POLYFUNCTIONAL SARS-CoV-2-SPECIFIC T CELLS PERSIST IN TISSUE OF COVID-19 CONVALESCENTS

Background: T cells play an essential role in SARS-CoV-2 immunity, including in defense against severe COVID-19. However, most studies analyzing SARSCoV- 2-specific T cells have been limited to analysis of blood. Furthermore, the role of T cells in SARS-CoV-2 immunity in pregnant women, which are at disproportionately higher risk of severe COVID-19, is poorly understood. Method(s): Here, we quantitated and deeply phenotyped SARS-CoV-2-specific T cells from convalescent women (n=12) that had mild (non-hospitalized) COVID-19 during pregnancy. Endometrial, maternal blood, and fetal cord blood specimens were procured at term, which ranged from 3 days to 5 months post-infection. SARS-CoV-2-specific T cells were deeply analyzed by CyTOF using a tailored phenotyping panel designed to assess the effector functions, differentiation states, and homing properties of the cells. Result(s): SARS-CoV-2-specific T cells were more abundant in the endometrium than in maternal or fetal cord blood. In a particularly striking example, in one donor sampled 5 months after infection, SARS-CoV-2-specific CD8+ T cells comprised 4.8% of total endometrial CD8+ T cells, while it only reached 1.4% in blood. Endometrial SARS-CoV-2-specific T cells were more frequently of the memory phenotype relative to their counterparts in maternal and fetal cord blood, which harbored higher frequencies of naive T cells. Relative to their counterparts in blood, endometrial SARS-CoV-2-specific T cells exhibited unique phenotypic features, including preferential expression of the T resident memory marker CD69, inflammatory tissue-homing receptor CXCR4, and the activation marker 4-1BB. Endometrial T cells were highly polyfunctional, and could secrete IFNg, TNFa, MIP1b, IL2, and/or IL4 in response to spike peptide stimulation. By contrast, their counterparts in blood preferentially produced the cytolytic effectors perforin and granzyme B. Conclusion(s): Polyfunctional SARS-CoV-2-specific T cells primed by prior exposure to the virus are abundant and persist in endometrial tissue for months after infection. These cells exhibit unique phenotypic features including preferential expression of select chemokine receptors and activation molecules. Compared to their blood counterparts, the effector functions of these cells are more cytokine-driven and less cytolytic. The long-term persistence of these cells in the endometrium may help protect future pregnancies from SARS-CoV-2 re-infection.

Unique Differentiation and Homing Features of T Cells in Individuals with Long Covid

Background: A significant portion of individuals experience persistent symptoms months after SARS-CoV-2 infection, broadly referred to as Long COVID (LC). Although the frequencies of subsets of SARS-CoV-2-specific T cells have been shown to differ in individuals with LC relative to those with complete recovery, a deep dive into phenotypic and functional features of total and SARSCoV- 2-specific T cells from individuals with LC has yet to be performed. Method(s): Here, we used CyTOF to characterize the phenotypes and effector functions of T cells from LIINC cohort. The median age was 46, the cohort was 55.8% female, and 9/43 had been hospitalized. Participants were reported a median of 7 LC symptoms at 8 months. SARS-CoV-2-specific total antibody levels were also measured in concurrent sera. Manual gating was used to define T cell subsets, SPICE analyses for polyfunctionality, T cell clustering for phenotypic features, and linear regression for correlation. Permutation tests, Student's t tests, and Welch's t test were used for statistical analysis. Result(s): SARS-CoV-2 total antibody responses were elevated in the LC group (p=0.043), and correlated with frequencies of SARS-CoV-2-specific T cells in those without LC (r=0.776, p< 0.001) but not those with LC. While the frequencies of total SARS-CoV-2-specific CD4+ and CD8+ T cells were similar between individuals with and without LC, those from individuals without LC tended to be more polyfunctional (co-expressing IFNgamma, TNFalpha, IL2, and/or MIP1beta). CD4+ T cells from individuals with LC harbored higher frequencies of Tcm (p=0.003), Tfh (p=0.037), and Treg subsets (p=0.0412), and preferentially expressed a variety of tissue homing receptors including CXCR4 and CXCR5 (p=0.037). SARS-CoV-2-specific CD4+ T cells producing IL6, albeit rare, were observed exclusively among those with LC (p=0.016). In addition, participants with LC harbored significantly higher frequencies of SARS-CoV-2-specific CD8+ T cells co-expressing exhaustion markers PD1 and CTLA4 (p=0.018). Conclusion(s): Long COVID is characterized by global phenotypic differences in the CD4+ T cell compartment in ways suggesting preferential migration of these cells to inflamed mucosal tissues. Individuals with LC also harbor higher numbers of exhausted SARS-CoV-2-specific CD8+ T cells, potentially implicating viral persistence. Finally, our data additionally suggest that individuals with LC may uniquely exhibit an uncoordinated T cell and antibody response during COVID-19 convalescence.

Cytokine Profile in Different Post-Covid-19 Condition Phenotypes

Background: At least 10% of SARS-CoV-2 infected patients suffer from persistent symptoms for >12 weeks, known as post-COVID-19 condition (PCC) or Long Covid. Reported symptomatology is diverse with >200 physical and neurological debilitating symptoms. Here, we analyzed pro-inflammatory cytokine levels as a potential mechanism underlying persistent symptomatology. Method(s): Clinical data and samples used belong to the KING cohort extension, which includes clinically well characterized PCC (N=358, 59 persistent symptoms evaluated), COVID-19 recovered and uninfected subjects. We used Gower distances to calculate symptom's similarity between PCC and Ward's hierarchical clustering method to identify different symptom patterns among PCC patients. Cytokine levels of randomly selected PCC, recovered and uninfected subjects (N=193) were measured on plasma samples collected >6 months after acute infection using the 30-Plex Panel for Luminex. Mann- Whitney t-test was used to compare PCC vs recovered groups and Kruskal-Wallis t-test for >2 groups comparisons (PCC vs recovered vs Uninfected and within PCC clusters). FDR correction was applied for statistical significance (p-adj). Result(s): Hierarchical clustering identified 5 different PCC clusters according to their symptomatology, where PCC3 and PCC5 clusters showed higher prevalence of women ( >80%) and more persistent symptoms, while acute COVID-19 was mild in >80% of the patients. We selected 91 PCC (belonging to each cluster), 57 recovered and 45 uninfected subjects for cytokine profiling (Table 1). 13 soluble markers were significantly elevated (IL-1beta, Eotaxin, MIP-1beta, MCP-1, IL-15, IL-5, HGF, IFN-alpha, IL-1RA, IL-7, MIG, IL-4 and IL-8) in PCC and recovered groups compared to uninfected subjects (all p-adj< 0.04). In addition, PCC subjects tended towards higher levels of IL-1RA compared to recovered group (padj= 0.071). Within PCC clusters, FGF-basic and RANTES were elevated while IL-2 and MIG were decreased in PCC3 and PCC5 compared to the other PCC clusters (all p-adj< 0.04). TNF-alpha, IP-10, G-CSF and MIP-1alpha were decreased in PCC3 and PCC5 not reaching statistical significance (all p-adj=0.07). Conclusion(s): Some cytokines remained altered in all SARS-CoV-2 infected subjects independently of persistent symptoms after 6 months from acute infection. Differences between PCC and recovered individuals are limited after correction. Importantly, PCC cytokine profiles showed differences between clusters, which suggests different PCC subsyndromes with distinct etiology. Subjects Characteristics (Table Presented).

Potential Predictors for Disease Severity in Hospitalised COVID-19 Patients

Background and Objectives Several studies have shown that SARS-CoV-2 can induce a cytokine release storm which is a major cause of disease severity and death. Therefore, cytokine levels in the serum may predict disease severity and survival in patients with COVID-19. Methods We included 88 COVID-19 patients who were hospitalised at the Division of Pulmonology of the Vienna General Hospital between January and May 2021 in this observational trial. Blood samples for serum peptide measurements were drawn at the time closest to hospitalisation, at day 5, 9 and 13( +/- 1). We correlated the type of ventilation (nasal oxygen therapy, high flow nasal canula, non-invasive ventilation or mechanical ventilation), occurrence of consolidations on chest X-ray or if available HRCT and the level of care (general ward, IMCU or ICU) with serum peptide values. We assessed the concentration of cytokines (IL-1a, IL-1b, IL-1RA, IL-6, L-7, L-10, IFN- gamma and TNF-alpha), chemokines (CCL-3, CCL-4 and CCL-7) and growth factors (G-CSF, GM-CSF and VEGF). Results Patients were 68 years of age (median) and stayed in hospital between 5-171 days. The peak inspiratory pressure in patients receiving non-invasive ventilation or mechanical ventilation was significantly associated with IL-1RA, G-CSF and IFN-gamma and the fraction of inspired oxygen in patients receiving highflow nasal canula oxygen therapy was significantly associated with IL-6, IL-7, IFN-gamma, and CCL-7. Results are shown in Table 1. No investigated cytokine correlated with the type of ventilation, occurrence of consolidations on imaging and in-hospital mortality. Conclusions In conclusion, concentrations of IL-1RA, G-CSF, IL-6, IL-7, IFN-gamma, and CCL-7 were associated with more severe disease progression in hospitalised COVID-19 patients.

Association of macrophage inflammatory protein-1beta with the recovery process in COVID-19

Background: Recovery from coronavirus disease 19 (COVID-19) is a gradual process that depends on the disease severity. Immunologic changes that precede and relate clinical symptoms may predict the course of COVID-19 and final outcome. Our goal was to determine prognostic markers of COVID-19 improvement. Method(s): The study included hospitalized patients from the ages 31-72 with moderate to severe COVID-19. All biomarkers were assessed at three checkpoints starting from the first day of hospitalization (day 0), continuing on day 8, and between 40-50 day. Luminex xMAP technology and the Bio-Plex Pro Human Cytokine 17-plex assay was used for quantitative evaluation cytokines and chemokines in peripheral blood of COVID-19 patients. The comparative study was done in combination with clinical data. Univariate and multivariate analyses of data were delivered. Finally, a fuzzy logic model for decision support was proposed and validated for explored data. Result(s): Macrophage inflammatory protein-1beta (MIP1b) was inversely related to COVID-19 evolution. MIP1b significantly higher on day 8 compared to day 0 (p < 0.0001) correlated with clinical improvement and predicted a successful course of the disease. It was also associated with the significant increase in TNF-alpha (p = 0.03), and decrease in IL-10 (p < 0.0001), and IL-6 (p = 0.01). The increase in MIP1b on day 8 correlated positively with eosinophil and lymphocytes counts and negatively with inflammatory mediators (ferritin, procalcitonin, fibrinogen, CRP). Moderately positive correlation between MIP-1b and TNF-alpha was noted, in parallel. Tested the statistical and machine learning predictors exhibited sensitivity to MIP1b input, improving the ROC curve compared to the classification models trained without MIP1b. Conclusion(s): This finding next to already known indicators such as IL-6, eosinophil and lymphocytes counts, highlight a role of MIP1b as a marker of good prognosis in COVID-19 and provide a novel insight into this as a potential diagnostic and therapeutic target.

Alterations in programmed death ligand expression in whole blood cells in COVID-19 patients

Background: Infections with SARS-CoV- 2 cause the coronavirus disease 2019 (COVID-19) pandemic. Alterations in immune cells of COVID-19 patients may predict the subsequent severity of disease. The changes in composition of immune cells in COVID-19 patients include lymphopenia, lower neutrophil to lymphocyte-ratios and an eosinopenia in about 50 to 80% of hospitalized patients. Eosinophils and neutrophils can interact with T cells via immune checkpoints receptors such as programmed death (PD)-1 on T cells and its counterpart PD-ligand 1 (PD-L1) on eosinophils or neutrophils. There are only limited studies on PD-1 and PD-L1 expressions in viral infections, we aimed to elucidate the interplay of T cells and other peripheral cells by analysing the immune checkpoints PD-1 and PD-L1 in expression during COVID-19. Method(s): Using flow cytometry, we have now analysed the immune checkpoint receptor expressions on whole blood cells from a total of 38 COVID-19 patients. The patient cohort comprises all ages and both sexes with the disease severity ranging from mild, moderate to severe, with ~10% mortality. We have further been investigating 21 biomarkers (G-CSF, GM-CSF, IFN-gamma, TGF-beta1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-23, IL-33, IP-10, MCP-1, MIP-1beta, TNF-alpha, and YKL-40) in plasma on a cohort of 76 COVID-19 patients using the MesoScale Multiplex Assay platform, with 48 healthy controls. Result(s): PD-L1 expression on eosinophils was significantly lower in COVID-19 patients in initial stages of infection, relative to healthy controls. There was an inverse relationship between disease progression and the expression of PD-1 on CD8+ T cells. These data suggests that analysis of PD-L1- PD1 cell networks in immune cells of EDTA blood of COVID-19 patients can predict disease outcomes. While most detectable biomarkers are strongly increased in COVID samples overall compared to healthy controls, the more severe the disease the higher the blood biomarker concentration. Conclusion(s): Taken together, the analysis of PD-L1- PD1 cell networks in immune cells together with plasma biomarkers of COVID-19 patients can predict disease outcomes.

The Significance of Epidermal Growth Factor and Other Urinary Cytokines since Subclinical Aki in Patients with Covid-19

Introduction: In hospitalized COVID-19 patients, disease progression leading to acute kidney injury (AKI) may be driven by immune dysregulation. We explored the role of urinary cytokines and their relationship with the kidney stress biomarkers neutrophil gelatinase-associated lipocalin (NGAL), tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor binding protein (IGFBP7) in COVID-19 patients without AKI at study entry. Method(s): Prospective, longitudinal cohort study included critically ill COVID-19 patients without AKI at the time they were enrolled to the study. Urine samples were collected on admission to critical care areas for determination of NGAL, [TIMP-2]*[IGFBP7] and cytokines concentrations with a second sample 5 days after the first urine sample. Demographic information, clinical and laboratory data were collected. Diagnosis and staging of AKI were based on KDIGO criteria using serum creatinine (sCr) levels and urine output. The urinary concentrations of TIMP-2 and IGFBP7 were determined ELISA in the same way NGAL. The concentrations of cytokines and chemokines in urine were measured with a Luminex 200 instrument. We performed descriptive statistics including means and standard deviations for normally distributed continuous variables;medians and interquartile ranges (IQR) for non-parametric distributions;and proportions for categorical variables. Logistic regression analysis was used to identify the association between relevant covariates with AKI. Principal component analysis (PCA) was performed to compress and simplify the size of the data set by keeping the most important information and analyzing the structure of the observations and the variables. Correlation of identified cytokines with kidney stress biomarkers was explored using the Spearman test. All statistical tests were two-sided, p values <0.05 were considered statistically significant. The analysis was conducted using R Studio 1.4.1717. Result(s): Of included 51 patients. Of those, 30 were males (58.8%);the median age was 53 years (IQR: 40-61);14 had systemic arterial hypertension (27.5%);16 had diabetes (31.4%);and 21 were obese (41.2%). 54.9% developed AKI. After adjusting for possible confounding variables, only EGF >4600 pg/ml remained associated with lower risk of AKI (OR=0.095, 95% CI: 0.01-0.81, p=0.031.In the PCA of day 1, Epidermal Growth Factor (EGF) and interferon (IFN)-alpha were associated with a lower risk of AKI (OR=0.24, 95% CI: 0.07-0.78, p=0.017), while Interleukin (IL)-12 and macrophage inflammatory protein (MIP)-1b were associated with a higher risk of AKI (OR=51.09, 95% CI: 2.12-1233, p=0.015). In the PCA of day 5, EGF and IFN-alpha remained associated with a lower risk of AKI (OR=0.09, 95% CI: 0.01-0.74;p=0.024), while IL-1 Receptor, granulocyte-colony stimulating factor (G-CSF), interferon-gamma-inducible protein 10 (IP-10) and IL-5 were associated with a higher risk of AKI (OR=7.7, 95% CI: 1.06-55.74, p=0.043). EGF had an inverse correlation with [TIMP2] x IGFBP7] (R-0.73, p=<0.001) and with NGAL (R= -0.63, p=0.002). Conclusion(s): Subclinical AKI was characterized by a significant up-regulation of NGAL, TIMP-2, IGFBP7 and proinflammatory cytokines. The lack of EGF regenerative effects and IFN-alpha antiviral activity seemed crucial for renal disease progression. AKI involved a proinflammatory urinary cytokine storm. No conflict of interestCopyright © 2023

Distinct profiles and kinetics of soluble infammatory markers in patients with acute and chronic HCV-infections and SARS-CoV-2-infections: Implications for the long-term consequences of viral infections?

Background and Aims Viral infections occur acutely but can also progress chronically, with the immune system having a central role in immunopathoge-nesis. The question arises whether all alterations in immune responses are reversible after viral elimination (spontaneously or by therapy). Therefore, the aim of this study is to compare soluble infammatory markers (SIM) during and after infection with SARS-CoV-2 and acute and chronic HCV-infections. Patients and Method Patients with acute HCV (n = 29), chronic HCV (n = 54), SARS-CoV-2 (n = 39) and 31 healthy-controls were included. Blood samples were tested at baseline, end of treatment/infection, and follow-up ( >= 9 months after baseline). IL-12p70, IL-1b, IL-4, IL-5, IL-6, IL-8, TNF, IFN-g, IL-10, IL-22, CXCL-10, MCP-1, MIP-1b, ITAC were quantified using the HD-SP-X Imaging and Analysis SystemTM. Results SIM profiles in patients with acute HCV were substantially elevated at baseline and the decrease during follow-up was considerably less compared to the SARS-CoV-2 cohort. In chronic HCV-patients, viral elimination by therapy resulted in a decrease in SIM, although not always to those of controls. Cirrhotic HCV patients had higher SIM levels after HCV elimination than non-cirrhotic chronic HCV-patients. In the SARS-CoV-2 cohort, most SIM returned to levels of controls 3 months after baseline. Conclusions SIM profiles and kinetics after viral elimination difer between blood-borne acute and chronic HCV- and respiratory SARS-CoV-2-infections. The immunologic imprint 9 months after cured HCV-infection (both acute and chronic) appears to be more pronounced than after SARS-CoV-2-infection. Further analysis is needed to correlate the SIM profle with the clinical pheno-type (long-HepC vs. long-COVID-19).

Predictive Value of Specific Cytokines for Lethal Covid-19 Outcome

In our study, we aimed to evaluate the significance of specific cytokines in blood plasma as predictive markers of COVID-associated mortality. Materials and methods. In plasma samples of 29 patients with PCR-confirmed COVID-19 we measured the concentrations of 47 molecules. These molecules included: interleukins and selected pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A/CTLA8, IL-17-E/IL-25, IL-17F, IL-18, IL-22, IL-27, IFNalpha2, IFNgamma, TNFalpha, TNFbeta/Lymphotoxin-alpha(LTA));chemokines (CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL11/Eotaxin, CCL22/MDC, CXCL1/GROalpha, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CX3CL1/Fractalkine);anti-inflammatory cytokines (IL-1Ra, IL-10);growth factors (EGF, FGF-2/FGF-basic, Flt-3 Ligand, G-CSF, M-CSF, GM-CSF, PDGF-AA, PDGFAB/BB, TGFalpha, VEGF-A);and sCD40L. We used multiplex analysis based on xMAP technology (Luminex, USA) using Luminex MagPix. As controls, we used plasma samples of 20 healthy individuals. Based on the results, we applied Receiver Operating Characteristic (ROC) analysis and Area Under Curve (AUC) values to compare two different predictive tests and to choose the optimal division point for disease outcome (survivors/non-survivors). To find optimal biomarker combinations, we as used cytokines concentrations as dependent variables to grow a regression tree using JMP 16 Software.Results. Out of 47 studied cytokines/chemokines/growth factors, we picked four pro-inflammatory cytokines as having high significance in evaluation of COVID-19 outcome: IL-6, IL-8, IL-15, and IL-18. Based on the results received, we assume that the highest significance in terms of predicting the outcome of acute COVID-19 belongs to IL-6 and IL-18. Conclusion. Analyzing concentrations of IL-6 and IL-18 before administering treatment may prove valuable in terms of outcome prognosis. Copyright © Arsentieva N.A. et al., 2022.

A Tempestade De Citocinas E O Desbalanco Imune No Covid-19 Grave

Objetivos: A infeccao pelo SARS-COV-2, responsavel pela COVID-19, se disseminou rapidamente. No final de 2019 a epidemia na China e, em marco de 2020, a pandemia mundial. Apresenta espectro clinico variavel, e em especial acomete o trato respiratorio. Os infectados podem ser assintomaticos ou ate evoluir com insuficiencia respiratoria aguda, disfuncao de multiplos orgaos e obito. Ha necessidade de internacao em 3%-20% e, destes, 10%-30% cuidados intensivos. Desta forma, ha uma preocupacao com a saturacao dos servicos de saude. A infeccao grave parece estar associada a um desequilibrio imune, desta forma, a melhor identificacao do perfil imune e fundamental para definicao prognostica e terapeutica. Materiais e Metodos: Avaliacao de adultos em UTI com COVID-19 grave, realizada de maio de 2020 a maio de 2021. Foram coletadas amostras de sangue total nas primeiras 24 horas de admissao (D0) na UTI e apos, a cada 4 dias por no maximo 20 dias. O grupo controle incluiu doadores de sangue voluntarios. Avaliamos 17 citocinas, dados clinicos e laboratoriais. O desfecho primario foi sobrevida livre de Ventilacao Mecanica (VM). Este projeto foi aprovado no Comite de etica em pesquisa. Resultados: Foram incluidos 62 pacientes com idade mediana de 58,7 anos e alta prevalencia de comorbidades. Observamos linfopenia associada a aumento na relacao neutrofilo-linfocito (NLR=9.8), niveis elevados de D-Dimero, DHL e PCR. A mortalidade foi de 8%, sendo necessario VM com uma mediana de 4 dias apos admissao UTI em 55%. A analise das citocinas no D0 em comparacao com o grupo controle evidenciou aumento nos niveis de sCD137, IL-10, Granzima-B, sFAS, IL-6, CCL-4, TNFalpha e perforina. Atraves da curva ROC foi possivel avaliar o papel preditivo para VM nos niveis de IL-6, TNFalpha, sCD137, sFAS, Perforina e Granzima-B. Discussao: O desequilibrio imune e o papel das citocinas vem sendo avaliado como fator causal da forma grave do COVID-19. A tempestade de citocinas e potencialmente fatal, uma doenca imune em que ha liberacao descontrolada destas celulas ativadas que desencadeia infiltrados celulares e liberacao de mediadores quimicos. E responsavel pela febre, fadiga e contribui tambem com extravasamento vascular, CIVD, disfuncao de multiplos orgaos, choque e obito. Em nosso estudo houve o aumento das citocinas IL-6, CCL4, TNFalpha, Granzima-B e perforina que sao marcadores pro inflamatorios, a IL-10 anti-inflamatoria e ainda, o sFAS e sCD137 responsaveis pela reducao da resposta celular T, por conseguinte, anti inflamatorios. Desta forma, representando a hiperativacao desequilibrada do sistema imune associada ao aumento de marcadores inflamatorios como D-Dimero e PCR. O aumento da NRL representa a gravidade da populacao, ja que estudos previos demonstraram como corte, acima de 3,63. O mecanismo de bloqueio imune atraves da ativacao de anti inflamatorios e apoptose de celulas auto reativas pode contribuir com o aumento de infeccoes secundarias tardias. O aumento das citocinas foi relacionado ao desfecho desfavoravel em diversos estudos, especialmente na manifestacao grave, sendo potenciais marcadores prognosticos. Conclusao: Nosso estudo ratifica a importancia das citocinas e o desequilibrio imune na forma grave do COVID-19. Alem disso, evidenciamos alguns potenciais marcadores preditivos de desfecho desfavoravel como TNFalpha, Perforina, Granzima-B, IL-6, sCD137 e sFAS. E ainda, estes podem ser estudados como potenciais alvos terapeuticos. Copyright © 2022

Analysis of Sars-Cov-2-Specific T-Cell Responses Using Novel Single- Cell Multiplexed Isolight in Covid-19 Convalescents

Purpose: COVID-19 has poor outcomes in transplant recipients with reduced antibody responses. However, the exact cellular immune response against SARS-CoV-2 remains largely unclear. We developed novel assays to analyze differential cellular immune responses in individual subjects and groups. Method(s): We assessed the T cell proliferative responses against spike, membrane, and nuclear proteins of SARS-CoV-2, or a mixture of all these peptides (mix) using 3H-thymidine incorporation and CFSE dilution assays. We have also established a SARS-CoV-2-specific multiplexed cytokine IsoLight at single-cell resolution. This is a very powerful technology that employs IsoPlexis' IsoCodechip with 12,000 micro-chambers. Each microchamber is pre-coated with a 32-plex antibody array to capture secreted cytokines. The results were evaluated using IsoSpeak software. Result(s): COVID-19 convalescent subjects (n=3) showed a very strong proliferative response to S/M/N and mix peptides of SARS-CoV-2 when compared to uninfected normal subjects who had only marginal proliferative responses. CFSE dilution assays demonstrated that spike and mix proteins markedly increased the proliferation of CD3 cells comprised of both CD4 and CD8 subsets. In the IsoLight assay, single-cell functional heterogeneity mapping 3D tSNE analysis showed a distinct combinatorial cytokine secretion pattern in stimulated cells compared to unstimulated controls (Fig.1). Polyfunctional activity topography-Principal component analysis (PAT-PCA) revealed that IFN-g, IL2, and MIP-1b drove the polyfunctional heterogeneity. When the percentage of polyfunctional cells (>=2 cytokines/cell), and polyfunctional strength index (PSI) were evaluated, CD8 cells secreted high levels of effector and chemoattractive cytokines while CD4 cells secreted effector and stimulatory cytokines. Most importantly, the depth and breadth of T cell responses, particularly the cytokine polyfunctionality correlated with the severity of the disease the patients had experienced. Conclusion(s): This novel COVID19-specific IsoLight cytokine assay is a powerful technology that can be utilized for in-depth analysis of T cell polyfunctionality at the single-cell level and for further differentiating the anti-SARS-CoV-2 immune capabilities of vulnerable individuals such as transplant patients.

Adenosine A2A Receptor (A2AR) Agonist Apadenoson Promotes Survival in K28-hACE2 Mice and Syrian Hamsters Infected with Sars-CoV-2

Purpose: To determine if Apadenoson or Regadenoson has a therapeutic effect in attenuating hyper-inflammation and improving survival rate in K18-hACE2mice or Syrian hamsters infected with SARS-CoV-2. Method(s): 6-8 weeks old male K18-hACE2mice were divided into Control group that received vehicle;Test group 1 that received the drug (Apadenoson or Regadenoson) 24hrs prior to challenge with SARS-CoV-2;and Test Group 2 (Drug-delay), that received the drug with a 5 hr delay post-viral infection (n=6/grp). Viral dose was 1250 PfuHong Kong/VM20001061/2020 delivered via intranasal route. Drug was delivered subcutaneously using 1007D ALZET pumps. 6 weeks old Syrian hamsters were divided into Control group that received Vehicle and Virus (n=4) and 2 test groups (n=5/group) that received Apadenoson+Virus and Regadenoson+Virus. Drugs were delivered by 2ML2 ALZET pumps (4ug/kg/hr). Hamsters were inoculated intratracheally with 750PFU SARS-CoV-2 WA1 strain prior to treatment. Mice were weighed and clinical scores recorded daily. Bronchoalveolar lavage fluid (BALF) and serum were collected along with lungs. Plethysmography was done on days 0, 2, 4 and 7. Result(s): Apadenoson administered post-infection was efficacious in decreasing weight loss, improving clinical score, and increasing the survival rate in K18-hACE2 mice, i.e. 50% survival was observed at Day 5 and at Day 7 post-infection for drug given before or after infection respectively. Apadenoson given post-infection improved the histopathology that was observed in the vehicle control group, decreased pro-inflammatory IL-6, IFN-gamma, MCCP-1, MIP-1beta, IP-10, and Rantes in serum, increased anti-inflammatory Ang1-7 levels, and decreased monocytes in BALF. 42% of mice that received Regadenoson pre-challenge survived infection compared to 6.25% in the vehicle or Drug delay (drug given post-infection) groups. Viral titers in the lungs of Regadenoson-treated mice were found decreased. Treatment also significantly decreased CD4+, CD8+T cells, eosinophils, and neutrophils in BALF. Plethysmography, in hamsters, showed significant improvement of pulmonary function parameters, Rpef and PenH, following treatment with Apadenoson given post-infection. Apadenoson cleared the virus from BALF and maintained Ang1-7 levels. Both drugs decreased plasma IFN-gamma levels. Conclusion(s): Treatment with Apadenoson attenuated inflammation, improved pulmonary function, decreased weight loss, and enhanced survival rate following infection with SARS-CoV-2 virus. The results demonstrate the translational significance of Apadenoson in the treatment of COVID-19.

SPECIALIZED PRO-RESOLVING MEDIATORS (SPMS) and INFLAMMATORY NETWORKS in PATIENTS AFFECTED by ADULT ONSET STILL'S DISEASE (AOSD) and COVID-19

Background: Uncontrolled systemic infammation characterizes COVID-19 and autoinfammatory diseases such as adult-onset Still's disease (AOSD). Biosynthesis of pro-resolving mediators (SPMs), i.e. lipoxins (LX), resolvins (Rv), pro-tectins (PD), and maresins (MaR), ensures infammation shutdown and tissue repair, limiting neutrophils recruitment and stimulating macrophages to remove apoptotic cells. Among protectins, reduction of PD1 was found in the lungs of mice infected with the H5N1 infuenza virus and experimental treatment with PD1 resulted in increased animals' survival (Morita M et al 2013). Objectives: We investigated the effects of SPMs in pathogenesis and clinical evolution of AOSD and compared these data with mild and severe COVID-19. Finally, we analyzed the potential role of PD1 in modulating the infammatory response of macrophages obtained from AOSD patients, COVID-19 patients and healthy donors (HDs). Methods: 21 patients hospitalized for COVID-19 (10 ICU and 11 hospitalized in medical clinical unit) and 13 patients with AOSD were enrolled. Plasma PD1 levels from patients and controls were analyzed by ELISA, and mono-cytes-derived macrophages were polarized into M1 and M2 phenotype. We analyzed the effect of PD-1 on macrophages differentiation. At 10 days, macrophages were analyzed for surface expression of subtypes markers by flow cytometry. Cytokines production was measured in supernatants by Bio-Plex Assays. Peripheral blood mononuclear cells (PBMCS) from 3 AOSD patients, 2 COVID-19 patients and 3 HDs were obtained. Next-generation deep sequencing was then performed to identify the differences in PBMCs transcripts profiles. Results: AOSD patients with systemic scored (SS) ≥1 showed an increase of PD1 levels compared to AOSD patients with lower systemic score (p=0.04) (Figure 1A). Similarly, plasma levels of PD1 were increased in COVID-19 patients independently from their clinical subsets, compared to HDs (p=0.02). In vitro treatment with PD1 of monocytes-derived macrophages from AOSD and COVID-19 patients induced a signifcant increase of M2 polarization vs control (p<0.05) (Figure 1B). Furthermore, a signifcant release of IL-10 and CCL4 from M2 macrophages was observed when compared to control (p<0.05) (Figure 1C). In the transcriptomes from 3 AOSD patients (2 mild and 1 severe), 2 COVID-19 patients (1 mild and 1 ICU) and 2 HDs, we observed that genes involved in infammation, lipid catabolism and monocytes activation were spe-cifcally dysregulated in AOSD and COVID-19 patients when compared to HDs. Among them pla2g15, pla2g12a, pla2g2d, involved in mobilization of SPMs precursors, were signifcant upregulated in patients compared to HDs (p<.01, ;log2FoldChange;>1.2) (Figure 1D). The largest part of the genes involved in infammation, lipid catabolism, and monocytes activation are less expressed in AOSD patients when compared to COVID19 patients, as reported in Table 1. Conclusion: The counterbalance by SPMs during infammation is still a largely unexplored pathway. Our study suggests that an imbalance of SPMs in autoin-fammatory diseases as well as COVID-19. The modulation of SPMs as observed in our experiments, might represent a new possible therapeutic strategy during AOSD and COVID-19.

COVID-19: DIARRHEA, INFLAMMATION AND INTESTINAL MICROBIOTA

Introduction: Increased inflammatory cytokines has been observed in COVID-19 patients and there is evidence showing an alteration in gut-microbiota composition. SARS-CoV-2 can cause gastrointestinal symptoms, such as diarrhea. Evidence of an altered gut-microbiota composition and cytokines levels in COVID-19 diarrhea patients is lacking. Objectives: To compare serum cytokine levels and gut microbiota between COVID-19 diarrhea (D-COVID- 19) and non-diarrhea (NonD-COVID-19) patients and non- COVID-19 controls (HC). Material and methods: We included 143 hospitalized COVID-19 patients (positive quantitative reverse transcription PCR) in a single University Hospital, and 53 ambulatory HC (negative rapid serological test) were included. Blood and stool samples were collected at hospital admission in COVID-19 patients and at the time of HC recruitment. 27- pro and anti-inflammatory cytokines (Bio-Plex Pro™, Bio- Rad) were measured. Gut microbiota composition and diversity profiles were characterized by sequencing the 16S rRNA gene V3-V4 region amplified using DNA extracted from stool samples. Bioinformatics analysis was performed with QIIME2 software. First, we compare cytokine levels between COVID- 19 and HC and then COVID-19 with and without diarrhea. All comparisons were adjusted for age, sex, and BMI with linear regression. Results: The mean age in COVID-19 patients was 54 +/- 15 years (F=50%) and 52 +/- 8 (F=62%) for HC. Diarrhea was present in 19 (13.29%) of COVID-19 patients. COVID-19 patients had significative higher levels of: IL- 1ra, IL-2, IL-6, IL-7, IL-8, IL-13, IP-10 and PDGF-bb. Significant lower values of: IL-9, FGF -basic, MIP-1β, TNF-α were observed in D-COVID-19 compared to NonD-COVID-19. COVID-19 patients had a significant reduction of bacterial species (p=0.0001), and diversity and complexity of the bacterial community (Shannon's index) (p=0.0001) compared to the HC. There was no difference between D-COVID-19 and NonD-COVID-19. There were also changes in the composition of the microbiota associated with COVID-19. At the phylum level, COVID-19 patients showed a significant decrease in Actinobacteria and Firmicutes, and an increase in Bacteroidetes. At species level, an increase of 4 species of the genus Bacteroides was observed in COVID-19 patients. 31 very diverse bacterial species were found, all decreased in D-COVID-19. Conclusions: An alteration in serum cytokine levels was observed between COVID-19 and HC. D-COVID-19 had a decrease in some proinflammatory cytokines. A significant decrease in richness and species diversity of gutmicrobiota was observed in COVID-19 patients compared to HC, but no significant differences were observed between D-COVID-19 and NonD-COVID-19. However, in D-COVID- 19, a decrease in some bacterial species was observed.(Table Presented)(Figure Presented)

Intrathecal inflammatory responses in the absence of SARS-CoV-2 nucleic acid in the CSF of COVID-19 hospitalized patients

Objective: Evaluate SARS-CoV-2 RNA and inflammatory cytokines and chemokines in the CSF of patients with acute COVID-19 and neurologic symptoms, and to compare these to controls and patients with known neurotropic pathogens. Background: Neurologic symptoms have been described in 30-60% of hospitalized patients with Coronavirus Disease 2019 (COVID-19). However, little is known about CSF profiles in these patients. Design/Methods: CSF from twenty-seven consecutive patients with COVID-19 and neurological symptoms was assayed for SARS-CoV-2 RNA using quantitative reverse transcription PCR (RT-qPCR) and unbiased metagenomic sequencing. Assays for blood brain barrier (BBB) breakdown (CSF:serum albumin ratio (Q-Alb)), and proinflammatory cytokines and chemokines (IL-6, IL-8, IL-15, IL-16, monocyte chemoattractant protein -1 (MCP-1) and monocyte inhibitory protein - 1β (MIP-1β)) were performed in 23 patients and compared to CSF from patients with HIV-1 (16 virally suppressed, 5 unsuppressed), West Nile virus (WNV) (n=4) and 16 healthy controls (HC). Results: Median CSF cell count for COVID-19 patients was 1 white blood cell/μL;two patients were infected with a second pathogen (Neisseria, Cryptococcus neoformans). No CSF samples had detectable SARS-CoV-2 RNA by either detection method. In patients with COVID-19 only, CSF IL-6, IL-8, IL-15, and MIP-1β levels were higher than HC and suppressed HIV (corrected-p < 0.05). MCP-1 and MIP-1β levels were higher, while IL-6, IL-8, IL-15 were similar in COVID-19 compared to WNV patients. Q-Alb correlated with all proinflammatory markers, with IL-6, IL-8, and MIP-1β (r≥0.6, p<0.01) demonstrating the strongest associations. Conclusions: Lack of SARS-CoV-2 RNA in CSF is consistent with pre-existing literature. Evidence of intrathecal proinflammatory markers in a subset of COVID-19 patients with BBB breakdown despite minimal CSF pleocytosis is atypical for neurotropic pathogens.

Umbilical cord-derived mesenchymal stem cell transplantation for COVID -19 patients: long-term benefits for lung regeneration

Coronavirus disease 2019 (COVID-19) has affected hundreds of millions of people worldwide. Currently, mortality due to COVID-19 is significantly reduced by vaccination, antiviral drugs, and some improved treatments. Mesenchymal stem cell (MSC) transplantation—particularly umbilical cord-derived MSC (UC-MSC)—has been used as an adjuvant therapy for COVID-19 with some clinical evidence (reviewed in the publication). Moreover, a recent piece published in eBiomedicine (part of The Lancet, https://doi.org/10.1016/j.ebiom.2021.103789) in the previous month showed the long-term effects of UC-MSC transplantation in COVID-19 in a 1-year follow-up randomized, double-blind, placebo-controlled trial, demonstrating significantly recovered lung lesions and symptoms compared to the control group (i.e., without UC-MSC transplantation). In this commentary, we would like to discuss the value of UC-MSC transplantation for COVID-19 patients based on the results from this study and suggest applying this therapy for COVID-19 patients.

Mucosal Cytokine Profiles in Children with COVID-19

Background. The mechanisms associated with COVID-19 in children are not well understood. We sought to define the differences in nasopharyngeal (NP) cytokine profiles according to clinical presentation in children with COVID-19. Methods. Single-center, prospective study in 137 children and adolescents < 21 years of age hospitalized with COVID-19, and 35 age, sex and race matched pre-pandemic (2016-2019) healthy controls. Children with COVID-19 were categorized according to their clinical presentation in: COVID-19-symptomatic;COVID-19-screening, and multisystem inflammatory syndrome (MIS-C). NP swabs were obtained within 24 hours of admission to measure SARS-CoV-2 loads by rt-PCR, and a 92-cytokine panel. Unsupervised and supervised analysis adjusted for multiple comparisons were performed. Results. From 3/2020 to 1/2021, we enrolled 76 COVID-19-symptomatic children (3.5 [0.2-15.75] years);45 COVID-19-screening (11.1 [4.2-16.1] years), and 16 MIS-C (11.2 [5.9-14.6] years). Median NP SARS-CoV-2 loads were higher in COVID-19-symptomatic versus screening and MIS-C (6.8 vs 3.5 vs 2.82 log10 copies/mL;p< 0.001). Statistical group comparisons identified 15 cytokines that consistently differed between groups and were clustered in three functional categories: (1) antiviral/regulatory, (2) pro-inflammatory/chemotactic, and (3) a combination of (1) and (2);(Fig 1). All 15 cytokines were higher in COVID-19-symptomatic versus controls (p< 0.05). Similarly, and except for TNF, CCL3, CCL4 and CCL23, which were comparable in COVID-19-symptomatic and screening patients, the remaining cytokines were higher in symptomatic children (p< 0.05). PDL-1 (p=0.01) and CCL3 (p=0.03) were the only cytokines significantly decreased in children with MIS-C versus symptomatic COVID-19 children. The 15 cytokines identified by multiple comparisons were correlated using Person's in R software. Red reflects a positive correlation and blue a negative correlation with the intensity of the color indicating the strength of the association. Conclusion. Children with symptomatic COVID-19 demonstrated higher viral loads and greater mucosal cytokines concentrations than those identified via screening, whereas in MIS-C concentrations of regulatory cytokines were decreased. Simultaneous evaluation of viral loads and mucosal immune responses using non-invasive sampling could aid with the stratification of children and adolescents with COVID-19 in the clinical setting.

Longitudinal Plasma Cytokine Profiles Differentiating COVID-19 Severity Groups

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infection with widely varying clinical severity. Severe COVID-19 was initially proposed to be secondary to cytokine storm syndrome (CSS). However, studies since showed that patients with severe COVID-19 rarely display CSS cytokine phenotypes, and may have more limited inflammatory responses instead. Methods. Prospective cohorts, aged 0-90 years of age who tested positive by polymerase chain reaction (PCR) for SARS-CoV-2 were enrolled from inpatient hospitals and outpatient testing centers in Memphis, TN from May 2020-January 2021. Longitudinal blood samples were obtained including acute, sub-acute and convalescent timepoints. Severity scores of asymptomatic, mild, moderate, and severe COVID-19 were assigned at time of convalescent assessment. Plasma was analyzed with a quantitative human magnetic 38-plex cytokine assay. Results. : 169 participants were enrolled, including 8 asymptomatic, 117 mild, 22 moderate and 17 severe cases, and 5 children with post-COVID-19 multisystem inflammatory syndrome in children (MIS-C). All moderate and severe patients were hospitalized and received treatment (39%). Clear distinctions were seen between asymptomatic-mild cases and moderate-severe cases at acute timepoints and during disease progression for GCSF, IL-8, IL-10, IL-15, IL-1Ra, IP-10, MIP-1a, MIP-1β, and TGFα. There was a significant difference between participants who did and did not require hospitalization for acute timepoint levels of IL-10, IL-15, MIP-1 β and TGFα (p< 0.01). Only 4 participants with active COVID-19 were found to meet criteria for CSS (2%), only 3 of which were severe. MIS-C participants showed nearly universally elevated cytokine levels compared to those with active COVID-19. Conclusion. Moderate and severe acute COVID-19 has a distinct cytokine profile from asymptomatic and mild cases, as detected from acute, subacute and convalescent plasma.

ORAL TANNINS REDUCE PROINFLAMMATORY CYTOKINES ASSOCIATED WITH DIARRHEA AND PNEUMONIA IN HOSPITALIZED COVID-19 PATIENTS

INTRODUCTION: There is evidence that the gut microbiota and its relationship with the immune system could be involved in the pathogenesis of COVID-19. SARS-CoV-2 can cause gastrointestinal symptoms during the early phases of the disease. Intestinal dysfunction induces changes in intestinal microbes, and an increase in inflammatory cytokines. Therefore, microbiota modulation could play a role in COVID-19 treatment. Tannins have been shown to work as prebiotics on the gastrointestinal microbiota. In particular, quebracho and chestnut tannins have shown to regulate the immune response and decrease in vitro-cytokines production, through microbiota fermentation-secondary metabolites, such as quercetin and SCFAs. OBJECTIVE: To evaluate the efficacy and the effect on cytokine levels of a tannin specific natural extract in COVID-19 patients. MATERIAL AND METHODS: This prospective, doubleblind, and randomized study was approved by the Hospital de Clínicas, José de San Martín (Buenos Aires, Argentina). Blood and stool samples were collected at baseline (Day 0) and after treatment (Day 14) during July-October 2020, with final follow-up in November 2020. We randomly assigned 124 RT-PCR confirmed COVID-19 cases (>18 years) to receive oral dry extracts of quebracho and chestnut tannins (240 mg) and B12 vitamin (0.72 μg) or placebo, twice daily for 14 days as adjunct treatment to their standard of care management. 27-pro and anti-inflammatory cytokines were measured on day 0 and 14 (Bio-Plex Pro™, Bio-Rad). Final enrollment of 140 patients with matched fecal microbiome characterization (16S, WGS and metabolites) is expected. RESULTS. Of 124 patients who were randomized (mean age 55+/-15, 63 [50.81%] male), 121 (97.58%) completed the trial. No adverse events were observed in the tannin group. Patients presenting with diarrhea (13%) had a trend to have elevated blood MIP-1α levels, which were significantly reduced by tannin treatment (Table 1). At baseline, higher levels of MIP-1α were also associated with diagnosis of pneumonia (Fig. 1), which was maintained after adjusting for confounders (age, sex, diabetes;p=0.04). Moreover, at baseline there was a positive correlation between MIP-1 α and IL-1ra, IL-2, MIP-1b and TNF-α, with all of these cytokines decreasing mostly with tannin treatment. CONCLUSION: To our knowledge, this clinical trial represents the first study to target the gut microbiome in hospitalized COVID-19 patients. Oral tannins as adjunct treatment with standard-of-care management of these patients significantly reduced proinflammatory cytokine levels that are generally associated with poor predictive outcomes, i.e. pneumonia and diarrhea. Further, our prospective studies will determine which microbiome-mediated mechanisms may attenuate the cytokine storm that is evident in COVID-19 disease pathogenesis. (Table presented) (Figure presented)

Evaluation of the Relationship between Clonal Hematopoiesis and Severe COVID-19 Disease in Rhesus Macaques

Clinical manifestations of infection with the novel SARS-CoV-2 in humans are widely varied, ranging from asymptomatic to COVID-19 respiratory failure and multiorgan damage. Profound inflammation is the hallmark of severe COVID-19 disease, and commonly does not occur until the second week of infection. Although risk factors for this late hyperinflammatory disease have been identified, most notably age and pre-existing co-morbidities, even within high-risk groups the specific factors leading to severe COVID-19 illness remain elusive. Acquired somatic mutations in hematopoietic stem and progenitor cells (HSPCs), termed clonal hematopoiesis (CH), are associated with advanced age, and loss of function (LOF) mutations in certain genes, most commonly DNMT3A and TET2, have been linked to a marked hyperinflammatory phenotype as well as clonal expansion of mutant HSPCs. Given the similar age range of frequent CH and severe COVID-19 disease, the presence of CH could impact the risk of severe COVID-19. Several human cohort studies have suggested this relationship may exist, but results to date are conflicting. Rhesus macaques (RM) have been established as a model for SARS-CoV infection and are being utilized to test therapies and vaccine development, but up to now, macaques have not been reported to develop late hyperinflammatory COVID-19 disease. We have created a robust RM model of CH by introducing LOF TET2 mutations into young adult HSPC via CRISPR/Cas9 followed by autologous transplantation, recapitulating the clonal expansion and hyperinflammatory phenotype. Thus, we hypothesized that macaques with CH could develop severe late COVID-19 disease and be utilized as a model to study disease pathophysiology or test therapeutic approaches. Macaques with either engineered (n=2) or natural CH (n=1) along with age-matched transplanted controls (n=3) were inoculated with SARS-CoV-2 and monitored clinically and via laboratory studies until 12 days post-inoculation (dpi). Macaques normally clear infection and symptoms within 3-5 days of infection. No significant differences in clinical symptoms and blood counts were noted, however, an aged animal with natural DNMT3A CH died on 10 dpi. IL-6 levels were somewhat higher in sera of the CH animals until 12 dpi, and in BAL, mean concentrations of MCP-1, IL-6, IL-8 and MIP-1b were consistently higher in CH macaques compared to controls. Interestingly, we found the median copy number of subgenomic SARS-CoV-2 RNA was higher at every timepoint in the CH group as compared with the control group, in both upper and lower respiratory samples. Lung sections from euthanasia at 10 or 12 dpi showed evidence of mild inflammation in all animals. However, in the immunohistochemical analysis, the viral antigen was detected in the lung tissues of all three animals in the CH group even at the time of autopsy, whereas only one animal of three controls had detectable viral antigen. Although the striking inflammation and serious disease have not been observed, data so far provide evidence of potential pathophysiological differences with or without CH upon SARS-CoV-2 infection. We continue to expand sample size and conduct further analyses to draw a solid conclusion, but we believe this model may be of benefit to understand the relationship between COVID-19 disease and CH. Disclosures: No relevant conflicts of interest to declare.