Inflammasome but Not Ifn-I/Iii Response Is Altered in Children with Long Covid

Background: Severe COVID-19 is less common in children than in adults. Increasing evidence show that distinct immune-pathological changes can persist weeks or months after SARS-CoV2 infection, leading to Long COVID (LC). We investigated the systemic type I/III interferon (IFN-I/III) and inflammation response in peripheral blood mononuclear cells (PBMCs) of children with and without LC symptoms. Method(s): Blood samples were collected from children attending Umberto I hospital of Rome, within 3-6 months after a SARS-CoV-2 positive test and from control children. RNA was extracted from PBMCs for determining the levels of IFN-I (IFN-Alpha2, -Beta, -Epsilon and -Omega), IFN-III (IFN-Lambda1-3), NLRP3 and IL-1beta genes, and miR-141 expression by quantitative RealTime-PCR assays, normalized to housekeeping GUS gene and RNU6B, respectively. Result(s): 28 participants (M 12.5y SD 3.0) with LC symptoms, 28 participants (M 11.8y SD 3.0) without LC symptoms and 18 children who've never had SARS-CoV- 2 infection (M 10.5y SD 3.1) were enrolled. Comparing the three study groups, we found reduced levels of IFN-Lambda1, IFN-Lambda2 and IFN-Lambda3 (p=0.006, p< 0.001, p=0.012, respectively;Kruskal-Wallis (KW) test) mRNA in patients who have had SARS-CoV-2 infection as opposed to control group, whereas transcript levels of IFN-Epsilon (p= 0.019;KW test) were increased in the former with respect to the latter group;as well, remaining IFN-I genes analyzed showed a tendency to be up-regulated. As far as NLRP3 and IL-1beta levels was concerned, these genes were increased in LC patients (p< 0.001 for both genes;KW test). Additionally, miR-141, which has been reported to regulate inflammasome response, was overexpressed in LC patients (p< 0.001;Mann-Whitney test). Conclusion(s): These results showed a decreased levels of IFN-III mRNAs and an overexpression of IFN-Epsilon in children after 3-6 months of SARS-CoV-2 infection regardless of development of LC symptoms, suggesting that SARSCoV- 2 could have caused dysregulation of IFN response through unknown mechanisms (e.g. epigenetic modifications). Also, we found an overexpression of miR-141, NLRP3 and IL-1beta mRNAs in LC patients, indicating that a prolonged activation of inflammasome pathways could be associated with the development of LC symptoms.

DIFFERENTIAL IFN GENES EXPRESSION in the UPPER AIRWAYS of CHILDREN and ADULTS

Background: Children generally develop a mild disease after SARS-CoV-2 infection;it has been shown (Loske J al., 2021) that higher basal expression of relevant pattern recognition receptors may result in a stronger early innate antiviral than in adults. However, how the early interferon (IFN) response differs from that in adults is not fully characterized. Hence, we aimed to investigate the expression of several IFN-related genes in nasopharyngeal (NP) cells from children and adults with asymptomatic or mild COVID-19, not requiring hospitalization. Methods: Children and adults attending emergency departments (ED) of Sapienza University Hospital, to perform SARS-CoV-2 molecular tests, were enrolled from November 2020 to February 2021, after informed consent was obtained. RNA from residual NP swabs was purified and 200 ng were reverse transcribed. Gene expression of genes coding for type I and III IFNs and for the well-known markers of IFNs' activation, ISG15 and ISG56, was measured by exonuclease-based Real time PCR assays with relative quantification to the invariant gene GUS (the 2-ΔCt method). Results: Residual NP cells from a total of 132 children and adults were included in the study;56 had SARS-CoV-2 positive results and 76 resulted negative. The expression of all tested genes showed a moderate significant inverse correlation with age, with the exception of ISG15. Participants were further stratified in age groups (< 16;16-35;36-65 years) resulting in: 25 SARS-CoV-2 negative and 26-positive children;14 SARS-CoV-2 negative and 16-positive young adults and 37 SARS-CoV-2 negative and 14-positive adults. In SARS-CoV-2 negative samples, higher levels of all study genes were found in children, while significantly decreasing in young and elderly adults. Among SARS-CoV-2 positive samples, those from children showed significantly higher levels of type I IFNs and of IFN lambda2 whereas ISG15 was far more elevated in adults. Moreover, levels of all type I IFNs, and of IFN lambda2, were significantly higher in individuals with no symptoms (65% of children and 44% of the young adults), whereas ISG15 was elevated in those with a mild COVID-19. Conclusion: The higher baseline expression of IFN-related genes in children may prompt a quicker activation of the IFN response after SARS-CoV-2 infection and contribute to effective control of viral replication;the higher ISG activation in adults may be caused by the inflammatory response and associated to COVID-19 symptoms.

DETECTION of CIRCULATING and AIRWAY AUTOANTIBODIES to IFN in SEVERE COVID-19 PATIENTS

Background: Evidence suggest that critically ill COVID-19 patients have an impairment of IFN-I response. This defect in antiviral mechanisms is explained in some patients by the presence of anti IFN-alpha neutralizing autoantibodies (NAbs). However, whether NAbs fluctuate longitudinally during COVID-19, and what are their specificity toward IFN-I subtypes and consequences on the IFN response remain elusive. Methods: Binding antibodies (BAbs) to IFN-alpha and IFN-beta were screened in serum samples (n=360) of COVID-19 patients using ELISA assays. All serum samples containing BAbs were processed to investigate NAbs using antiviral bioassay. Respiratory samples (n=17) were also included for the NAbs analysis. Transcript levels of IFN-alpha, IFN-beta, IFN-omega and IFN stimulated genes (ISGs) were analyzed through RT/Real Time PCR. Results: Results showed that 16.94% (61/360) of COVID-19 patients had circulating BAbs against IFN-alpha and IFN-beta. Further, 21% (13/61) of critically ill subjects had NAbs with a variable titer against all the IFN-alpha subtypes (70-71680 TRU/ml) while only 1 patient had anti IFN-beta NAbs. About 70% of these serum samples showed cross reactivity to IFN-omega at different extent (27-106667 TRU/ml). Longitudinal evaluation at different time points after hospitalization indicate the persistence of high NAbs titer throughout the time. NAbs to IFN-alpha (10-20 TRU/ml) were also detected in 17.64% of respiratory samples. Patients with NAbs had severe disease and exhibited alterations in the levels of many hematological indicators [(white blood cells, neutrophils, platelets, neutrophils to lymphocytes ratio, platelets to lymphocytes ratio, D-dimer, C-reactive protein and lactate dehydrogenase;p<0.05)]. Transcriptomic analysis indicated that levels of IFN genes were lower in NAbs patients than in healthy donors (p≤0.05). However, only the ISGs levels were reduced compared to those found in the NAbs negative patients. Of note, expression of ISGs, was abolished during hospitalization in all patients with persistent high titer of NAbs. Conclusion: Our finding demonstrate that NAbs with a broad specificity to IFN-I can be found in blood and respiratory samples from severe COVID-19 patients. NAbs detection was associated with a defective IFN response and with an increased levels of markers of disease severity.

ORAL BACTERIOTHERAPY IN PATIENTS WITH COVID-19: A RETROSPECTIVE COHORT STUDY

Oral bacteriotherapy with SAB51 has been previously evidenced as support of standard care in treating patients hospitalized for SARS-CoV2 pneumonia and associated gastrointestinal symptoms leading to a significantly decreased risk of developing respiratory failure. This study aimed to compare the rate of mortality, the need for ICU hospitalization, and the length of hospitalization in patients with severe COVID-19 pneumonia who received the best available therapy in Italy (BAT) versus patients treated with BAT and supplemented with SLAB51. 200 adults (≥18 years) hospitalized from March 6 until April 26, 2020 with severe COVID-19 pneumonia and needing oxygen support were investigated. All patients received therapeutic regimens including hydroxychloroquine (200 mg twice a day for 7 days), azithromycin (500 mg once a day for 7 days), lopinavir–ritonavir (400/100 mg twice a day) or darunavir–cobicistat (800/150 mg once a day) for 14 days, and low molecular weight heparin. Out of the 200 patients, 112 received BAT without oral bacteriotherapy and 88 BAT with SLAB51. Crude mortality was 22%. Significantly higher mortality has been observed in the group of patients managed only with BAT (30%) to that treated with BAT plus SLAB51 (11%). In both groups, the highest mortality was registered for the combination of Tocilizumab and hydroxychloroquine and Tocilizumab. By multivariate analysis, the age >65 years, PCR >41.8 mg/L, Platelets <150.000 mmc, and CV events were associated with increased mortality risk. Overall results evidenced that oral bacteriotherapy with SLAB51 was an independent variable associated with a reduced risk for death.(Image presented) Death probability. Kaplan-Meier curves displaying Best Available therapy (BAT) versus BAT and oral bacteriotherapy.

Oral Bacteriotherapy In The Managemente Of Fatigue In Covid-19 Patients

Prolonged Fatigue, have been reported in many Covid-19 patients. There is a mounting suspicion that SARS-CoV2 acts as a catalyst for a condition known as chronic fatigue syndrome (CFS). Oral bacteriotherapy with SAB51 has been previously evidenced as support of standard care in ameliorating SARS-CoV2 pneumonia and associated gastrointestinal symptoms. This study aimed to evaluate the effectiveness of SLAB51 in preventing or contrasting CFS in patients hospitalized for SARS-CoV2 infection. 65 hospitalized adults with severe COVID-19 pneumonia have been enrolled. All patients received the best available therapy (BAT), including hydroxychloroquine, azithromycin, lopinavir–ritonavir, or daru-navir–cobicistat for 7 days, and low molecular weight heparin. In addition to the above treatments, 32 randomly chosen patients initiated oral bacteriotherapy with the multistrain probiotic SLAB51. Before starting therapies, a venous blood sample was collected to evaluate a complete set of biochemical features, including asparagine and lactate levels. A second blood sample has been collected after 7 days of treatment. At hospital discharge, patients were administered with the Fatigue Assessment Scale (FAS) according to that previously reported by De Vries and colleagues in 2004. Discharged subjects were asked to compile a second FAS relative to conditions experimented at admittance. A value of FAS≥21 was considered to assess the presence of chronic fatigue. At admittance, no significant differences were determined between the group of patients treated only with BAT and that additionally administered with oral bacteriotherapy for age, sex, the prevalence of Fatigue, and levels of biochemical features. Obtained results evidenced a significant increase of asparagine and lactate levels in the group of patients administered with SLAB51 after a 7 days treatment. In contrast, no significant differences were observed for the group of subjects receiving only BAT. Observed levels of asparagine and lactate were significantly higher in subjects administered with oral bacteriotherapy to those observed for the patients treated with BAT only. Notably, at hospital discharge, the group of patients receiving only BAT, presented a considerable increase in the proportion of subjects positive for CFS. In comparison, an invariant prevalence of CFS positive patients were observed in the group administered with SLAB51. Furthermore, a significant association between CFS development and the lack of oral bacteriotherapy supplementation has been observed. Obtained results strongly evidenced that oral bacteriotherapy could represent a promising therapeutic strategy in reducing fatigue and preventing CFS development above all in fragile subjects. Furthermore, our findings contribute to stress the importance of the modulation of gut microbiota in influencing the outcome of Covid-19 patients. (Figure Presented)

Compartmental t-cell profile and IFN response in SARS-CoV-2-infected subjects

Background: A severe SARS-CoV-2 related immunopathology may be the driver cause underlying the deleterious clinical manifestations observed in COVID-19 patients. To identify possible tissue-specific immune responses patterns, a compartmental immunophenotyping analysis of CD4+ and CD8+ T lymphocytes and IFN response has been performed in SARS-CoV-2 infected subjects with acute respiratory distress syndrome. Methods: Bronchoalveolar lavage (BAL) and Peripheral Blood Mononuclear Cells (PBMC) samples were collected from 13 SARS-CoV-2 infected subjects (9 males and 4 females) consecutively admitted to intensive care unit (ICU) of Policlinico Umberto I, Sapienza University Hospital in Rome (Italy). The frequencies of CD4+, CD8+ T lymphocytes and those expressing immune activation markers (CD38, HLADR), naïve, central memory (CMEM), and effector memory (TEM) T cell subsets were evaluated in both anatomical sites by multiparametric flow cytometry. Gene expression levels of Interferon regulatory factor 7 (IRF7) and the Interferon Stimulated Gene 15 (ISG15) were evaluated in BAL and PBMC by Real-time PCR. Results: Critically SARS-CoV-2 infected patients exhibited a lung compartmentalization of CD8+ T cells (p=0.003), with a lower CD4/CD8 ratio in BAL compared to blood district (p<0.01). However, higher frequencies of CD8+ T cells were recorded in PBMC of female SARS-CoV-2 infected patients (p=0.04) and the same trend was observed in the lung compartment. By contrast, a trend of increasing CD4+ T cells frequencies was observed in BAL samples of male patients, as opposed to blood compartment. Additionally, an increased expression of immune activation markers CD38 and HLADR has been detected in BAL CD8+ T cells (p<0.01) as well as in blood CD4+ T cells (p=0.03). An increased frequency of CD4+ and CD8+ TEM cells has been documented in BAL of SARS-CoV-2 infected patients (p<0.05), as opposed to higher frequencies of CD4+ and CD8+ TCM cells recorded in the blood compartment (p<0.01). Notably, higher levels of ISG15 and IRF7 found in BAL were inversely associated to activated CD8+ T cell frequencies in the lung compartment compared to blood district (ISG15: r=-0.570, p<0.05) (IRF7: r=-0.683, p=0.01). Conclusion: Our findings provide new insight into a distinct T cells profile and IFN genes expression in the lung and in the blood compartment of SARS-CoV-2 infected patients, that might be highly relevant for the clinical course of COVID-19.

Comparison of clinical features and outcomes in COVID-19 and influenza pneumonia patients requiring intensive care unit admission.

BACKGROUND: Little is known in distinguishing clinical features and outcomes between coronavirus disease-19 (COVID-19) and influenza (FLU). MATERIALS/METHODS: Retrospective, single-centre study including patients with COVID-19 or FLU pneumonia admitted to the Intensive care Unit (ICU) of Policlinico Umberto I (Rome). Aims were: (1) to assess clinical features and differences of patients with COVID-19 and FLU, (2) to identify clinical and/or laboratory factors associated with FLU or COVID-19 and (3) to evaluate 30-day mortality, bacterial superinfections, thrombotic events and invasive pulmonary aspergillosis (IPA) in patients with FLU versus COVID-19. RESULTS: Overall, 74 patients were included (19, 25.7%, FLU and 55, 74.3%, COVID-19), median age 67 years (58-76). COVID-19 patients were more male (p = 0.013), with a lower percentage of COPD (Chronic Obstructive Pulmonary Disease) and chronic kidney disease (CKD) (p = 0.001 and p = 0.037, respectively) than FLU. SOFA score was higher (p = 0.020) and lymphocytes were significantly lower in FLU than in COVID-19 [395.5 vs 770.0 cells/mmc, p = 0.005]. At multivariable analysis, male sex (OR 6.1, p < 0.002), age > 65 years (OR 2.4, p = 0.024) and lymphocyte count > 725 cells/mmc at ICU admission (OR 5.1, p = 0.024) were significantly associated with COVID-19, whereas CKD and COPD were associated with FLU (OR 0.1 and OR 0.16, p = 0.020 and p < 0.001, respectively). No differences in mortality, bacterial superinfections and thrombotic events were observed, whereas IPA was mostly associated with FLU (31.5% vs 3.6%, p = 0.0029). CONCLUSIONS: In critically ill patients, male sex, age > 65 years and lymphocytes > 725 cells/mmc are related to COVID-19. FLU is associated with a significantly higher risk of IPA than COVID-19.