Plasma cytokine profile in patients with severe COVID-19

Background/Objectives: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease (COVID-19) enters the lung tissue through exocytosis, leading to the release of a large amount of pro-inflammatory cytokines called 'cytokine storm'. The aim was to provide more insight into relationship between plasma cytokines profile and fatal outcome of COVID-19. Method(s): Plasma cytokines (IL-17F,GM-CSF,IFNg,IL-10,CCL20/ MIP3a,IL-12P70,IL-13, IL-15,IL-17A,IL-22,IL-9,IL-1b,IL-33,IL-2,IL-21,IL-4,IL-23,IL-5,IL-6,IL-17E/IL-25,IL-27,IL-31,TNFa,TNFb,IL-28A) were detected in 30 patients with severe COVID-19 by a Luminex assay system with Milliplex Human Th17 Magnetic Premix 25 Plex Kit (HT17MG-14K-PX-25, Merk-Millipore, USA) according to the instructions. Patients were followed up for 30 days since admission to intensive care. 18 patients died and 12 patients survived during the period of observation. The control group comprised 10 individuals who had never been diagnosed with COVID-19. Result(s): IL-10 and CCL20/MIP3a plasma levels were elevated in non-survivors patients with COVID-19 compared to controls (p = 0.0027, p = 0.012, respectively). IL-15, IL-6, IL-27 plasma levels were higher in survivors with COVID-19 compared to controls (p = 0.049, p = 0.026, p = 0.00032, respectively). Interestingly, IL-15, IL-27 plasma levels were increased in non-survivors with COVID-19 compared to controls and survivors with severe COVID-19 (IL-15: p = 0.00098, p = 0.00014, respectively;IL-27: p = 0.011, p < 0.0001, respectively). Receiver operating characteristic (ROC) analysis has been conducted for IL-15 and IL-27. Cut-off value was estimated as 25.50 pg/ml for IL-15 and 1.51 pg/ml for IL-27. Conclusion(s): Our study demonstrated a more pronounced immune response in non-surviving patients with severe COVID-19. IL-15, IL-27 could be considered as a sensitive biomarker of the fatal outcome from COVID-19.

The role of interleukin-21 in COVID-19 vaccine-induced B cell-mediated immune responses in patients with kidney disease and kidney transplant recipients.

T-cell-mediated help to B cells is required for the development of humoral responses, in which the cytokine interleukin (IL)-21 is key. Here, we studied the mRNA-1273 vaccine-induced SARS-CoV-2-specific memory T-cell IL-21 response, memory B cell response, and immunoglobulin (Ig)G antibody levels in peripheral blood at 28 days after the second vaccination by ELISpot and the fluorescent bead-based multiplex immunoassay, respectively. We included 40 patients with chronic kidney disease (CKD), 34 patients on dialysis, 63 kidney transplant recipients (KTR), and 47 controls. We found that KTR, but not patients with CKD and those receiving dialysis, showed a significantly lower number of SARS-CoV-2-specific IL-21 producing T cells than controls (P < .001). KTR and patients with CKD showed lower numbers of SARS-CoV-2-specific IgG-producing memory B cells when compared with controls (P < .001 and P = .01, respectively). The T-cell IL-21 response was positively associated with the SARS-CoV-2-specific B cell response and the SARS-CoV-2 spike S1-specific IgG antibody levels (both Pearson r = 0.5; P < .001). In addition, SARS-CoV-2-specific B cell responses were shown to be IL-21 dependent. Taken together, we show that IL-21 signaling is important in eliciting robust B cell-mediated immune responses in patients with kidney disease and KTR.

AUTOPHAGY AND Acyl-CoA-BINDING PROTEIN INFLUENCE COVID-19 SEVERITY

Background: Autophagy, a cytosolic-structure degradation pathway, allows production of IL21 by CD4 T-cells and efficient cytolytic responses by CD8 T-cells. Autophagy is in part regulated by acyl-CoA-binding protein (ACBP) which has two functions. Intracellular ACBP favors autophagy, whereas secreted extracellular ACBP inhibits autophagy. Herein, we assessed whether autophagy and the ACBP pathway were associated with COVID-19 severity. Method(s): Through the BQC-19 Quebec biobank, somalogic proteomic analysis was performed on 5200 proteins in plasma samples collected between March 2020 and December 2021. Plasma from 903 patients (all data available) during the acute phase of COVID-19 were assessed. COVID-19 severity was stratified using WHO criteria. In vitro, ACBP intracellular levels, autophagy levels (LC3II) and IL21 production were assessed by flow in PBMCs after a 24h stimulation with IL6, phorbol myristate acetate (PMA)+ionomycin or lipopolysaccharide (LPS). Plasma levels of anti-SARS-CoV-2 (full spike protein or RBD) IgG were assessed by ELISA. Result(s): Median age of the cohort was 62 yo, 48% were female, 55% had comorbidities (see table). Increasing plasma levels of ACBP were found with severity (mild, moderate, severe and fatal groups having 5.3, 7.3, 9.5 and 10.6 RFU/50muL of plasma, respectively, p< 0.001 for all comparisons). Patients with comorbidities had higher plasma ACBP levels (7.4 vs 6.4 RFU/50muL, p< 0.001). Plasma ACBP levels were higher during the delta and omicron-variant periods (8.4 vs 6.8 RFU/50muL;p< 0.001). Plasma ACBP levels correlated with LC3II levels (r=0.51, P< 0.001) and IL6 (r=0.41, p< 0.001), but neither with markers IL1beta nor IL8. ACBP levels negatively correlated with IL21 levels (r=-0.27, p< 0.001), independently of age, sex, and severity. ACBP levels were not associated with levels of anti-SARS-CoV-2 IgG levels. In vitro, IL6 stimulation of healthy control PBMC induced extracellular ACBP release. Moreover, adding recombinant ACBP: 1) reduced autophagy in lymphocytes and monocytes upon polyclonal stimulation with PMA/ionomycin or LPS;2) reduced intracellular production of IL21 in T-cells after PMA/ ionomycin stimulation. Conclusion(s): Plasma ACBP levels were inversely linked with IL21 levels, suggesting that autophagy and IL21 allow control of SARS-CoV-2 infection, independently of the level of SARS-CoV-2 antibody secretion. ACBP is a targetable autophagy checkpoint and its extracellular inhibition may improve SARS-CoV-2 immune control. (Table Presented).

Clinical and immunological impact of booster immunization with recombinant mRNA vaccines for SARS-CoV-2 in patients with pemphigus and bullous pemphigoid

Vaccines based on recombinant mRNA technology helped to control the pandemic caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2). Clinical trials for showed that these vaccines are safe and effective and promote a strong type 1 driven T cell response. Yet, several reports claimed that SARS-CoV-2 mRNA vaccination might favor the onset, worsening or the reactivation of autoimmune disorders like pemphigus and bullous pemphigoid. However, no study demonstrated a direct immunological link between mRNA vaccination and disease appearance/worsening. We aimed to analyze the immunological and clinical effects exerted by mRNA booster vaccinations for SARS-CoV-2 in a cohort of patients with pemphigus (n=9), bullous pemphigoid (n=4) and in healthy individuals (n=5). Patients and healthy individuals were monitored at baseline, and after two and four weeks of mRNA vaccination. We assessed the clinical disease status, antibodies against the SARS-CoV-2 spike protein, antibody levels for BP180/230, DSG1/3 and tetanustoxoid. We also determined the distribution of peripheral T helper / T follicular cell subsets, intracellular cytokine production of T cells and cytokine serum levels. Our results show that booster vaccination increased anti spike protein IgG, while tetanustoxoid igC and skin-specific autoantibody titers were not or minimally affected. We observed an increase in Th1/Th17.1 cells, together with an increase in the intracellular production of IFN-gamma, IL-4 and IL-21 in peripheral T cells of pemphigus patients. Importantly, clinical activity in both remittent patients and in patients with active disease remained stable. In summary, vaccination with mRNA vaccines induced a specific activation of the humoral system with production of protective antibodies against the Sars-CoV-2 spike protein without affecting autoimmune disease activity in patients with pemphigus and bullous pemphigoid.Copyright © 2023

COVID-19 Vaccination Induces a Poor Interleukin-21 Memory T-cell Response in Kidney Transplant Recipients

Purpose: COVID-19-related morbidity and mortality is high among kidney patients. Several studies recently suggested low humoral and cellular immune responses after two doses of mRNA-1273 (Moderna) in these patients. Interleukin (IL)-21 is key in orchestrating an effective immune response against viral infections, is mainly produced by activated CD4+ T-cells and stimulates both humoral and cellular immunity. However, T-cell function may be impaired in kidney patients and this may explain the poor response to vaccination. Currently, there is limited data available on the vaccine-induced IL-21 memory T-cell response in these patients. We studied the induction of SARS-CoV-2-specific IL-21 memory T-cell response after mRNA- 1273 vaccination in 3 groups of kidney patients. Method(s): 113 participants were randomly selected from a prospective controlled multicenter cohort study, including 38 controls, 19 chronic kidney disease (CKD) stages G4/5 (eGFR <30 mL/min/1.73m2), 20 dialysis and 36 kidney transplant patients. All participants received 2 doses of mRNA-1273. To assess the vaccineinduced IL-21 memory T-cell response, we performed an IL-21 ELISpot (per 3.105 PBMCs) in these participants at baseline and 28 days after the second vaccination. SARS-CoV-2 S1-specific IgG antibody levels were already measured in the context of the multicenter cohort study. Result(s): Kidney transplant recipients had a significantly lower number of SARSCoV- 2-specific IL-21 producing memory T-cells when compared to controls (median of 46 versus 146, P<0.001). Participants with CKD G4/5 or on dialysis also had reduced SARS-CoV-2-specific IL-21 producing memory T-cells compared to controls (median of 128 [19-658] and 124 [7-654] versus 146 [10-635], p=0.43 and p=0.45, respectively), but the difference was less pronounced. In addition, a positive correlation was found between the number of SARS-CoV-2-specific IL-21 producing memory T-cells and SARS-CoV-2 S1-specific IgG antibody levels for all groups (Pearson correlation coefficient of 0.2, p=0.028). Conclusion(s): Kidney transplant recipients have an impaired antibody response after two doses of mRNA-1273 (Moderna), which correlates with poor SARS-CoV- 2-specific T-cell reactivity. These findings suggest that poor IL-21 memory T-cell response might hamper protection against COVID-19.

Differences in Strength and Longevity of SARS-CoV-2-Specific Adaptive Immunity Between Convalescent and Vaccinated SOT Patients and Immunocompetent Individuals

Purpose: Short-term adaptive immune memory has been reported among immunocompetent (IC) and convalescent Solid Organ Transplant (SOT) individuals following SARS-CoV-2 infection as well as after active vaccination. However, quality and longevity of anti-viral immune memory comparisons between natural and active immunization has not been thoroughly assessed among SOT. Method(s): SARS-CoV-2-specific adaptive immune memory was assessed at different compartments (serological, memory B cells [mBC] and cytokine [Th1: IFN-gamma, IL-2, IFN-gamma/IL-2 and Th2: IL-21 and IL-5] producing T cells) by ELISA and FluoroSpotbased assays, respectively, in 41 convalescent patients with severe COVID-19 (22 SOT and 19 IC) and 39 vaccinated patients (19 SOT and 20 IC) with a mRNA-based vaccine) at different time-points post immunization (T1=21days after infection/1st dose;T2=3months after infection/2nd dose;T3=6months after infection/2nd dose). Additionally, a group of convalescent mild (19 SOT and 19 IC) and asymptomatic patients (9 SOT and 10 IC) were also evaluated at T3. Result(s): Overall, statistically significant higher immune responses in all immune compartments were observed in convalescent patients than among those after vaccination. After vaccination, low seropositivity rates (5,88%) were observed among SOT after 1st dose, whereas seroconversion was fully achieved in IC patients and SOT with severe COVID-19 (p<0.001). Similarly, while the presence of mBc after vaccination progressively increased over time, it was less pronounced and significantly delayed among SOT than convalescent patients in all time points (p<0.001 T1, T2 and T3). SARS-CoV-2-specific Th1 and Th2 frequencies were significantly higher among vaccinated IC patients than SOT, being these responses significantly lower than those observed in convalescent among SOTT and IC patients (p<0.001 T1, T2 and T3). At 6 months after vaccination, IgG titers, mBc frequencies and Th1/ Th2 T-cell responses after two-dose vaccination in SOT mimicked those observed in convalescent SOT with an asymptomatic/mild clinical COVID-19 infection. Conclusion(s): The type of immunization against SARS-CoV-2, either natural or active after vaccination, clearly differentiates the quality and length of adaptive immune memory, with a clear weaker immune response observed among SOT.

IS PSORIATIC ARTHRITIS A RISK FACTOR for SEVERE COVID-19 INFECTION? DATA from the ARGENTINIAN REGISTRY SAR-COVID

Background: Comorbidities, particularly cardio-metabolic disorders, are highly prevalent in patients with psoriatic arthritis (PsA) and they were associated with an increased risk of atherosclerotic cardiovascular disease, which have been associated with higher morbidity and mortality. Whether PsA enhances the risk of SARS-CoV-2 infection or affects the disease outcome remains to be ascertained. Objectives: To describe the sociodemographic, clinical and treatment characteristics of patients with PsA with confrmed SARS-CoV-2 infection from the SAR-COVID registry and to identify the variables associated with poor COVID-19 outcomes, comparing them with those with rheumatoid arthritis (RA). Methods: Cross-sectional observational study including patients ≥18 years old, with diagnosis of PsA (CASPAR criteria) and RA (ACR/EULAR 2010 criteria), who had confrmed SARS-CoV-2 infection (RT-PCR or serology) from the SAR-COVID registry. Recruitment period was between August 13, 2020 and July 31, 2021. Sociodemographic variables, comorbidities, and treatments were analyzed. To assess the severity of the infection, the ordinal scale of the National Institute of Allergy and Infectious Diseases (NIAID)1 was used, and it was considered that a patient met the primary outcome, if they presented criteria of categories 5 or higher on the severity scale. For this analysis, Chi2 test, Fisher's test, Student's test or Wilcoxon test, and binomial logistic regression using NIAID>=5 as dependent variable were performed. Results: A total of 129 PsA patients and 808 with RA were included. Clinical characteristics are shown in Table 1. Regarding PsA treatment, 12.4% of PsA were receiving IL-17 inhibitors, 5.4% IL12-23 inhibitors, one patient apremilast and one abatacept. The frequency of NIAID≥5 was comparable between groups (PsA 19.5% vs RA 20.1%;p=0.976). (Figure 1). PsA patients with NIAID≥5 in comparison with NIAID<5 were older (58.6±11.4 vs 50±12.5;p=0.002), had more frequently hypertension (52.2% vs 23%;p=0.011) and dyslipidemia (39.1% vs 15%;p=0.017). In the multivariate analysis, age (OR 1.06;95% CI 1.02-1.11) was associated with a worse outcome of the COVID-19 (NIAID≥5) in patients with PsA, while those who received methotrexate (OR 0.34;95% CI 0.11-0.92) and biological DMARDs (OR 0.28;95% CI 0.09-0.78) had a better outcome. Conclusion: Although PsA patients have a higher frequency of cardiovascular and metabolic comorbidities than those with RA, the COVID-19 severity was similar. Most of the patients had mild SARS-CoV-2 infection and a low death rate.

COVID vaccination associated immunological cutaneous reactions

While our knowledge about the short-term side-effects of COVID-19 vaccination in adults has rapidly evolved, data about the long-term systemic side-effects and potential new onset autoimmune disorders has been limited. Here we present a case series of patients with new onset autoimmune skin conditions between 10 days and 4 weeks post mRNA COVID-19 vaccination and discuss the underlying pathophysiological changes contributing to these side-effects. Exclusions included any patients who have previously tested positive for COVID-19 or had COVID-19 symptoms. Our cases include new onset discoid lupus, localized cutaneous lupus, dermatomyositis, linear IgA bullous disease, pemphigus vulgaris, bullous pemphigoid, lichen planus pemphigoides, erosive lichen planus, psoriasis and vitiligo. In addition, we are reporting significant flare-up of pre-existing autoimmune skin conditions after a long period of remission. These include three cases of psoriasis, two cases of systemic lupus, one pemphigus vulgaris koebnerizing within a previous shingles site, and a case of pyoderma gangrenosum flare. The BNT162b2 vaccine is a potent activator of the T- and B-cell pathways. The production of interleukin (IL)-17 and IL- 21 seems to play an important role in vaccine-induced immunological protection, which is also linked to germinal centre activation linked to autoimmune disorders. This report improves our knowledge regarding some rarer potential sideeffects associated with these new vaccines and highlights the importance of further studies.

CD64 GENE-MODIFIED PRIMARY NK CELLS LOADED with ANTI-HIV bNAbs TRIGGER HIGH ADCC

Background: As part of a combined HIV CURE immuno-therapy strategy, we transduced primary human NK cells with the high affinity CD64 Fc receptor and pre-loaded them with HIV-specific bNAbs. We named these chimeric NK cells "NuKES" (NK Enhancement Strategy) for their augmented capacity to mediate ADCC and their potential clinical application as an autologous primary NK cell immuno-therapy against HIV. Methods: We transduced primary NK cells from control donors with a lentivirus expressing human CD64 in the presence or absence of irradiated K562 feeder cells expressing co-stimulatory molecules (CD40, 4-1BB) and/or cytokine pre-stimulation (IL-2, IL-21, IL-15). CD64 expressing NK cells were CFSE labeled and expanded ex vivo or FACS sorted at various times post transduction to high purity. CD64 expressing NK cells were then pre-loaded with HIV-specific bNAbs and tested in a functional ADCC CD107a degranulation assay against HIV-1 infected autologous CD4+ primary T cells. Results: After pre-stimulation with cytokines and/or irradiated K562 Feeder Cells, we could routinely achieve (n=5) greater than 40% CD64 expression in primary human NK cells (Day 14 post-transduction shown in Figure 1A). NK cells maintained strong proliferation potential with greater than 6 cells divisions beyond 10 days post transduction as determined by CFSE dilution (Day 10 post-transduction shown in Figure 1B). Phenotypically, CD64 transduced NK cells were similar to control NK cells and possessed strong expression of CD56, CD16, CD69 with intermediate levels of the NK maturation marker CD57. CD64 transduced NK cells could be successfully pre-loaded with HIV-specific bNAbs and possessed an enhanced capacity (GMFI of 2,014 versus 276) to retain 10-1074 for several hours as compared to control NK cells (Figure 1C). Functionally, CD64 transduced NK cells showed a significant two-fold increase in ADCC-triggered degranulation capacity against autologous HIV-1 infected CD4+ primary T cells compared to control NK cells after pre-loading with HIV-specific bNAbs (27.6% versus 13.2% CD107a). Conclusion: Primary human NK cells can be successfully transduced with CD64 and expanded ex vivo to high purity. Preparation of bNAbs specific NuKES represent a viable autologous NK immuno-therapy approach against HIV-1 with potential adaptation for added disease targets (i.e., COVID, Cancer) moving forward.

Intracellular Cytokine Profiling by High-Dimensional Flow Cytometry Demonstrates Highly Augmented Cytotoxicity and Overly Activated NK cells in COVID-19 Patients

Background: COVID-19 pandemic has caused more than 4.7 million deaths worldwide to date and still continues globally unabated. Numerous studies have linked the mortality in COVID-19 to aggressive immune response and cytokine storm. However, little is known about the cytokine profiles of individual immune cells that are directly involved in tissue damage. Here we investigate intracellular cytokines in individual T and NK cells of COVID-19 patients. Design: We studied 50 blood samples from 22 COVID-19 patients, 4 with mild, 6 moderate and 12 severe disease. There were 6 healthy controls. We performed high-dimensional 30-color spectral flow cytometry to characterize the immune cell subsets. For cytokine study, cells were stimulated for 6 hours, and stained for surface antigens and intracellular cytokines (IL1b, IL2, IL4, IL6, IL8, IL10, IL12, IL17a, IL21, INFg, GnzB, TNFa, and GMCSF). Data ware acquired on FACSymphony 50-parameter analyzer and analysis performed using FlowJo. Results: Our studies revealed significant differences in lymphocyte cytokine profiles between COVID+ and healthy controls (Fig 1). CD4+ and CD8+ T-cells exhibited increased percentages of IL2+ and IFNg+ cells, indicating a shift towards Th1 reaction. Granzyme B is highly upregulated in all T and NK cell subsets, demonstrating highly armed cytotoxic cells in COVID patients. The most prominent changes were noted in NK cells, 7 cytokines were highly expressed, most are proinflammatory cytokines. Of particular interest are IL-21 and GMCSF, both are known to play important roles in inflammatory cell recruitment, activation and renewal, which can lead to augmented tissue inflammation and injury. These changes were already evident in patients with mild disease, but there is heightened cytokine production in severe cases. Conclusions: Using high-dimensional flow cytometry we demonstrated for the first time significantly increased production of multiple proinflammatory cytokines and cytotoxic molecules in individual T and NK cells of COVID-19 patients. NK cells are most drastically activated. It is conceivable that when recruited to the target tissue such as lung, these highly primed cells will play a major role in tissue injury and ultimately organ failure via their direct cytotoxicity and cytokine secretion. This is consistent with previous reports of increased NK cells in the COVID lungs. Analysis of NK cell cytokine profiles may serve to predict disease progression, and reveal new targets for immune-therapy for severe COVID patients. (Table Presented).

The impact of methotrexate and targeted immunosuppression on cellular and humoral immune responses to COVID-19 vaccine BNT162b2: a cohort study

Individuals on immunosuppression were excluded from COVID-19 vaccine trials. We evaluated immune responses to COVID-19 vaccine BNT162b2 (Pfizer-BioNTech) in people taking methotrexate and biologics. Given the roll out of extended interval vaccination programmes to maximise population coverage, we present findings following the first dose. We recruited individuals with psoriasis (n=84) established on methotrexate or biologic monotherapy (TNF, IL-17 or IL-23 inhibitors) and healthy controls (n=17). Immunogenicity was evaluated pre and post (day 28) vaccine. Seroconversion rates were lower in patients taking immunosuppression (78%, 95%CI 67-87%) compared to controls (100%, 95%CI 79-100%), with the lowest rate in those on methotrexate (50%, 95%CI 26-74%). Neutralising activity to wild-type SARS-CoV-2 was lower in patients receiving methotrexate (median ID50 152, IQR 47-257) compared to controls (median ID50 316, IQR 212-481, p<0.01), but preserved in those receiving biologics (median ID50 280, IQR 137-428). Neutralising titres against B.1.1.7 were comparably low in all participants. Spike-specific T cell responses (including IFNγ, IL-2, IL-21) were induced in all groups, and were equivalent among individuals receiving methotrexate, biologics and controls. Functional humoral immunity to a single dose of BNT162b2 is impaired by methotrexate but not by biologics, while cellular responses are unaffected. Seroconversion alone may not adequately reflect vaccine immunogenicity in individuals with immune-mediated disease receiving immunosuppression. Real-world pharmacovigilance studies will determine whether these findings translate to clinical effectiveness.

Longitudinal Transcriptional Analysis of Peripheral Blood Leukocytes in COVID Convalescent Donors

Introduction Severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) can induce a strong host immune response. Several groups have investigated the course of antibody responses in patients recovering from SARS-CoV-2 infections but little is known about the recovery of cellular immunity. This study investigated the cellular immune response in people who had recovered from SARS-CoV2 infection. Methods 162 coronavirus disease 2019 (COVID-19) convalescent plasma donors (CCD) and 40 healthy donor (HD) controls were enrolled prospectively in an IRB-approved protocol (Clinical Trials Number: NCT04360278) and provided written informed consent to participate in the study. Using the nCounter platform and host response panel with 785 genes across more than 50 pathways, we compared transcriptomic profiles on RNA samples obtained from the peripheral blood leukocytes of these 162 CCD and 40 HD. Additionally, in 69 of the 162 CCD samples, we evaluated transcriptomic trends at more than one-time point during the convalescent period. Results Age, sex, ethnicity, and body mass index distributions were similar among the CCD and HD. With respect to baseline complete blood counts, hemoglobin, platelets, and absolute basophil and eosinophil counts, all were similar among CCD and HD (Table 1). However, despite sample collections occurring several days after convalescence, mean counts for absolute neutrophil counts, absolute monocyte counts, and absolute lymphocyte counts were significantly higher among CCD compared to HD. 30-90 days after diagnosis, 19 of 773 genes differed (FDR < 0.05) between the average CCD and HD samples. Up-regulated genes included MAFB, CTLA4, PTGS2, and the chemokine signaling genes CXCR4, CXCL5, CXCL2 and CCR4. Down-regulated genes included PTGER2, CASP8, and the interleukins IL36A, IL31, IL20 and IL21 (Figure 1 a,b). Differential gene expression persisted for months. At 90-120 days, 13 genes were differentially regulated, including again MAFB CXCR4, PTGS2, CXCL2 and PTGER2, plus SMAD4. At 120-150 days post-diagnosis, 58 genes were differentially expressed (FDR < 0.05) compared to HD. Pathways with up-regulated genes included Treg differentiation, type III interferon signaling and chemokine signaling. 150-360 days post-diagnosis, 4 genes remained up-regulated on average (FDR < 0.05): PTGS2, PIK3CR, CXCL1 and SMAD4 (Figure 1 c,d). Individual patients varied considerably from the mean trend. Scoring samples by their similarity to the gene expression profile of the mean HD sample, 21 CCD samples from 20 unique patients (12%) were identified as highly perturbed from HD. 84% of these highly perturbed samples were collected > 90 days post-diagnosis. Of these 21 samples, 6 were distinguished by > 2-fold up-regulation of a cluster of interleukin and type-1 interferon genes (Figure 2). Conclusions Overall, our study identified important gene expression trends in CCD compared to HD in the post-acute period. The changes varied with time and among donors. As the expression of T-cell inhibitory molecule CTLA4 fell, the number of differentially expressed increased with the most marked changes occurring 120 to 150 days post-diagnosis in genes in chemokine signaling, type III interferon signaling and Treg pathways. Persistent alterations in inflammatory pathways and T-cell activation/exhaustion markers for months after active infection may help shed light on the pathophysiology of a prolonged post-viral syndrome observed in individuals following recovery from COVID-19 infection. Our data may serve as the basis for risk modification strategies in the period of active infection. Future studies may inform the ability to identify druggable targets involving these pathways to mitigate the long-term effects of COVID-19 infection. [Formula presented] Disclosures: Danaher: NanoString Technologies: Current Employment, Current holder of individual stocks in a privately-held company.

Interleukin-21: a potential biomarker for diagnosis and predicting prognosis in COVID-19 patients

Background/aim: COVID-19 patients have a wide spectrum of disease severity. Several biomarkers were evaluated as predictors for progression towards severe disease. IL-21 is a member of common γ-chain cytokine family and creates some specific effects during programming and maintenance of antiviral immunity. We aimed to assess IL-21 as a biomarker for diagnosis and outcome prediction in patients hospitalized with COVID-19. Materials and methods: Patients with a preliminary diagnosis of COVID-19 and pneumonia other than COVID-19 admitted to a tertiary care hospital were included consecutively in this comparative study. Results: The study population consisted of 51 patients with COVID-19 and 11 patients with non-COVID-19 pneumonia. Serum IL-21 concentration was markedly higher, and serum CRP concentration was significantly lower in COVID-19 patients compared to non-COVID-19 pneumonia patients. Within COVID-19 patients, 10 patients showed radiological and clinical progression. Patients with clinical worsening had lower lymphocyte count and haemoglobin. In addition to that, deteriorating patients had higher urea, LDH levels, and elevated concentration of both IL-6 and IL-21. The cut-off value of 106 ng/L for IL-21 has 80.0% sensitivity, %60.9 specificity for discriminating patients with clinical worsening. Multivariable analysis performed to define risk factors for disease progression identified IL-6 and IL-21 as independent predictors. Odds ratio for serum IL-6 concentrations ≥ 3.2 pg/mL was 8.07 (95% CI: 1.37-47.50, p = 0.04) and odds ratio for serum IL-21 concentrations ≥ 106 ng/L was 6.24 (95% CI: 1.04 ­ 37.3, p = 0.02). Conclusion: We identified specific differences in serum IL-21 between COVID-19 and non-COVID-19 pneumonia patients. Serum IL-21 measurement has promising predictive value for disease progression in COVID-19 patients. High serum IL-6 and IL-21 levels obtained upon admission are independent risk factors for clinical worsening.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.

A clinical trial of IL-15 and IL-21 combination therapy for COVID-19 is warranted.

Previous studies of SARS-CoV-2 viral infection suggest that both the humoral and cytotoxic arms of the immune system are weak in patients with severe COVID-19 disease when compared to mild disease. A cytokine storm is also induced in severe disease. IL-15 has been shown to support the cytotoxic arm of the immune response. IL-21 has been shown to support both the cytotoxic and humoral arms of the immune response. In addition, in some settings, Il-21 has been shown to actually decrease IL-6 and TNF-alpha production, reducing the inflammatory proteins involved in the cytokine storm. Furthermore, in other settings, the combination of IL-15 and IL-21 has been shown to be more effective than either interleukin alone in promoting an effective immune response. Therefore, a clinical trial that examines the use of the combination of IL-15 and IL-21 for COVID-19 patients is warranted.