Emergence of transmissible SARS-CoV-2 variants with decreased sensitivity to antivirals in immunocompromised patients with persistent infections.

We investigated the impact of antiviral treatment on the emergence of SARS-CoV-2 resistance during persistent infections in immunocompromised patients (n=15). All patients received remdesivir and some also received nirmatrelvir-ritonavir or monoclonal antibodies. Sequence analysis showed that nine patients carried viruses with mutations in the nsp12 (RNA dependent RNA polymerase), while four had viruses with nsp5 (3C protease) mutations. Infectious SARS-CoV-2 with a double mutation in nsp5 (T169I) and nsp12 (V792I) was recovered from respiratory secretions 77 days after initial COVID-19 diagnosis from a patient treated with remdesivir and nirmatrelvir-ritonavir. In vitro characterization confirmed its decreased sensitivity to remdesivir and nirmatrelvir, which was overcome by combined antiviral treatment. Studies in golden Syrian hamsters demonstrated efficient transmission to contact animals. This study documents the isolation of SARS-CoV-2 carrying resistance mutations to both nirmatrelvir and remdesivir from a patient and demonstrates its transmissibility in vivo .

Early immune factors associated with the development of post-acute sequelae of SARS-CoV-2 infection in hospitalized and non-hospitalized individuals.

Background: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to post-acute sequelae of SARS-CoV-2 (PASC) that can persist for weeks to years following initial viral infection. Clinical manifestations of PASC are heterogeneous and often involve multiple organs. While many hypotheses have been made on the mechanisms of PASC and its associated symptoms, the acute biological drivers of PASC are still unknown. Methods: We enrolled 494 patients with COVID-19 at their initial presentation to a hospital or clinic and followed them longitudinally to determine their development of PASC. From 341 patients, we conducted multi-omic profiling on peripheral blood samples collected shortly after study enrollment to investigate early immune signatures associated with the development of PASC. Results: During the first week of COVID-19, we observed a large number of differences in the immune profile of individuals who were hospitalized for COVID-19 compared to those individuals with COVID-19 who were not hospitalized. Differences between individuals who did or did not later develop PASC were, in comparison, more limited, but included significant differences in autoantibodies and in epigenetic and transcriptional signatures in double-negative 1 B cells, in particular. Conclusions: We found that early immune indicators of incident PASC were nuanced, with significant molecular signals manifesting predominantly in double-negative B cells, compared with the robust differences associated with hospitalization during acute COVID-19. The emerging acute differences in B cell phenotypes, especially in double-negative 1 B cells, in PASC patients highlight a potentially important role of these cells in the development of PASC.

Distinct temporal trajectories and risk factors for Post-acute sequelae of SARS-CoV-2 infection.

Background: The understanding of Post-acute sequelae of SARS-CoV-2 infection (PASC) can be improved by longitudinal assessment of symptoms encompassing the acute illness period. To gain insight into the various disease trajectories of PASC, we assessed symptom evolution and clinical factors associated with the development of PASC over 3 months, starting with the acute illness period. Methods: We conducted a prospective cohort study to identify parameters associated with PASC. We performed cluster and case control analyses of clinical data, including symptomatology collected over 3 months following infection. Results: We identified three phenotypic clusters associated with PASC that could be characterized as remittent, persistent, or incident based on the 3-month change in symptom number compared to study entry: remittent (median; min, max: -4; -17, 3), persistent (-2; -14, 7), or incident (4.5; -5, 17) (p = 0.041 remittent vs. persistent, p < 0.001 remittent vs. incident, p < 0.001 persistent vs. incident). Despite younger age and lower hospitalization rates, the incident phenotype had a greater number of symptoms (15; 8, 24) and a higher proportion of participants with PASC (63.2%) than the persistent (6; 2, 9 and 52.2%) or remittent clusters (1; 0, 6 and 18.7%). Systemic corticosteroid administration during acute infection was also associated with PASC at 3 months [OR (95% CI): 2.23 (1.14, 4.36)]. Conclusion: An incident disease phenotype characterized by symptoms that were absent during acute illness and the observed association with high dose steroids during acute illness have potential critical implications for preventing PASC.

Interferon ɛ restricts Zika virus infection in the female reproductive tract.

Interferon ɛ (IFNɛ) is a unique type I IFN that has been implicated in host defense against sexually transmitted infections. Zika virus (ZIKV), an emerging pathogen, can infect the female reproductive tract (FRT) and cause devastating diseases, particularly in pregnant women. How IFNɛ contributes to protection against ZIKV infection in vivo is unknown. In this study, we show that IFNɛ plays a critical role in host protection against vaginal ZIKV infection in mice. We found that IFNɛ was expressed not only by epithelial cells in the FRT but also by immune and stromal cells at baseline or after exposure to viruses or specific Toll-like receptor (TLR) agonists. IFNɛ-deficient mice exhibited abnormalities in the epithelial border and underlying tissue in the cervicovaginal tract, and these defects were associated with increased susceptibility to vaginal but not subcutaneous ZIKV infection. IFNɛ deficiency resulted in an increase in magnitude, duration, and depth of ZIKV infection in the FRT. Critically, intravaginal administration of recombinant IFNɛ protected Ifnɛ-/- mice and highly susceptible Ifnar1-/- mice against vaginal ZIKV infection, indicating that IFNɛ was sufficient to provide protection even in the absence of signals from other type I IFNs and in an IFNAR1-independent manner. Our findings reveal a potentially critical role for IFNɛ in mediating protection against the transmission of ZIKV in the context of sexual contact.

Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.

Gastrointestinal fungal dysbiosis is a hallmark of several diseases marked by systemic immune activation. Whether persistent pathobiont colonization during immune alterations and impaired gut barrier function has a durable impact on host immunity is unknown. We found that elevated levels of Candida albicans immunoglobulin G (IgG) antibodies marked patients with severe COVID-19 (sCOVID-19) who had intestinal Candida overgrowth, mycobiota dysbiosis and systemic neutrophilia. Analysis of hematopoietic stem cell progenitors in sCOVID-19 revealed transcriptional changes in antifungal immunity pathways and reprogramming of granulocyte myeloid progenitors (GMPs) for up to a year. Mice colonized with C. albicans patient isolates experienced increased lung neutrophilia and pulmonary NETosis during severe acute respiratory syndrome coronavirus-2 infection, which were partially resolved with antifungal treatment or by interleukin-6 receptor blockade. sCOVID-19 patients treated with tocilizumab experienced sustained reductions in C. albicans IgG antibodies titers and GMP transcriptional changes. These findings suggest that gut fungal pathobionts may contribute to immune activation during inflammatory diseases, offering potential mycobiota-immune therapeutic strategies for sCOVID-19 with prolonged symptoms.

Influenza Defective Interfering Virus Promotes Multiciliated Cell Differentiation and Reduces the Inflammatory Response in Mice.

Influenza defective interfering (DI) viruses have long been considered promising antiviral candidates because of their ability to interfere with replication-competent viruses and induce antiviral immunity. However, the mechanisms underlying DI-mediated antiviral immunity have not been extensively explored. Here, we demonstrated the interferon (IFN)-independent protection conferred by the influenza DI virus against homologous virus infection in mice deficient in type I and III IFN signaling. We identified unique host signatures responding to DI coinfection by integrating transcriptional and posttranscriptional regulatory data. DI-treated mice exhibited reduced viral transcription, less intense inflammatory and innate immune responses, and primed multiciliated cell differentiation in their lungs at an early stage of infection, even in the absence of type I or III IFNs. This increased multiciliogenesis could also be detected at the protein level via the immunofluorescence staining of lung tissue from DI-treated mice. Overall, our study provides mechanistic insight into the protection mediated by DIs, implying a unifying theme involving inflammation and multiciliogenesis in maintaining respiratory homeostasis and revealing their IFN-independent antiviral activity. IMPORTANCE During replication, the influenza virus generates genetically defective viruses. These are found in natural infections as part of the virus population within the infected host. Some versions of these defective viruses are thought to have protective effects through their interference with replication-competent viruses and induction of antiviral immunity. To better determine the mechanisms underlying the protective effects of these defective interfering (DI) viruses, we tested a DI that we previously identified in vitro with mice. Mice that were infected with a mix of wild-type influenza and DI viruses had less intense inflammatory and innate immune responses than did mice that were infected with the wild-type virus only, even when type I or III interferons, which are cytokines that play a prominent role in defending the respiratory epithelial barrier, were absent. More interestingly, the DI-infected mice had primed multiciliated cell differentiation in their lungs, indicating the potential promotion of epithelial repair by DIs.

Interferon ε restricts Zika virus infection in the female reproductive tract.

Interferon ε (IFNε) is a unique type I IFN that has been implicated in host defense against sexually transmitted infections (STIs). Zika virus (ZIKV), an emerging pathogen, can infect the female reproductive tract (FRT) and cause devastating diseases, particularly in pregnant women. How IFNε contributes to protection against ZIKV infection in vivo is unknown. Here, we show that IFNε plays a critical role in host protection against vaginal ZIKV infection in mice. We found that IFNε was expressed not only by epithelial cells in the FRT, but also by certain immune and other cells at baseline or after exposure to viruses or specific TLR agonists. IFNε-deficient mice exhibited abnormalities in the epithelial border and underlying tissue in the cervicovaginal tract, and these defects were associated with increased susceptibility to vaginal, but not subcutaneous ZIKV infection. IFNε-deficiency resulted in an increase in magnitude, duration, and depth of ZIKV infection in the FRT. Critically, intravaginal administration of recombinant IFNε protected Ifnε-/- mice and highly susceptible Ifnar1-/- mice against vaginal ZIKV infection, indicating that IFNε was sufficient to provide protection even in the absence of signals from other type I IFNs and in an IFNAR1-independent manner. Our findings reveal a potentially critical role for IFNε in mediating protection against transmission of ZIKV in the context of sexual contact.

A metagenomic DNA sequencing assay that is robust against environmental DNA contamination.

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present Sample-Intrinsic microbial DNA Found by Tagging and sequencing (SIFT-seq) a metagenomic sequencing assay that is robust against environmental DNA contamination introduced during sample preparation. The core idea of SIFT-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied SIFT-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of sepsis and inflammatory bowel disease in blood.

System-wide transcriptome damage and tissue identity loss in COVID-19 patients.

The molecular mechanisms underlying the clinical manifestations of coronavirus disease 2019 (COVID-19), and what distinguishes them from common seasonal influenza virus and other lung injury states such as acute respiratory distress syndrome, remain poorly understood. To address these challenges, we combine transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues to define body-wide transcriptome changes in response to COVID-19. We then match these data with spatial protein and expression profiling across 357 tissue sections from 16 representative patient lung samples and identify tissue-compartment-specific damage wrought by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, evident as a function of varying viral loads during the clinical course of infection and tissue-type-specific expression states. Overall, our findings reveal a systemic disruption of canonical cellular and transcriptional pathways across all tissues, which can inform subsequent studies to combat the mortality of COVID-19 and to better understand the molecular dynamics of lethal SARS-CoV-2 and other respiratory infections.

Interferon mediated prophylactic protection against respiratory viruses conferred by a prototype live attenuated influenza virus vaccine lacking non-structural protein 1.

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

A metagenomic DNA sequencing assay that is robust against environmental DNA contamination.

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied Coffee-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of inflammatory bowel disease in blood.

Sex-Related Differences in Clinical Presentation and Risk Factors for Mortality in Patients Hospitalized With Coronavirus Disease 2019 in New York City.

We evaluated sex-related differences in symptoms and risk factors for mortality in 4798 patients hospitalized with coronavirus disease 2019 in New York City. When adjusted for age and comorbidities, being male was an independent predictor of death with mortality significantly higher than females, even with low severe acute respiratory syndrome coronavirus 2 viral load at admission.

Prophylactic Protection Against Respiratory Viruses Conferred by a Prototype Live Attenuated Influenza Virus Vaccine.

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an immediate antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

Prophylactic protection against respiratory viruses conferred by a prototype live attenuated influenza virus vaccine.

The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 (ΔNS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of ΔNS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a ΔNS1 virus results in an immediate antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that ΔNS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on ΔNS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.

Social and Clinical Determinants of COVID-19 Outcomes: Modeling Real-World Data from a Pandemic Epicenter.

IMPORTANCE: As the United States continues to accumulate COVID-19 cases and deaths, and disparities persist, defining the impact of risk factors for poor outcomes across patient groups is imperative. OBJECTIVE: Our objective is to use real-world healthcare data to quantify the impact of demographic, clinical, and social determinants associated with adverse COVID-19 outcomes, to identify high-risk scenarios and dynamics of risk among racial and ethnic groups. DESIGN: A retrospective cohort of COVID-19 patients diagnosed between March 1 and August 20, 2020. Fully adjusted logistical regression models for hospitalization, severe disease and mortality outcomes across 1-the entire cohort and 2- within self-reported race/ethnicity groups. SETTING: Three sites of the NewYork-Presbyterian health care system serving all boroughs of New York City. Data was obtained through automated data abstraction from electronic medical records. PARTICIPANTS: During the study timeframe, 110,498 individuals were tested for SARS-CoV-2 in the NewYork-Presbyterian health care system; 11,930 patients were confirmed for COVID-19 by RT-PCR or covid-19 clinical diagnosis. MAIN OUTCOMES AND MEASURES: The predictors of interest were patient race/ethnicity, and covariates included demographics, comorbidities, and census tract neighborhood socio-economic status. The outcomes of interest were COVID-19 hospitalization, severe disease, and death. RESULTS: Of confirmed COVID-19 patients, 4,895 were hospitalized, 1,070 developed severe disease and 1,654 suffered COVID-19 related death. Clinical factors had stronger impacts than social determinants and several showed race-group specificities, which varied among outcomes. The most significant factors in our all-patients models included: age over 80 (OR=5.78, p= 2.29x10-24) and hypertension (OR=1.89, p=1.26x10-10) having the highest impact on hospitalization, while Type 2 Diabetes was associated with all three outcomes (hospitalization: OR=1.48, p=1.39x10-04; severe disease: OR=1.46, p=4.47x10-09; mortality: OR=1.27, p=0.001). In race-specific models, COPD increased risk of hospitalization only in Non-Hispanics (NH)-Whites (OR=2.70, p=0.009). Obesity (BMI 30+) showed race-specific risk with severe disease NH-Whites (OR=1.48, p=0.038) and NH-Blacks (OR=1.77, p=0.025). For mortality, Cancer was the only risk factor in Hispanics (OR=1.97, p=0.043), and heart failure was only a risk in NH-Asians (OR=2.62, p=0.001). CONCLUSIONS AND RELEVANCE: Comorbidities were more influential on COVID-19 outcomes than social determinants, suggesting clinical factors are more predictive of adverse trajectory than social factors.

Systemic Tissue and Cellular Disruption from SARS-CoV-2 Infection revealed in COVID-19 Autopsies and Spatial Omics Tissue Maps.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has infected over 115 million people and caused over 2.5 million deaths worldwide. Yet, the molecular mechanisms underlying the clinical manifestations of COVID-19, as well as what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome (ARDS), remains poorly understood. To address these challenges, we combined transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues, matched with spatial protein and expression profiling (GeoMx) across 357 tissue sections. These results define both body-wide and tissue-specific (heart, liver, lung, kidney, and lymph nodes) damage wrought by the SARS-CoV-2 infection, evident as a function of varying viral load (high vs. low) during the course of infection and specific, transcriptional dysregulation in splicing isoforms, T cell receptor expression, and cellular expression states. In particular, cardiac and lung tissues revealed the largest degree of splicing isoform switching and cell expression state loss. Overall, these findings reveal a systemic disruption of cellular and transcriptional pathways from COVID-19 across all tissues, which can inform subsequent studies to combat the mortality of COVID-19, as well to better understand the molecular dynamics of lethal SARS-CoV-2 infection and other viruses.

Profiling of immune dysfunction in COVID-19 patients allows early prediction of disease progression.

With a rising incidence of COVID-19-associated morbidity and mortality worldwide, it is critical to elucidate the innate and adaptive immune responses that drive disease severity. We performed longitudinal immune profiling of peripheral blood mononuclear cells from 45 patients and healthy donors. We observed a dynamic immune landscape of innate and adaptive immune cells in disease progression and absolute changes of lymphocyte and myeloid cells in severe versus mild cases or healthy controls. Intubation and death were coupled with selected natural killer cell KIR receptor usage and IgM+ B cells and associated with profound CD4 and CD8 T-cell exhaustion. Pseudo-temporal reconstruction of the hierarchy of disease progression revealed dynamic time changes in the global population recapitulating individual patients and the development of an eight-marker classifier of disease severity. Estimating the effect of clinical progression on the immune response and early assessment of disease progression risks may allow implementation of tailored therapies.

Metabolic and Immune Markers for Precise Monitoring of COVID-19 Severity and Treatment.

Deep understanding of the SARS-CoV-2 effects on host molecular pathways is paramount for the discovery of early biomarkers of outcome of coronavirus disease 2019 (COVID-19) and the identification of novel therapeutic targets. In that light, we generated metabolomic data from COVID-19 patient blood using high-throughput targeted nuclear magnetic resonance (NMR) spectroscopy and high-dimensional flow cytometry. We find considerable changes in serum metabolome composition of COVID-19 patients associated with disease severity, and response to tocilizumab treatment. We built a clinically annotated, biologically-interpretable space for precise time-resolved disease monitoring and characterize the temporal dynamics of metabolomic change along the clinical course of COVID-19 patients and in response to therapy. Finally, we leverage joint immuno-metabolic measurements to provide a novel approach for patient stratification and early prediction of severe disease. Our results show that high-dimensional metabolomic and joint immune-metabolic readouts provide rich information content for elucidation of the host's response to infection and empower discovery of novel metabolic-driven therapies, as well as precise and efficient clinical action.

Integrase-Defective Lentiviral Vectors for Delivery of Monoclonal Antibodies against Influenza.

Delivering rapid protection against infectious agents to non-immune populations is a formidable public health challenge. Although passive immunotherapy is a fast and effective method of protection, large-scale production and administration of monoclonal antibodies (mAbs) is expensive and unpractical. Viral vector-mediated delivery of mAbs offers an attractive alternative to their direct injection. Integrase-defective lentiviral vectors (IDLV) are advantageous for this purpose due to the absence of pre-existing anti-vector immunity and the safety features of non-integration and non-replication. We engineered IDLV to produce the humanized mAb VN04-2 (IDLV-VN04-2), which is broadly neutralizing against H5 influenza A virus (IAV), and tested the vectors' ability to produce antibodies and protect from IAV in vivo. We found that IDLV-transduced cells produced functional VN04-2 mAbs in a time- and dose-dependent fashion. These mAbs specifically bind the hemagglutinin (HA), but not the nucleoprotein (NP) of IAV. VN04-2 mAbs were detected in the serum of mice at different times after intranasal (i.n.) or intramuscular (i.m.) administration of IDLV-VN04-2. Administration of IDLV-VN04-2 by the i.n. route provided rapid protection against lethal IAV challenge, although the protection did not persist at later time points. Our data suggest that administration of mAb-expressing IDLV may represent an effective strategy for rapid protection against infectious diseases.

Remdesivir Treatment for Severe COVID-19 in Third-Trimester Pregnancy: Case Report and Management Discussion.

We report a case of COVID-19 in third-trimester pregnancy, who required support in an intensive care unit and received remdesivir. After discharge, she had an uncomplicated vaginal delivery at term. COVID-19 in pregnancy may be managed without emergent delivery; a multispecialty team is critical in caring for these patients.