Cell therapy is a key tool of regenerative medicine, but until the beginning of the last decade, products based on viable human cells were used primarily to repair damaged tissues and organs. Currently, the field of application of biomedical cell products has expanded significantly, but researchers still show considerable interest in the use of human cells in regenerative medicine. The stage of development of cell products varies significantly depending on the type of tissue and pathology, and ranges from preclinical and pilot clinical trials to authorised drugs with a long history of use. On the one hand, this may be attributed to methodological differences in the production and use of cell products, and on the other, to specific aspects of differentiation of cell types used in regenerative medicine, primarily mesenchymal stem cells. The aim of this study was to analyse current trends in the use of cell therapy in regenerative medicine and prospects for using available technologies. The paper summarises the main achievements in the use of cell therapy for regeneration of skin, bone and cartilage, nervous and cardiovascular systems. The key mechanisms of cell therapy effect are determined, on the one hand, by the differentiation potential of multipotent cells, and on the other, by the complex (immunomodulating, angiogenic, proliferative) action of the proteome expressed by the administered cells. The paper describes viable cell-based products currently authorised for each indication, and analyses the level of their clinical use. It might be promising to use directed cell differentiation technologies, as well as induced pluripotent cells in regenerative medicine.

The main objective of the present research was to review the studies that look into ways of improving production of anti-rabies immunoglobulin in terms of bioethical principles: ensuring patients’ right to receive quality pharmaceutical care, and compliance with the 3R principles. At the same time, compliance with bioethical principles should contribute to the improvement of production technology and the product quality, which is especially important for antirabies immunoglobulin due to the existing high demand for it. The paper analyses the current trends in avoidance of animal use in the production of rabies immunoglobulin. It summarises the main methods of production of serum products for post-exposure prophylaxis of rabies. The example of heterologous rabies immunoglobulin is used to substantiate the need to improve quality control of drugs by following the 3R principles. The paper highlights the potential use of cell cultures for determination of rabies immunoglobulin specific activity. The authors formulated the objectives that include development and use of pyrogen detection methods which do not involve animal use and are consistent with the current pharmacopoeial standards. They assessed the possibility and feasibility of removing Abnormal Toxicity Test for heterologous rabies immunoglobulin in accordance with the current international trends. The formulated objectives imply optimisation of production of heterologous rabies immunoglobulin in Russia in order to improve the quality of antirabies measures. The objectives include higher production volumes to ensure the availability of the product to patients, reduction of the product reactogenicity by using cell-culture technologies for obtaining rabies antigen, as well as development and implementation of in vitro test methods for product quality control in terms of Specific activity, Pyrogenicity, and Abnormal toxicity

There has been a sharp increase in the number of diseases associated with potentially pathogenic microorganisms of the genus Mycobacterium, which differ from Mycobacterium tuberculosis. These bacteria are known as atypical mycobacteria or nontuberculosis mycobacteria (NTM), and the diseases they cause are called mycobacteriosis. NTMs include more than 20 species of acid-resistant microorganisms that are widespread in the environment and that are not members of the M. tuberculosis complex. However, the role of certain types of NTMs in the pathogenesis of human diseases is rather ambiguous. The aim of the paper was to analyse the current rise in the incidence of NTM diseases, as well as the main areas of research on early diagnosis of mycobacteriosis and the detection and testing of drug susceptibility of these microorganisms. The paper summarises current views on NTM species differences, their prevalence and pathogenicity for humans and animals. The authors analysed the main efforts aimed at diagnosis and treatment of NTM diseases. The paper cites the results of the study of NTM susceptibility/resistance to anti-tuberculosis drugs. The diagnosis of mycobacteriosis remains extremely difficult, mainly because of the similarity of the clinico-radiological evidence with that of tuberculosis. Detection of NTM multiple and extensive drug resistance to the majority of anti-tuberculosis drugs complicates the treatment of the NTM disease. Further study of various aspects of NTM diseases is especially important given the increase in the incidence and prevalence of mycobacteriosis all over the world, challenging differential diagnosis, and detection of NTM extensive drug resistance.

The area affected by the disease caused by Madariaga virus has been expanding recently. Given the geographic location of the endemic regions that overlap with tourist destinations, as well as the confirmed possibility of importing the disease, we cannot rule out the risk of appearance of this disease in Russia. The aim of the study was to analyse the properties of Madariaga virus (Alphavirus genus, eastern equine encephalomyelitis virus complex) and some epidemiological and epizootological characteristics of the virus-associated disease which manifests itself in diffuse inflammation of brain and spinal cord. Based on both ecological and molecular genetic characteristics of Madariaga virus, it is classified as an individual virus which is part of the eastern equine encephalomyelitis virus complex. There is evidence that this pathogen can cause epizootic outbreaks in horses, infect other types of mammals (rats, bats), as well as, potentially, birds and reptiles. The reservoir of infection is the short-tailed cane mouse (Zygodontomys brevicauda). Human cases have also been described, and the potential route of infection was identified as transmission via mosquito bites. Vectors of pathogen transmission are mosquitoes of the Culex, Aedes, and Psorophora genera. Serological studies carried out in Panama revealed the presence of antibodies to Madariaga virus in 2–5% of the examined patients, which indicates the existence of asymptomatic infection along with clinically apparent infection. Phylogenetic analysis of strains isolated from infected people showed that the strains belong to lineage III of the eastern equine encephalomyelitis virus subtype common in Central and South America. The paper summarises the results of assessment of potential risk factors of Madariaga virus infection in endemic regions that was performed using the Akaike information criterion. Risk groups in endemic regions are represented by farm workers and fishermen. The results of the studies show that the area affected by the disease is expanding, and the strains that belong to genetic lineage III of the eastern equine encephalomyelitis virus pose the greatest epidemic risk.

Rabies is an acute viral disease caused by a virus of the Rhabdoviridae family of the Lyssavirus genus, which affects the central nervous system and is characterised by absolute mortality. Vaccination is the only way to prevent the disease in humans. One of the products used for vaccination is a cultural concentrated purified inactivated dry rabies vaccine produced by the Federal State Budgetary Institution of Science “Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences” (hereinafter—Chumakov Center).

The aim of the study was to examine the structure of the working virus seed of Vnukovo-32 strain used by the Chumakov Center for rabies vaccine production, to assess its genetic stability during production, to explore the possibility of using molecular genetic methods for identification of the production strain in the finished dosage form, and to study the nucleotide sequence of the CVS strain.

Materials and methods: Vnukovo-32 rabies virus production strain, working virus seeds, finished batches of the rabies vaccine, CVS fixed rabies virus strain used in the assessment of specific immunity. The molecular genetic study was performed using RT-PCR followed by restriction and sequencing.

Results: the paper presents the results of nucleotide sequence analysis of the G gene fragment obtained from the Vnukovo-32 production strain, batches of the working virus seed, and finished batches of the rabies vaccine produced in 2012, 2018, and 2019, and the CVS fixed rabies virus strain used in the assessment of the vaccine’s specific immunity. The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form.

Conclusion: Vnukovo-32 and CVS strains used by the Chumakov Center are rabies viruses. Analysis of the nucleotide sequence of the G gene fragment showed that the Vnukovo-32 strain remains stable throughout different production stages. The obtained nucleotide sequence of gene G of the Vnukovo-32 strain was deposited in GenBank (accession number MN116503). The study demonstrated that restriction analysis could be used for Vnukovo-32 strain identification at all production stages, including the finished dosage form. 

Development and implementation in clinical practice of recombinant human erythropoietins (rhEPOs) remain a priority task today. Additional studies were performed in order to obtain clinical trial authorisation for rhEPO tablets for oral use. The studies were aimed to demonstrate the suitability of the erythropoietin producer strain based on Chinese hamster ovary cells (CHOpE) for the production of rhEPO, and the compliance of the substance characteristics with the requirements for erythropoietin (EPO).

The aim of the study was to characterise the rhEPO substance obtained from the CHOpE strain cells in accordance with the requirements for EPO.

Materials and methods: the rhEPO substance was obtained by culturing the strain of Chinese hamster ovary cells—CHOpE. The expression construct of the producer strain was evaluated using methods for determination of nucleotide and amino acid sequences. The Sanger method was used to perform sequencing of the nucleotide sequence encoding the human EPO gene. The amino acid sequences of the rhEPO molecule C- and N-termini were determined by the Edman method. The copy number of the EPO gene in CHOpE cells was determined by real-time polymerase chain reaction. The properties of the rhEPO substance were evaluated in accordance with the requirements for EPO. Isoelectric focusing, peptide mapping, and polyacrylamide gel electrophoresis were used for identification of the rhEPO substance. The ratio of isoform composition was determined by capillary electrophoresis. Dimer impurities and high molecular weight related substances were determined by high-pressure liquid chromatography. The content of protein and residual nucleic acids was determined by spectrophotometry. The concentration of the rhEPO substance was assessed by enzyme immunoassay.

The results of the study confirmed genetic stability of the CHOpE producer strain and demonstrated identity of N- and C-terminal amino acid sequences of the rhEPO molecule to those of the natural EPO. The CHOpE producer strain was used to obtain a rhEPO substance which is homogenous and does not contain impurities of EPO oligomeric forms. Dimers and high molecular weight related substances account for less than 0.5%. The rhEPO molecular weight ranges from 32 to 38 kDa, and the isoelectric point is within 2.8—4.15. The study identified the peaks of isoforms 1–8, the isoform composition of the rhEPO substance corresponds to that of EPO. It was determined that 1 mol of the substance contains 13.75 mols of sialic acids.

Conclusions: the study confirmed the suitability of the CHOpE producer strain for the production of rhEPO. The obtained rhEPO substance meets requirements for EPO. 

Prophylactic immunisation against brucellosis is part of the National Immunisation Schedule for Epidemic Settings. The immunisation is performed with a live vaccine—a lyophilized suspension of the Brucella abortus strain 19 BА in a stabilizing medium. The paper presents the results of quality evaluation of 9 batches of live brucellosis vaccine that were submitted to the Testing Centre for Evaluation of Medicinal Immunobiological Products’ Quality of the Federal State Budgetary Institution “Scientific Centre for Expert Evaluation of Medicinal Products” of the Ministry of Health of the Russian Federation for assessment of the product’s compliance with the established specifications. The paper also presents the results of evaluation of the passport information provided by the manufacturer for these batches. There is no doubt about the need for objective quality evaluation of brucellosis vaccines as well as about the significance of its improvement.

The aim of study was to assess the prospects for improving quality evaluation of live brucellosis vaccines in terms of Specific activity (concentration of microbial cells, number of living microbial cells, number of cutaneous doses).

Materials and methods: specific activity (concentration of microbial cells and number of living microbial cells) was determined by visual and microbiological methods using the industrial reference standard of brucellosis vaccine OSO 42-28-396-2018, batch 6 and the bacterial suspension of the Brucella abortus strain 19 BА acquired from the joint stock company Scientific and Production Association “Microgen” in 2016. The number of cutaneous doses in the brusellosis vaccine was determined by the calculation method. Statistical processing of the results was performed using Microsoft Excel.

Results: there was a mismatch between the brucella concentration coefficient of 1.7×109 microbial cells/mL determined by comparison with the industrial reference standard of bacterial suspension turbidity, 10 IU and the actual concentration of microbial cells obtained in the study. According to preliminary results, the brucella concentration coefficient corresponding to the industrial reference standard of bacterial suspension turbidity, 10 IU can reach 3.0×109 microbial cells/mL.

Conclusions: the obtained results can serve as a basis for amending the data on the brucella concentration coefficient in the Passport and the Instructions for use of the industrial reference standard of bacterial suspension turbidity, 10 IU, as well as the Specific activity section (concentration of microbial cells, number of living microbial cells, number of cutaneous doses) of the established specifications for the brucellosis vaccine. Before amending the information on the brucella concentration corresponding to 10 IU in the Passport and the Instructions for use of the reference standard of bacterial suspension turbidity (OSO 42-28-85P), additional studies should be performed with other types of brucella.