T-cell immune response is extremely important in protecting human body from diverse viral infections. It is known that it can ensure viral clearance and complete recovery in patients with humoral immunodeficiency. COVID-19 patients were found to have T-cell response primarily directed against SARS-CoV-2 structural S, M, N, E proteins, with nucleocapsid protein being most conserved. To assess patients immunity against coronavirus infection and evaluate an effectiveness of vaccine candidates, it is necessary to develop an optimal diagnostic antigen to evaluate arising T-cell response against SARS-CoV-2 antigenic determinants. A diagnostic test to determine host specific susceptibility to SARS-CoV-2 infection should target conserved regions of global SARS-CoV-2 variants. The study was aimed to develop a structure of an antigen bearing conserved and immunogenic sequences derived from SARS-CoV-2 structural proteins and to obtain an Escherichia coli producer strain containing a recombinant protein to be subsequently used for assessing antiviral T-cell immunity. Developing of the antigen was performed in silico: TepiTool and NetMHCIIpan were used to predict and identify high affinity epitopes spanning SARS-CoV-2 E, M, N, S proteins and MHC II binding. Several variants of recombinant antigen proteins were constructed, from which one was selected based on its physicochemical properties: isoelectric point, hydrophobicity index and aliphatic index, as well as 3D representation built by using the I-TASSER. The sequence was synthesized and cloned into the pET24a(+) vector. The resulting plasmid pCorD_PS was transformed into E. coli DH5 followed by Rosetta (DE3). The strain-producer of the recombinant E. coli protein CorD_PS was assessed for the presence and stability of IPTG-induced antigen protein expression and elimination of recombinant coronavirus antigen-bearing plasmid. Based on the study data, an antigen was developed consisting of conserved regions from SARS-CoV-2 S, M, N, E proteins. A 53 kDa recombinant protein was predicted to be stable in aqueous solutions with isoelectric point of 9.56 potentially allowing to simplify protein purification from E. coli cells. Plasmid DNA pCorD_PS (6695 bp) encoding final recombinant coronavirus antigen cloned into pET24a(+) vector was obtained. A stable, productive E. coli CorD_PS strain was obtained. The obtained strain-producer resulting in recombinant E. coli CorD_PS antigen is stable allowing to move on to design antigen purification technique and further develop SARS-CoV-2-specific diagnostic test system.

Due to the development of the pandemic and the need for mass vaccination, the development of next-generation vaccines against COVID 19 coronavirus infection is a major challenge for medicine and biotechnology. Due to the prevalence of COVID 19, it is still urgent to develop a safe and protective vaccine, especially one that stimulates T-cell immune response. This work presents the design and production of a DNA immunogen based on plasmid DNA encoding a hybrid protein containing immunogenic fragments of structural proteins of SARS-CoV 2 β-coronavirus. As a result, a vector construct based on plasmid DNA encoding a hybrid protein containing the most immunogenic parts of structural proteins M, S, N, E of SARS-CoV 2 β-coronavirus was created by genetic engineering methods for transient expression in mammalian cells; a strain of E. coli, a producer of DNA-coronavirus β-coronavirus, was created by electroporation. coli, a DNA-immunogen producer; a method of purification of recombinant plasmid DNA was developed on the basis of a sequential step-by-step process: this method allowed to obtain 100 mg of DNA-immunogen pCMV 3Tag 3a-CVVVV3 in a solution with a concentration of 1 mg/mL (100 ml), which corresponds to the concentration established in the State Pharmacopoeia of the Russian Federation (OFS.1.7.1.0013.0013.1). .7.1.0013.18 DNA-vaccines) quality parameters - the obtained result is stably reproduced in laboratory conditions; methods of plasmid DNA quality control were worked out and expression of hybrid protein was tested by Western blotting. The CVVV3 antigen was shown to bind specifically to IgG immunoglobulins from rabbit sera after immunization with pCMV 3Tag 3A-CVVVV3 DNA-immunogen. The proportion of superhelicalized plasmid DNA in the sample of DNA-immunogen pCMV 3Tag 3A-CVVVV3 was 85.64 %, the content of endotoxins was less than 25 U/mg; the concentration of residual proteins of the strain-producer in a solution of 1 mg/ml of DNA-immunogen pCMV 3Tag 3A-CVVVV3 was less than 100ng per 1 ml (mg of total recombinant DNA).

The aim of this work is an experimental study of the radioprotective effect of shortened recombinant flagellin under conditions of total body irradiation of mice by survival rates and the effect on hemo- and immunopoiesis. A new recombinant shortened derivative of Salmonella enterica, the dFliC protein obtained by structure-oriented reengineering of a previously developed molecule, was used. The radioprotective effect of dFliC was studied in the mouse model of hematopoietic acute radiation syndrome. The 30-day survival of irradiated mice was analyzed using the Kaplan–Meier method. The effect of the dFliC protein on the number of splenic colony-forming units (CFUs) and myelokaryocytes in the bone marrow, peripheral blood parameters, and the cytokine profile in the blood serum of mice was assessed. Statistica 8.0 software was used for statistical processing of the results. A purification test record for the recombinant flagellin dFliC was developed, and protein samples with a purity of 92.79% according to HPLC were obtained. Administration of dFliC at a dose of 1 mg/kg 15–30 min prior to 7.8 Gy X-rays increased 30-day survival of mice by 38% (p < 0.05 compared to the vehicle control). On day 9 after X-ray irradiation at a dose of 7 Gy, the number of colonies increased 2.8 times in dFliC-treated mice (p < 0.05), the viability of hematopoietic progenitor cells increased and the severity of thrombocytopenia decreased. An increase in the production of IL-3 and GM-CSF involved in the regulation of the early stages of hematopoietic cell development and the macrophage chemoattractant IL-12p40 was recorded; the content of proinflammatory cytokines IL-1β and IL-33 was maintained at a lower level than during irradiation without the use of dFliC. Deimmunization and structural rearrangement of the flagellin molecule did not lead to a decrease in the radioprotective effect of the recombinant protein. The radioprotective efficacy of dFliC is provided by a protective effect on bone marrow hematopoietic cells and stimulation of postradiation restoration of hemo- and immunopoiesis by regulating the expression of cytokines with a wide range of biological activity.

Full-length ACTH molecule and ACTH-related hexapeptide H-PKKRRP-OH are demonstrated by the patch-clamp method to decrease the Na_V1.8 channel activation gating system effective charge in the nociceptive neuron membrane, while ACTH-related tetrapeptide Ac-KKRR-NH₂ has no effect. ACTH(1–24), a fully functional ACTH mimetic, and H-PKKRRP-OH show analgesic effects in the formalin test in vivo. All peptides contain the cationic KKRR motif, but only H-PKKRRP-OH and ACTH(1–24) relieve acute pain, targeting the Na_V1.8 channel as a receptor. This seemingly controversial result is explained by application of conformational analysis and blind docking. Though conformational analysis indicates that both H-PKKRRP-OH and Ac-KKRR-NH₂ contain the cationic functional groups at the earlier suggested characteristic distance of 9–12 Å, Ac-KKRR-NH₂ does not interact with the S4_I voltage sensor of the Na_V1.8 channel activation gating system. The docking demonstrates that an extensive network of ligand–receptor ionic and hydrogen bonds involving D151, E157, R218, and R221 VSD_I residues, essential for the analgesic tripeptide Ac-KKK-NH₂ binding, is formed upon the H-PKKRRP-OH binding. Particularly important are the ionic bonds between the H-PKKRRP-OH C-terminal carboxylate anion and the S4_I R218 and R221 guanidinium groups. The described mechanism of Na_V1.8 channel modulation is fundamentally different from the effect of channel blockers.

Purpose of the study: To determine the possibility of a non-invasive evaluation of the biologically active substances (BAS) effect on the rate of a pH level restoration in a muscle after a maximum load using 1H magnetic resonance spectroscopy (MRS). Materials and methods. Creatine monohydrate and beta-alanine were taken as tested biologically active substances, used according to the manufacturer's recommendations. At the first stage, calibration curves of a pH dependence on the magnitude of chemical shifts were plotted during assigning 1H spectra of model carnosine dipeptide solutions for non-invasive determination of intramuscular pH. Further experiments were carried out on laboratory animals (mice) using a 9 T NMR spectrometer Bruker Advance III WB 400MHz WB (Bruker, Germany). In experiments on volunteers the functional test pwc170 was used for assessing the ergogenic effects of biologically active substances on rectus quadriceps femoris. The test allows to achieve the level of myocytes cytoplasm acidification with lactate, and the effectiveness of functional biologically active substances on endurance, and also the function of aerobic systems by the muscle pH rate of recovery. Detection was performed using a high-field magnetic resonance imaging scanner (Philips Healthcare, Achieva 3.0T, North Braband, The Netherlands) and two SENSE Flex-L surface ring radiofrequency coils. Results. The effect of oral intake of creatine and beta-alanine on the restoration of rectus quadriceps femoris muscle pH after an acidification of the myocytes cytoplasm with lactate was evaluated using the 1H MRS method. Reproducible results with optimal signal-to-noise ratios and width of carnosine spectral peaks were achieved in volunteers using individual protocols and 1H MRS at 3T in the quadriceps femoris. Animal experiments have highlighted the need to develop and use more accurate techniques for voxel extraction and fat suppression during in vivo 1H spectroscopy to reliably capture the chemical shifts of carnosine peaks. Conclusion. The data obtained using 1H MRS on volunteers allow us to conclude that the developed method makes it possible to non-invasively assess the effect of biologically active substances on the rate of restoration of pH level in a muscle after a critical load in real time in vivo .