Gut microbial disruption in critically ill patients with COVID-19 associated pulmonary aspergillosis

ObjectivesCOVID-19 disease can be exacerbated by Aspergillus superinfection (CAPA). The causes of CAPA are not yet fully understood. Recently, alterations in the gut microbiome have been associated with a complicating course and increasing severity of COVID-19 disease, most likely via immunological mechanisms. Aim of this study was to investigate a potential association between severe CAPA and alterations in the gut and bronchial microbiota. MethodsWe performed 16S rRNA gene amplicon sequencing of stool and bronchial samples from a total of 16 COVID-19 patients with CAPA and 26 patients without CAPA. All patients were admitted to the intensive care unit. Results were carefully tested for potential influences on the microbiome during hospitalization. ResultsWe found that late in COVID-19 disease, CAPA patients exhibited a trend towards reduced gut microbial diversity. Furthermore, late stage CAPA disease showed an increased presence of Staphylococcus epidermidis in the gut. This is not found in late non-CAPA cases or early disease. The analysis of bronchial samples did not show significant results. ConclusionsThis is the first study showing alterations in the gut microbiome accompany severe CAPA and possibly influence the hosts immunological response. In particular, an increase of Staphylococcus epidermidis in the intestine could be of importance. SummaryThe composition of intestinal bacteria in severe CAPA disease is altered with an increase in Staphylococcus epidermidis in the gut. Alterations in the composition of intestinal bacteria in severe CAPA may indicate immunologic involvement of the gut in the disease.

Improved SARS-CoV-2 neutralization of Delta and Omicron variants of concern after fourth vaccination in hemodialysis patients

BackgroundHemodialysis patients are exposed to a markedly increased risk when infected with SARS-CoV-2. To date it is unclear if hemodialysis patients benefit from a fourth vaccination. MethodsA total of 142 hemodialysis patients (median age 72.6 years, 33.8% female) received four COVID-19 vaccinations between December 2020 and March 2022. RDB binding antibody titers were determined in a competitive surrogate neutralization assay. Vero-E6 cells were infected with SARS-CoV-2 variants of concern (VoC) Delta (B.1.617.2) or Omicron (B.1.1.529, sub lineage BA.1) in a biosafety level 3 laboratory to determine serum infection neutralization capacity before and after vaccination. ResultsAfter the fourth vaccination serum infection neutralization capacity significantly increased from a 50% inhibitory concentration (IC50, serum dilution factor 1:x) of 247.0 (46.3-1560.8) to 2560.0 (1174.0-2560.0) for the Delta VoC, and from 37.5 (20.0-198.8) to 668.5 (182.2-2560.0) for the Omicron VoC (each p<0.001). A significant increase of the neutralization capacity was even observed for patients who had high antibody titers after three vaccinations (p<0.001). Univariate regression analysis indicated immunosuppressive medication (p=0.001) and hepatitis B vaccination non-response (p=0.046), and multivariate analysis immunosuppressive medication as the only factor associated with a reduced effect against Delta (p<0.001). Ten patients with SARS-CoV-2 breakthrough infection before the fourth vaccination had by trend lower prior neutralization capacity for Omicron (p=0.051). ConclusionsOur findings suggest that hemodialysis patients benefit from a fourth vaccination in particular in the light of the highly infectious SARS-CoV-2 Omicron variant. A routinely applied four-time vaccination seems to broaden immunity against variants and would be recommended in hemodialysis patients.

CMV seropositivity is a potential novel risk factor for severe COVID-19 in non-geriatric patients

BackgroundCOVID-19 has so far affected more than 250 million individuals worldwide, causing more than 5 million deaths. Several risk factors for severe disease have been identified, most of which coincide with advanced age. In younger individuals, severe COVID-19 often occurs in the absence of obvious comorbidities. Guided by the finding of cytomegalovirus (CMV)-specific T cells with some cross-reactivity to SARS-CoV-2 in a COVID-19 intensive care unit (ICU) patient, we decided to investigate whether CMV seropositivity is associated with severe or critical COVID-19. MethodsNational German COVID-19 bio-sample and data banks were used to retrospectively analyze the CMV serostatus of patients who experienced mild (n=101), moderate (n=130) or severe to critical (n=80) disease by CMV IgG serology. We then investigated the relationship between disease severity and CMV serostatus via statistical models. ResultsNon-geriatric patients (< 70 years) with severe COVID-19 were found to have a very high prevalence of CMV-seropositivity, while CMV status distribution in individuals with mild disease was similar to the prevalence in the German population; interestingly, this was not detectable in older patients. Prediction models support the hypothesis that the CMV serostatus might be a strong biomarker in identifying younger individuals with a higher risk of developing severe COVID-19. ConclusionsWe identified CMV-seropositivity as a potential novel risk factor for severe COVID-19 in non-geriatric individuals in the studied cohorts. More mechanistic analyses as well as confirmation of similar findings in cohorts representing the currently most relevant SARS-CoV-2 variants should be performed shortly.

Gut Bacterial Dysbiosis and Instability is Associated with the Onset of Complications and Mortality in COVID-19

ObjectiveThere is a growing debate about the involvement of the gut microbiome in COVID-19, although it is not conclusively understood whether the microbiome has an impact on COVID-19, or vice versa, especially as analysis of amplicon data in hospitalized patients requires sophisticated cohort recruitment and integration of clinical parameters. Here, we analyzed fecal and saliva samples from SARS-CoV-2 infected and post COVID-19 patients and controls considering multiple influencing factors during hospitalization. Design16S rRNA gene sequencing was performed on fecal and saliva samples from 108 COVID-19 and 22 post COVID-19 patients, 20 pneumonia controls and 26 asymptomatic controls. Patients were recruited over the first and second corona wave in Germany and detailed clinical parameters were considered. Serial samples per individual allowed intra-individual analysis. ResultsWe found the gut and oral microbiota to be altered depending on number and type of COVID-19-associated complications and disease severity. The occurrence of individual complications was correlated with low-risk (e.g., Faecalibacterium prausznitzii) and high-risk bacteria (e.g., Parabacteroides). We demonstrated that a stable gut bacterial composition was associated with a favorable disease progression. Based on gut microbial profiles, we identified a model to estimate mortality in COVID-19. ConclusionGut microbiota are associated with the occurrence of complications in COVID-19 and may thereby influencing disease severity. A stable gut microbial composition may contribute to a favorable disease progression and using bacterial signatures to estimate mortality could contribute to diagnostic approaches. Importantly, we highlight challenges in the analysis of microbial data in the context of hospitalization.

Early Reduction of SARS-CoV-2 Replication in Bronchial Epithelium by Kinin B2 Receptor Antagonism

BackgroundSARS-CoV2 has evolved to enter the host via the ACE2 receptor which is part of the Kinin-kallirein pathway. This complex pathway is only poorly understood in context of immune regulation but critical to control infection. This study examines SARS-CoV2 infection and epithelial mechanisms of the kinin-kallikrein system at the kinin B2 receptor level in SARS-CoV-2 infection that is of direct translational relevance. MethodsFrom acute SARS-CoV-2-positive patients and -negative controls, transcriptomes of nasal brushings were analyzed. Primary airway epithelial cells (NHBEs) were infected with SARS-CoV-2 and treated with the approved B2R antagonist icatibant. SARS-CoV-2 RNA RT-qPCR, cytotoxicity assays, plaque assays and transcriptome analyses were performed. The treatment effect was further studied in a murine airway inflammation model in vivo. ResultsHere, we report a broad and strong upregulation of kallikreins and the kinin B2 receptor (B2R) in the nasal mucosa of acutely symptomatic SARS-CoV-2-positive patients. A B2R antagonist impeded SARS-CoV-2 replication and spread in NHBEs, as determined in plaque assays on Vero E6 cells. B2R antagonism reduced the expression of SARS-CoV-2 entry receptor ACE2 in vitro and in a murine airway inflammation model in vivo. In addition, it suppressed gene expression broadly, particularly genes involved in G-protein-coupled-receptor signaling and ion transport. ConclusionsIn summary, this study provides evidence that treatment with B2R antagonists protects airway epithelial cells from SARS-CoV-2 by inhibiting its replication and spread, through the reduction of ACE2 levels and the interference with several cellular signaling processes. Future clinical studies need to shed light on the airway protection potential of approved B2R antagonists, like icatibant, in the treatment of early-stage COVID-19.

Recruitment of highly functional SARS-CoV-2-specific CD8+ T cell receptors mediating cytotoxicity of virus-infected target cells in non-severe COVID-19

T cell immunity is crucial for the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and has been widely characterized on a quantitative level. In contrast, the quality of such T cell responses has been poorly investigated, in particular in the case of CD8+ T cells. Here, we explored the quality of SARS-CoV-2-specific CD8+ T cell responses in individuals who recovered from mild symptomatic infections, through which protective immunity should develop, by functional characterization of their T cell receptor (TCR) repertoire. CD8+ T cell responses specific for SARS-CoV-2-derived epitopes were low in frequency but could be detected robustly early as well as late - up to twelve months - after infection. A pool of immunodominant epitopes, which accurately identified previous SARS-CoV-2 infections, was used to isolate TCRs specific for epitopes restricted by common HLA class I molecules. TCR-engineered T cells showed heterogeneous functional avidity and cytotoxicity towards virus-infected target cells. High TCR functionality correlated with gene signatures of T cell function and activation that, remarkably, could be retrieved for each epitope:HLA combination and patient analyzed. Overall, our data demonstrate that highly functional HLA class I TCRs are recruited and maintained upon mild SARS-CoV-2 infection. Such validated epitopes and TCRs could become valuable tools for the development of diagnostic tests determining the quality of SARS-CoV-2-specific CD8+ T cell immunity, and thereby investigating correlates of protection, as well as to restore functional immunity through therapeutic transfer of TCR-engineered T cells.

New susceptibility loci for severe COVID-19 by detailed GWAS analysis in European populations

Given the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), a deeper analysis of the host genetic contribution to severe COVID-19 is important to improve our understanding of underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of a well-characterized cohort of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany/Austria, including stratified analyses based on age, sex and disease severity, as well as targeted analyses of chromosome Y haplotypes, the human leukocyte antigen (HLA) region and the SARS-CoV-2 peptidome. By inversion imputation, we traced a reported association at 17q21.31 to a highly pleiotropic [~]0.9-Mb inversion polymorphism and characterized the potential effects of the inversion in detail. Our data, together with the 5th release of summary statistics from the COVID-19 Host Genetics Initiative, also identified a new locus at 19q13.33, including NAPSA, a gene which is expressed primarily in alveolar cells responsible for gas exchange in the lung.

Heterologous prime-boost vaccination with ChAdOx1 nCoV-19 and BNT162b2 mRNA

Administration of a first dose of the COVID-19 vaccine ChAdOx1 nCoV-19 (Vaxzevria(R), AstraZeneca) is associated with a certain risk for vaccine-induced immune thrombotic thrombocytopenia. Therefore, several countries have recommended replacing the second dose of ChAdOx1 nCoV-19 with an mRNA-based vaccine as a precautionary measure, although data on safety and efficacy of such heterologous prime-boost regimen are sparse. Therefore, vaccinees, who had received a heterologous vaccination using ChAdOx1 nCoV-19 as prime and BNT162b2 (Comirnaty(R), BioNTech-Pfizer) mRNA as boost vaccination were offered SARS-CoV-2 antibody testing to quantify their vaccine-induced neutralizing antibody response5. The results were compared to cohorts of healthcare workers or volunteers, who received homologous BNT162b2 or homologous ChAdOx1 nCoV-19 vaccination regimens, respectively. A striking increase of vaccine-induced SARS-CoV-2 neutralizing antibody activity was observed in 229 vaccinees that received a BNT162b2 boost 9 to 12 weeks after ChAdOx1 nCoV-19 prime. In our cohort comprising over 480 individuals, the heterologous vaccination scheme induced significantly higher neutralizing antibody titers than homologous ChAdOx1 nCoV-19 and even than homologous BNT162b2 vaccination. This proves that a single dose of a COVID-19 mRNA vaccine after ChAdOx1 nCoV-19 prime vaccination is sufficient to achieve high neutralizing antibody levels predicting immune protection from SARS-CoV-2 infection, and may even increase vaccine efficacy offering an alternative in a setting of vaccine shortage.

Efficient inhibition of SARS-CoV-2 strains by a novel ACE2-IgG4-Fc fusion protein with a stabilized hinge region

The novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) enters its host cells after binding to the angiotensin-converting enzyme 2 (ACE2) via its spike glycoprotein. This interaction is critical for virus entry and virus-host membrane fusion. Soluble ACE2 ectodomains bind and neutralize the virus but the short in vivo half-lives of soluble ACE2 limits its therapeutic use. Fusion of the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain can prolong the in vivo half-life but bears the risk of unwanted Fc-receptor activation and antibody-dependent disease enhancement. Here, we describe optimized ACE2-Fc fusion constructs that avoid Fc-receptor binding by using IgG4-Fc as a fusion partner. The engineered ACE2-IgG4-Fc fusion proteins described herein exhibit promising pharmaceutical properties and a broad antiviral activity at single-digit nanomolar concentration. In addition, they allow to maintain the beneficial enzymatic activity of ACE2 and thus are very promising candidate antivirals broadly acting against coronaviruses.

Strategies for infection control and prevalence of anti-SARS-CoV-2 IgG in 4,554 employees of a university hospital in Munich, Germany

BackgroundHospital staff are at high risk of infection during the coronavirus disease (COVID-19) pandemic. We analysed the exposure characteristics, efficacy of protective measures, and transmission dynamics in this hospital-wide prospective seroprevalence study. Methods and FindingsOverall, 4554 individuals were tested for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies using a chemiluminescent immunoassay. Individual risk factors, use of personal protective equipment (PPE), occupational exposure, previous SARS-CoV-2 infection, and symptoms were assessed using a questionnaire and correlated to anti-SARS-CoV-2 IgG antibody titres and PCR testing results. Odds ratios with corresponding exact 95% confidence intervals were used to evaluate associations between individual factors and seropositivity. Spatio-temporal trajectories of SARS-CoV-2-infected patients and staff mobility within the hospital were visualised to identify local hotspots of virus transmission. The overall seroprevalence of anti-SARS-CoV-2-IgG antibody was 2.4% [95% CI 1.9-2.9]. Patient-facing staff, including those working in COVID-19 areas, had a similar probability of being seropositive as non-patient-facing staff. Prior interaction with SARS-CoV-2-infected co-workers or private contacts and unprotected exposure to COVID-19 patients increased the probability of seropositivity. Loss of smell and taste had the highest positive predictive value for seropositivity. The rate of asymptomatic SARS-CoV-2 infections was 25.9%, and higher anti-SARS-CoV-2 IgG antibody titres were observed in symptomatic individuals. Spatio-temporal hotspots of SARS-CoV-2-positive staff and patients only showed partial overlap. ConclusionsPatient-facing work in a healthcare facility during the SARS-CoV-2 pandemic may be safe if adequate PPE and hygiene measures are applied. The high numbers of asymptomatic SARS-CoV-2 infections that escaped detection by symptomatic testing underline the value of cross-sectional seroprevalence studies. Unprotected contact is a major risk factor for infection and argues for the rigorous implementation of hygiene measures.

Multi-level proteomics reveals host-perturbation strategies of SARS-CoV-2 and SARS-CoV

The global emergence of SARS-CoV-2 urgently requires an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. We therefore conducted a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactome of both viruses, as well as their influence on transcriptome, proteome, ubiquitinome and phosphoproteome in a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon SARS-CoV-2 and SARS-CoV infections at different layers and identified unique and common molecular mechanisms of these closely related coronaviruses. The TGF-{beta} pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org) highlights many hotspots that can be targeted by existing drugs and it can guide rational design of virus- and host-directed therapies, which we exemplify by identifying kinase and MMPs inhibitors with potent antiviral effects against SARS-CoV-2.

Intensive care risk estimation in COVID-19 pneumonia based on clinical and imaging parameters: experiences from the Munich cohort

The evolving dynamics of coronavirus disease 2019 (COVID-19) and the increasing infection numbers require diagnostic tools to identify patients at high risk for a severe disease course. Here we evaluate clinical and imaging parameters for estimating the need of intensive care unit (ICU) treatment. We collected clinical, laboratory and imaging data from 65 patients with confirmed COVID-19 infection based on PCR testing. Two radiologists evaluated the severity of findings in computed tomography (CT) images on a scale from 1 (no characteristic signs of COVID-19) to 5 (confluent ground glass opacities in over 50% of the lung parenchyma). The volume of affected lung was quantified using commercially available software. Machine learning modelling was performed to estimate the risk for ICU treatment. Patients with a severe course of COVID-19 had significantly increased IL-6, CRP and leukocyte counts and significantly decreased lymphocyte counts. The radiological severity grading was significantly increased in ICU patients. Multivariate random forest modelling showed a mean {+/-} standard deviation sensitivity, specificity and accuracy of 0.72 {+/-} 0.1, 0.86 {+/-} 0.16 and 0.80 {+/-} 0.1 and a ROC-AUC of 0.79 {+/-} 0.1. The need for ICU treatment is independently associated with affected lung volume, radiological severity score, CRP and IL-6.