Free-Breathing Low-Field MRI of the Lungs Detects Functional Alterations Associated With Persistent Symptoms After COVID-19 Infection.

OBJECTIVES: With the COVID-19 pandemic, repetitive lung examinations have become necessary to follow-up symptoms and associated alterations. Low-field MRI, benefiting from reduced susceptibility effects, is a promising alternative for lung imaging to limit radiations absorbed by patients during CT examinations, which also have limited capability to assess functional alterations. The aim of this investigative study was to explore the functional abnormalities that free-breathing 0.55 T MRI in combination with the phase-resolved functional lung (PREFUL) analysis could identify in patients with persistent symptoms after COVID-19 infection. MATERIALS AND METHODS: Seventy-four COVID-19 patients and 8 healthy volunteers were prospectively scanned in free-breathing with a balanced steady-state free-precession sequence optimized at 0.55 T, 5 months postinfection on average. Normalized perfusion (Q), fractional ventilation (FV), and flow-volume loop correlation (FVLc) maps were extracted with the PREFUL technique. Q, FV, and FVLc defects as well as defect overlaps between these metrics were quantified. Morphological turbo-spin-echo images were also acquired, and the extent of abnormalities was scored by a board-certified radiologist. To investigate the functional correlates of persistent symptoms, a recursive feature elimination algorithm was applied to find the most informative variables to detect the presence of persistent symptoms with a logistic regression model and a cross-validation strategy. All MRI metrics, sex, age, body mass index, and the presence of preexisting lung conditions were included. RESULTS: The most informative variables to detect persistent symptoms were the percentage of concurrent Q and FVLc defects and of areas free of those defects. A detection accuracy of 71.4% was obtained with these 2 variables when fitting the model on the entire dataset. Although none of the single variables differed between patients with and without persistent symptoms ( P > 0.05), the combined score of these 2 variables did ( P < 0.02). This score also showed a consistent increase from healthy volunteers (7.7) to patients without persistent symptoms (8.2) and with persistent symptoms (8.6). The morphological abnormality score showed poor correlation with the functional parameters. CONCLUSIONS: Functional pulmonary examinations using free-breathing 0.55 T MRI with PREFUL analysis revealed potential quantitative markers of impaired lung function in patients with persistent symptoms after COVID-19 infection, potentially complementing morphologic imaging. Future work is needed to explore the translational relevance and clinical implication of these findings.

Successful treatment of COVID-19 infection with convalescent plasma in B-cell-depleted patients may promote cellular immunity.

Treatment with convalescent plasma has been shown to be safe in coronavirus disease in 2019 (COVID-19) infection, although efficacy reported in immunocompetent patients varies. Nevertheless, neutralizing antibodies are a key requisite in the fight against viral infections. Patients depleted of antibody-producing B cells, such as those treated with rituximab (anti-CD20) for hematological malignancies, lack a fundamental part of their adaptive immunity. Treatment with convalescent plasma appears to be of general benefit in this particularly vulnerable cohort. We analyzed clinical course and inflammation markers of three B-cell-depleted patients suffering from COVID-19 who were treated with convalescent plasma. In addition, we measured serum antibody levels as well as peripheral blood CD38/HLA-DR-positive T-cells ex vivo and CD137-positive T-cells after in vitro stimulation with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides in these patients. We observed that therapy with convalescent plasma was effective in all three patients and analysis of CD137-positive T-cells after stimulation with SARS-CoV-2 peptides showed an increase in peptide-specific T-cells after application of convalescent plasma. In conclusion, we here demonstrate efficacy of convalescent plasma therapy in three B-cell-depleted patients and present data that suggest that while application of convalescent plasma elevates systemic antibody levels only transiently, it may also boost specific T-cell responses.

Diagnostic performance of four SARS-CoV-2 antibody assays in patients with COVID-19 or with bacterial and non-SARS-CoV-2 viral respiratory infections.

SARS-CoV-2 antibody assays are used for epidemiological studies and for the assessment of vaccine responses in highly vulnerable patients. So far, data on cross-reactivity of SARS-CoV-2 antibody assays is limited. Here, we compared four enzyme-linked immunosorbent assays (ELISAs; Vircell SARS-CoV-2 IgM/IgA and IgG, Euroimmun SARS-CoV-2 IgA and IgG) for detection of anti-SARS-CoV-2 antibodies in 207 patients with COVID-19, 178 patients with serological evidence of different bacterial infections, 107 patients with confirmed viral respiratory disease, and 80 controls from the pre-COVID-19 era. In COVID-19 patients, the assays showed highest sensitivity in week 3 (Vircell-IgM/A and Euroimmun-IgA: 78.9% each) and after week 7 (Vircell-IgG: 97.9%; Euroimmun-IgG: 92.1%). The antibody indices were higher in patients with fatal disease. In general, IgM/IgA assays had only limited or no benefit over IgG assays. In patients with non-SARS-CoV-2 respiratory infections, IgG assays were more specific than IgM/IgA assays, and bacterial infections were associated with more false-positive results than viral infections. The specificities in bacterial and viral infections were 68.0 and 81.3% (Vircell-IgM/IgA), 84.8 and 96.3% (Euroimmun-IgA), 97.8 and 86.0% (Vircell-IgG), and 97.8 and 99.1% (Euroimmun-IgG), respectively. Sera from patients positive for antibodies against Mycoplasma pneumoniae, Chlamydia psittaci, and Legionella pneumophila yielded particularly high rates of unspecific false-positive results in the IgM/IgA assays, which was revealed by applying a highly specific flow-cytometric assay using HEK 293 T cells expressing the SARS-CoV-2 spike protein. Positive results obtained with anti-SARS-CoV-2 IgM/IgA ELISAs require careful interpretation, especially if there is evidence for prior bacterial respiratory infections.

Human CD4+ T cells specific for dominant epitopes of SARS-CoV-2 Spike and Nucleocapsid proteins with therapeutic potential.

Since December 2019, Coronavirus disease-19 (COVID-19) has spread rapidly throughout the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both short-lived neutralizing antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID-19 patients with a potentially protective response to two major antigens of the SARS-CoV-2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus-reactive CD4+ T cells, we were able to confirm a set of nine immunodominant epitopes and characterize T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR-αß sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR-transgenic CD4+ T cell therapy of COVID-19.

SARS-CoV-2 vaccination responses in untreated, conventionally treated and anticytokine-treated patients with immune-mediated inflammatory diseases.

OBJECTIVES: To better understand the factors that influence the humoral immune response to vaccination against SARS-CoV-2 in patients with immune-mediated inflammatory diseases (IMIDs). METHODS: Patients and controls from a large COVID-19 study, with (1) no previous history of COVID-19, (2) negative baseline anti-SARS-CoV-2 IgG test and (3) SARS-CoV-2 vaccination at least 10 days before serum collection were measured for anti-SARS-CoV-2 IgG. Demographic, disease-specific and vaccination-specific data were recorded. RESULTS: Vaccination responses from 84 patients with IMID and 182 controls were analysed. While all controls developed anti-SARS-CoV-2 IgG, five patients with IMID failed to develop a response (p=0.003). Moreover, 99.5% of controls but only 90.5% of patients with IMID developed neutralising antibody activity (p=0.0008). Overall responses were delayed and reduced in patients (mean (SD): 6.47 (3.14)) compared with controls (9.36 (1.85); p<0.001). Estimated marginal means (95% CI) adjusted for age, sex and time from first vaccination to sampling were 8.48 (8.12-8.85) for controls and 6.90 (6.45-7.35) for IMIDs. Significantly reduced vaccination responses pertained to untreated, conventionally and anticytokine treated patients with IMID. CONCLUSIONS: Immune responses against the SARS-CoV-2 are delayed and reduced in patients with IMID. This effect is based on the disease itself rather than concomitant treatment.

Circulating Adaptive Immune Cells Expressing the Gut Homing Marker α4ß7 Integrin Are Decreased in COVID-19.

Background: Infection with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a wide range of symptoms including gastrointestinal manifestations, and intestinal epithelial cells are a target of the virus. However, it is unknown how the intestinal immune system contributes to systemic immune responses in coronavirus disease 2019 (COVID-19). Methods: We characterized peripheral blood lymphocytes from patients with active COVID-19 and convalescent patients as well as healthy controls by flow cytometry. Results: The frequency and absolute number of circulating memory T and B cells expressing the gut homing integrin α4ß7 integrin was reduced during COVID-19, whether gastrointestinal symptoms were present or not. While total IgA-expressing B cells were increased, gut-imprinted B cells with IgA expression were stable. Conclusion: COVID-19 is associated with a decrease in circulating adaptive immune cells expressing the key gut homing marker α4ß7 suggesting that these cells are preferentially recruited to extra-intestinal tissues independently of α4ß7 or that the systemic immune response against SARS-CoV-2 is at least numerically dominated by extraintestinal, particularly pulmonary, immune cell priming.

Lack of antibodies against seasonal coronavirus OC43 nucleocapsid protein identifies patients at risk of critical COVID-19.

BACKGROUND: The vast majority of COVID-19 patients experience a mild disease. However, a minority suffers from critical disease with substantial morbidity and mortality. OBJECTIVES: To identify individuals at risk of critical COVID-19, the relevance of a seroreactivity against seasonal human coronaviruses was analyzed. METHODS: We conducted a multi-center non-interventional study comprising 296 patients with confirmed SARS-CoV-2 infections from four tertiary care referral centers in Germany and France. The ICU group comprised more males, whereas the outpatient group contained a higher percentage of females. For each patient, the serum or plasma sample obtained closest after symptom onset was examined by immunoblot regarding IgG antibodies against the nucleocapsid protein (NP) of HCoV 229E, NL63, OC43 and HKU1. RESULTS: Median age was 60 years (range 18-96). Patients with critical disease (n=106) had significantly lower levels of anti-HCoV OC43 nucleocapsid protein (NP)-specific antibodies compared to other COVID-19 inpatients (p=0.007). In multivariate analysis (adjusted for age, sex and BMI), OC43 negative inpatients had an increased risk of critical disease (adjusted odds ratio (AOR) 2.68 [95% CI 1.09 - 7.05]), higher than the risk by increased age or BMI, and lower than the risk by male sex. A risk stratification based on sex and OC43 serostatus was derived from this analysis. CONCLUSIONS: Our results suggest that prior infections with seasonal human coronaviruses can protect against a severe course of COVID-19. Therefore, anti-OC43 antibodies should be measured for COVID-19 inpatients and considered as part of the risk assessment for each patient. Hence, we expect individuals tested negative for anti-OC43 antibodies to particularly benefit from vaccination against SARS-CoV-2, especially with other risk factors prevailing.

Upregulation of CCR4 in activated CD8+ T cells indicates enhanced lung homing in patients with severe acute SARS-CoV-2 infection.

COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected are to date not well understood. Comparing the immunological phenotype in healthy donors and patients with mild versus severe COVID-19 shows that in COVID-19 patients, NK-/B-cell activation and proliferation are enhanced independent of severity. As an important precondition for effective antibody responses, T-follicular helper cells and antibody secreting cells are increased both in patients with mild and severe SARS-CoV-2 infection. Beyond this, T cells in COVID-19 patients exhibit a stronger activation profile with differentiation toward effector cell phenotypes. Importantly, when looking at the rates of pulmonary complications in COVID-19 patients, the chemokine receptor CCR4 is higher expressed by both CD4 and CD8 T cells of patients with severe COVID-19. This raises the hypothesis that CCR4 upregulation on T cells in the pathogenesis of COVID-19 promotes stronger T-cell attraction to the lungs leading to increased immune activation with presumably higher pulmonary toxicity. Our study contributes significantly to the understanding of the immunological changes during COVID-19, as new therapeutic agents, preferentially targeting the immune system, are highly warranted.

Interleukin-3 is a predictive marker for severity and outcome during SARS-CoV-2 infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide health threat. In a prospective multicentric study, we identify IL-3 as an independent prognostic marker for the outcome during SARS-CoV-2 infections. Specifically, low plasma IL-3 levels is associated with increased severity, viral load, and mortality during SARS-CoV-2 infections. Patients with severe COVID-19 exhibit also reduced circulating plasmacytoid dendritic cells (pDCs) and low plasma IFNα and IFNλ levels when compared to non-severe COVID-19 patients. In a mouse model of pulmonary HSV-1 infection, treatment with recombinant IL-3 reduces viral load and mortality. Mechanistically, IL-3 increases innate antiviral immunity by promoting the recruitment of circulating pDCs into the airways by stimulating CXCL12 secretion from pulmonary CD123+ epithelial cells, both, in mice and in COVID-19 negative patients exhibiting pulmonary diseases. This study identifies IL-3 as a predictive disease marker for SARS-CoV-2 infections and as a potential therapeutic target for pulmunory viral infections.

IgA2 Antibodies against SARS-CoV-2 Correlate with NET Formation and Fatal Outcome in Severely Diseased COVID-19 Patients.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to an adaptive immune response in the host and the formation of anti-SARS-CoV-2 specific antibodies. While IgG responses against SARS-CoV-2 have been characterized quite well, less is known about IgA. IgA2 activates immune cells and induces inflammation and neutrophil extracellular trap (NET) formation which may contribute to organ injury and fatal outcome in SARS-CoV-2-infected patients. SARS-CoV-2 spike protein specific antibody levels were measured in plasma samples of 15 noninfected controls and 82 SARS-CoV-2-infected patients with no or mild symptoms, moderate symptoms (hospitalization) or severe disease (intensive care unit, ICU). Antibody levels were compared to levels of C-reactive protein (CRP) and circulating extracellular DNA (ecDNA) as markers for general inflammation and NET formation, respectively. While levels of SARS-CoV-2-specific IgG were similar in all patient groups, IgA2 antibodies were restricted to severe disease and showed the strongest discrimination between nonfatal and fatal outcome in patients with severe SARS-CoV-2 infection. While anti-SARS-CoV-2 IgG and IgA2 levels correlated with CRP levels in severely diseased patients, only anti-SARS-CoV-2 IgA2 correlated with ecDNA. These data suggest that the formation of anti-SARS-CoV-2 IgA2 during SARS-CoV-2 infection is a marker for more severe disease related to NET formation and poor outcome.

Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2.

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.

Vascular occlusion by neutrophil extracellular traps in COVID-19.

BACKGROUND: Coronavirus induced disease 2019 (COVID-19) can be complicated by severe organ damage leading to dysfunction of the lungs and other organs. The processes that trigger organ damage in COVID-19 are incompletely understood. METHODS: Samples were donated from hospitalized patients. Sera, plasma, and autopsy-derived tissue sections were examined employing flow cytometry, enzyme-linked immunosorbent assays, and immunohistochemistry. PATIENT FINDINGS: Here, we show that severe COVID-19 is characterized by a highly pronounced formation of neutrophil extracellular traps (NETs) inside the micro-vessels. Intravascular aggregation of NETs leads to rapid occlusion of the affected vessels, disturbed microcirculation, and organ damage. In severe COVID-19, neutrophil granulocytes are strongly activated and adopt a so-called low-density phenotype, prone to spontaneously form NETs. In accordance, markers indicating NET turnover are consistently increased in COVID-19 and linked to disease severity. Histopathology of the lungs and other organs from COVID-19 patients showed congestions of numerous micro-vessels by aggregated NETs associated with endothelial damage. INTERPRETATION: These data suggest that organ dysfunction in severe COVID-19 is associated with excessive NET formation and vascular damage. FUNDING: Deutsche Forschungsgemeinschaft (DFG), EU, Volkswagen-Stiftung.

Patients with immune-mediated inflammatory diseases receiving cytokine inhibitors have low prevalence of SARS-CoV-2 seroconversion.

Immune-mediated inflammatory diseases (IMIDs) of the joints, gut and skin are treated with inhibitors of inflammatory cytokines. These cytokines are involved in the pathogenesis of coronavirus disease 2019 (COVID-19). Investigating anti-SARS-CoV-2 antibody responses in IMIDs we observe a reduced incidence of SARS-CoV-2 seroconversion in IMID patients treated with cytokine inhibitors compared to patients receiving no such inhibitors and two healthy control populations, despite similar social exposure. Hence, cytokine inhibitors seem to at least partially protect from SARS-CoV-2 infection.