIV status was strongly associated with PASC (odds ratio 4.01, p=0.008), and the proportion of PD-1+ CD4+ T cells and levels of certain inflammatory markers (IL-6, TNF-alpha, and IP-10) were associated with persistent symptoms. Conclusions: We identified potentially important differences in SARS-CoV-2-specific CD4+ and CD8+ T cells that might have implications for long-term immunity conferred by natural infection. HIV status strongly predicted the presence of PASC. Larger and more detailed studies of PASC in PWH are urgently needed.

Plasma markers of neurologic injury and systemic inflammation in individuals with self-reported neurologic post-acute sequelae of SARS-CoV-2 infection (PASC) (preprint)

Background: The biologic mechanisms underlying neurologic post-acute-sequelae of SARS-CoV-2 infection (PASC) are incompletely understood. Methods: We measured markers of neuronal injury (glial fibrillary acidic protein [GFAP], neurofilament light chain [NfL]) and soluble markers of inflammation among a cohort of people with prior confirmed SARS-CoV-2 infection at early and late recovery following the initial illness (defined as less than and greater than 90 days, respectively). The primary clinical outcome was the presence of self-reported central nervous system (CNS) PASC symptoms during the late recovery timepoint. We compared fold-changes in marker values between those with and without CNS PASC symptoms using linear mixed effects models and examined relationships between neurologic and immunologic markers using rank linear correlations. Results: Of 121 individuals, 52 reported CNS PASC symptoms. During early recovery, those who went on to report CNS PASC symptoms had elevations in GFAP (1.3-fold higher mean ratio, 95% CI 1.04-1.63, p=0.02), but not NfL (1.06-fold higher mean ratio, 95% CI 0.89-1.26, p=0.54). During late recovery, neither GFAP nor NfL levels were elevated among those with CNS PASC symptoms. Although absolute levels of NfL did not differ, those who reported CNS PASC symptoms demonstrated a stronger downward trend over time in comparison to those who did not report CNS PASC symptoms (p=0.041). Those who went on to report CNS PASC also exhibited elevations in IL-6 (48% higher during early recovery and 38% higher during late recovery), MCP-1 (19% higher during early recovery), and TNF-alpha (19% higher during early recovery and 13% higher during late recovery). GFAP and NfL correlated with levels of several immune activation markers during early recovery; these correlations were attenuated during late recovery. Conclusions: Self-reported neurologic symptoms present >90 days following SARS-CoV-2 infection are associated with elevations in markers of neurologic injury and inflammation at early recovery timepoints, suggesting that early injury can result in long-term disease. The correlation of GFAP and NfL with markers of systemic immune activation suggests one possible mechanism that might contribute to these symptoms. Additional work is needed to better characterize these processes and to identify interventions to prevent or treat this condition.

SARS-CoV-2 serology across scales: a framework for unbiased seroprevalence estimation incorporating antibody kinetics and epidemic recency (preprint)

Serosurveys are a key resource for measuring SARS-CoV-2 cumulative incidence. A growing body of evidence suggests that asymptomatic and mild infections (together making up over 95% of all infections) are associated with lower antibody titers than severe infections. Antibody levels also peak a few weeks after infection and decay gradually. We developed a statistical approach to produce adjusted estimates of seroprevalence from raw serosurvey results that account for these sources of spectrum bias. We incorporate data on antibody responses on multiple assays from a post-infection longitudinal cohort, along with epidemic time series to account for the timing of a serosurvey relative to how recently individuals may have been infected. We applied this method to produce adjusted seroprevalence estimates from five large-scale SARS-CoV-2 serosurveys across different settings and study designs. We identify substantial differences between reported and adjusted estimates of over two-fold in the results of some surveys, and provide a tool for practitioners to generate adjusted estimates with pre-set or custom parameter values. While unprecedented efforts have been launched to generate SARS-CoV-2 seroprevalence estimates over this past year, interpretation of results from these studies requires properly accounting for both population-level epidemiologic context and individual-level immune dynamics.

Genetic regulation of OAS1 nonsense-mediated decay underlies association with risk of severe COVID-19 (preprint)

Genomic regions have been associated with COVID-19 susceptibility and outcomes, including the chr12q24.13 locus encoding antiviral proteins OAS1-3. Here, we report genetic, functional, and clinical insights into genetic associations within this locus. In Europeans, the risk of hospitalized vs. non-hospitalized COVID-19 was associated with a single 19Kb-haplotype comprised of 76 OAS1 variants included in a 95% credible set within a large genomic fragment introgressed from Neandertals. The risk haplotype was also associated with impaired spontaneous but not treatment-induced SARS-CoV-2 clearance in a clinical trial with pegIFN-{lambda}1. We demonstrate that two exonic variants, rs10774671 and rs1131454, affect splicing and nonsense-mediated decay of OAS1. We suggest that genetically-regulated loss of OAS1 expression contributes to impaired spontaneous clearance of SARS-CoV-2 and elevated risk of hospitalization for COVID-19. Our results provide the rationale for further clinical studies using interferons to compensate for impaired spontaneous SARS-CoV-2 clearance, particularly in carriers of the OAS1 risk haplotypes.

Markers of immune activation and inflammation in individuals with post-acute sequelae of SARS-CoV-2 infection (preprint)

BACKGROUND: The biological processes associated with post-acute sequelae of SARS-CoV-2 infection (PASC) are unknown. METHODS: We measured soluble markers of inflammation in a SARS-CoV-2 recovery cohort at early (<90 days) and late (>90 days) timepoints. We defined PASC as the presence of one or more COVID-19-attributed symptoms beyond 90 days. We compared fold-changes in marker values between those with and without PASC using mixed effects models with terms for PASC and early and late recovery time periods. RESULTS: During early recovery, those who went on to develop PASC generally had higher levels of cytokine biomarkers including TNF-alpha (1.14-fold higher mean ratio, 95%CI 1.01-1.28, p=0.028) and IP-10 (1.28-fold higher mean ratio, 95%CI 1.01-1.62, p=0.038). Among those with PASC, there was a trend toward higher IL-6 levels during early recovery (1.28-fold higher mean ratio, 95%CI 0.98-1.70, p=0.07) which became more pronounced in late recovery (1.44-fold higher mean ratio, 95%CI: 1.11-1.86, p<0.001). These differences were more pronounced among those with a greater number of PASC symptoms. CONCLUSIONS: Persistent immune activation may be associated with ongoing symptoms following COVID-19. Further characterization of these processes might identify therapeutic targets for those experiencing PASC.

multiSero: open multiplex-ELISA platform for analyzing antibody responses to SARS-CoV-2 infection (preprint)

Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL-1 RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.

Rapid implementation of a cohort for the study of post-acute sequelae of SARS-CoV-2 infection/COVID-19 (preprint)

BACKGROUND: As the coronavirus disease 2019 (COVID-19) pandemic continues and millions remain vulnerable to infection with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), attention has turned to characterizing post-acute sequelae of SARS-CoV-2 infection (PASC). METHODS: From April 21 to December 31, 2020, we assembled a cohort of consecutive volunteers who a) had documented history of SARS-CoV-2 RNA-positivity; b) were [≥] 2 weeks past onset of COVID-19 symptoms or, if asymptomatic, first test for SARS-CoV-2; and c) were able to travel to our site in San Francisco. Participants learned about the study by being identified on medical center-based registries and being notified or by responding to advertisements. At 4-month intervals, we asked participants about physical symptoms that were new or worse compared to the period prior to COVID-19, mental health symptoms and quality of life. We described 4 time periods: 1) acute illness (0-3 weeks), 2) early recovery (3-10 weeks), 3) late recovery 1 (12-20 weeks), and 4) late recovery 2 (28-36 weeks). Blood and oral specimens were collected at each visit. RESULTS: We have, to date, enrolled 179 adults. During acute SARS-CoV-2 infection, 10 had been asymptomatic, 125 symptomatic but not hospitalized, and 44 symptomatic and hospitalized. In the acute phase, the most common symptoms were fatigue, fever, myalgia, cough and anosmia/dysgeusia. During the post-acute phase, fatigue, shortness of breath, concentration problems, headaches, trouble sleeping and anosmia/dysgeusia were the most commonly reported symptoms, but a variety of others were endorsed by at least some participants. Some experienced symptoms of depression, anxiety, and post-traumatic stress, as well as difficulties with ambulation and performance of usual activities. The median visual analogue scale value rating of general health was lower at 4 and 8 months (80, interquartile range [IQR]: 70-90; and 80, IQR 75-90) compared to prior to COVID-19 (85; IQR 75-90). Biospecimens were collected at nearly 600 participant-visits. CONCLUSION: Among a cohort of participants enrolled in the post-acute phase of SARS-CoV-2 infection, we found many with persistent physical symptoms through 8 months following onset of COVID-19 with an impact on self-rated overall health. The presence of participants with and without symptoms and ample biological specimens will facilitate study of PASC pathogenesis. Similar evaluations in a population-representative sample will be needed to estimate the population-level prevalence of PASC.