Features of the Immune Response in COVID-19

BACKGROUND This review is devoted to the analysis of the features of the immune response in COVID-19. The review indicates the clinical manifestations of COVID-19, modern data on the immunopathogenesis of the disease and its complications are considered. AIM OF STUDY To clarify some pathogenetic mechanisms of the immune response in COVID-19, which can help in creating an algorithm for examining patients for early prognosis and prevention of severe course and complications of the disease. MATERIAL AND METHODS To achieve this goal, the results of domestic and foreign scientific studies on the pathogenesis, diagnosis and treatment of COVID-19 were analyzed. The literature search was carried out in electronic search engines Scopus and PubMed. For the analysis, scientific articles published in the period from 2019 to 2021 were selected;88% of analyzed works are not older than 5 years. CONCLUSION The late production of type I IFN, an increase in the level of pro-inflammatory monocytes, a decrease in the expression of HLA-DR on monocytes, violation of the presentation of the virus and the formation of specific lymphocytes, the death of T-lymphocytes and profound immunosuppression are of greatest importance for the development of a severe form of COVID-19. © 2023 Sklifosovsky Research Institute for Emergency Medicine. All rights reserved.

Evaluation of expression and titration of recombinant nucleocapsid protein as an immunogenic candidate against SARS-CoV2

Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate. Materials and Methods: B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Results: The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high- frequency distribution was improved to 85%. Expression of recombinant protein in E. coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000. Conclusion: According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.

CD71+ erythroid cells suppress T-cell effector functions and predict immunotherapy outcomes in patients with virus-associated solid tumors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer. However, only a portion of patients respond to such treatments. Therefore, it remains a prevailing clinical need to identify factors associated with acquired resistance or lack of response to ICIs. We hypothesized that the immunosuppressive CD71+ erythroid cells (CECs) within the tumor and/or distant 'out-of-field' may impair antitumor response. METHODS: We studied 38 patients with cancer through a phase II clinical trial investigating the effects of oral valproate combined with avelumab (anti-programmed death-ligand 1 (PD-L1)) in virus-associated solid tumors (VASTs). We quantified the frequency/functionality of CECs in blood and biopsies of patients. Also, we established an animal model of melanoma (B16-F10) to investigate the possible effects of erythropoietin (EPO) treatment on anti-PD-L1 therapy. RESULTS: We found a substantial expansion of CECs in the blood of patients with VAST compared with healthy controls. We noted that the frequency of CECs in circulation was significantly higher at the baseline and throughout the study in non-responders versus responders to PD-L1 therapy. Moreover, we observed that CECs in a dose-dependent manner suppress effector functions of autologous T cells in vitro. The subpopulation of CD45+CECs appears to have a more robust immunosuppressive property compared with their CD45- counterparts. This was illustrated by a stronger expression of reactive oxygen species, PD-L1/PD-L2, and V-domain Ig suppressor of T-cell activation in this subpopulation. Lastly, we found a higher frequency of CECs in the blood circulation at the later cancer stage and their abundance was associated with anemia, and a poor response to immunotherapy. Finally, we report the expansion of CECs in the spleen and tumor microenvironment of mice with melanoma. We found that although CECs in tumor-bearing mice secret artemin, this was not the case for VAST-derived CECs in humans. Notably, our results imply that EPO, a frequently used drug for anemia treatment in patients with cancer, may promote the generation of CECs and subsequently abrogates the therapeutic effects of ICIs (eg, anti-PD-L1). CONCLUSIONS: Our results demonstrate that anemia by the expansion of CECs may enhance cancer progression. Notably, measuring the frequency of CECs may serve as a valuable biomarker to predict immunotherapy outcomes.

Effector memory T cell subset CD45RA-CCR7-CD27-CD28- EM3 increases in direct proportion to the disease severity of COVID-19

COVID-19, which emerged in December 2019 and continues to wreak havoc, has led to the death of many people around the world. In this study, we aimed to uncover the variables underlying the exacerbation of the disease by considering the changes in T cell subsets in adults and juveniles with different disease severity of COVID-19. Peripheral blood samples of 193 patients (128 adults and 65 juveniles) diagnosed with COVID-19 were evaluated in a flow cytometer, and a broad T cell profile was revealed by examining T cell subsets in terms of exhaustion and senescence. We found remarkable differences in the effector memory (EM;CD45RA-CCR7-) cell subsets of severe pneumonia cases. The frequencies of EM2 CD4+ T, EM3 CD4+ T, EM3 CD8+ T, EM2 DN T and EM3 DN T cells were found to increase in severe pneumonia cases. Consistently, these cells were found in juveniles and uncomplicated adults in similar or lower proportions to healthy controls. The findings of our study provide a view of the T cell profile that may underlie differences in the course of COVID-19 cases in juveniles and adults and may provide new insights into the development of effective treatment strategies.

T cells and subgroups in children with COVID-19

Objective: In COVID-19 disease, it was observed that T lymphocytes decreased numerically, both CD4+ and CD8+ could decrease, and sometimes the CD8+ level increased significantly. The virus-specific CD8+ T-cells are thought to be TEM or TEMRA cells. However, the characteristics of these cells, particularly their role in the pathogenesis of SARS-CoV-2 infection or COVID-19 disease, are unclear. Therefore, this study aimed to examine the flow cytometric changes observed in T helper, T cytotoxic cells, and subtypes during diagnosis in pediatric patients diagnosed with COVID-19 infection with SARS-CoV-2 PCR positivity. Methods: Twenty-two children aged 0-18, diagnosed with COVID-19, with flow cytometry;T Helper Cell (TH), T Cytotoxic Cell (TC), T Naive Cells (TN), Central Memory (TCM), Effector Memory (TEM), RA + Effector memory (TEMRA) and Recent Thymic Emigrants (RTEs) were studied. Results: T cell counts were found to be expected in all age groups. The CD4/CD8 ratio increased in the under-five and over 16 age group. While TCM among CD4+T cells decreased in the group above 16 years of age, TEM decreased in all age groups. RTEs decreased in all except the age group 16+. Naive CD8+ T cells (TN) were found to be high in all age groups. Conclusion: A low number of CD4+ and CD8+ lymphocytes have been reported as a distinctive laboratory finding in 2019 Coronavirus disease (COVID-19). Having enough naive T cells is essential for the immune system to respond consistently to unknown pathogens. This study found that these cells were higher than expected in children.

Modern aspects of COVID-19 in HIV-positive patients (literature review)

The article is devoted to the global problems of modern medicine - HIV infection and the COVID-19 pandemic. The review of the literature highlights current ideas about the pathogenesis and course of COVID-19 in patients with HIV infection, and also touches upon the problems of concomitant pathology and mental health of patients with HIV in the setting of the COVID-19 pandemic. It has been shown that HIV-positive patients are a risk group for the severe course of COVID-19, in particular, individuals with severe immunodeficiency (CD4+ T lymphocytes 200 cells/l) due to the development of synergetic lung damage by SARS-CoV-2 and secondary infectious agents such as cytomegalovirus and Pneumocystis carinii. It has been proven that one of the targets of the SARS-CoV-2 virus is CD4+ T cells, which in COVID-19 leads to a more rapid progression of immunodeficiency in patients with HIV infection and, thus, significantly increases the risk of secondary diseases and death. Particular attention should be paid to middle-aged and elderly people living with HIV, who, compared with HIV-negative patients, are more likely to have concomitant pathology - arterial hypertension, cardiomyopathy and diabetes mellitus, which are the risk factors for severe COVID-19. The results of studies on the effect of antiretroviral drugs on the course of COVID-19 showed that HIV-infected patients receiving tenofovir + emtricitabine have a lower risk of severe COVID-19 and associated hospitalization than patients receiving other HIV treatment regimens. Clinical and preclinical data support the potential use of tenofovir in the treatment of novel coronavirus infection.

Alterations in immunophenotype and metabolic profile of mononuclear cells during follow up in children with multisystem inflammatory syndrome (MIS-C).

Introduction: Although children seem to be less susceptible to COVID-19, some of them develop a rare but serious hyperinflammatory condition called multisystem inflammatory syndrome in children (MIS-C). While several studies describe the clinical conditions of acute MIS-C, the status of convalescent patients in the months after acute MIS-C is still unclear, especially the question of persistence of changes in the specific subpopulations of immune cells in the convalescent phase of the disease. Methods: We therefore analyzed peripheral blood of 14 children with MIS-C at the onset of the disease (acute phase) and 2 to 6 months after disease onset (post-acute convalescent phase) for lymphocyte subsets and antigen-presenting cell (APC) phenotype. The results were compared with six healthy age-matched controls. Results: All major lymphocyte populations (B cells, CD4 + and CD8+ T cells, and NK cells) were decreased in the acute phase and normalized in the convalescent phase. T cell activation was increased in the acute phase, followed by an increased proportion of γ/δ-double-negative T cells (γ/δ DN Ts) in the convalescent phase. B cell differentiation was impaired in the acute phase with a decreased proportion of CD21 expressing, activated/memory, and class-switched memory B cells, which normalized in the convalescent phase. The proportion of plasmacytoid dendritic cells, conventional type 2 dendritic cells, and classical monocytes were decreased, while the proportion of conventional type 1 dendritic cells was increased in the acute phase. Importantly the population of plasmacytoid dendritic cells remained decreased in the convalescent phase, while other APC populations normalized. Immunometabolic analysis of peripheral blood mononuclear cells (PBMCs) in the convalescent MIS-C showed comparable mitochondrial respiration and glycolysis rates to healthy controls. Conclusions: While both immunophenotyping and immunometabolic analyzes showed that immune cells in the convalescent MIS-C phase normalized in many parameters, we found lower percentage of plasmablasts, lower expression of T cell co-receptors (CD3, CD4, and CD8), an increased percentage of γ/δ DN Ts and increased metabolic activity of CD3/CD28-stimulated T cells. Overall, the results suggest that inflammation persists for months after the onset of MIS-C, with significant alterations in some immune system parameters, which may also impair immune defense against viral infections.

The Study of the Immune Status in Post-Covid Syndrome by Flow Cytometry

A novel coronavirus infection was described in 2019 in Wuhan, China. From the first months of the spread of the infection around the world, evidence began to appear that patients after recovery had various symptoms. Duration, intensity, and variability of symptoms vary among patients and are often not associated with the severity of the most acute illness. Recently the concept of post-COVID syndrome (postCOVID or long-COVID in the English-language literature) has acquired increasingly clear diagnostic criteria. Persistent symptoms and / or the appearance of delayed complications after 4 weeks or more from the onset of symptoms of an acute illness are commonly called post-COVID syndrome. The wide range of symptoms that can occur in patients with post-COVID syndrome is now a major health concern worldwide. A proper clinical evaluation will help determine the etiology and build a treatment plan. Longer studies aimed at identifying the effects of COVID-19, possible risk factors for their development, a detailed study of the pathogenetic mechanisms of SARS-CoV-2, and the development of treatment and rehabilitation methods to improve the mental and physical health of surviving patients are relevant elements of study for the foreseeable future. T-lymphocytes are a poorly studied population of T lymphocytes. These cells are more often localized in the mucous membranes of the body which have the properties of innate and acquired immunity. The main biological functions are cytolysis, immunoregulation which indicates an important immunocompetent role of this type of cell population in severe infectious diseases. This article provides information on the fraction of T-lymphocytes during the formation of adaptive immunity in patients with post-COVID syndrome.

A systematic review and meta-analysis of the relationship between T-lymphocytes and respiratory tract infection in children.

The objective of this paper was to investigate the relationship between T-lymphocytes and respiratory tract infection in children. A meta-analysis was performed of studies related to virus-infected respiratory illnesses in children, and the change in the ratio of their T-lymphocyte subsets CD4+/CD8+. A systematic literature review was performed using MEDLINE (through PubMed), CINAHL (via Ebsco), Scopus, and Web of Science, for studies describing change in T-lymphocyte levels in children suffering from acute respiratory illnesses. Studies were included as per the Population, Intervention, Comparison, Outcomes and Study (PICOS) criteria, and relevant event data were extracted. A risk of publication bias and a risk of bias assessment were performed, and a funnel plot was designed using RevMan software. A column histogram was designed to compare the adverse effects. A total of 12 studies from the years 2000-2022 were included in the meta-analysis, containing information about 1111 patients. The current meta-analysis has a low risk of publication bias with the Egger's test p-value being 0.583 (p > 0.05) and the Begg's test p-value being 0.772 (p > 0.05). The odds ratio (OR) value was 3.66 (95% confidence interval (95% CI): 1.08-12.43), the risk ratio (RR) value was 1.91 (95% CI: 1.07-3.40) and the significance level was p < 0.05, which indicates that an alteration in T-lymphocyte levels occurs in respiratory infections. T-lymphocyte levels are altered during infection, and the association between T-lymphocytes and respiratory diseases in children was investigated in this study. Based on statistically significant data (p < 0.05), we concluded that T-lymphocyte levels are adjusted in the event of viral respiratory sickness in children to alleviate the infection.

Enhanced inhibition of MHC-I expression by SARS-CoV-2 Omicron subvariants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) possess mutations that confer resistance to neutralizing antibodies within the Spike protein and are associated with breakthrough infection and reinfection. By contrast, less is known about the escape from CD8+ T cell-mediated immunity by VOC. Here, we demonstrated that all SARS-CoV-2 VOCs possess the ability to suppress major histocompatibility complex class I (MHC-I) expression. We identified several viral genes that contribute to the suppression of MHC I expression. Notably, MHC-I upregulation was strongly inhibited after SARS-CoV-2 but not influenza virus infection in vivo. While earlier VOCs possess similar capacity as the ancestral strain to suppress MHC-I, the Omicron subvariants exhibited a greater ability to suppress surface MHC-I expression. We identified a common mutation in the E protein of Omicron that further suppressed MHC-I expression. Collectively, our data suggest that in addition to escaping from neutralizing antibodies, the success of Omicron subvariants to cause breakthrough infection and reinfection may in part be due to its optimized evasion from T cell recognition.

Analysis of clinical characteristics and warning factors in SARS-CoV-2 variants infection

Objective: To investigate the influence of the variation of SARS-CoV-2 on the clinical feature, and to provide early warning signs for the variation of SARS-CoV-2 in clinical work. Methods: From Jan 2, 2021 to Jun 30, 2021, a total of 105 COVID-19 patients were included in the study using a case-control method. Nasal swab samples were collected from the study subjects, the viral genes were sequenced, and patients were divided into Delta variant group and non-Delta variant group according to their gene sequences. Clinically relevant data were collected from the two groups, and indicators such as days of hospitalization, age distribution, lymphocytes, neutrophils, B lymphocytes, NK cells, IL-4, and IL-10 were compared;subgroup analysis was performed based on the number of days of viral negativity in the study subjects as the basis for grouping, and differences in immunological characteristics were compared, including lymphocytes, neutrophils, B lymphocytes, NK cells, IL-4, IL-10, etc. Results: The theoretical hospitalization days of Delta variant group were (22.2..8.33) d, which were significantly longer than (17.6 .. 10.50) d of non-Delta variant group (t = 2.396, P < 0.05). The total lymphocyte count and IL-4 of Delta variant group were (1.22..0.86) ..109/L and (0.80 .. 0.23) ng/mL, which were significantly lower than corresponding (1.91 .. 0.70) ..109/L and (1.59 .. 0.59) ng/mL of non-Delta variant group (t = 4.329, 9.072, P < 0.05), while IL-10 was (7.16 .. 7.77) ng/mL, which was significantly higher than (4.26 .. 3.91) ng/mL of non-Delta mutation group (t = 1.980, P < 0.05). Subgroup analysis showed that the total lymphocyte count and IL-4 concentration in Delta variant group were (1.04 .. 0.60) ..109/L and (0.74 .. 0.25) ng/ml, which were significantly lower than corresponding (1.62..0.56) ..109/L and (1.56 .. 0.52) ng/mL in non-Delta variant group, in patients with delayed discharge (P < 0.05). Conclutions SARS-CoV-2 variant has an impact on clinical manifestations. The patient's B cell count and IL-10 concentration increased or IL-2 and IL-4 concentration decreased within 12 hours of admission indicated variant virus infection. The decrease of total lymphocyte count, especially T lymphocyte reduction, strongly suggests discharge delay due to viral clearance disorder.

Natural killer (NK) cells: an innate defense shield against respiratory viral infections

In general, B and T lymphocytes, which are involved in adaptive immunity, are in charge of cell-mediated response and antibody-mediated immunity, respectively. Another subset of lymphocytes, known as natural killer (NK) cells, are innate effector cells. They serve as the body's initial line of defence against viral infections. They perform the task of eliminating stressed cells and are crucial for tumour immunity. These cells are capable of performing their killing function without clonal expansion and differentiation following activation. The NK cells will immediately eliminate infected host cells but other lymphocytes need lymphocyte proliferative response which takes several days and further differentiate into effector cells, so that they eliminate host cells infected by the viral pathogen. The NK cells also form a bridge between the adaptive and innate immunity and play significant roles during respiratory infection. Number and the role of NK cells correlate with the severity of severe acute respiratory syndrome (SARS);the number and the percentage of CD158b+ NK cells in severe SARS infection were significantly less in number than those with mild cases. Innate defence mechanisms, particularly NK cells, are able to control SARS infection even in the absence of T cells and antibodies, according to cellular immunological responses to SARS infection in mice. As a result, NK cells are crucial in the fight against viral infections of the respiratory system. As an innate immune system, they serve as the initial line of virus protection. It is possible to do additional research to take advantage of this NK cell trait and develop a cutting-edge therapeutic approach to fight developing respiratory viral diseases.

Synthesis of killer T cell epitopes for peptide-based vaccine of SARS-CoV-2. (Special Issue: Chemistry and global challenges (Part B).)

COVID-19 is a current global pandemic, which has prompted many countries to develop ways to deal with it. Peptides have many medicinal and diagnostic benefits, so recently, many researchers have been developing peptide-based vaccines against COVID-19. In peptide-based vaccines, peptides act as specific antigens that will provide a faster immune response because they do not go through the process of cutting proteins in the Major Histocompatibility complex (MHC) antigen-presenting cells (APC) and can be directly presented outside the cells so that they can be recognized by the host killer T cells (CTL). Vaccine development can be accelerated with the help of immunoinformatic to predict specific epitopes to induce the CTL. We have predicted the CTL epitope through the immunoinformatic method. This study aims to synthesize candidate CTL epitopes as a candidate for the SARS-CoV-2 vaccine using the SPPS method with the Fmoc/t-Bu strategy. In this study, two CTL epitopes were synthesized through a conventional solid-phase peptide synthesis (SPPS) method, and another CTL epitope was synthesized using a semi-automated peptide synthesizer. The SPPS method is faster because the purification is only carried out at the final stage, while the Fmoc/t-Bu strategy was applied because it provides a mild reaction condition. Both synthetic approaches were compared. The semi-automated peptide synthesizer made the synthesis faster and more efficient due to the use of an inert gas (N2) during the synthesis. The synthetic peptides were characterized by TOF-ESI-MS. The three peptides showed ion peaks at m/z 1137.5509 (M+H)+, 1064.3468 (M+H)+, and 916.5859 (M+H)+, indicating correct molecular ion peaks for EILDITPCSF, IPIGAGICASY, and FIAGLIAIV, respectively.

Cerebral Cryptococcosis Associated with CD4+ T-lymphocytopenia in Non-HIV Patients after SARS-CoV-2 Infection: Case Series in a Specialized Institute in Lima, Peru.

Cases of cryptococcosis have been reported in patients with COVID-19. The majority are in patients with severe symptoms or who received immunosuppressants. However, there is still no clear association between COVID-19 and cryptococcosis. We report eight cases of cerebral cryptococcosis associated with CD4+ T lymphocytopenia in non-HIV patients after SARS-CoV-2 infection. The median age was 57 years and 5/8 were male. In addition, 2/8 of patients had diabetes, and 8/8 had a history of mild COVID-19, with a median of 75 days before diagnosis of cerebral cryptococcosis. All patients denied having received prior immunosuppressive therapy. The most frequent symptoms were confusion (8/8), headache (7/8), vomiting (6/8), and nausea (6/8) All patients were diagnosed by isolating Cryptococcus in cerebrospinal fluid. The median CD4+ and CD8+ T lymphocytes were 247 and 173.5, respectively. Other causes of immunosuppression, such as HIV or HTLV infection, were excluded in all patients. Finally, three patients died, and one presented long-term visual and auditory sequelae. The CD4+/CD8+ T lymphocyte count normalized during follow-up in those patients who survived. We hypothesize that CD4+ T lymphocytopenia in the patients in this case series could increase the risk of cryptococcosis after SARS-CoV-2 infection.

Comparison of T Lymphocyte Subsets and Natural Killer Lymphocytes in Moderate Versus Severe COVID-19 Patients.

Severe respiratory involvement that follows a process of immune dysregulation and intense cytokine production remains to be the most dreaded complication of Coronavirus Disease-2019 (COVID-19) infection. The aim of this study was to analyze T lymphocyte subsets and natural killer (NK) lymphocytes in moderate and severe cases of COVID-19 infection and assess their significance in disease severity and prognosis. Twenty moderate cases and 20 severe cases of COVID-19 were studied and compared regarding blood picture, biochemical markers, T lymphocyte population subsets, and NK lymphocytes, which were determined by flow cytometric analysis. On analyzing the flow cytometric data of T lymphocyte cells and their subsets and NK cells in two groups of COVID-19 infection (one group moderate and the other severe cases), some immature NK lymphocyte relative and absolute counts were higher in the severe patients with worse outcome and death, while some mature NK lymphocyte relative and absolute counts were depressed in both groups. Also, interleukin (IL)-6 was significantly higher in severe cases when compared to moderate cases, and there was a positive significant correlation between immature NK lymphocyte relative and absolute counts and IL-6. There was no statistically significant difference between T lymphocyte subsets (T helper and T cytotoxic) with disease severity or outcome. Some immature NK lymphocyte subsets contribute to the widespread inflammatory response that complicates severe cases of COVID-19; therapeutic approaches directed to enhancing NK maturation or drugs that block NK cell inhibitory receptors have a potential role in controlling COVID-19 induced cytokine storm.

Correlation of Lymphocyte Subpopulations, Clinical Features and Inflammatory Markers during Severe COVID-19 Onset.

BACKGROUND: Dysregulation of the immune response in the course of COVID-19 has been implicated in critical outcomes. Lymphopenia is evident in severe cases and has been associated with worse outcomes since the early phases of the pandemic. In addition, cytokine storm has been associated with excessive lung injury and concomitant respiratory failure. However, it has also been hypothesized that specific lymphocyte subpopulations (CD4 and CD8 T cells, B cells, and NK cells) may serve as prognostic markers for disease severity. The aim of this study was to investigate possible associations of lymphocyte subpopulations alterations with markers of disease severity and outcomes in patients hospitalized with COVID-19. MATERIALS/METHODS: A total of 42 adult hospitalized patients were included in this study, from June to July 2021. Flow-cytometry was used to calculate specific lymphocyte subpopulations on day 1 (admission) and on day 5 of hospitalization (CD45, CD3, CD3CD8, CD3CD4, CD3CD4CD8, CD19, CD16CD56, CD34RA, CD45RO). Markers of disease severity and outcomes included: burden of disease on CT (% of affected lung parenchyma injury), C-reactive protein and interleukin-6 levels. PO2/FiO2 ratio and differences in lymphocytes subsets between two timepoints were also calculated. Logistic and linear regressions were used for the analyses. All analyses were performed using Stata (version 13.1; Stata Corp, College Station, TX, USA). RESULTS: Higher levels of CD16CD56 cells (Natural Killer cells) were associated with higher risk of lung injury (>50% of lung parenchyma). An increase in CD3CD4 and CD4RO cell count difference between day 5 and day 1 resulted in a decrease of CRP difference between these timepoints. On the other hand, CD45RARO difference was associated with an increase in the difference of CRP levels between the two timepoints. No other significant differences were found in the rest of the lymphocyte subpopulations. CONCLUSIONS: Despite a low patient number, this study showed that alterations in lymphocyte subpopulations are associated with COVID-19 severity markers. It was observed that an increase in lymphocytes (CD4 and transiently CD45RARO) resulted in lower CRP levels, perhaps leading to COVID-19 recovery and immune response homeostasis. However, these findings need further evaluation in larger scale trials.

Manufacture and Characterization of Good Manufacturing Practice-Compliant SARS-COV-2 Cytotoxic T Lymphocytes.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 virus-specific cytotoxic T-cell lymphocytes (vCTLs) could provide a promising modality in COVID-19 treatment. We aimed to screen, manufacture, and characterize SARS-CoV-2-vCTLs generated from convalescent COVID-19 donors using the CliniMACS Cytokine Capture System (CCS). METHODS: Donor screening was done by stimulation of convalescent COVID-19 donor peripheral blood mononuclear cells with viral peptides and identification of interferonγ (IFN-γ)+ CD4 and CD8 T cells using flow cytometry. Clinical-grade SARS-CoV-2-vCTLs were manufactured using the CliniMACS CCS. The enriched SARS-CoV-2-vCTLs were characterized by T-cell receptor sequencing, mass cytometry, and transcriptome analysis. RESULTS: Of the convalescent donor blood samples, 93% passed the screening criteria for clinical manufacture. Three validation runs resulted in enriched T cells that were 79% (standard error of the mean 21%) IFN-γ+ T cells. SARS-CoV-2-vCTLs displayed a highly diverse T-cell receptor repertoire with enhancement of both memory CD8 and CD4 T cells, especially in CD8 TEM, CD4 TCM, and CD4 TEMRA cell subsets. SARS-CoV-2-vCTLs were polyfunctional with increased gene expression in T-cell function, interleukin, pathogen defense, and tumor necrosis factor superfamily pathways. CONCLUSIONS: Highly functional SARS-CoV-2-vCTLs can be rapidly generated by direct cytokine enrichment (12 hours) from convalescent donors. CLINICAL TRIALS REGISTRATION: NCT04896606.

The Function of Memory CD8+ T Cells in Immunotherapy for Human Diseases.

Memory T (Tm) cells protect against Ags that they have previously contacted with a fast and robust response. Therefore, developing long-lived Tm cells is a prime goal for many vaccines and therapies to treat human diseases. The remarkable characteristics of Tm cells have led scientists and clinicians to devise methods to make Tm cells more useful. Recently, Tm cells have been highlighted for their role in coronavirus disease 2019 vaccines during the ongoing global pandemic. The importance of Tm cells in cancer has been emerging. However, the precise characteristics and functions of Tm cells in these diseases are not completely understood. In this review, we summarize the known characteristics of Tm cells and their implications in the development of vaccines and immunotherapies for human diseases. In addition, we propose to exploit the beneficial characteristics of Tm cells to develop strategies for effective vaccines and overcome the obstacles of immunotherapy.

Quantification of Vitamin D at Different Levels of Clinical Worsening of COVID-19.

INTRODUCTION AND AIM: Vitamin D is the name given to a group of lipid-soluble steroidal substances of physiological importance in the body, especially in bone metabolism. The active form of vitamin D is believed to have immunomodulatory effects on immune system cells, especially T lymphocytes, as well as on the production and action of several cytokines and on the expression of potent antimicrobial peptides in epithelial cells that line the respiratory tract, playing an important role in protecting the lung from infections. The aim of this study was to assess vitamin D levels in patients with COVID-19 in healthcare service and to verify that these levels are adequate to protect the progression of this infection. METHODS: The aim of this observational study was to evaluate the serum concentration of vitamin D in 300 patients suspected of being infected with COVID-19, treated at Basic Health Units (BHUs) and at the Hospital Complex in the municipality of São Bernardo do Campo. RESULTS: 294 patients were included, 195 (66%) of which tested positive for COVID-19 and 99 (34%) negative for COVID-19. Among the patients in the positive group, 163 patients were in the mild group (84%); 22 patients in the moderate group (11%); 8 patients in the severe group (4%), and 2 patients in the deceased group (1%). CONCLUSION: For the patients in this study, no association was observed for the protective factor of vitamin D against COVID-19 infection, and its role in controlling the clinical staging of the disease was not verified.

T cell maturation is significantly affected by SARS-CoV-2 infection.

Coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An adequate T cell response is essential not only for fighting disease but also for the creation of immune memory. Thus, the present study aims to evaluate the T cells of patients with moderate, severe and critical COVID-19 not only at the time of illness but also 2 months after diagnosis to observe whether changes in this compartment persist. In this study, 166 COVID-19 patients were stratified into moderate/severe and critical disease categories. The maturation and activation of T cells were evaluated through flow cytometry. In addition, Treg cells were analysed. Until 15 days after diagnosis, patients presented a reduction in absolute and relative T lymphocyte counts. After 2 months, in moderate/severe patients, the counts returned to a similar level as that of the control group. In convalescent patients who had a critical illness, absolute T lymphocyte values increased considerably. Patients with active disease did not show differentiation of T cells. Nonetheless, after 2 months, patients with critical COVID-19 showed a significant increase in CD4+ EMRA (CD45RA+ effector memory) T lymphocytes. Furthermore, COVID-19 patients showed delayed T cell activation and reduced CD8+ suppressor T cells even 2 months after diagnosis. A reduction in CD4+ Treg cells was also observed, and their numbers returned to a similar level as that of healthy controls in convalescent patients. The results demonstrate that COVID-19 patients have a delayed activation and differentiation of T cells. In addition, these patients have a great reduction of T cells with a suppressor phenotype.