ABSTRACT
Background: The nucleocapsid protein (N-protein) of SARS-CoV- 2 regulates transcription, replication and packaging of the viral genome. Potentially, each of the structural proteins can act as an antigen for the production of specific antibodies and T-cell stimulation. This study evaluates T-cellular immunity to N-protein after infection caused by the SARS-CoV- 2 for up to 9 months. Method(s): Patients who had COVID-19 in 2020 were divided by severity into 3 groups: mild (n = 41), moderate (n = 46), and severe (n = 29). Blood samples were taken 3 times: at 3, 6 and 9 months after infection. Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stained with Tag-it Violet vital dye. PBMCs were stimulated with PepTivator SARS-CoV- 2 ProtN (pool of peptides, covering the complete sequence of N-protein);unstimulated cells served as negative control. PBMCs were cultured in AIM-V media for 5-7 days. Cell colonies were detected by microscopy. Relative content (%) of proliferating live T cells (CD3+7AAD-) were evaluated by flow cytometry. Samples were counted positive for antigen-specific T cells (ASCs) to N-protein when both proliferation was >1% compared with negative control and microcolony were observed. Result(s): There were 58.5% positive samples with ASCs to the N-protein in mild, 60.9% in moderate and 48.3% in severe group in 3 months after recovery from COVID-19. There was no difference between groups at this point (p > 0.05, chi2). After 6 months results were: 26.7% positive samples in mild, 21.9% in moderate and 35.0 % in severe group. No differences between groups were observed after 6 months, but percentages of positive samples were less in mild and moderate groups when compared 3d and 6th months (p3-6 < 0.05, chi2). Drastic decreases of ASCs-positives in the mild group was observed after 9 month -only 4.2% (p6-9 < 0.05, chi2). Further decrease in moderate (25.0%) and severe (41.7%) groups was not observed after 9 months. Conclusion(s): T cell immunity to the N-protein of SARS-CoV- 2 was formed after mild, moderate and severe forms of COVID-19 with different effectiveness. The duration of immunity depends on the form of the disease and was stronger in severe group. By the 9th month, the number of positive samples in the group with mild COVID-19 was significantly lower when compared with the moderate and severe groups.
ABSTRACT
Background: Belarus started developing a vaccine against SARS-CoV- 2 in 2021. The aim of the first stage of investigation was to evaluate the immunogenicity of the vaccine prototypes (VP) in vitro. Method(s): SARS-CoV- 2 strains (n = 7) were isolated using Vero E6 cells, inactivated with beta-propiolactone and purified. Antigens (Ag) were adsorbed on adjuvants: Al(OH)3 (Ag+AH group) or Al3PO4 (Ag+AP group). The single dose of VP (500 ul) was composed of 10 mug of Ag adsorbed on adjuvants (200 mug of Al3+). Blood samples from SARS-CoV- 2 recovered donors (n = 18) and healthy controls having no history of COVID-19 infection (n = 5) were used. Whole blood and Tag-it Violet labeled PBMCs were cultivated with VP, pure Ag, adjuvants (0.25-1 mug of Ag, 20 mug of Al3+ for probe) or pool of peptides, covering sequence of SARS-CoV- 2 N, S, M-proteins (PP), for 6 h and 7 days respectively. INF-gamma production and proliferation of CD3+ T-cells were assayed by FACS. Result(s): Counts of CD3+IFN-gamma+ T cells were 3.14(2.72-5.13)/ 1x105 CD3+ T cells in negative control (NC), and 12.73(10.09-33.95)/ 1x105 CD3+ T cells in specific positive control (PP) (n = 18, p < 0.0001), proving presence of antigen-specific T cells (ASCs) in donor blood. Samples were considered positive for VP and Ag immunogenicity when numbers of CD3+IFN-gamma+ T cells were 1.5 times greater compared with NC. Both VP types (Ag+AP, Ag+AH) and pure SARS-CoV- 2 isolates stimulated the production of INF-gamma by ASCs, responses ranged from 1 to 4 isolates of 7 studied per donor. Immunogenicity of Ag+AP, Ag+AH was confirmed by proliferation assay. Proliferation level was 1.07(0.97-2.38)% in Ag group with no differences from NC (n = 7, p > 0.5). Proliferation was significantly greater in VP groups compared with Ag: 2.47(1.65-2.68)% in Ag+AH, 4.03(2.56-4.61)% in Ag+AP (p = 0.009 and 0.002, respectively), stimulation of T cell was stronger by Ag+AP compared with Ag+AH (p = 0.009, M-U test). Pure adjuvants did not induce T cells response. There was no T-cell stimulation by Ag and VP in samples obtained from COVID-19 negative donors. Conclusion(s): The VP against SARS-CoV- 2 infection composed of inactivated virus adsorbed on Al(OH)3 and Al3PO4 adjuvants has immunogenic properties proven in two different immunological assays. VP stimulated activation and proliferation of ASCs in vitro suggesting this VP can be used for further preclinical in vivo evaluation.
ABSTRACT
Background: COVID-19 is a disease caused by the SARS-CoV-2 virus that is often associated with pneumonia and acute respiratory distress syndrome (ARDS). Pathogenesis of COVID-19 is closely related to the host ' s response to the virus. Impaired regulation of immunity has been observed in patients with severe COVID-19 pneumonia. Mesenchymal stem cells (MSCs), having multipotency, ability to selfrenew, and ability to evade immune response may be useful in in the treatment of COVID-19 pneumonia. MSCs have an immunomodulatory effect on almost all types of immunocompetent cells: T-and B-lymphocytes, natural killer cells, monocytes and macrophages, dendritic cells, neutrophils. This study assesses the safety and tolerability of pooled allogeneic mesenchymal stem cells (poolMSC) in COVID 19 pneumonia. Method: The study included 5 patients with PCR confirmed COVID-19 pneumonia-4 men and 1 woman, average age 62.4 years. Pooled MSC (pMSC) was a mixture of 3 cultures of olfactory mucosa-derived MSCs (OM-MSCs) obtained from the mucous membrane of the middle nasal passage of healthy volunteers. MSCs were tested for viability (>95%), immunophenotype CD90 + CD105 + CD73 + CD31-CD45-HLA-DR-, and sterility. pMSC at a dosage of 1×10 6 cells per kg of body weight were suspended in 100 ml of saline and injected intravenously over 60 minutes. Results: All patients were carefully examined at baseline before pMSC infusion. Clinical examination was done on the day of infusion of pMSC with a skin test to pMSC was performed. In the absence of systemic and local allergic reactions pMSC were injected. Three patients received one pMSC infusion, and two patients received two pMSC infusions at an interval of 4 days. None of the patients had any adverse reactions to the pMSC skin test or infusion. Conclusion: Assessment of pMSC infusion demonstrated good tolerance and safety of intravenous use in patients with severe pneumonia caused by the SARS-CoV-2 and complicated by ARDS.