Study of the adjuvant properties of preparations containing recombinant human granulocyte-macrophage colony stimulating factor

The relevance of the search for new vaccine adjuvants is growing along with the increase in the number of current vaccine preparations, especially those developed on the basis of proteins. Some cytokines are known to exert adjuvant properties. The present work is devoted to the study of adjuvant activity of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and constructs based on it. Earlier, we developed a technology for isolation and purification of GM-CSF from the E. coli SG20050/p280_2GM producer strain, as well as a technology for conjugating polyglucin:spermidine complexes with rhGM-CSF. Double-stranded RNA was used to obtain molecular constructs on the basis of rhGM-CSF conjugate. To assemble constructs, the ratio of the components was calculated for one dose of the preparation to contain 5-40 mg of rhGM-CSF and 100 mg of double-stranded RNA. The effectiveness of the formation of molecular constructs was evaluated by dsRNA electrophoretic mobility shift in a 1% agarose gel. The effectiveness of the resulting adjuvants was determined in ELISA assays by measuring the titers of specific antibodies in mouse sera against ovalbumin or recombinant receptor-binding domain of the surface S protein of the severe acute respiratory syndrome coronavirus 2 (Delta variant (B.1.617.2). The experiments were carried out in 100 male BALB/c mice weighing 16-18 g. Mice were immunized twice, with a 14-day interval, by intramuscular injection of 200 mL per animal. Recombinant receptor-binding domain of the surface protein of SARS-CoV-2 was administered at a dose of 50 mg/animal, ovalbumin – at two doses – 1 mg or 5 mg/animal. Corresponding antigen was used as a positive control, a saline solution – as a negative control. It was shown that the maximum effect was achieved by immunization with a construct based on double-stranded RNA and rhGM-CSF conjugated to polyglucin-spermidine. The use of a conjugate without double-stranded RNA as an adjuvant also improved humoral response. The use of native rhGM-CSF did not increase the titers of specific antibodies. Thus, it was found that rhGM-CSF being a part of a polysaccharide conjugate or a molecular construct exerted an ability to enhance the humoral immune response to protein antigens

Development of a vaccine adjuvant based on squalene and study of its adjuvant properties

The use of modern subunit vaccines involves adjuvant introduction into their composition. Currently, the search for new and improvement of existing adjuvant systems is actively underway. Squalene- based adjuvants are well-known and approved in a number of countries for clinical use in influenza vaccines. Our study was devoted to the development of an adjuvant composition on the basis of squalene. The resulting adjuvants were composed in a form of oil emulsion containing a hydrophilic and hydrophobic phase. The stability of the emulsion was achieved by treating it with ultrasound at a frequency of 22 kHz. Particle sizes of the obtained emulsions were examined with the use of an electron microscope. The particle size was calculated to be 50-80 nm for the majority of particles (84%). Adjuvant activity was evaluated in 100 male Balb/C mice, weighing 16-18 g. To assess the humoral immune response, immunization was performed twice, with a 14-day interval, by intramuscular injection of 200 mL per animal. The receptor-binding domain (RBD) of the surface S protein of the severe acute respiratory syndrome coronavirus 2 (Delta variant (B.1.617.2)) or ovalbumin (OVA) from chicken eggs were used as antigens. RBD was administered at a dose of 50 mg/animal; OVA was administered at two doses (1 mg or 5 mg/animal). An antigen with aluminum hydroxide was used as a positive control; a saline solution was used as a negative control. The effectiveness of the obtained adjuvants was determined by measuring the titers of specific antibodies in mouse sera in ELISA assays using the recombinant RBD of SARS-CoV-2 S-protein or ovalbumin from chicken eggs. It was shown that the use of squalene-based adjuvants increased the antigens’ immunogenicity. The average titers of specific antibodies against RBD in the experimental group were 4 times higher than in the group immunized with RBD adjuvanted with aluminum hydroxide. An increase in immunogenicity of the antigen adjuvanted with squalene was also observed in the experimental OVA-group. Thus, it was shown that the developed squalene-based adjuvant compositions could be an alternative to the traditional adjuvants based on aluminum salts

Клинико-иммунологическая оценка эффективности применения рекомбинатного интерферона альфа-2b в терапии детей, больных корью

The aim of the study was to assess the clinical and immunological efficacy of recombinant interferon Alfa-2b. Examined 34 children aged from 1 year to 7 years carrying medium to heavy degree of measles. All patients were not vaccinated against measles. In the treatment of 18 people (Control group) were used only basic therapy (pathogenic, symptomatic). At 16 people (Study group) used a combination therapy (basic + Viferon). As an antiviral agent used and immunotropic preparation of human recombinant interferon alfa-2b in the form of rectal suppositories – Viferon (OOO «Feron», Russia). Immunological patient survey was conducted in the dynamics of the disease – during the height (in 1-6 days the rash) and in convalescence period (10-16 days rash). Indicators of cellular immunity (leukocytes, lymphocytes, CD3 +, CD3 + CD (16 + 56) +, CD3 + CD4 +, CD3 + CD8 +, CD4 + CD8 +, CD3-CD8 +, CD3-CD (16 + 56), CD19 +, CD25 +, CD3 + HLA DR +, CD (16 + 56) + HLA DR +, HLA DR +, CD95 +) were evaluated by flow cytometry. To assess the production of IFN-α and IFN-γ in the samples of peripheral blood were cultured in the CO2 incubator for 18 hours at 37 C and 4% CO2. The supernatants of cell culture were investigated with the purpose of studying spontaneous and induced production of interferon alpha and gamma. The concentrations of interferon α and γ (IFNα, IFNγ) in the blood serum, as well as levels of spontaneous and induced cytokine production data of blood cells of sick children was determined by ELISA. All the children at the peak of the disease found a dramatic inhibition of cellular immunity and the production of cytokines, which is consistent with the concept of «measles anergy» accepted in the scientific literature. The period of convalescence oppression immunity indices remained, but it was less pronounced in the group of children treated with the drug of human recombinant interferon alfa-2b -Viferon. Clinical efficacy of Viferon in the treatment of patients with measles children characterized by rapid positive dynamics of symptoms of acute period (normalization of body temperature, reducing intoxication, catarrhal symptoms and severity of the syndrome exanthema). It was also found reduction in the incidence of complications, reduction in the average bedday and smooth during the period of convalescence later.Цель исследования – оценка клинико-иммунологической эффективности рекомбинантного интерферона альфа-2b. Проведено обследование 34 детей в возрасте от 1 года до 7 лет, переносивших среднетяжелую степень кори. Все пациенты против кори не привитые. В терапии 18 человек (группа сравнения) применяли только базисную терапию (патогенетическая, симптоматическая), у 16 человек (основная группа) применяли комбинированную терапию (базисная + Виферон). В качестве противовирусного и иммунотропного средства использовали препарат человеческого рекомбинантного интерферона альфа-2b в виде ректальных суппозиториев – Виферон (ООО «ФЕРОН», Россия). Иммунологическое обследование пациентов проводили в динамике заболевания – в периоде разгара (на 1–6-е сут высыпания) и в периоде реконвалесценции (на 10–16-е сут высыпания). Показатели клеточного звена иммунитета (лейкоциты, лимфоциты, CD3+, CD3+CD(16+56)+, CD3+CD4+,CD3+CD8+, CD4+CD8+, CD3-CD8+, CD3-CD(16+56), CD19+, CD25+, CD3+HLA DR+, CD(16+56)+ HLA DR+, HLA DR+, CD95+) оценивались методом проточной цитометрии. Для оценки продукции IFN-α и IFN-γ образцы периферической крови культивировали в СО2 инкубаторе в течение 18 ч при 37° С и 4% СО2. Супернатанты клеточной культуры исследовались с целью изучения спонтанной и индуцированной продукции интерферона альфа и гамма. Концентрации IFNα, IFNγ в сыворотке крови, а также уровни спонтанной и индуцированной продукции данных цитокинов клетками крови больных детей определяли иммуноферментным методом. У всех детей в периоде разгара болезни установлено резкое угнетение показателей клеточного иммунитета и продукции цитокинов, что согласуется с принятым в научной литературе понятием «коревая анэргия». В периоде реконвалесценции угнетение показателей иммунитета сохранялось, однако было менее выражено в группе  детей, получавших препарат Виферон. Клиническая эффективность применения Виферона в комплексной терапии больных корью детей характеризовалась быстрой положительной динамикой симптомов острого периода (нормализация температуры тела, снижение интоксикации, выраженности катарального синдрома и синдрома экзантемы). Установлено также снижение частоты развития осложнений, сокращение среднего койко-дня и гладкое течение периода поздней реконвалесценции

Результаты 5-летнего мониторинга за циркуляцией сезонных коронавирусов у госпитализированных детей в препандемическом периоде

Coronaviruses can cause damage to various parts of the respiratory system, gastrointestinal tract, and other organs and systems.The aim of the study: to monitor the circulation of seasonal coronaviruses in hospitalized children in the pre-pandemic period.Materials and methods: real-time multiplex PCR was used to test samples of nasopharyngeal mucus from 2188 patients aged 1 monthto 17 years, hospitalized with acute respiratory infection in 2014—2018. The results are presented with the indication of the fractions (%) and the calculation of the 95% confidence interval according to Klopper-Pearson. The differences between the groups were evaluated using the Pearson χ2 test. The differences in the groups were considered statistically significant at the level of the criterion p< 0.05.Results: monitoring of the circulation of pathogens of acute respiratory viral infection (ARVI) during 5 epidemic seasons showed that the appearance of a new subtype of coronavirus in 2019 was preceded by a gradual displacement of influenza, RS-and bocavirus infections from the circulation due to a statistically significant increase in the proportion of seasonal coronaviruses from 3.6% in 2014—2015 to 10.8% in the prepandemic season 2018—2019 (p= 0.007). The circulation of seasonal coronaviruses had a distinct seasonality (november-april)with the peak of registration in february (28.4%) and march (36.7%). Seasonal coronaviruses were detected in 7.3% of hospitalized children with ARVI, with a predominance in the age groups under 2 years (58.2%) and 3—6 years (25.4%). Hospitalization was more often required for patients with lower respiratory tract lesions (58.2%), a fifth of which was pneumonia (21.8%). In most children, ARVI caused by coronaviruses occurred as a monoinfection (79.9%), combined infection with other pathogens was observed in 20.1% of cases with fluctuations from 18.2% to 28.6% in different epidemic seasons. Viral associations are most common in young children (85.2%).Коронавирусы могут вызвать поражение различных отделов дыхательной системы, желудочно-кишечного тракта, а также других органов и систем.Цель исследования: провести мониторинг за циркуляцией сезонных коронавирусов у госпитализированных детей в препандемическом периоде.Материалы и методы: методом мультиплексной ПЦР в реальном времени проведено тестирование образцов носоглоточной слизи 2188 пациентов в возрасте от 1 мес. до 17 лет, госпитализированных с острой респираторной инфекцией в 2014—2018 гг. Результаты представлены с указанием долей (%) и расчетом 95% доверительного интервала по Клопперу-Пирсону. Различия между группами оценивали с помощью критерия χ² Пирсона. Различия в группах считались статистически значимыми при уровне критерия p< 0,05.Результаты: мониторинг за циркуляцией возбудителей острой респираторной вирусной инфекции (ОРВИ) в течение 5-ти эпидемических сезонов показал, что появлению нового подтипа коронавируса в 2019 г. предшествовало постепенное вытеснение из циркуляции возбудителей гриппа, РС- и бокавирусной инфекции за счет статистически значимого увеличения доли сезонных коронавирусов с 3,6% в 2014—2015 гг. до 10,8% в препандемическом сезоне 2018—2019 гг. (р= 0,007). Циркуляция сезонных коронавирусов имела отчетливую сезонность (ноябрь-апрель) с пиком регистрации в феврале (28,4%) и марте (36,7%). Сезонные коронавирусы выявлялись у 7,3% госпитализированных детей с ОРВИ, с преобладанием в возрастных группах до 2 лет (58,2%) и 3—6 лет (25,4%).Госпитализация чаще требовалась пациентам с поражением нижних отделов респираторного тракта (58,2%), пятую часть из которых составляла пневмония (21,8%). У большинства детей ОРВИ, обусловленная коронавирусами, протекала в виде моноинфекции (79,9%), сочетанное инфицирование с другими возбудителями наблюдалось в 20,1% случаев с колебаниями от 18,2% до 28,6% в различные эпидемические сезоны. Вирусные ассоциации наиболее характерны для детей раннего возраста (85,2%

Особенности новой коронавирусной инфекции у детей разного возраста

The new coronavirus infection (COVID-1 9) is a socially significant problem around the world. According to available statistics, complications are less common among children, asymptomatic or mild forms of the disease prevail more often.This article presents the features of the viral landscape of the upper respiratory tract in children with ARVI in a pandemic, the clinical and laboratory features of the course of COVID-1 9 in children of different ages.It was found that SARS-CoV-2 is detected only in a third (32.9%) of hospitalized patients with respiratory symptoms, in 4.3% of cases — in combination with seasonal CoV-OC43 / CoV-229E, in 1 1.6% — with other respiratory viruses. The most frequent source of infection with the SARS-Cov-2 were family members. Children with a moderate form of the disease predominated among the patients. The leading symptoms of COVID-19 were fever, catarrhal symptoms, as well as gastrointestinal manifestations and anosmia. A feature of the new coronavirus infection in newborns and children of the first month of life was the absence of fever and intoxication, the lack of expression of catarrhal manifestations when the colon is involved in the pathological process (colitis, rarely — hemocolitis). In the compete blood test in children under the age of one year, monocytosis prevailed, in children over 7 years old — leukopenia and accelerated ESR. Among the changes in the biochemical analysis of blood, the most common was an increased C-reactive protein.Новая коронавирусная инфекция (COVID-19) является социально-значимой проблемой во всем мире. По имеющейся статистике, среди детей реже встречаются осложнения и чаще преобладают бессимптомные или легкие формы заболевания.В данной статье представлены особенности вирусного пейзажа верхних дыхательных путей у детей с ОРВИ в условиях пандемии, клинико-лабораторные особенности течения COVID-19 у детей разного возраста.Установлено, что SARS-CoV-2 обнаруживается только у трети (32,9%) госпитализированных пациентов с респираторными симптомами, в 4,3% случаях — в сочетании с сезонными CoV-OC43/CoV-229E, в 11,6% — с другими респираторными вирусами. Источником заражения SARS-Cov-2 чаще являлись члены семьи. Среди пациентов преобладали дети со среднетяжелой формой заболевания. Ведущими симптомами COVID-19 являлись лихорадка, катаральные симптомы, а также гастроинтестинальные проявления и аносмия. Особенностью новой коронавирусной инфекции у новорожденных и детей первого месяца жизни являлось отсутствие лихорадки и интоксикации, невыраженность катаральных проявлений при вовлечении в патологический процесс толстой кишки (колит, редко — гемоколит). В гемограмме у детей в возрасте до года преобладал моноцитоз, у детей старше 7 лет — лейкопения и ускорение СОЭ. Среди изменений в биохимическом анализе крови наиболее часто встречалось повышение С-реактивного белка

ЭТИОТРОПНАЯ ТЕРАПИЯ ОСТРЫХ РЕСПИРАТОРНЫХ ВИРУСНЫХ ИНФЕКЦИЙ СО СТЕНОЗИРУЮЩИМИ ЛАРИНГОТРАХЕИТАМИ

Clinical-laboratory analysis of the effectiveness of VIFERON® (suppositories rectal) in children from 1 month up to 7 years with acute respiratory viral infections (ARVI), accompanied by acute stenosing laryngotracheitis (ASLT), hospitalized in Children's City Clinical Hospital №5 named after N.F. Filatov. We have found that the drug VIFERON® helps statistically significant reduction in the duration of all major clinical manifestations of the disease as a whole, and also reduces the severity and duration of clinical symptoms ASLT, and significantly contributes to the faster elimination of viral antigens.Проведен клиническо-лабораторный анализ эффективности препарата ВИФЕРОН® (суппозитории ректальные) у детей в возрасте от 1 мес. до 7 лет с острыми респираторными вирусными инфекциями (ОРВИ), сопровождающимися острым стенозирующим ларинготрахеитом (ОСЛТ), госпитализированных в ДГКБ №5 им. Филатова. Нами установлено, что препарат ВИФЕРОН® способствует статистически значимому сокращению продолжительности основных клинических проявлений заболевания, уменьшает выраженность и продолжительность клинических симптомов ОСЛТ, способствует более быстрой элиминации вирусных антигенов

ОСТРЫЙ СТЕНОЗИРУЮЩИЙ ЛАРИНГОТРАХЕИТ ПРИ РЕСПИРАТОРНЫХ ВИРУСНЫХ МОНОИНФЕКЦИЯХ У ДЕТЕЙ, ГОСПИТАЛИЗИРОВАННЫХ В СТАЦИОНАРЫ САНКТ-ПЕТЕРБУРГА

A comparative analysis of the development of acute stenosing laryngotracheitis (LSLT) in respiratory viral monoinfections in 1050 children, hospitalized in St. Petersburg hospitals. It was established that the clinical manifestations, severity and outcome of OSLT in viral monoinfections depend highly on the etiologic factor, the patient's age and his premorbid background. More often, stenoses of the larynx are provoked by parainfluenza viruses (26,7%), the next most important are the PC virus (22,3%) and adenoviruses (18,8%), but during epidemics the number of lesions of the larynx sharply increases, with influenza viruses as the main pathogen, reaching up to 31,1%. Boys (71,2%) suffer from LSLT more often than girls (28,8%). Burdened premorbid background were 88,1% of children.  Проведен сравнительный анализ развития острого стенозирующего ларинготрахеита (ОСЛТ) при респираторно-вирусных моноинфекциях у 1050 детей, госпитализированных в стационары Санкт-Петербурга. Установлено, что клинические проявления, тяжесть и исход ОСЛТ при вирусных моноинфекциях зависят с высокой долей вероятности от этиологического фактора, возраста пациента и его преморбидного фона. Чаще других стенозы гортани провоцируют вирусы парагриппа (26,7%), следующими по значимости являются РС-вирус (22,3%) и аденовирусы (18,8%), но в периоды эпид. подъема гриппа число поражений гортани возрастает, а основными возбудителями ОСЛТ становятся вирусы гриппа (31,1%). У мальчиков ОСЛТ регистрируется чаще, чем у девочек (71,2 и 28,8%). Отягощенный преморбидный фон был выявлен в 88,1% детей

Clinical and immunological efficacy of combination therapy of children with measles

The aim of the study was to assess the clinical and immunological efficacy of recombinant interferon Alfa-2b. Examined 34 children aged from 1 year to 7 years carrying medium to heavy degree of measles. All patients were not vaccinated against measles. In the treatment of 18 people (Control group) were used only basic therapy (pathogenic, symptomatic). At 16 people (Study group) used a combination therapy (basic + Viferon). As an antiviral agent used and immunotropic preparation of human recombinant interferon alfa-2b in the form of rectal suppositories – Viferon (OOO «Feron», Russia). Immunological patient survey was conducted in the dynamics of the disease – during the height (in 1-6 days the rash) and in convalescence period (10-16 days rash). Indicators of cellular immunity (leukocytes, lymphocytes, CD3 +, CD3 + CD (16 + 56) +, CD3 + CD4 +, CD3 + CD8 +, CD4 + CD8 +, CD3-CD8 +, CD3-CD (16 + 56), CD19 +, CD25 +, CD3 + HLA DR +, CD (16 + 56) + HLA DR +, HLA DR +, CD95 +) were evaluated by flow cytometry. To assess the production of IFN-α and IFN-γ in the samples of peripheral blood were cultured in the CO2 incubator for 18 hours at 37 C and 4% CO2. The supernatants of cell culture were investigated with the purpose of studying spontaneous and induced production of interferon alpha and gamma. The concentrations of interferon α and γ (IFNα, IFNγ) in the blood serum, as well as levels of spontaneous and induced cytokine production data of blood cells of sick children was determined by ELISA. All the children at the peak of the disease found a dramatic inhibition of cellular immunity and the production of cytokines, which is consistent with the concept of «measles anergy» accepted in the scientific literature. The period of convalescence oppression immunity indices remained, but it was less pronounced in the group of children treated with the drug of human recombinant interferon alfa-2b -Viferon. Clinical efficacy of Viferon in the treatment of patients with measles children characterized by rapid positive dynamics of symptoms of acute period (normalization of body temperature, reducing intoxication, catarrhal symptoms and severity of the syndrome exanthema). It was also found reduction in the incidence of complications, reduction in the average bedday and smooth during the period of convalescence later

ETIOTROPIC THERAPY FOR ACUTE RESPIRATORY VIRAL INFECTIONS WITH STENOSING LARYNGOTRACHEITIS

Clinical-laboratory analysis of the effectiveness of VIFERON® (suppositories rectal) in children from 1 month up to 7 years with acute respiratory viral infections (ARVI), accompanied by acute stenosing laryngotracheitis (ASLT), hospitalized in Children's City Clinical Hospital №5 named after N.F. Filatov. We have found that the drug VIFERON® helps statistically significant reduction in the duration of all major clinical manifestations of the disease as a whole, and also reduces the severity and duration of clinical symptoms ASLT, and significantly contributes to the faster elimination of viral antigens