SARS-CoV-2 infection elucidates unique features of pregnancy-specific immunity (preprint)

Pregnancy is a risk factor for increased severity of SARS-CoV-2 and other respiratory infections. The mechanisms underlying this risk have not been well-established, partly due to a limited understanding of how pregnancy shapes immune responses. To gain insight into the role of pregnancy in modulating immune responses at steady state and upon perturbation, we collected peripheral blood mononuclear cells (PBMC), plasma, and stool from 226 women, including 152 pregnant individuals (n = 96 with SARS-CoV-2 infection and n = 56 healthy controls) and 74 non-pregnant women (n = 55 with SARS-CoV-2 and n = 19 healthy controls). We found that SARS-CoV-2 infection was associated with altered T cell responses in pregnant compared to non-pregnant women. Differences included a lower percentage of memory T cells, a distinct clonal expansion of CD4-expressing CD8+ T cells, and the enhanced expression of T cell exhaustion markers, such as programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain-3 (Tim-3), in pregnant women. We identified additional evidence of immune dysfunction in severely and critically ill pregnant women, including a lack of expected elevation in regulatory T cell (Treg) levels, diminished interferon responses, and profound suppression of monocyte function. Consistent with earlier data, we found maternal obesity was also associated with altered immune responses to SARS-CoV-2 infection, including enhanced production of inflammatory cytokines by T cells. Certain gut bacterial species were altered in pregnancy and upon SARS-CoV-2 infection in pregnant individuals compared to non-pregnant women. Shifts in cytokine and chemokine levels were also identified in the sera of pregnant individuals, most notably a robust increase of interleukin-27 (IL-27), a cytokine known to drive T cell exhaustion, in the pregnant uninfected control group compared to all non-pregnant groups. IL-27 levels were also significantly higher in uninfected pregnant controls compared to pregnant SARS-CoV-2-infected individuals. Using two different preclinical mouse models of inflammation-induced fetal demise and respiratory influenza viral infection, we found that enhanced IL-27 protects developing fetuses from maternal inflammation but renders adult female mice vulnerable to viral infection. These combined findings from human and murine studies reveal nuanced pregnancy-associated immune responses, suggesting mechanisms underlying the increased susceptibility of pregnant individuals to viral respiratory infections.

Modeling the emergence of viral resistance for SARS-CoV-2 during treatment with an anti-spike monoclonal antibody (preprint)

The COVID-19 pandemic has led to over 760 million cases and 6.9 million deaths worldwide. To mitigate the loss of lives, emergency use authorization was given to several anti-SARS-CoV-2 monoclonal antibody (mAb) therapies for the treatment of mild-to-moderate COVID-19 in patients with a high risk of progressing to severe disease. Monoclonal antibodies used to treat SARS-CoV-2 target the spike protein of the virus and block its ability to enter and infect target cells. Monoclonal antibody therapy can thus accelerate the decline in viral load and lower hospitalization rates among high-risk patients with susceptible variants. However, viral resistance has been observed, in some cases leading to a transient viral rebound that can be as large as 3-4 orders of magnitude. As mAbs represent a proven treatment choice for SARS-CoV-2 and other viral infections, evaluation of treatment-emergent mAb resistance can help uncover underlying pathobiology of SARS-CoV-2 infection and may also help in the development of the next generation of mAb therapies. Although resistance can be expected, the large rebounds observed are much more difficult to explain. We hypothesize replenishment of target cells is necessary to generate the high transient viral rebound. Thus, we formulated two models with different mechanisms for target cell replenishment (homeostatic proliferation and return from an innate immune response anti-viral state) and fit them to data from persons with SARS-CoV-2 treated with a mAb. We showed that both models can explain the emergence of resistant virus associated with high transient viral rebounds. We found that variations in the target cell supply rate and adaptive immunity parameters have a strong impact on the magnitude or observability of the viral rebound associated with the emergence of resistant virus. Both variations in target cell supply rate and adaptive immunity parameters may explain why only some individuals develop observable transient resistant viral rebound. Our study highlights the conditions that can lead to resistance and subsequent viral rebound in mAb treatments during acute infection.

SARS-CoV-2 Viral Clearance and Evolution Varies by Extent of Immunodeficiency (preprint)

Despite vaccination and antiviral therapies, immunocompromised individuals are at risk for prolonged SARS-CoV-2 infection, but the immune defects that predispose to persistent COVID- 19 remain incompletely understood. In this study, we performed detailed viro-immunologic analyses of a prospective cohort of participants with COVID-19. The median time to nasal viral RNA and culture clearance in the severe hematologic malignancy/transplant group (S-HT) were 72 and 21 days, respectively, which were significantly longer than clearance rates in the severe autoimmune/B-cell deficient (S-A), non-severe, and non-immunocompromised groups (P<0.001). Participants who were severely immunocompromised had greater SARS-CoV-2 evolution and higher risk of developing antiviral treatment resistance. Both S-HT and S-A participants had diminished SARS-CoV-2-specific humoral, while only the S-HT group had reduced T cell-mediated responses. This highlights the varied risk of persistent COVID-19 across immunosuppressive conditions and suggests that suppression of both B and T cell responses results in the highest contributing risk of persistent infection.

SARS-CoV-2 virologic rebound with nirmatrelvir-ritonavir therapy (preprint)

Abstract Objective: To compare the frequency of replication-competent virologic rebound with and without nirmatrelvir-ritonavir treatment for acute COVID-19. Secondary aims were to estimate the validity of symptoms to detect rebound and the incidence of emergent nirmatrelvir-resistance mutations after rebound. Design: Observational cohort study. Setting: Multicenter healthcare system in Boston, Massachusetts. Participants: We enrolled ambulatory adults with a positive COVID-19 test and/or a prescription for nirmatrelvir-ritonavir. Exposures: Receipt of 5 days of nirmatrelvir-ritonavir treatment versus no COVID-19 therapy. Main Outcome and Measures: The primary outcome was COVID-19 virologic rebound, defined as either (1) a positive SARS-CoV-2 viral culture following a prior negative culture or (2) two consecutive viral loads [≥]4.0 log10 copies/milliliter after a prior reduction in viral load to <4.0 log10 copies/milliliter. Results: Compared with untreated individuals (n=55), those taking nirmatrelvir-ritonavir (n=72) were older, received more COVID-19 vaccinations, and were more commonly immunosuppressed. Fifteen individuals (20.8%) taking nirmatrelvir-ritonavir experienced virologic rebound versus one (1.8%) of the untreated (absolute difference 19.0% [95%CI 9.0-29.0%], P=0.001). In multivariable models, only N-R was associated with VR (AOR 10.02, 95%CI 1.13-88.74). VR occurred more commonly among those with earlier nirmatrelvir-ritonavir initiation (29.0%, 16.7% and 0% when initiated days 0, 1, and [≥]2 after diagnosis, respectively, P=0.089). Among participants on N-R, those experiencing rebound had prolonged shedding of replication-competent virus compared to those that did not rebound (median: 14 vs 3 days). Only 8/16 with virologic rebound reported worsening symptoms (50%, 95%CI 25%-75%); 2 were completely asymptomatic. We detected no post-rebound nirmatrelvir-resistance mutations in the NSP5 protease gene. Conclusions and Relevance: Virologic rebound occurred in approximately one in five people taking nirmatrelvir-ritonavir and often occurred without worsening symptoms. Because it is associated with replication-competent viral shedding, close monitoring and potential isolation of those who rebound should be considered.

Statistical Challenges when Analyzing SARS-CoV-2 RNA Measurements Below the Assay Limit of Quantification in COVID-19 Clinical Trials (preprint)

Most clinical trials evaluating COVID-19 therapeutics include assessments of antiviral activity. In recently completed outpatient trials, changes in nasal SARS-CoV-2 RNA levels from baseline were commonly assessed using analysis of covariance (ANCOVA) or mixed models for repeated measures (MMRM) with single-imputation for results below assay lower limits of quantification (LLoQ). Analyzing changes in viral RNA levels with singly-imputed values can lead to biased estimates of treatment effects. In this paper, using an illustrative example from the ACTIV-2 trial, we highlight potential pitfalls of imputation when using ANCOVA or MMRM methods, and illustrate how these methods can be used when considering values

Viral and immunologic evaluation of smokers with severe COVID-19 (preprint)

Smoking negatively affects B cell function and immunoglobulin levels, but it is unclear if this immune dysfunction contributes to the risk of severe COVID-19 in smokers. We evaluated binding IgM, IgA and IgG antibodies to spike and receptor binding domain antigens, and used a pseudovirus assay quantify neutralization titers in a set of 27 patients with severe COVID-19. We found no significant differences between binding and neutralization antibody responses for people with a smoking history and people who never smoked. High plasma viral load, but not antibody titers, was linked to an increased risk of death. Humoral immune dysfunction was not a major driver of severe COVID-19 in smokers.

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1Omicron (preprint)

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date1-7. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes3,8. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor-binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

Viral and Symptom Rebound in Untreated COVID-19 Infection (preprint)

Background: There are reports of viral RNA and symptom rebound in people with COVID-19 treated with nirmatrelvir/ritonavir. Since the natural course of viral and symptom trajectories of COVID-19 has not been well described, we evaluated the incidence of viral and symptom rebound in untreated outpatients with mild-moderate COVID-19. Methods: The study population included 568 participants enrolled in the ACTIV-2/A5401 platform trial who received placebo. Anterior nasal swabs were collected for SARS-CoV-2 RNA testing on days 0-14, 21 and 28. Participants recorded the severity of 13 targeted symptoms daily from day 0 to 28. Viral rebound was defined as [≥]0.5 log10 viral RNA copies/mL increase and symptom rebound was defined as a 4-point total symptom score increase from baseline. Baseline was defined as study day 4 (primary analysis) or 8 days from symptom onset (secondary analysis). Findings: In both the primary and secondary analyses, 12% of participants had viral rebound. Viral rebounders were older than non-rebounders (median 54 vs 47 years, P=0.04). Symptom rebound occurred in 27% of participants after initial symptom improvement and in 10% of participants after initial symptom resolution. The combination of high-level viral rebound to [≥]5.0 log10 RNA copies/mL and symptom rebound after initial improvement was observed in 1-2% of participants. Interpretation: Viral RNA rebound or symptom relapse in the absence of antiviral treatment is common, but the combination of high-level viral and symptom rebound is rare. Funding: This study was supported by the National Institute of Allergy and Infectious Diseases; ACTIV-2/A5401 ClinicalTrials.gov number NCT04518410.

Virologic characterization of symptom rebound following nirmatrelvir-ritonavir treatment for COVID-19 (preprint)

We enrolled seven individuals with recurrent symptoms following nirmatrelvir-ritonavir treatment. High viral loads (median 6.1 log10 copies/mL) were detected at enrollment and for a median of 17 days after initial diagnosis. Three of seven had culturable virus for up to 16 days after initial diagnosis. No known resistance-associated mutations were identified.

Duration of viable virus shedding in SARS-CoV-2 omicron variant infection (preprint)

Clinical features of SARS-CoV-2 Omicron variant infection, including incubation period and transmission rates, distinguish this variant from preceding variants. However, whether the duration of shedding of viable virus differs between omicron and previous variants is not well understood. To characterize how variant and vaccination status impact shedding of viable virus, we serially sampled symptomatic outpatients newly diagnosed with COVID-19. Anterior nasal swabs were tested for viral load, sequencing, and viral culture. Time to PCR conversion was similar between individuals infected with the Delta and the Omicron variant. Time to culture conversion was also similar, with a median time to culture conversion of 6 days (interquartile range 4-8 days) in both groups. There were also no differences in time to PCR or culture conversion by vaccination status.

Monoclonal antibody treatment drives rapid culture conversion in SARS-CoV-2 infection (preprint)

Monoclonal antibodies (mAbs) are the treatment of choice for high-risk ambulatory persons with mild to moderate COVID-19. We studied viral culture dynamics post-treatment in a subset of participants receiving the mAb bamlanivimab in the ACTIV-2 trial. Viral load by qPCR and viral culture were performed from anterior nasal swabs collected on study days 0 (day of treatment), 1, 2, 3, and 7. Treatment with mAb resulted in rapid clearance of culturable virus in participants without treatment-emergent resistance. One day after treatment, 0 of 28 (0%) participants receiving mAb and 16 of 39 (41%) receiving placebo still had culturable virus (p <0.0001); nasal viral loads were only modestly lower in the mAb-treated group at days 2 and 3. Recrudescence of culturable virus was detected in three participants with emerging mAb resistance and viral load rebound. The rapid reduction in shedding of viable SARS-CoV-2 after mAb treatment highlights the potential role of mAbs in preventing disease transmission.

Bamlanivimab reduces nasopharyngeal SARS-CoV-2 RNA levels but not symptom duration in non-hospitalized adults with COVID-19 (preprint)

Importance: The antiviral activity and efficacy of anti-SARS-CoV-2 monoclonal antibody (mAb) therapies to accelerate recovery from COVID-19 is important to define. Objective: To determine safety and efficacy of the mAb bamlanivimab to reduce nasopharyngeal (NP) SARS-CoV-2 RNA levels and symptom duration. Design: ACTIV-2/A5401 is a randomized, blinded, placebo-controlled platform trial. Two dose cohorts were enrolled between August 19 and November 17, 2020 for phase 2 evaluation: in the first, participants were randomized 1:1 to bamlanivimab 7000 mg versus placebo, and in the second to bamlanivimab 700 mg versus placebo. Randomization was stratified by time from symptom onset ([≤] or >5 days) and risk of progression to severe COVID-19 ('higher' vs 'lower'). Setting: Multicenter trial conducted at U.S. sites. Participants: Non-hospitalized adults [≥]18 years of age with positive SARS-CoV-2 antigen or nucleic acid test within 7 days, [≤]10 days of COVID-19 symptoms, and with oxygen saturation [≥]92% within 48 hours prior to study entry. Intervention: Single infusion of bamlanivimab (7000 or 700 mg) or placebo. Main Outcomes and Measures: Detection of NP SARS-CoV-2 RNA at days 3, 7, 14, 21, and 28, time to improvement of all of 13 targeted COVID-19 symptoms by daily self-assessment through day 28, and grade 3 or higher treatment emergent adverse events (TEAEs) through day 28. Secondary measures included quantitative NP SARS-CoV-2 RNA, all-cause hospitalizations and deaths (composite), area under the curve of symptom scores from day 0 through day 28, plasma bamlanivimab concentrations, plasma and serum inflammatory biomarkers, and safety through week 24. Results: Ninety-four participants were enrolled to the 7000 mg cohort and 223 to the 700 mg cohort and initiated study intervention. The proportion meeting protocol criteria for 'higher' risk for COVID-19 progression was 42% and 51% for the 7000 and 700 mg cohort, respectively. Median time from symptom onset at study entry for both cohorts was 6 days. There was no difference in the proportion with undetectable NP SARS-CoV-2 RNA at any post-treatment timepoints (risk ratio compared to placebo, 0.82-1.05 for 7000 mg dose [overall p=0.88] and 0.81-1.21 for 700 mg dose [overall p=0.49]), time to symptom improvement (median of 21 vs 18.5 days, p=0.97, for 7000 mg bamlanivimab vs placebo and 24 vs 20.5 days, p=0.08, for 700 mg bamlanivimab vs placebo), or grade 3+ TEAEs with either dose compared to placebo. Median NP SARS-CoV-2 RNA levels were lower at day 3 and C-reactive protein, ferritin, and fibrinogen levels significantly reduced at days 7 and 14 for bamlanivimab 700 mg compared to placebo, with similar trends observed for bamlanivimab 7000 mg. Viral decay modeling supported more rapid decay with bamlanivimab compared to placebo. Conclusions and Relevance: Treatment with bamlanivimab 7000 mg and 700 mg was safe and compared to placebo led to more rapid reductions in NP SARS-CoV-2 RNA and inflammatory biomarkers, but did not decrease time to symptom improvement. The clinical utility of mAbs for outcomes other than hospitalizations and deaths is uncertain. Trial Registration: ClinicalTrials.gov Identifier: NCT04518410

Emergence of SARS-CoV-2 Resistance with Monoclonal Antibody Therapy (preprint)

Resistance mutations to monoclonal antibody (mAb) therapy has been reported, but in the non-immunosuppressed population, it is unclear if in vivo emergence of SARS-CoV-2 resistance mutations alters either viral replication dynamics or therapeutic efficacy. In ACTIV-2/A5401, non-hospitalized participants with symptomatic SARS-CoV-2 infection were randomized to bamlanivimab (700mg or 7000mg) or placebo. Treatment-emergent resistance mutations were significantly more likely detected after bamlanivimab 700mg treatment than placebo (7% of 111 vs 0% of 112 participants, P=0.003). There were no treatment-emergent resistance mutations among the 48 participants who received bamlanivimab 7000mg. Participants with emerging mAb resistant virus had significantly higher pre-treatment nasopharyngeal and anterior nasal viral load. Intensive respiratory tract viral sampling revealed the dynamic nature of SARS-CoV-2 evolution, with evidence of rapid and sustained viral rebound after emergence of resistance mutations, and worsened symptom severity. Participants with emerging bamlanivimab resistance often accumulated additional polymorphisms found in current variants of concern/interest and associated with immune escape. These results highlight the potential for rapid emergence of resistance during mAb monotherapy treatment, resulting in prolonged high level respiratory tract viral loads and clinical worsening. Careful virologic assessment should be prioritized during the development and clinical implementation of antiviral treatments for COVID-19.

Simultaneous detection of SARS-CoV-2 RNA and host antibodies enabled by a multiplexed electrochemical sensor platform (preprint)

There continues to be a great need for rapid, accurate, and cost-effective point-of-care devices that can diagnose the presence of SARS-CoV-2 virus and development of IgG and IgM antibody responses in early and late stages of COVID-19 disease. Here, we describe a versatile multiplexed electrochemical (EC) sensor platform modified with an antifouling nanocomposite coating that enables single-molecule CRISPR/Cas-based molecular detection of SARS-CoV-2 viral RNA with on-chip signal validation as well as multiplexed serological detection of antibodies against three SARS-CoV-2 viral antigens. The CRISPR-based EC platform achieved 100% accuracy for detection of viral RNA and showed an excellent correlation with RT-qPCR using 30 clinical saliva samples. The serology EC platform obtained 100% sensitivity and 100% specificity for anti-SARS-CoV-2 IgG, as well as 94% specificity and 82% sensitivity for anti-SARS-CoV-2 IgM with 112 clinical plasma samples. These data demonstrate that integration of CRISPR-based RNA detection and serological assays with antifouling nanocomposite-based EC sensors enables performance as good or better than traditional laboratory-based techniques.

Sexually dimorphic placental responses to maternal SARS-CoV-2 infection (preprint)

There is a persistent male bias in the prevalence and severity of COVID-19 disease. Underlying mechanisms accounting for this sex difference remain incompletely understood. Interferon responses have been implicated as a modulator of disease in adults, and play a key role in the placental anti-viral response. Moreover, the interferon response has been shown to alter Fc-receptor expression, and therefore may impact placental antibody transfer. Here we examined the intersection of viral-induced placental interferon responses, maternal-fetal antibody transfer, and fetal sex. Placental interferon stimulated genes (ISGs), Fc-receptor expression, and SARS-CoV-2 antibody transfer were interrogated in 68 pregnancies. Sexually dimorphic placental expression of ISGs, interleukin-10, and Fc receptors was observed following maternal SARS-CoV-2 infection, with upregulation in males. Reduced maternal SARS-CoV-2-specific antibody titers and impaired placental antibody transfer were noted in pregnancies with a male fetus. These results demonstrate fetal sex-specific maternal and placental adaptive and innate immune responses to SARS-CoV-2.

Expansion of Cytotoxic CD4+ T cells in the lungs in severe COVID-19 (preprint)

The contributions of T cells infiltrating the lungs to SARS-CoV-2 clearance and disease progression are poorly understood. Although studies of CD8+ T cells in bronchoalveolar lavage and blood have suggested that these cells are exhausted in severe COVID-19, CD4+ T cells have not been systematically interrogated within the lung parenchyma. We establish here that cytotoxic CD4+ T cells (CD4+CTLs) are prominently expanded in the COVID-19 lung infiltrate. CD4+CTL numbers in the lung increase with disease severity and progression is accompanied by widespread HLA-DR expression on lung epithelial and endothelial cells, increased apoptosis of epithelial cells and tissue remodeling. Based on quantitative evidence for re-activation in the lung milieu, CD4+ CTLs are as likely to drive viral clearance as CD8+ T cells and may also be contributors to lung inflammation and eventually to fibrosis in severe COVID-19.

Type I, II, and III interferon signatures correspond to COVID-19 disease severity (preprint)

We analyzed the plasma levels of interferons and cytokines, and the expression of interferon-stimulated genes in peripheral blood mononuclear cells in COVID-19 patients with different disease severity. Mild patients exhibited transient type I interferon responses, while ICU patients had prolonged type I interferon responses with hyper-inflammation mediated by interferon regulatory factor 1. Type II interferon responses were compromised in ICU patients. Type III interferon responses were induced in the early phase of SARS-CoV-2 infection, even in convalescent patients. These results highlight the importance of type I and III interferon responses during the early phase of infection in controlling COVID-19 progression.

SARS-CoV-2 Sequence Characteristics of COVID-19 Persistence and Reinfection (preprint)

BackgroundBoth SARS-CoV-2 reinfection and persistent infection have been described, but a systematic assessment of mutations is needed. We assessed sequences from published cases of COVID-19 reinfection and persistence, characterizing the hallmarks of reinfecting sequences and the rate of viral evolution in persistent infection. MethodsA systematic review of PubMed was conducted to identify cases of SARS-CoV-2 reinfection and persistent infection with available sequences. Amino acid changes in the reinfecting sequence were compared to both the initial and contemporaneous community variants. Time-measured phylogenetic reconstruction was performed to compare intra-host viral evolution in persistent COVID-19 to community-driven evolution. ResultsFourteen reinfection and five persistent infection cases were identified. Reports of reinfection cases spanned a broad distribution of ages, baseline health status, reinfection severity, and occurred as early as 1.5 months or >8 months after the initial infection. The reinfecting viral sequences had a median of 9 amino acid changes with enrichment of changes in the S, ORF8 and N genes. The number of amino acid changes did not differ by the severity of reinfection and reinfecting variants were similar to the contemporaneous sequences circulating in the community. Patients with persistent COVID-19 demonstrated more rapid accumulation of mutations than seen with community-driven evolution with continued viral changes during convalescent plasma or monoclonal antibody treatment. ConclusionsSARS-CoV-2 reinfection does not require an unusual set of circumstances in the host or virus, while persistent COVID-19 is largely described in immunosuppressed individuals and is associated with accelerated viral evolution as measured by clock rates.

SARS-CoV-2 Viremia is Associated with Distinct Proteomic Pathways and Predicts COVID-19 Outcomes (preprint)

BackgroundSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) plasma viremia has been associated with severe disease and death in coronavirus disease 2019 (COVID-19) in small-scale cohort studies. The mechanisms behind this association remain elusive. MethodsWe evaluated the relationship between SARS-CoV-2 viremia, disease outcome, inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using qRT-PCR based platform. Proteomic data were generated with Proximity Extension Assay (PEA) using the Olink platform. ResultsThree hundred participants with nucleic acid test-confirmed COVID-19 were included in this study. Levels of plasma SARS-CoV-2 viremia at the time of presentation predicted adverse disease outcomes, with an adjusted odds ratio (aOR) of 10.6 (95% confidence interval [CI] 4.4, 25.5, P<0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and aOR of 3.9 (95%CI 1.5, 10.1, P=0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, endothelium/vasculature and alterations in coagulation pathways. ConclusionsThese results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes.

Ultrasensitive measurement of both SARS-CoV2 RNA and serology from saliva (preprint)

Tests for COVID-19 generally measure SARS-CoV2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and serology from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive serology test based on Single Molecule Array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with serology identifies more patients as infected than either method alone. Our results suggest the utility of combining viral RNA and serology testing from saliva, a single easily accessible biofluid.