Effects of Toll Like Receptor Agonists and SARS-Cov-2 Antigens on Interferon (IFN) Expression by Peripheral Blood CD3+ T Cells in COVID-19 Patients (preprint)

Background: Signaling by toll like receptors (TLRs) initiates important immune responses against viral infection. The role of TLRs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is not well elucidated. Thus, we investigated the interaction of TLRs agonists and SARS-COV-2 antigens with immune cells in vitro. Material & methods: 30 coronavirus disease 2019 (COVID-19) patients (15 severe and 15 moderate) and 10 age and sex matched control (HC) were enrolled. Peripheral blood mononuclear cells (PBMCs) were isolated and activated with TLR3, 7, 8 and 9 agonists, the spike protein (SP) of SARS-CoV-2 and the Receptor Binding Domain (RBD) unit of SP. Frequencies of CD3+IFN-β+ T cells, and CD3+IFN-g+ T cells was evaluated by flow cytometry. Interferon (IFN)-b gene expression was assessed by qRT-PCR. Results: The frequency of CD3+IFN-β+ T cells was higher in moderate and severe patients at baseline in comparison with HCs. Stimulation of PBMCs from moderate patients with SP and TLR8 agonist significantly upregulated the frequency of CD3+IFN-β+ T cells (P=0.0005 and 0.0024, respectively) when compared to non-stimulated (NS) samples. The greatest increase in CD3+IFN-b+ T cell frequency in PBMCs from severe patients was seen with TLR8 and TLR7 agonists when compared to NS (P= 0.003 and 0.0167, respectively). TLR stimulation did not significantly enhance the frequency of CD3+IFN-g+ T cells generated from PBMCs from moderate and severe patients compared with unstimulated controls. However, the frequency of CD3+IFN-ɣ+ T cells in PBMCs from moderate patients was upregulated by agonists of TLR3, 8 and 9, SP and RBD when compared with NS samples from HCs. The expression of the IFN-β gene after stimulation of CD3+T cells with the TLR8 agonist was also up-regulated in moderate than severe patients (moderate vs. severe: p=0.0006). In addition, stimulation of CD3+ T cells with SP, up-regulated the expression of IFN-β gene expression in cells from patients with moderate disease (moderate vs. severe: p=0.01). Conclusion: Stimulation of PBMCs from COVID-19 patients with a TLR8 agonist and with SP enhanced IFN-b protein and gene levels. This may potentiate immune responses against SARS-CoV-2 infection and prevent viral replication and spread.

Long-standing COVID-19 disease in immunedeficient patients; case reports and literature review (preprint)

Introduction: Patients with primary or secondary immunodeficiency are at higher risk of severe disease and death following SARS-CoV-2 infection compared with the general population. We describe here the effect of rituximab therapy in 5 patients with humoral and cellular immune deficiencies (1 patient with thymoma or Good`syndrome, 1 HIV/AIDS positive patient, 2 patients with Multiple Sclerosis (MS) and 1 patient with chronic lymphocytic leukemia (CLL). T cell responses were evaluated using the QuantiFERON SARS-CoV-2 assay following incubation with the SARS-CoV-2 Ag1, Ag2 and Ag3 viral antigens. Immunephenotyping of T cells (TCD4+, TCD8+) and B cells (CD19+ and CD20+) was determined by flow cytometry. Results: All studied immunocompromised patients showed reduced cellular immune responses (release of interferon (IFN)-g) to SARS-CoV-2 antigens than healthy controls [patients; Ag1, Ag2 and Ag3 and Nil (Median 5-95% percentile) (12 (1-95), 12 (1.5-78), 13.5 (12-95)  and 3 (1-98) U/ml)], ]controls; Ag1,Ag2 and Ag3 and Nil (Median 5-95% percentile) 24.5 (7-89), 65 (31-173), 53.5 (13-71.5) and 3 (1-14) U/ml)]. The frequency of peripheral blood B cells was also reduced in these patients compared to healthy control subjects (p=0.0282). Conclusion: T-cell dependent antibody responses require the activation of B cells by helper T cells. Reduced B cell numbers in immunocompromised patients infected with SARS-CoV-2 indicates the need for these patients to take additional precautions to prevent COVID-19 infection

Decreased Serum Levels of Angiotensin Converting Enzyme (ACE)2 and Enhanced Cytokine Levels with Severity of COVID-19: Normalisation Upon Disease Recovery (preprint)

Background:SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). Circulating soluble angiotensin-converting enzyme (sACE2), the main receptor for SARS-CoV-2, together with components of the renin-angiotensin system promote infection and disease severity. Objective: In this pilot study we followed the time-course of sACE2 levels in relation to systemic cytokines in severe and moderate COVID-19 patients treated with remdesivir/dexamethasone in combination. Methods: Peripheral blood was obtained upon admission from 30 patients (12 with moderate disease and 18 with severe disease) and 14 patients with PCR-confirmed mild COVID-19. Severe and moderate patients were treated with remdesivir (200mg/first day and 100mg/day for the remaining days ) and dexamethasone (100mg/day ). 6 healthy control subjects (HC) were also enrolled. Serum interleukin (IL)-6 and IL-8 and sACE2 levels were measured by ELISA at baseline and during treatment in severe and moderate patients and at baseline in mild and HCs. Results: Baseline sACE2 levels were lower in severe (p=0.0005) and moderate (p=0.0022) patients than in patients with mild COVID-19 and in HC (p=0.0023 and p=0.0012 respectively). Serum sACE2 levels increased in patients with severe disease recovered over time compared with moderate (p=0.0021) and severe (p=0.0411) COVID-19 subjects at baseline. Systemic IL-6 and IL-8 levels were higher in all patient groups compared with HC and were not significantly affected over time or by remdesivir/dexamethasone treatment for 5 days. Conclusion: Serum sACE2 levels increase in severe COVID-19 patients as they recover over time whilst circulating cytokines are unaffected. Future studies should link these results to clinical outcomes.Funding: IMA is financially supported by the Welcome Trust (093080/Z/10/Z), the EPSRC (EP/T003189/1), and the Community Jameel Imperial College COVID-19 Excellence Fund (G26290) and by the UK MRC (MR/T010371/1). SM is supported by EU project 853850.Declaration of Interests: The authors declare that there is no conflict of interest to this article.Ethics Approval Statement: The study was approved by the institutional ethics board of the Masih Daneshvari Hospital (Ethics number SBMU.NRITLD.REC.1399.226).

Safety and Efficacy of the Combination of BCc1 and Hep-S Nanochelating-Based Medicines in Hospitalized COVID-19 Adult Patients: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial (preprint)

Background: The mortality and morbidity of COVID‐19 disease as well as the lack of a proper medication has forced researchers and clinicians to employ urgent efficient technologies to overcome this current pandemic. In the severe forms of COVID-19, the patients develop a cytokine storm syndrome (CSS) where pro-inflammatory cytokines such as IL-6 and TNF-α play a key role in the development of this serious process. The efficiency of nanomedicines - as efficient immunomodulators - that are synthesized based on nanochelating technology have been proved in the previous studies. In the present study, the therapeutic effect of the combination of BCc1 and Hep-S nanomedicines on hospitalized COVID-19 patients was evaluated. Method: Laboratory-confirmed moderate COVID-19 patients at Masih Daneshvari Hospital were enrolled to participate in a randomized, double-blind, placebo-controlled study in two separate groups: combination of BCc1 and Hep-S (N=62) (treatment) or placebo (N=60) (placebo). The primary outcome of the study was evaluating the safety of the nanomedicines combination and its effect on the number of deceased patients, while the secondary outcome was decrease in inflammatory cytokines. Results: : The evaluation of blood biochemical indices as well as clinical symptoms showed that adding the combination of BCc1 and Hep-S nanomedicines to the standard protocol of the treatment caused no adverse effects. The results analysis revealed that 28-day consumption of the nanomedicines led to a significant decrease in the mean level of IL-6 cytokine of the patients in the treatment group (p < 0.05). In addition, the patients in the treatment group had lower TNF-α levels compared to those in the control (p > 0.05) and they also showed less need for oxygen therapy. Finally, the number of the deceased patients in the treatment group was 30% lower than that of the control (p > 0.05). Conclusion: The combination of BCc1 and Hep-S, as safe nanomedicines, inhibits IL-6 as a highly important and well-known cytokine in COVID-19 pathophysiology, and presents a promising view for immunomodulation that can manage CSS and reduce mortality rate in COVID19 patients.Trial registration IRCTID, IRCT20170731035423N2. Registered 12 Jun 2020, http://www.irct.ir/ IRCT20170731035423N2.

Myeloid-derived Suppressor Cells in the Blood of Iranian COVID-19 Patients (preprint)

Background: A cytokine storm and lymphopenia are reported in coronavirus disease 2019 (COVID-19). Myeloid-derived suppressive cells (MDSCs) exist in two different forms, granulocyte (G-MDSCs) and monocytic (M-MDSCs) that both suppress T-cell function. Serum IL-6 and IL-8 levels seem to correlate with the number of blood MDSCs. Objective: In the current study we aimed to find MDSCs frequency in severe COVID-19 patients from Iran and their correlations with serum IL-8 levels. Methods: : 37 severe (8 on ventilation, 29 without ventilation) and 13 moderate COVID-19 patients together with 8 healthy subjects were enrolled at the Masih Daneshvari Hospital, Tehran-Iran between 10th April 2020- 9th March 2021. Clinical and biochemical features, serum and whole blood were obtained. CD14, CD15, CD11b and HLA-DR expression on MDSCs was measured by flow cytometry. Results: : M-MDSCs (P≤0.0001) and G-MDSCs (P≤0.0001) frequency were higher in Iranian COVID-19 patients compared to healthy subjects. M-MDSC frequency was higher in non-ventilated compared to moderate COVID-19 subjects (P=0.004). IL-8 levels were higher in patients serum with COVID-19 than in normal healthy subjects (P=0.03). IL8 level was significant difference in ventilated, non-ventilated and moderate patients (P=0.005). The frequency of G-MDSCs correlated negatively with INR (r=-0.39, P=0.02). Conclusion: Serum IL-8 levels did not correlate with the number of systemic MDSCs in COVID-19 patients. The highest levels of M-MDSCs were seen in the blood of severe non-ventilated patients. MDSC frequency in blood in the current study did not predict the survival and severity of COVID-19 patients.

Evaluation of lymphocyte subtypes in COVID-19 patients (preprint)

BackgroundAlthough the many aspects of COVID-19 have not been yet recognized, it seems that the dysregulation of the immune system has a very important role in the progression of the disease. In this study the lymphocyte subsets were evaluated in COVID-19 patients with different severity. MethodsIn this prospective study, the levels of peripheral lymphocyte subsets (CD3+, CD4+, CD8+ T cells; CD19+ and CD20+ B cells; CD16+/CD56+ NK cells, and CD4+/CD25+/FOXP3+ regulatory T cells) were measured in 67 confirmed patients with COVID-19 on the first day of admission. ResultsThe mean age of cases was 51.3 {+/-} 14.8 years. Thirty-two patients (47.8%) were classified as severe cases and 11 (16.4%) patients were categorized as critical. The frequency of blood lymphocytes, CD3+ cells, CD25+FOXP3+ T cells; and absolute count of CD3+ T cells, CD25+FOXP3+ T cells, CD4+ T cells, CD8+ T cells, CD16+56+ lymphocytes were lower in more severe cases in comparison to milder cases. Percentages of lymphocytes, T cells, and NK cells were significantly lower inthe patients who died (p= 0.002 and P= 0.042, p=0.006, respectively). ConclusionFindings of this cohort study suggests that the frequency of CD4+, CD8+, CD25+FOXP3+ T cells, and NK cells were difference in the severe COVID-19 patients. Moreover, lower frequency of, T cells, and NK cells are predictors of mortality of these patients.

Myeloid-derived suppressor cells in the blood of Iranian COVID-19 patients (preprint)

Background A cytokine storm and lymphopenia are reported in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection associated with coronavirus disease 2019 (COVID-19). Myeloid-derived suppressive cells (MDSCs) exist in two different forms, granulocyte (G-MDSCs) and monocytic (M-MDSCs) that both suppress T-cell function. Serum IL-6 and IL-8 levels seem to correlate with the number of blood MDSCs. Objective To determine the frequency of MDSCs in severe COVID-19 patients from Iran and their correlations with serum IL-8 levels. Methods 37 severe (8 on ventilation, 29 without ventilation) and 13 moderate COVID-19 patients together with 8 healthy subjects were enrolled at the Masih Daneshvari Hospital, Tehran-Iran between 10th April 2020-9th March 2021. Clinical and biochemical features, serum and whole blood were obtained. CD14, CD15, CD11b and HLA-DR expression on MDSCs was measured by flow cytometry. Results M-MDSCs (P≤0.0001) and G-MDSCs (P≤0.0001) frequency were higher in Iranian COVID-19 patients compared to healthy subjects. M-MDSC frequency was higher in non-ventilated compared to moderate COVID-19 subjects (P=0.004). Serum IL-8 levels were higher in patients with COVID-19 than in normal healthy subjects (P=0.03). IL8 level was significant difference in ventilated, non-ventilated and moderate patients (P=0.005). The frequency of G-MDSCs correlated negatively with INR (r=-0.39, P=0.02). Conclusion Serum IL-8 levels did not correlate with the number of systemic MDSCs in COVID-19 patients. The highest levels of M-MDSCs were seen in the blood of severe non-ventilated patients. MDSC frequency in blood in the current study did not predict the survival and severity of COVID-19 patients.

Decreased neutrophil phagocytosis and killing of bacteria in COVID-19 patients (preprint)

A new coronavirus disease was described in December 2019 (COVID-19) in Wuhan City, Hubei Province, China and has reached pandemic status. According to the World Health Organization, the incubation time from being infected to symptom emergence averages 5-6 days for COVID-19 but can be up to 14 days. The mortality rate varies in different countries but is greater in elderly people and in patients with cardiovascular disease, diabetes and chronic respiratory diseases. Patients with chronic respiratory diseases often have reduced neutrophil function. We sought to measure neutrophil phagocytosis and bacterial killing in COVID-19 patients. 30 COVID-19 patients and 9 healthy individuals were recruited from the Masih Daneshvari Hospital (Tehran, Iran) from March-May 2020. Polymorphonuclear (PMN) cells were isolated from whole fresh blood and incubated with green fluorescent protein (GFP) labelled methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Phagocytosis was determined by measuring the florescence of co-cultures of bacteria and neutrophils and reported as the lag time before exponential growth. The number of viable bacteria was determined after 70 h by the Colony-Forming Unit (CFU). Bacterial phagocytosis of SA (22±0.9 versus 9.2±0.5h, p<0.01) and PA (12.4±0.6 versus 4.5±0.22, p<0.01) was significantly reduced in COVID-19 patients compared with healthy control subjects. After 70h there was a significant increase in CFU in COVID-19 subjects compared with healthy control subjects for both SA (2.6±0.09 x 108 versus 0.8±0.04 x 108 CFU/ml, p<0.001) and PA (2.2±0.09 x 109 versus 1.0±0.06 x 109 CFU/ml, p<0.001).These results suggests a defect in bacterial clearance by neutrophils in COVID-19 patients.  

Increased serum levels of soluble TNF-α receptor is associated with mortality of ICU COVID-19 patients (preprint)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) has spread to almost 100 countries, infected over 10M patients and resulted in 505K deaths worldwide as of 30th June 2020. The major clinical feature of severe COVID-19 requiring ventilation is acute Respiratory Distress Syndrome (ARDS) with multi-functional failure as a result of a cytokine storm with increased serum levels of cytokines such as TNF- and IL-6 being reported. TNF- levels are increased during the cytokine storm in very ill patients and soluble receptors for IL-6 and IL-2 are present in the blood of COVID-19 patients, Objectives: To elucidate the involvement of serum levels of soluble TNF-Receptor of severe and mild COVID-19 patients to determine for severity of disease. Method: We recruited 16 severe COVID-19 patients in the ICU on ventilator support and 26 milder COVID-19 patients who were hospitalised but not within the intensive care unit (ICU) between March-May 2020 at the Masih Daneshvari Hospital Tehran, Iran. After harvesting of whole blood the serum was isolated and soluble TNF-Receptor levels measured by ELISA. Results: Serum levels of the usually inhibitory soluble TNF receptor 1 (sTNFaR1) were significantly elevated in severe COVID-19 patients at admission to ICU. High serum levels of sTNFaR1 were associated with mortality of severe COVID-19 patients treated within ICU. Conclusions: This pilot study demonstrates for role of STNF-aR1 receptor in severity of disease. Future studies should examine whether lower levels of systemic sTNFaR1 at admission may indicate a better disease outcome.