A proper and representative monitoring of SARS-CoV-2 herd immunity including a long-term health impact on recovered patients and vaccinated individuals is of great importance. For this, a monitoring campaign should assesses both humoral and T-cell immune arms. Upon that, analyzing antigen specific-cell activation and cellular phenotype are informative. We developed a flow cytometry method for detection of intracellular IFN-producing antigen-reactive T cells after exposure of human peripheral blood mononuclear cells (PBMC) to SARS-CoV-2 virus antigens. The method was validated according to the following characteristics: sensitivity, specificity, precision, and robustness. We used positive samples from donors recovered from COVID-19 and negative samples from donors who had no contact with COVID-19 patients and lacking antibodies to SARS-CoV-2. All samples were tested by laboratory methods. Peripheral blood mononuclear cells were isolated from donor blood by centrifugation in a Ficoll density gradient. Specific T cells were stimulated with S-protein as well as N, M, ORF3a, and ORF7a protein peptides to count IFN-producing T cells by flow cytometer. The data were statistically analyzed. The area limited by ROC-curve and false positive rate (AUC) for CD4 and CD8 cells was from 0.97 to 1.00. Precision was considered acceptable because the coefficient of variation for all PBCM did not exceed 20%. Robustness was confirmed for frozen and freshly prepared PBMC samples. The thresholds levels to recognize immune and non-immune samples were defined for CD4-positive T-cells (0.029%) and CD8-positive T-cells (0.0640.068%). Also, acceptance criteria for positive and negative controls were defined. Based on the validation, the suitability of the method Evaluation of antigen-reactive T cells that produce intracellular IFN in response to SARS-CoV-2 virus antigens by flow cytometry was confirmed. The method allows for reliable data that was used to characterize standard control samples for internal quality control of TigraTest SARS-CoV-2 kits.

In this work, we set out to compare the immunogenicity of the Gam-COVID-Vac two-component vaccine produced by GENERIUM JSC (Russia) following its intranasal or intramuscular administration of the first or both components to BALB/c mice. The immunogenicity was evaluated according to antigen-specific IgG and IgA antibodies in the blood and bronchoalveolar fluid, the number of antigen-specific IFN-γ-producing T-lymphocytes, the number of antigen-specific IFN-γ-producing CD4 and CD8 T-lymphocytes. Intranasal administration was shown to induce the mucosal immunity, significantly exceeding both qualitatively and quantitatively the effect of intramuscular administration (by the number of animals with antigen-specific IgA antibodies and the titer of antibodies in blood serum and bronchoalveolar lavage). At the same time, intramuscular administration slightly exceeded the effect obtained under intranasal administration in terms of the total number of CD8-IFN-γ-producing lymphocytes.

With the onset of the COVID-19 pandemic, a number of molecular-based tests have been developed to diagnose SARS-CoV-2 infection. However, numerous available serological tests lack sufficient sensitivity or specificity. They do not detect specific antibodies in a significant proportion of patients with PCR-confirmed COVID-19. There is evidence that some convalescents have a relatively short-lived humoral immunity. In contrast, a number of publications have shown that T-cell response to human coronaviruses, including SARS-CoV-1, MERS, and SARS-CoV-2, can be strong and long-term. Assessment of T-cell immunity to SARS-CoV-2 is important not only for stratification of risks and identification of potentially protected populations with immunity acquired as a result of previous infection, but also for determining immunogenicity and potential efficacy of vaccines under development. The existing methods of quantitative or semi-quantitative assessment of specific T-cell response are mainly used in scientific research and are not standardised. The aim of the study was to develop and verify experimentally a test kit to be used in a standardised procedure for in vitro determination of T-cells specific to SARS-CoV-2 antigens, in human peripheral blood. Materials and methods: the TigraTest® SARS-CoV-2 kit developed by GENERIUM, which determines the number of T-cells secreting interferon gamma in vitro, was tested in the study. Samples of venous blood of volunteers from three different groups were analysed in the study: presumably healthy volunteers; COVID-19 convalescents; individuals vaccinated against SARS-CoV-2. Results: the authors developed the TigraTest® SARS-CoV-2 kit for in vitro determination of T-cells specific to SARS-CoV-2 antigens in human peripheral blood, demonstrated its specificity and performed preliminary assessment of its sensitivity. The study analysed the range and magnitude of the T-cell response in convalescent and vaccinated individuals. A pronounced T-cell response was also shown in some individuals with no symptoms or with unconfirmed diagnosis. It was discovered that the mean T-cell response to peptides of the spike protein (S-protein) was higher in the vaccinated individuals than in the convalescent patients. A correlation was determined between the severity of the disease and the level of T-cell response. Specific contributions of various groups of antigens to the T-cell response after COVID-19 infection were also determined. Conclusions: the TigraTest® SARS-CoV-2 kit is a specific and sensitive tool for the assessment of T-cell immunity to the SARS-CoV-2 virus, which can also be used for vaccinated individuals. The kit may be used in clinical practice for comprehensive assessment of immunity to SARS-CoV-2.

Enzyme replacement therapy (ERT) is one of the most efficient treatments for lysosomal storage diseases. Type 1 Gaucher disease is caused by β-glucocerebrosidase enzyme deficiency, which may be compensated for by intravenous infusions of imiglucerase—a recombinant enzyme. Imiglucerase targets macrophages and enters these cells via interaction with mannose receptors on the cell membrane. Characterisation of internalization of enzymes by target cells is important in the context of the development of new medicines and production of existing ERT medicines. The peritoneal and alveolar macrophages, as well as macrophages of the spleen of small laboratory animals (rats and mice) are widely used in such studies. However, isolation of cells from animal sources raises ethical issues, and therefore continuous mammalian cell lines may offer an attractive alternative.The aim of thestudy:to conduct comparative studies on the internalization of recombinant imiglucerase into mouse peritoneal macrophages and L929 mouse fibroblasts.Materials and methods:CerezymeR batches 7HV0913, C6214H05, 7HV0888 (Genzyme Ltd., UK); Glurazim batches 020416, 011117, 021117 (LLC “IBC “Generium”, Russia). We used peritoneal macrophages obtained from BALB/c mice and L929 mouse fibroblasts. The cells were cultured in DMEM/F12 complete growth medium with 10% fetal bovine serum. The activity of imiglucerase internalized into the cells was evaluated spectrophotometrically by hydrolysis of the artificial substrate—4-methylumbelliferyl-β-Dglucopyranoside.Results:the study compared internalization of recombinant imiglucerase (the active ingredient of CerezymeR and Glurazim) by mouse peritoneal macrophages and L929 mouse fibroblasts. It was demonstrated that the medicines activity in the lysates of peritoneal macrophages is comparable with that in the lysates of L929 mouse fibroblasts. Regardless of the model system, the activity of Glurazim stayed within the acceptable range (80–125%) established for biosimilar products.Conclusions:the experiments proved that L929 mouse fibroblasts could be recommended for assessment of internalization of recombinant imiglucerase.

African swine fever (ASF) caused by African swine fever virus (ASFV) of Asfivirus genus, Asfarviridae family, can occur in peracute, acute, subacute, chronic or asymptomatic form. At early stages of epizootics, the infection usually occurs in its acute form eventually becoming chronic and/or asymptomatic. Seven to ten days post infection the survived pigs develop virus-specific antibodies which persist for a long time. An assumption is reasonable that in the near future, due to repeated passaging ASFV in wild boar populations in European countries, the ASFV isolates may appear which will cause chronic or asymptomatic rather than the acute forms of the disease. In our study we compared different tests to find those the most effective to reveal latent carriers when no apparent symptoms of the disease observed. Thus, our research was aimed at investigation of some special aspects of the laboratory diagnostics of chronic and asymptomatic forms of ASF. The chronic form of the disease was observed in a pig experimentally inoculated with an attenuated ASF virus Stavropol 01/08 A4S2/9k (at passage 33) at a dose of 106.0 HAU50. On day 5 to 7 post inoculation the signs typical of chronic forms of the infection were registered including depression and fever up to 40.5 С. The antiviral antibody was detected in the swine blood serum from day 7. After the animal was killed on day 21, a haemadsorption assay revealed ASF virus present at low titers in spleen and mandibular lymph node samples while in liver and lung samples it was not found. Based on the results of polymerase chain reaction (PCR), the viral DNA was determined in the mandibular lymph node sample only. Furthermore, immunoblotting assay identified ASF antibody titers of 1:20 to 1:160 in all the organs examined. The asymptomatic forms of ASF were observed in a wild boar yearling which has been intramuscularly inoculated with an attenuated ASF virus strain MK-200 at a dose of 107.0 HAU50. The antiviral antibody was observed in the wild boar serum from day 8. After the animal was killed on day 25, no pathological signs typical of ASF were found, nor was ASF virus found in the organ samples examined using haemadsorption assay or its DNA was detected in PCR. In immunoblotting assay, virus-specific antibodies were identified in liver, spleen, lung and mandibular lymph node samples at dilutions of 1:40 to 1:320. The opportunity of detecting antibodies in spleen, lung and/or liver samples facilitates the monitoring for ASF to be carried out under the infection control campaigns, especially with respect to wild boars shot in game husbandries. Animals sequentially infected with an ASFV low-virulent isolate and a virulent one can survive, in which case it is quite possible to diagnose the disease using both PCR and serological methods. For making laboratory diagnosis of ASF chronic and/or asymptomatic forms, as well as carrying out monitoring studies, serological methods are recommended.

Особенности протекания африканской чумы свиней (АЧС, возбудитель ДНК-со-держащий вирус семейства Asfarviridae, поражает как диких кабанов Sus scrofa, так и домашних свиней Sus scrofa domestica ) и ее формы следует учитывать для эффективного использования различных методов диагностики АЧС, направленных на выявление возбудителя или специфических антител. Особенно важна диагностика АЧС у диких кабанов. Восприимчивость к вирусу АЧС дикого кабана хорошо известна, заболевание неоднократно воспроизводилось в эксперименте, а также было установлено в естественных условиях у дикого европейского кабана. При отстреле диких кабанов очень часто в лаборатории доставляются только пробы органов, поэтому мы приняли решение изучить диагностическое значение антител, выявленных в тканях, и определить эффективность применения разработанной ранее тест-системы при таких исследованиях. В статье представлены результаты валидации непрямого варианта ИФА (коммерческий набор «ВНИИВВиМ АЧС-ИФА Ат/Аг») для обнаружения специфических антител против вируса АЧС в сыворотках крови и экстрактах тканей селезенки. В качестве метода сравнения использовали реакцию непрямой иммунофлуоресценции (РНИФ). Для оценки полученных данных применили ROC-анализ. Тестирование с помощью непрямого ИФА положительных ( n = 66) и отрицательных ( n = 410) проб сывороток крови свиней показало высокую чувствительность и специфичность ИФА относительно РНИФ. Из 476 проб сывороток, исследованных методом непрямого ИФА, только 8 (1,6 %) попали в область положительной/отрицательной отсечки. Самые высокие показатели чувствительности (100 %) и специфичности (99,27 %) непрямого ИФА при исследовании сывороток крови от домашних свиней и диких кабанов установлены при величине отсечки 0,264. Пробы экстрактов селезенки были отобраны после отстрела клинически здоровых диких кабанов из неблагополучной по АЧС Смоленской области (2013-2014 годы). С учетом характера течения острой формы болезни мы можем предположить у положительно реагировавших животных состояние, соответствующее инкубационному периоду развития патологии. При тестировании положительных ( n = 59) и отрицательных ( n = 678) проб экстрактов ткани селезенки мы также установили высокую чувствительность и специфичность непрямого ИФА относительно РНИФ. Из проверенных проб ( n = 737) 10 проб (1,3 %) попали в область положительной/отрицательной отсечки. Самая высокая чувствительность (100 %) и специфичность (98,82 %) установлены при величине отсечки 0,284. Таким образом, тестовый набор показал хорошие результаты при исследовании сывороток крови свиней чувствительность 100 % (94,6-100 %) и специфичность 99,27 % (97,90-99,80 %), при исследовании проб экстрактов ткани селезенки чувствительность 100 % (93,90-100 %), специфичность 98,82 % (97,70-99,50 %).

African swine fever (ASF) caused by African swine fever virus (ASFV) of Asfivirus genus, Asfarviridae family, can occur in peracute, acute, subacute, chronic or asymptomatic form.At early stages of epizootics, the infection usually occurs in its acute form eventually becoming chronic and/or asymptomatic.Seven to ten days post infection the survived pigs develop virus-specific antibodies which persist for a long time.An assumption is reasonable that in the near future, due to repeated passaging ASFV in wild boar populations in European countries, the ASFV isolates may appear which will cause chronic or asymptomatic rather than the acute forms of the disease.In our study we compared different tests to find those the most effective to reveal latent carriers when no apparent symptoms of the disease observed.Thus, our research was aimed at investigation of some special aspects of the laboratory diagnostics of chronic and asymptomatic forms of ASF.The chronic form of the disease was observed in a pig experimentally inoculated with an attenuated ASF virus Stavropol 01/08 A 4 S 2 /9k (at passage 33) at a dose of 10 6.0 HAU 50 .On day 5 to 7 post inoculation the signs typical of chronic forms of the infection were registered including depression and fever up to 40.5 С.The antiviral antibody was detected in the swine blood serum from day 7.After the animal was killed on day 21, a haemadsorption assay revealed ASF virus present at low titers in spleen and mandibular lymph node samples while in liver and lung samples it was not found.Based on the results of polymerase chain reaction (PCR), the viral DNA was determined in the mandibular lymph node sample only.Furthermore, immunoblotting assay identified ASF antibody titers of 1:20 to 1:160 in all the organs examined.The asymptomatic forms of ASF were observed in a wild boar yearling which has been intramuscularly inoculated with an attenuated ASF virus strain MK-200 at a dose of 10 7.0 HAU 50 .The antiviral antibody was observed in the wild boar serum from day 8.After the animal was killed on day 25, no pathological signs typical of ASF were found, nor was ASF virus found in the organ samples examined using haemadsorption assay or its DNA was detected in PCR.In immunoblotting assay, virus-specific antibodies were identified in liver, spleen, lung and mandibular lymph node samples at dilutions of 1:40 to 1:320.The opportunity of detecting antibodies in spleen, lung and/or liver samples facilitates the monitoring for ASF to be carried out under the infection control campaigns, especially with respect to wild boars shot in game husbandries.Animals sequentially infected with an ASFV low-virulent isolate and a virulent one can survive, in which case it is quite possible to diagnose the disease using both PCR and serological methods.For making laboratory diagnosis of ASF chronic and/or asymptomatic forms, as well as carrying out monitoring studies, serological methods are recommended.

The causal agent of African swine fever (ASF) is a DNA virus belonging to Asfaviridae family which affects both wild boar Sus scrofa and domestic pig Sus scrofa domestica. Special features of the course of (ASF and its forms should be considered for the effective use of various ASF diagnostic methods aimed at the pathogen or specific antibody identification. ASF diagnosis in the wild boar is of special importance. The wild boar susceptibility to ASF virus is well known, and the disease has been repeatedly reproduced in experiments and detected in European wild boars in natural conditions. It is not unfrequently that when shooting wild boars, only organ samples are delivered to laboratories, so we decided to estimate the diagnostic value of the antibodies detected in tissues and evaluate an earlier developed test system for its efficacy when used for the assay. This report represents the results of validation of an indirect ELISA (a commercial kit «VNIIVViM ASF-ELISA Ab/Ag») ASF virus specific antibody detection in blood serum and spleen tissue extracts. For comparison, an indirect immunofluorescence assay (indirect IFA) was used. To estimate the obtained results, ROC analysis was applied. Examination of positive (n = 66) and negative (n = 410) porcine blood serum samples using indirect ELISA showed high sensitivity and specificity of the method with reference to IFA. Among the 476 serum samples examined in indirect ELISA, only 8 sera (1.6 %) were within the positive/negative cutoff area. The highest sensitivity (100 %) and specificity (99.27 %) for indirect ELISA when examining both domestic pig and wild boar blood sera were determined at a cutoff value of 0.264. The samples of spleen extracts to be used for the study were collected from clinically healthy wild boars in ASF-affected Smolensk region in 2013 to 2014. In view of an acute form of the disease, we can suspect that the animals exhibiting positive reaction were in a latency period of the pathogeny. When examining the positive (n = 59) and negative (n = 678) spleen extracts, we also determined high sensitivity and specificity levels of indirect ELISA with reference to indirect IFA. Among the samples examined (n = 737), 10 (1.3 %) samples of spleen extracts were within the positive/negative cutoff area. The highest sensitivity (100 %) and specificity (98.82 %) were observed at a cutoff value of 0.284. Thus, we confirmed the assay to be effective for porcine blood sera with 100 % sensitivity (94.6 to 100 %) and 99.27 % specificity (97.90 to 99.80 %) and for spleen extracts with 100 % sensitivity (93.90 to 100 %) and 98.82 % specificity (97.70 to 99.50 %).

Epizootic diarrhea fever (EDF), characterized by acute enteritis and hard intestinal disorders, is caused by the virus of the same group of С oronavirus ( Coronaviridae ) as the virus of transmissible gastroenteritis of pigs, coronavirus of cats and dogs, and human 229E strain. From 2005, at the territory of the Russian Federation the outbreaks of the disease were recorded, clinically similar to pig transmissible gastroenteritis. The infections caused a loss of piglets, in some cases up to 80 %. However, the sows in such farms have been vaccinated against the transmissible gastroenteritis of pigs. The aim of our study was to isolate in cell culture and identify an infectious agent that causes the outbreaks with diarrheal syndrome in pigs. From the pathological material of 3-5-day-old piglets with the pathology a RNA virus was isolated and identified as the virus of epizootic diseases of pigs. Virus infectivity in the fifth passage in Vero cell culture was 10 3,50 ТCID 50/sm 3. The isolate was named as BS-08. To date, there are 25 successive passages of the virus in cell culture. A citopatic effect is registered in 24-48 hours, and the infectious titer (titer of citopatic effect — TCE) makes 10 4,75-10 5,50 TCID 50/sm 3.