Contrasting Effects of SARS-CoV-2 Vaccination vs. Infection on Antibody and TCR Repertoires (preprint)

Antibodies and helper T cells play important roles in SARS-CoV-2 infection and vaccination. We sequenced B- and T-cell receptor repertoires (BCR/TCR) from the blood of 251 infectees, vaccinees, and controls to investigate whether features of these repertoires could predict subjects' SARS-CoV-2 neutralizing antibody titer (NAbs), as measured by enzyme-linked immunosorbent assay (ELISA). We sequenced recombined immunoglobulin heavy-chain (IGH), TCRbeta (TRB), and TCRdelta (TRD) genes in parallel from all subjects, including select B- and T-cell subsets in most cases, with a focus on their hypervariable CDR3 regions, and correlated this AIRRseq data with demographics and clinical findings from subjects' electronic health records. We found that age affected NAb levels in vaccinees but not infectees. Intriguingly, we found that vaccination, but not infection, has a substantial effect on non-productively recombined IGHs, suggesting a vaccine effect that precedes clonal selection. We found that repertoires' binding capacity to known SARS-CoV-2-specific CD4+ TRBs performs as well as the best hand-tuned fuzzy matching at predicting a protective level of NAbs, while also being more robust to repertoire sample size and not requiring hand-tuning. The overall conclusion from this large, unbiased, clinically well annotated dataset is that B- and T-cell adaptive responses to SARS-CoV-2 infection and vaccination are surprising, subtle, and diffuse. We discuss methodological and statistical challenges faced in attempting to define and quantify such strong-but-diffuse repertoire signatures and present tools and strategies for addressing these challenges.

SARS-CoV-2 live virus culture and sample freeze-thaw stability (preprint)

The COVID-19 pandemic has presented unique diagnostic challenges including the need to store and test large number of samples for clinical and research studies. While SARS CoV-2 diagnosis relies on RT-qPCR and antigen testing, live virus culture remains an important surrogate for viral 'infectiousness', as we previously described in 'SARS-CoV-2 Antigen Tests Predict Infectivity Based on Viral Culture: Comparison of Antigen, PCR Viral Load and Viral Culture Testing on a Large Sample Cohort' (Clin Microbiol Infect, 2022, PMC9293398). Live virus isolation and characterization has also been important to the SARS CoV-2 research community, to assess viral fitness, cellular tropism, and live virus neutralization, particularly with the emergence of new variants. Many clinical and research studies make use of samples that are frozen in transport media and investigated at later dates. The effect of freezing on RT-qPCR results is well established. However, the effect of freeze-thaw on viral viability has not been. Here, we therefore examined the effect of freeze-thaw on viral culture isolation from a large number of clinical samples that were split, and then cultured either fresh or after being frozen for 7 or 17-18 days. Samples represented the range of viral loads (genome copies/mL) observed in our patient population. We found that freeze-thaw did not significantly affect viral culture isolation. Therefore, the ability to assess infectiousness of samples previously frozen in transport medium appears to be maintained.

The Coviral Portal: Multi-Cohort Viral Loads and Antigen-Test Virtual Trials for COVID-19 (preprint)

Background: Regulatory approval of new over-the-counter tests for infectious agents such as SARS-CoV-2 has historically required that clinical trials include diverse groups of specific patient populations, making the approval process slow and expensive. Showing that populations do not differ in their viral loads---the key factor determining test performance---could expedite the evaluation of new tests. Methods. 46,726 RT-qPCR-positive SARS-CoV-2 viral loads were annotated with patient demographics and health status. Real-world performance of two commercially available antigen tests was evaluated over a wide range of viral loads. An open-access web portal was created allowing comparisons of viral-load distributions across patient groups and application of antigen-test performance characteristics to patient distributions to predict antigen-test performance on these groups. Findings. In several cases distributions were surprisingly similar where a difference was expected (e.g. smokers vs. non-smokers); in other cases there was a difference that was the opposite direction from expectations (e.g. higher in patients who identified as White vs. Black). Sensitivity and specificity of antigen tests for detecting contagiousness were similar across most groups. The portal is at https://arnaoutlab.org/coviral/. Conclusions. In silico analyses of large-scale, real-world clinical data repositories can serve as a timely evidence-based proxy for dedicated trials of antigen tests for specific populations. Free availability of richly annotated data facilitates large-scale hypothesis generation and testing.

A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients (preprint)

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

Limit of Detection for Rapid Antigen Testing of the SARS-CoV-2 Omicron Variant (preprint)

There has been debate in the literature about the ability of antigen tests to detect the SARS-CoV-2 Omicron variant including indication on the US Food and Drug administration website that antigen tests may have lower sensitivity for the Omicron variant without provision of data or the potential scale of the issue (see https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-viral-mutations-impact-covid-19-tests - omicronvariantimpact, accessed 1/27/2022). Here we determined the limit of detection (LoD) for the Omicron variant compared with the WA1 strain used for LoD studies described in the Instructions for Use for all Emergency Use Authorization (EUA)-approved antigen tests. Using live virus (to avoid artifactual findings potentially obtained with gamma-irradiated or heat-killed virus) quantified by plaque forming units (PFU), we examined the analytical sensitivity of three antigen tests widely used in the United States: the Abbott Binax Now, the AccessBio CareStart , and LumiraDx antigen tests. We found that the 95% detection threshold (LoD) for antigen tests was at least as good for Omicron as for the WA1 strain. Furthermore, the relationship of genome copies to plaque forming units for Omicron and WA1 overlap. Therefore, the LoD equivalency also applies if the quantitative comparator is genome copies determined from live virus preparations. Taken together, our data support the continued ability of the antigen tests examined to detect the Omicron variant.

SARS-CoV-2 Antigen Tests Predict Infectivity Based on Viral Culture: Comparison of Antigen, PCR Viral Load, and Viral Culture Testing on a Large Sample Cohort (preprint)

The relationship of SARS-CoV-2 antigen testing results, viral load, and viral culture detection remains to be fully defined. Presumptively, viral culture can provide a surrogate measure for infectivity of sampled individuals, and thereby inform how and where to most appropriately deploy available diagnostic testing modalities. We therefore determined the relationship of antigen testing results from three lateral flow and one microfluidics assay to viral culture performed in parallel in 181 nasopharyngeal swab samples positive for SARS-CoV-2. Sample viral loads, determined by RT-qPCR, were distributed across the range of viral load values observed in our testing population. We found that antigen tests were predictive of viral culture positivity, with the LumiraDx method showing enhanced sensitivity (90%; 95% confidence interval (95% CI) 83-94%) compared with the BD Veritor (74%, 95% CI 65-81%), CareStart (74%, 95% CI 65-81%) and Oscar Corona (74%, 95% CI 65-82%) lateral flow antigen tests. Antigen and viral culture positivity were also highly correlated with sample viral load, with areas under the receiver?operator characteristic curves (ROCs) of 0.94-0.97 and 0.92, respectively. In particular, a viral load threshold of 100,000 copies/mL was 95% sensitive (95% CI, 90-98%) and 72% specific (95% CI, 60-81%) for predicting viral culture positivity. Taken together, the detection of SARS-CoV-2 antigen identified highly infectious individuals, some of whom may harbor 10,000-fold more virus in their samples than those with any detectable infectious virus. As such, our data support use of antigen testing in defining infectivity status at the time of sampling.

Saliva Is Comparable to Nasopharyngeal Swabs for Molecular Detection of SARS-CoV-2 (preprint)

Abstract Background. The continued need for molecular testing for SARS-CoV-2 and promise of self-collected saliva as an alternative to nasopharyngeal (NP) swabs for sample acquisition led us to compare saliva to NP swabs in an outpatient setting, without special restrictions to avoid food, drink, smoking, or toothbrushing. Methods. A total of 385 pairs of NP and saliva specimens were obtained, the majority from individuals presenting for initial evaluation, and were tested on two high-sensitivity RT-PCR platforms: the Abbott m2000 and Abbott Alinity m, both with limits of detection (LoD) of 100 copies of viral RNA/mL. Results. Concordance between saliva and NP was excellent overall (Cohen's {kappa}=0.93) for initial as well as followup testing on both platforms, and for specimens treated with guanidinium transport medium as preservative and for untreated saliva ({kappa}=0.88-0.95). Viral loads were on average 16x higher in NP specimens than saliva specimens, suggesting that only the relatively small fraction of outpatients (~8% in this study) who present with very low viral loads (<1,600 copies/mL from NP swabs) would be missed by saliva testing relative to NP testing, for sensitive testing platforms. Special attention was necessary to ensure leak-resistant specimen collection and transport. Conclusions. The advantages of self-collection without additional restrictions will likely outweigh a minor potential decrease in clinical sensitivity in individuals less likely to pose an infectious risk to others for many real-world scenarios, especially for initial testing.

Verification of the Abbott Alinity m Resp-4-Plex Assay for detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus (preprint)

COVID-19 symptomology may overlap with those of other circulating respiratory virus. In the future, hospital epidemiology will be focused on viruses with the potential for causation of severe disease, and there will be additional need for detection of respiratory viruses with potential sequelae for which there are specific and potential life-saving treatments. Therefore, there will be increasing need for multiplex respiratory assays to detection such virus. The Abbott Alinity m Resp-4-Plex assay is a multiplex PCR assay that simultaneously detects and differentiates infection with SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV). Based on perceived assay utility, we characterized the new assay in a series of validation studies to characterize its accuracy, precision, and analytical sensitivity. All were found to be robust within the measures examined. In the context of sample-to-answer, near random access automation on the Alinity m platform, we believe that the Resp-4-Plex assay offers significant utility in addressing the needs of the current SARS-CoV-2 pandemic and especially in the future with anticipated endemic circulation of SARS-CoV-2 with other respiratory viruses.

A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2 (preprint)

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.