Evidence for broad cross-reactivity of the SARS-CoV-2 NSP12-directed CD4+ T-cell response with pre-primed responses directed against common cold coronaviruses.

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC). Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs. Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4+ T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4+ T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro. However, the NSP12 peptide-specific CD4+ T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection. Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs.

TNFα aggravates detrimental effects of SARS-CoV-2 infection in the liver.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus does not only lead to pulmonary infection but can also infect other organs such as the gut, the kidney, or the liver. Recent studies confirmed that severe cases of COVID-19 are often associated with liver damage and liver failure, as well as the systemic upregulation of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα). However, the impact these immune mediators in the liver have on patient survival during SARS-CoV-2 infection is currently unknown. Here, by performing a post-mortem analysis of 45 patients that died from a SARS-CoV-2 infection, we find that an increased expression of TNFA in the liver is associated with elevated mortality. Using publicly available single-cell sequencing datasets, we determined that Kupffer cells and monocytes are the main sources of this TNFα production. Further analysis revealed that TNFα signaling led to the upregulation of pro-inflammatory genes that are associated with an unfavorable outcome. Moreover, high levels of TNFA in the liver were associated with lower levels of interferon alpha and interferon beta. Thus, TNFα signaling in the infected SARS-CoV-2 liver correlates with reduced interferon levels and overall survival time.

Interferon-induced IL-10 drives systemic T-cell dysfunction during chronic liver injury.

BACKGROUND & AIMS: Patients with chronic liver disease (CLD), including cirrhosis, are at increased risk of intractable viral infections and are hyporesponsive to vaccination. Hallmarks of CLD and cirrhosis include microbial translocation and elevated levels of type I interferon (IFN-I). We aimed to investigate the relevance of microbiota-induced IFN-I in the impaired adaptive immune responses observed in CLD. METHODS: We combined bile duct ligation (BDL) and carbon tetrachloride (CCl4) models of liver injury with vaccination or lymphocytic choriomeningitis virus infection in transgenic mice lacking IFN-I in myeloid cells (LysM-Cre IFNARflox/flox), IFNAR-induced IL-10 (MX1-Cre IL10flox/flox) or IL-10R in T cells (CD4-DN IL-10R). Key pathways were blocked in vivo with specific antibodies (anti-IFNAR and anti-IL10R). We assessed T-cell responses and antibody titers after HBV and SARS-CoV-2 vaccinations in patients with CLD and healthy individuals in a proof-of-concept clinical study. RESULTS: We demonstrate that BDL- and CCL4-induced prolonged liver injury leads to impaired T-cell responses to vaccination and viral infection in mice, subsequently leading to persistent infection. We observed a similarly defective T-cell response to vaccination in patients with cirrhosis. Innate sensing of translocated gut microbiota induced IFN-I signaling in hepatic myeloid cells that triggered excessive IL-10 production upon viral infection. IL-10R signaling in antigen-specific T cells rendered them dysfunctional. Antibiotic treatment and inhibition of IFNAR or IL-10Ra restored antiviral immunity without detectable immune pathology in mice. Notably, IL-10Ra blockade restored the functional phenotype of T cells from vaccinated patients with cirrhosis. CONCLUSION: Innate sensing of translocated microbiota induces IFN-/IL-10 expression, which drives the loss of systemic T-cell immunity during prolonged liver injury. IMPACT AND IMPLICATIONS: Chronic liver injury and cirrhosis are associated with enhanced susceptibility to viral infections and vaccine hyporesponsiveness. Using different preclinical animal models and patient samples, we identified that impaired T-cell immunity in BDL- and CCL4-induced prolonged liver injury is driven by sequential events involving microbial translocation, IFN signaling leading to myeloid cell-induced IL-10 expression, and IL-10 signaling in antigen-specific T cells. Given the absence of immune pathology after interference with IL-10R, our study highlights a potential novel target to reconstitute T-cell immunity in patients with CLD that can be explored in future clinical studies.

High-resolution analysis of individual spike peptide-specific CD4+ T-cell responses in vaccine recipients and COVID-19 patients.

Objectives: Potential differences in the breadth, distribution and magnitude of CD4+ T-cell responses directed against the SARS-CoV-2 spike glycoprotein between vaccinees, COVID-19 patients and subjects who experienced both ways of immunisation have not been comprehensively compared on a peptide level. Methods: Following virus-specific in vitro cultivation, we determined the T-cell responses directed against 253 individual overlapping 15-mer peptides covering the entire SARS-CoV-2 spike glycoprotein using IFN-γ ELISpot and intracellular cytokine staining. In vitro HLA binding was determined for selected peptides. Results: We mapped 955 single peptide-specific CD4+ T-cell responses in a cohort of COVID-19 patients (n = 8), uninfected vaccinees (n = 16) and individuals who experienced both infection and vaccination (n = 11). Patients and vaccinees (two-time and three-time vaccinees alike) had a comparable number of CD4+ T-cell responses (median 26 vs. 29, P = 0.7289). Most of these specificities were conserved in B.1.1.529 and the BA.4 and BA.5 sublineages. The highest magnitude of these in vitro IFN-γ CD4+ T-cell responses was observed in COVID-19 patients (median 0.35%), and three-time vaccinees showed a higher magnitude than two-time vaccinees (median 0.091% vs. 0.175%, P < 0.0001). Twelve peptide specificities were each detected in at least 40% of subjects. In vitro HLA binding showed promiscuous presentation by DRB1 molecules for several peptides. Conclusion: Both SARS-CoV-2 infection and vaccination prime broadly directed T-cell responses directed against the SARS-CoV-2 spike glycoprotein. This comprehensive high-resolution analysis of spike peptide specificities will be a useful resource for further investigation of spike-specific T-cell responses.

Three Separate Spike Antigen Exposures by COVID-19 Vaccination or SARS-CoV-2 Infection Elicit Strong Humoral Immune Responses in Healthcare Workers.

BACKGROUND: The immunogenicity of different COVID-19 vaccine regimens and combinations in naïve and convalescent individuals has not been formally tested in controlled studies, and real-life observational studies are scarce. METHODS: We assessed the SARS-CoV-2 infection and COVID-19 vaccination-induced immunity of 697 hospital workers at the University Medical Center Hamburg-Eppendorf between 17 and 31 January 2022. RESULTS: The overall prevalence of anti-NC-SARS-CoV-2 antibodies indicating prior infection was 9.8% (n = 68) and thus lower than the seroprevalence in the general population. All vaccinated individuals had detectable anti-S1-RBD-SARS-CoV-2 antibodies (median AU/mL [IQR]: 13,891 [8505-23,543]), indicating strong protection against severe COVID-19. Individuals who received three COVID-19 vaccine doses (median AU/mL [IQR]: 13,856 [8635-22,705]) and those who resolved a prior SARS-CoV-2 infection and had received two COVID-19 vaccine doses (median AU/mL [IQR] 13,409 [6934-25,000]) exhibited the strongest humoral immune responses. CONCLUSIONS: The current study indicates that three exposures to the viral spike protein by either SARS-CoV-2 infection or COVID-19 vaccination are necessary to elicit particularly strong humoral immune responses, which supports current vaccination recommendations.

High and Sustained Ex Vivo Frequency but Altered Phenotype of SARS-CoV-2-Specific CD4+ T-Cells in an Anti-CD20-Treated Patient with Prolonged COVID-19.

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145-164) epitope-specific CD4+ T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4+ T-cell response in both patients, with higher frequencies of virus-specific CD4+ T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4+ T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4+ T-cells with CD45RA- CXCR5+ PD-1+ circulating T follicular helper cell (cTFH) phenotype. Furthermore, we observed a delayed contraction of CD127- virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4+ T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell-T-cell interaction, a robust virus-specific CD4+ T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection.

Molecular consequences of SARS-CoV-2 liver tropism.

Extrapulmonary manifestations of COVID-19 have gained attention due to their links to clinical outcomes and their potential long-term sequelae1. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) displays tropism towards several organs, including the heart and kidney. Whether it also directly affects the liver has been debated2,3. Here we provide clinical, histopathological, molecular and bioinformatic evidence for the hepatic tropism of SARS-CoV-2. We find that liver injury, indicated by a high frequency of abnormal liver function tests, is a common clinical feature of COVID-19 in two independent cohorts of patients with COVID-19 requiring hospitalization. Using autopsy samples obtained from a third patient cohort, we provide multiple levels of evidence for SARS-CoV-2 liver tropism, including viral RNA detection in 69% of autopsy liver specimens, and successful isolation of infectious SARS-CoV-2 from liver tissue postmortem. Furthermore, we identify transcription-, proteomic- and transcription factor-based activity profiles in hepatic autopsy samples, revealing similarities to the signatures associated with multiple other viral infections of the human liver. Together, we provide a comprehensive multimodal analysis of SARS-CoV-2 liver tropism, which increases our understanding of the molecular consequences of severe COVID-19 and could be useful for the identification of organ-specific pharmacological targets.

Disturbed lipid and amino acid metabolisms in COVID-19 patients.

The Coronavirus disease 2019 (COVID-19) pandemic is overwhelming the healthcare systems. Identification of systemic reactions underlying COVID-19 will lead to new biomarkers and therapeutic targets for monitoring and early intervention in this viral infection. We performed targeted metabolomics covering up to 630 metabolites within several key metabolic pathways in plasma samples of 20 hospitalized COVID-19 patients and 37 matched controls. Plasma metabolic signatures specifically differentiated severe COVID-19 from control patients. The identified metabolic signatures indicated distinct alterations in both lipid and amino acid metabolisms in COVID-19 compared to control patient plasma. Systems biology-based analyses identified sphingolipid, tryptophan, tyrosine, glutamine, arginine, and arachidonic acid metabolism as mostly impacted pathways in COVID-19 patients. Notably, gamma-aminobutyric acid (GABA) was significantly reduced in COVID-19 patients and GABA plasma levels allowed for stratification of COVID-19 patients with high sensitivity and specificity. The data reveal large metabolic disturbances in COVID-19 patients and suggest use of GABA as potential biomarker and therapeutic target for the infection.

Low incidence of COVID-19 in a prospective cohort of patients with liver cirrhosis and hepatocellular carcinoma treated at a tertiary medical center during the 2020 pandemic.

BACKGROUND AND AIMS: Patients with liver cirrhosis (LC) are considered to be at increased risk for mortality when acquiring SARS-CoV-2 infection and subsequently developing Corona Virus Disease 2019 (COVID-19). During the COVID-19 pandemic, hospitals are regarded as sites with increased risk of infection. Therefore, patient contacts are often limited to urgent indications, which could negatively affect disease monitoring. However, data regarding actual infection rates in cirrhotic patients is limited. The aim of this prospective study was to assess the incidence of COVID-19 in patients with LC with/without hepatocellular carcinoma (HCC) with physical presentation at our University Medical Center. METHODS: Patients were enrolled between 1st April and 30th June 2020 at the University Medical Center Hamburg-Eppendorf, Germany. Symptoms of upper airway infection at baseline and presence of SARS-CoV-2 antibodies (IgG/IgM/IgA) were assessed at baseline and follow-up (FU) using an Electro-chemiluminescence immunoassay (Roche Elecsys). FU visits, including liver function test, clinical assessment and symptom questionnaire, were conducted after 6-8 weeks (FU-1) and 6 months (FU-2). Prior to inclusion of the first patient, obligatory face masks and personal distance were implemented as protective measures. RESULTS: A total of 150 patients were enrolled, 23% (n = 35) also had diagnosis of HCC (median age: 64 years, range: 19-86), 69% were male. Liver function according to Child-Pugh score (CPS) was: CPS A: 46% (n = 62); CPS B: 37% (n = 50); CPS C: 17% (n = 23). Clinical symptoms indicating upper airway infection were present in 53% (n = 77): shortness of breath (n = 40) and coughing (n = 28) were the most frequent. For the 150 patients enrolled, 284 outpatient visits were registered and 33 patients were admitted to the University Medical Center during the follow-up period. After a median of 52 days, n = 110 patients completed FU-1 and n = 72 completed FU-2 after a median of 6.1 months. Only in one patient, an 80-year-old man with stable liver function (CPS A) and advanced HCC, SARS-CoV-2 antibodies were detected at baseline and FU-1, while antibody testing was negative in the remaining patients at baseline, FU-1 and FU-2. CONCLUSION: The incidence of COVID-19 at our tertiary medical center during the pandemic was low in LC and HCC patients, when simple protective measures were implemented. Therefore, a routine care for patients with chronic liver diseases does not increase the risk of SARS-CoV-2 infection and should be maintained with protective measures.

Sustained Response After Remdesivir and Convalescent Plasma Therapy in a B-Cell-Depleted Patient With Protracted Coronavirus Disease 2019 (COVID-19).

We provide detailed clinical, virological, and immunological data of a B-cell-depleted patient treated with obinutuzumab for follicular lymphoma with protracted coronavirus disease 2019 (COVID-19) and viremia. A sustained response was achieved after 2 courses of remdesivir and subsequent convalescent plasma therapy. Immunocompromised patients might require combined and prolonged antiviral treatment regimens.

Validation of a Prospective Urinalysis-Based Prediction Model for ICU Resources and Outcome of COVID-19 Disease: A Multicenter Cohort Study.

In COVID-19, guidelines recommend a urinalysis on hospital admission as SARS-CoV-2 renal tropism, post-mortem, was associated with disease severity and mortality. Following the hypothesis from our pilot study, we now validate an algorithm harnessing urinalysis to predict the outcome and the need for ICU resources on admission to hospital. Patients were screened for urinalysis, serum albumin (SA) and antithrombin III activity (AT-III) obtained prospectively on admission. The risk for an unfavorable course was categorized as (1) "low", (2) "intermediate" or (3) "high", depending on (1) normal urinalysis, (2) abnormal urinalysis with SA ≥ 2 g/dL and AT-III ≥ 70%, or (3) abnormal urinalysis with SA or AT-III abnormality. Time to ICU admission or death served as the primary endpoint. Among 223 screened patients, 145 were eligible for enrollment, 43 falling into the low, 84 intermediate, and 18 into high-risk categories. An abnormal urinalysis significantly elevated the risk for ICU admission or death (63.7% vs. 27.9%; HR 2.6; 95%-CI 1.4 to 4.9; p = 0.0020) and was 100% in the high-risk group. Having an abnormal urinalysis was associated with mortality, a need for mechanical ventilation, extra-corporeal membrane oxygenation or renal replacement therapy. In conclusion, our data confirm that COVID-19-associated urine abnormalities on admission predict disease aggravation and the need for ICU (ClinicalTrials.gov number NCT04347824).

Multi-dimensional and longitudinal systems profiling reveals predictive pattern of severe COVID-19.

COVID-19 is a respiratory tract infection that can affect multiple organ systems. Predicting the severity and clinical outcome of individual patients is a major unmet clinical need that remains challenging due to intra- and inter-patient variability. Here, we longitudinally profiled and integrated more than 150 clinical, laboratory, and immunological parameters of 173 patients with mild to fatal COVID-19. Using systems biology, we detected progressive dysregulation of multiple parameters indicative of organ damage that correlated with disease severity, particularly affecting kidneys, hepatobiliary system, and immune landscape. By performing unsupervised clustering and trajectory analysis, we identified T and B cell depletion as early indicators of a complicated disease course. In addition, markers of hepatobiliary damage emerged as robust predictor of lethal outcome in critically ill patients. This allowed us to propose a novel clinical COVID-19 SeveriTy (COST) score that distinguishes complicated disease trajectories and predicts lethal outcome in critically ill patients.

Patient Characteristics and Clinical Course of COVID-19 Patients Treated at a German Tertiary Center during the First and Second Waves in the Year 2020.

In this study, we directly compared coronavirus disease 2019 (COVID-19) patients hospitalized during the first (27 February-28 July 2020) and second (29 July-31 December 2020) wave of the pandemic at a large tertiary center in northern Germany. Patients who presented during the first (n = 174) and second (n = 331) wave did not differ in age (median [IQR], 59 years [46, 71] vs. 58 years [42, 73]; p = 0.82) or age-adjusted Charlson Comorbidity Index (median [IQR], 2 [1, 4] vs. 2 [0, 4]; p = 0.50). During the second wave, a higher proportion of patients were treated as outpatients (11% [n = 20] vs. 20% [n = 67]), fewer patients were admitted to the intensive care unit (43% [n = 75] vs. 29% [n = 96]), and duration of hospitalization was significantly shorter (median days [IQR], 14 [8, 34] vs. 11 [5, 19]; p < 0.001). However, in-hospital mortality was high throughout the pandemic and did not differ between the two periods (16% [n = 27] vs. 16% [n = 54]; p = 0.89). While novel treatment strategies and increased knowledge about the clinical management of COVID-19 may have resulted in a less severe disease course in some patients, in-hospital mortality remained unaltered at a high level. These findings highlight the unabated need for efforts to hamper severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) transmission, to increase vaccination coverage, and to develop novel treatment strategies to prevent mortality and decrease morbidity.

Malaria in the Time of COVID-19: Do Not Miss the Real Cause of Illness.

We report a case of Plasmodium falciparum malaria in a patient asymptomatically co-infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the current ongoing coronavirus pandemic, co-infections with unrelated life-threatening febrile conditions may pose a particular challenge to clinicians. The current situation increases the risk for cognitive biases in medical management.

Clonal expansion and activation of tissue-resident memory-like Th17 cells expressing GM-CSF in the lungs of severe COVID-19 patients.

Hyperinflammation contributes to lung injury and subsequent acute respiratory distress syndrome (ARDS) with high mortality in patients with severe coronavirus disease 2019 (COVID-19). To understand the underlying mechanisms involved in lung pathology, we investigated the role of the lung-specific immune response. We profiled immune cells in bronchoalveolar lavage fluid and blood collected from COVID-19 patients with severe disease and bacterial pneumonia patients not associated with viral infection. By tracking T cell clones across tissues, we identified clonally expanded tissue-resident memory-like Th17 cells (Trm17 cells) in the lungs even after viral clearance. These Trm17 cells were characterized by a a potentially pathogenic cytokine expression profile of IL17A and CSF2 (GM-CSF). Interactome analysis suggests that Trm17 cells can interact with lung macrophages and cytotoxic CD8+ T cells, which have been associated with disease severity and lung damage. High IL-17A and GM-CSF protein levels in the serum of COVID-19 patients were associated with a more severe clinical course. Collectively, our study suggests that pulmonary Trm17 cells are one potential orchestrator of the hyperinflammation in severe COVID-19.

Seroprevalence of SARS-CoV-2 antibodies among hospital workers in a German tertiary care center: A sequential follow-up study.

We sequentially assessed the presence of SARS-CoV-2 IgG antibodies in 1253 hospital workers including 1026 HCWs at the University Medical Center Hamburg-Eppendorf at three time points during the early phase of the epidemic. By the end of the study in July 2020, the overall seroprevalence was 1.8% (n = 22), indicating the overall effectiveness of infection control interventions in mitigating coronavirus disease 2019 (COVID-19) in hospital workers.

Defining the CD39/CD73 Axis in SARS-CoV-2 Infection: The CD73- Phenotype Identifies Polyfunctional Cytotoxic Lymphocytes.

The ectonucleotidases CD39 and CD73 regulate immune responses by balancing extracellular ATP and adenosine in inflammation and are likely to be involved in the pathophysiology of COVID-19. Here, we analyzed CD39 and CD73 on different lymphocyte populations in a small cohort of COVID-19 patients and in healthy individuals. We describe a significantly lower level of expression of CD73 on cytotoxic lymphocyte populations, including CD8+ T, natural killer T (NKT), and natural killer (NK) cells, during COVID-19. Interestingly, the decrease of CD73 on CD8+ T cells and NKT cells correlated with serum ferritin levels. Furthermore, we observed distinct functional differences between the CD73+ and CD73- subsets of CD8+ T cells and NKT cells with regard to cytokine/toxin secretion. In COVID-19 patients, the majority of the CD73-CD8+ T cells were capable of secreting granzyme B, perforin, tumor necrosis factor (TNF-α) or interferon-gamma (IFN-γ). To conclude, in this first study of CD39 and CD73 expression of lymphocytes in COVID-19, we show that CD8+ T cells and NKT cells lacking CD73 possess a significantly higher cytotoxic effector functionality compared to their CD73+ counterparts. Future studies should investigate differences of cellular CD39 and CD73 expression in patients at different disease stages and their potential as prognostic markers or targets for immunomodulatory therapies.