A 9-mRNA signature measured from whole blood by a prototype PCR panel predicts 28-day mortality upon admission of critically ill COVID-19 patients.

Immune responses affiliated with COVID-19 severity have been characterized and associated with deleterious outcomes. These approaches were mainly based on research tools not usable in routine clinical practice at the bedside. We observed that a multiplex transcriptomic panel prototype termed Immune Profiling Panel (IPP) could capture the dysregulation of immune responses of ICU COVID-19 patients at admission. Nine transcripts were associated with mortality in univariate analysis and this 9-mRNA signature remained significantly associated with mortality in a multivariate analysis that included age, SOFA and Charlson scores. Using a machine learning model with these 9 mRNA, we could predict the 28-day survival status with an Area Under the Receiver Operating Curve (AUROC) of 0.764. Interestingly, adding patients' age to the model resulted in increased performance to predict the 28-day mortality (AUROC reaching 0.839). This prototype IPP demonstrated that such a tool, upon clinical/analytical validation and clearance by regulatory agencies could be used in clinical routine settings to quickly identify patients with higher risk of death requiring thus early aggressive intensive care.

Dynamics of viral shedding during ancestral or Omicron BA.1 SARS-CoV-2 infection and enhancement of pre-existing immunity during breakthrough infections.

Omicron variant is circulating in the presence of a globally acquired immunity unlike the ancestral SARS-CoV-2 isolate. Herein, we investigated the normalized viral load dynamics and viral culture status in 44 fully vaccinated healthcare workers (HCWs) infected with the Omicron BA.1 variant. Viral load dynamics of 38 unvaccinated HCWs infected with the 20A variant during the first pandemic wave was also studied. We then explored the impact of Omicron infection on pre-existing immunity assessing anti-RBD IgG levels, neutralizing antibody titres against 19A, Delta and Omicron isolates, as well as IFN-γ release following cell stimulation with SARS-CoV-2 peptides. We reported that two weeks after diagnosis a greater proportion of HCWs infected with 20A (78.9%, 15/19) than with Omicron BA.1 (44.7%, 17/38; p = 0.02) were still positive by RT-qPCR. We found that Omicron breakthrough infections led to an overall enhancement of vaccine-induced humoral and cellular immunity as soon as a median [interquartile range] of 8 [7-9] days post symptom onset. Among samples with similar high viral loads, non-culturable samples exhibited higher neutralizing antibody titres and anti-RBD IgG levels than culturable samples. Additionally, Omicron infection led to an enhancement of antibodies neutralization capacity against other SARS-CoV-2 isolates. Taken together, the results suggest that Omicron BA.1 vaccine breakthrough infection is associated with a faster viral clearance than that of the ancestral SARS-CoV-2, in addition this new variant leads to a rapid enhancement of the humoral response against multiple SARS-CoV-2 variants, and of the cellular response.

Evaluation of EPISEQ SARS-CoV-2 and a Fully Integrated Application to Identify SARS-CoV-2 Variants from Several Next-Generation Sequencing Approaches.

Whole-genome sequencing has become an essential tool for real-time genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide. The handling of raw next-generation sequencing (NGS) data is a major challenge for sequencing laboratories. We developed an easy-to-use web-based application (EPISEQ SARS-CoV-2) to analyse SARS-CoV-2 NGS data generated on common sequencing platforms using a variety of commercially available reagents. This application performs in one click a quality check, a reference-based genome assembly, and the analysis of the generated consensus sequence as to coverage of the reference genome, mutation screening and variant identification according to the up-to-date Nextstrain clade and Pango lineage. In this study, we validated the EPISEQ SARS-CoV-2 pipeline against a reference pipeline and compared the performance of NGS data generated by different sequencing protocols using EPISEQ SARS-CoV-2. We showed a strong agreement in SARS-CoV-2 clade and lineage identification (>99%) and in spike mutation detection (>99%) between EPISEQ SARS-CoV-2 and the reference pipeline. The comparison of several sequencing approaches using EPISEQ SARS-CoV-2 revealed 100% concordance in clade and lineage classification. It also uncovered reagent-related sequencing issues with a potential impact on SARS-CoV-2 mutation reporting. Altogether, EPISEQ SARS-CoV-2 allows an easy, rapid and reliable analysis of raw NGS data to support the sequencing efforts of laboratories with limited bioinformatics capacity and those willing to accelerate genomic surveillance of SARS-CoV-2.

Recombinant human interleukin-7 reverses T cell exhaustion ex vivo in critically ill COVID-19 patients

BackgroundLymphopenia is a hallmark of severe coronavirus disease 19 (COVID-19). Similar alterations have been described in bacterial sepsis and therapeutic strategies targeting T cell function such as recombinant human interleukin 7 (rhIL-7) have been proposed in this clinical context. As COVID-19 is a viral sepsis, the objectives of this study were to characterize T lymphocyte response over time in severe COVID-19 patients and to assess the effect of ex vivo administration of rhIL-7.ResultsPeripheral blood mononuclear cells from COVID-19 patients hospitalized in intensive care unit (ICU) were collected at admission and after 20 days. Transcriptomic profile was evaluated through NanoString technology. Inhibitory immune checkpoints expressions were determined by flow cytometry. T lymphocyte proliferation and IFN-γ production were evaluated after ex vivo stimulation in the presence or not of rhIL-7. COVID-19 ICU patients were markedly lymphopenic at admission. Mononuclear cells presented with inhibited transcriptomic profile prevalently with impaired T cell activation pathways. CD4 + and CD8 + T cells presented with over-expression of co-inhibitory molecules PD-1, PD-L1, CTLA-4 and TIM-3. CD4 + and CD8 + T cell proliferation and IFN-γ production were markedly altered in samples collected at ICU admission. These alterations, characteristic of a T cell exhaustion state, were more pronounced at ICU admission and alleviated over time. Treatment with rhIL-7 ex vivo significantly improved both T cell proliferation and IFN-γ production in cells from COVID-19 patients.ConclusionsSevere COVID-19 patients present with features of profound T cell exhaustion upon ICU admission which can be reversed ex vivo by rhIL-7. These results reinforce our understanding of severe COVID-19 pathophysiology and opens novel therapeutic avenues to treat such critically ill patients based of immunomodulation approaches. Defining the appropriate timing for initiating such immune-adjuvant therapy in clinical setting and the pertinent markers for a careful selection of patients are now warranted to confirm the ex vivo results described so far.Trial registration ClinicalTrials.gov identifier: NCT04392401 Registered 18 May 2020, http:// clinicaltrials.gov/ct2/show/NCT04392401.

T cell response against SARS-CoV-2 persists after one year in patients surviving severe COVID-19.

BACKGROUND: In critically ill COVID-19 patients, the initial response to SARS-CoV-2 infection is characterized by major immune dysfunctions. The capacity of these severe patients to mount a robust and persistent SARS-CoV-2 specific T cell response despite the presence of severe immune alterations during the ICU stay is unknown. METHODS: Critically ill COVID-19 patients were sampled five times during the ICU stay and 9 and 13 months afterwards. Immune monitoring included counts of lymphocyte subpopulations, HLA-DR expression on monocytes, plasma IL-6 and IL-10 concentrations, anti-SARS-CoV-2 IgG levels and T cell proliferation in response to three SARS-CoV-2 antigens. FINDINGS: Despite the presence of major lymphopenia and decreased monocyte HLA-DR expression during the ICU stay, convalescent critically ill COVID-19 patients consistently generated adaptive and humoral immune responses against SARS-CoV-2 maintained for more than one year after hospital discharge. Patients with long hospital stays presented with stronger anti-SARS-CoV-2 specific T cell response but no difference in anti-SARS-CoV2 IgG levels. INTERPRETATION: Convalescent critically ill COVID-19 patients consistently generated a memory immune response against SARS-CoV-2 maintained for more than one year after hospital discharge. In recovered individuals, the intensity of SARS-CoV-2 specific T cell response was dependent on length of hospital stay. FUNDING: This observational study was supported by funds from the Hospices Civils de Lyon, Fondation HCL, Claude Bernard Lyon 1 University and Région Auvergne Rhône-Alpes and by partial funding by REACTing (Research and ACTion targeting emerging infectious diseases) INSERM, France and a donation from Fondation AnBer (http://fondationanber.fr/).

Seroconversion in septic ICU patients presenting with COVID-19: necessary but not sufficient.

BACKGROUND: As COVID-19 pandemic and vaccination effects progress, research now focuses on adaptive immunological response to SARS-CoV-2. Few studies specifically investigated intensive care unit (ICU) patients, and little is known about kinetics of humoral response in such critically ill patients. In this context, the main objective of the present work was to perform a longitudinal analysis of the humoral response in critically ill COVID-19 patients with prolonged ICU stays in regard with initial inflammatory response, disease severity and mortality. METHODS: Over a 3 week period, circulating immunoglobulins (Ig) against SARS-CoV-2 along with several immunological and clinical parameters were measured in 64 ICU COVID-19 patients. RESULTS: Critically ill COVID-19 patients mounted a dynamic and sustained antibody response of both IgM and IgG as soon as the first day of ICU hospitalization. This serological response was not associated with any of the classical immunological parameters measured at ICU admission or with initial severity clinical scores. IgM and IgG levels and seroconversion trajectories were not associated with unfavourable outcome. CONCLUSION: Despite rapid seroconversion and elevated humoral response, COVID-19 patients are still characterized by elevated mortality. Additional studies, including cytotoxic T cell functions, are mandatory to understand the immunological mechanisms contributing to long stay of COVID-19 patients in ICU.

Early nasal type I IFN immunity against SARS-CoV-2 is compromised in patients with autoantibodies against type I IFNs.

IFN-I and IFN-III immunity in the nasal mucosa is poorly characterized during SARS-CoV-2 infection. We analyze the nasal IFN-I/III signature, namely the expression of ISGF-3-dependent IFN-stimulated genes, in mildly symptomatic COVID-19 patients and show its correlation with serum IFN-α2 levels, which peak at symptom onset and return to baseline from day 10 onward. Moreover, the nasal IFN-I/III signature correlates with the nasopharyngeal viral load and is associated with the presence of infectious viruses. By contrast, we observe low nasal IFN-I/III scores despite high nasal viral loads in a subset of critically ill COVID-19 patients, which correlates with the presence of autoantibodies (auto-Abs) against IFN-I in both blood and nasopharyngeal mucosa. In addition, functional assays in a reconstituted human airway epithelium model of SARS-CoV-2 infection confirm the role of such auto-Abs in abrogating the antiviral effects of IFN-I, but not those of IFN-III. Thus, IFN-I auto-Abs may compromise not only systemic but also local antiviral IFN-I immunity at the early stages of SARS-CoV-2 infection.

Clinical and laboratory characteristics of symptomatic healthcare workers with suspected COVID-19: a prospective cohort study.

A comprehensive clinical and microbiological assessments of COVID-19 in front-line healthcare workers (HCWs) is needed. Between April 10th and May 28th, 2020, 319 HCWs with acute illness were reviewed. In addition to SARS-CoV-2 RT-PCR screening, a multiplex molecular panel was used for testing other respiratory pathogens. For SARS-CoV-2 positive HCWs, the normalized viral load, viral culture, and virus neutralization assays were performed weekly. For SARS-CoV-2 negative HCWs, SARS-CoV-2 serological testing was performed one month after inclusion. Among the 319 HCWs included, 67 (21.0%) were tested positive for SARS-CoV-2; 65/67 (97.0%) developed mild form of COVID-19. Other respiratory pathogens were found in 6/66 (9.1%) SARS-CoV-2 positive and 47/241 (19.5%) SARS-Cov-2 negative HCWs (p = 0.07). The proportion of HCWs with a viral load > 5.0 log10 cp/mL (Ct value < 25) was less than 15% at 8 days after symptom onset; 12% of HCWs were positive after 40 days (Ct > 37). More than 90% of cultivable virus had a viral load > 4.5 log10 cp/mL (Ct < 26) and were collected within 10 days after symptom onset. Among negative HCWs, 6/190 (3.2%) seroconverted. Our data suggest that the determination of viral load can be used for appreciating the infectiousness of infected HCWs. These data could be helpful for facilitating their return to work.

Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories.

A reliable diagnostic assay is crucial to early detect new COVID-19 cases and limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Since the onset of the COVID-19 pandemic, the World Health Organization has published several diagnostic molecular approaches developed by referral laboratories, including Charité (Germany), HKU (Hong Kong), China CDC (China), US CDC (United States), and Institut Pasteur, Paris (France). We aimed to compare the sensitivity and specificity of these different RT-PCR assays using SARS-CoV-2 cell culture supernatants and clinical respiratory samples. Overall, the different RT-PCR assays performed well for SARS-CoV-2 detection and were all specific except the N Charité (Germany), and N2 US CDC (United States) assays. RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC were found to be the most sensitive assays. The data presented herein are of prime importance to facilitate the equipment choice of diagnostic laboratories, as well as for the development of marketed tests.