Cellular immune response in infected mice to NSP protein encoded by the negative strand NS RNA of influenza A virus

Influenza A virus belongs to a family of enveloped viruses with an RNA genome of negative polarity consisting of 8 RNA segments. The transcription of this RNA genome results in the synthesis of positive-sense mRNAs that translate up to 16 unique viral proteins with the help of splicing and translational shift mechanisms. The 8th NS segment encodes the NS1 protein (27 kDa), which is an active interferon antagonist, and the nuclear export protein NEP (14 kDa) through the standard negative polarity pathway. In addition, an alternative open reading frame for the synthesis of a third viral protein (NSP, negative-strand protein) by means of a direct translation of genome polarity RNA (the so-called positive polarity genome strategy) was identified in the NS segment. Since it is unknown as to whether the NSP protein can be synthesized in the infected organism post viral infection, the generation of spleen leucocytes specific to this protein was studied in mice after two sequential infections with influenza A viruses of H1N1 and H3N2 subtypes. It was found that leucocyte clones specifically recognizing a peptide domain in the central region of the NSP protein (amino acid positions 82-119) were generated in mice infected with influenza A viruses. In silico prediction has shown strong major histocompatibility complex-1 (MHC-I) and MHC-II specific epitopes in this central domain of the NSP. Comparative analysis of the influenza H3N2 viruses circulating in humans during 1968-2018 has shown high NSP variability, which was similar to that shown for the hemagglutinin (HA) and neuraminidase (NA) proteins. The highest variability was found to be in the N- and C-terminal parts of the NSP. These observations suggest that synthesis of the NSP protein occurs in infected animals and further support a bipolar (ambisense) strategy of the RNA genome of human influenza A virus.Influenza A virus belongs to a family of enveloped viruses with an RNA genome of negative polarity consisting of 8 RNA segments. The transcription of this RNA genome results in the synthesis of positive-sense mRNAs that translate up to 16 unique viral proteins with the help of splicing and translational shift mechanisms. The 8th NS segment encodes the NS1 protein (27 kDa), which is an active interferon antagonist, and the nuclear export protein NEP (14 kDa) through the standard negative polarity pathway. In addition, an alternative open reading frame for the synthesis of a third viral protein (NSP, negative-strand protein) by means of a direct translation of genome polarity RNA (the so-called positive polarity genome strategy) was identified in the NS segment. Since it is unknown as to whether the NSP protein can be synthesized in the infected organism post viral infection, the generation of spleen leucocytes specific to this protein was studied in mice after two sequential infections with influenza A viruses of H1N1 and H3N2 subtypes. It was found that leucocyte clones specifically recognizing a peptide domain in the central region of the NSP protein (amino acid positions 82-119) were generated in mice infected with influenza A viruses. In silico prediction has shown strong major histocompatibility complex-1 (MHC-I) and MHC-II specific epitopes in this central domain of the NSP. Comparative analysis of the influenza H3N2 viruses circulating in humans during 1968-2018 has shown high NSP variability, which was similar to that shown for the hemagglutinin (HA) and neuraminidase (NA) proteins. The highest variability was found to be in the N- and C-terminal parts of the NSP. These observations suggest that synthesis of the NSP protein occurs in infected animals and further support a bipolar (ambisense) strategy of the RNA genome of human influenza A virus

Structure and phase composition of complex refractory coating and of the reaction zone of interaction with single-crystal alloy ZhS36-VI after high-temperature holds

A complex coating consisting of three layers, i.e., a gas-circulation coating and a two-layer ion-plasma coating with an internal Ni - Cr - Al - Ta - Re - Y layer and an external Al - Ni - Cr - Y layer for protecting single crystal turbine rotor blades from high-temperature oxidation is considered. The fine structure and the phase composition of the complex coating and of the reaction zone of interaction with single-crystal alloy ZhS36-VI after high-temperature holds in a range of 1050 - 1300 C for 1-1000 h are studied. © 2013 Springer Science+Business Media New York

Long-term strength of a composition of complex refractory coating and single-crystal rhenium-alloyed nickel alloy after high-temperature holds

The long-term strength of a system of single-crystal nickel alloy ZhS36-VI with Cr - Al gas circulation coating (GCC) + Ni - Cr - Al - Ta - Re - Yion-plasma coating (IPC) + Al - Ni - Cr - Yion-plasma coating (IPC) is analyzed under conditions close to the operating ones after a hold for 1 - 1000 h in a temperature range of 1050 - 1300 C. © 2013 Springer Science+Business Media New York

Клеточный иммунный ответ у инфицированных животных против белка NSP, кодируемого негативной цепью NS RNA вируса гриппа А

Influenza A virus belongs to a family of enveloped viruses with an RNA genome of negative polarity consisting of 8 RNA segments. The transcription of this RNA genome results in the synthesis of positive-sense mRNAs that translate up to 16 unique viral proteins with the help of splicing and translational shift mechanisms. The 8th NS segment encodes the NS1 protein (27 kDa), which is an active interferon antagonist, and the nuclear export protein NEP (14 kDa) through the standard negative polarity pathway. In addition, an alternative open reading frame for the synthesis of a third viral protein (NSP, negative-strand protein) by means of a direct translation of genome polarity RNA (the so-called positive polarity genome strategy) was identified in the NS segment. Since it is unknown as to whether the NSP protein can be synthesized in the infected organism post viral infection, the generation of spleen leucocytes specific to this protein was studied in mice after two sequential infections with influenza A viruses of H1N1 and H3N2 subtypes. It was found that leucocyte clones specifically recognizing a peptide domain in the central region of the NSP protein (amino acid positions 82-119) were generated in mice infected with influenza A viruses. In silico prediction has shown strong major histocompatibility complex-1 (MHC-I) and MHC-II specific epitopes in this central domain of the NSP. Comparative analysis of the influenza H3N2 viruses circulating in humans during 1968-2018 has shown high NSP variability, which was similar to that shown for the hemagglutinin (HA) and neuraminidase (NA) proteins. The highest variability was found to be in the N- and C-terminal parts of the NSP. These observations suggest that synthesis of the NSP protein occurs in infected animals and further support a bipolar (ambisense) strategy of the RNA genome of human influenza A virus.Вирус гриппа А относится к оболочечным RNA-содержащим вирусам с негативно-полярным геномом, состоящим из 8 сегментов RNA, каждый из которых является матрицей для синтеза позитивно-полярных mRNA, которые, в свою очередь, служат матрицей для синтеза 16 вирусных белков с использованием для некоторых механизма сплайсинга или трансляционного шифта. Восьмой сегмент NS посредством классической негативной стратегии генома кодирует белок NS1, обладающий анти-интерфероновой активностью (27 кДа), и белок ядерного экспорта NEP (NS2) (nuclear export protein, 14 кДа). Кроме этого, в сегменте NS обнаружена дополнительная открытая рамка для синтеза третьего вирусного белка по альтернативному пути посредством прямой трансляции негативно полярной вирусной RNA. Эта стратегия генома получила название позитивно-полярной. Образование данного белка, названного NSP (negative strand protein), при инфекции вируса гриппа А в целостном организме пока не обнаружено. Анализ белка NSP in silico выявил наличие доменов MHC-I (major histocompatibility complex-1) и MHC-II. Сравнительный анализ вирусов гриппа подтипа H3N2, циркулировавших среди людей в период с 1968 по 2018 гг., показал высокую изменчивость гена белка NSP, которая сходна с изменчивостью поверхностных белков гемагглютинина (HA) и нейраминидазы (NA). Наибольшая изменчивость обнаруживалась в зонах, соответствующих N- и C-концевым участкам белка NSP. В настоящей работе изучена возможность образования иммунных лейкоцитов, специфичных к белку NSP, у мышей после инфекции вирусом гриппа А. Показано, что у мышей после заражения последовательно двумя вирусами гриппа А разных сероподтипов обнаруживаются иммунные лейкоциты, специфически распознающие вирусные домены в центральной зоне белка NSP (позиции аминокислот 82-119). Полученные данные с большой вероятностью позволяют утверждать, что при инфекции вирусом гриппа А в организме животных имеет место экспрессия гена NSP, что в свою очередь подтверждает концепцию о биполярной (амбисенс) стратегии генома вируса гриппа А

ОПРЕДЕЛЕНИЕ СРОКОВ НАЧАЛА ГЕМОДИАЛИЗА: РАЗРАБОТКА И ПОДТВЕРЖДЕНИЕ ШКАЛЫ START

Aim. The optimal time for initiating of chronic dialysis remains unknown. The scale for mortality risk assessment could help in decision-making concerning dialysis start timing.Methods. We randomly divided 1856 patients started dialysis in 2009–2016 into developmental and validation group (1:1) to create and validate scoring system «START» predicting mortality risk at dialysis initiation in order to fi nd unmodifi able and modifi able factors which could help in the decision-making of dialysis start. In the series of univariate regression models in the developmental set, we evaluated the mortality risk linked with available parameters: age, eGFR, serum phosphate, total calcium, hemoglobin, Charlson comorbidity index, diabetes status, urgency of start (turned to be signifi cant) and gender, serum sodium, potassium, blood pressure (without impact on survival). Similar hazard ratios were converted to score points.Results. The START score was highly predictive of death: C-statistic was 0.82 (95% CI 0.79–0.85) for the developmental dataset and 0.79 (95% CI 0.74–0.84) for validation dataset (both p < 0.001). On applying the cutoff between 7–8 points in the developmental dataset, the risk score was highly sensitive 81.1% and specifi c 67.9%; for validation dataset, the sensitivity was 78.9%, specifi city 67.9%. We confi rmed the similarity in survival prediction in the validation set to developmental set in low, medium and high START score groups. The difference in survival between three levels of START-score in validation set remained similar to that of developmental set: Wilcoxon = 8.78 (p = 0.02) vs 15.31 (p < 0.001) comparing low–medium levels and 25.18 (p < 0.001) vs 39.21 (p < 0.001) comparing medium–high levels.Conclusion. Developed START score system including modifi able factors showed good mortality prediction and could be used in dialysis start decision-making. Цель. Оптимальное время начала лечения гемодиализом (ГД) остается неопределенным. Создание шкалы для оценки рисков для пациента, начинающего диализ, поможет в принятии решения о сроках его начала.Методы. По данным регистра пациентов на заместительной почечной терапии проанализированы результаты лечения 1856 пациентов, начавших диализ в 2009–2016 гг. Случайным образом их разделили в соотношении 1:1 на группу разработки и группу подтверждения для создания прогностической шкалы оценки вероятности летального исхода при лечении программным гемодиализом (шкала START). При этом учитывали модифицируемые и немодифицируемые факторы риска. В серии регрессионных моделей с одной переменной в группе разработки шкалы мы оценивали риски смерти, связанные с доступными для анализа модифицируемыми и немодифицируемыми параметрами. Среди них значимыми оказались возраст, расчетная скорость клубочковой фильтрации (рСКФ), уровни фосфатов, общего кальция, гемоглобина, индекс коморбидности Чарлсон, наличие сахарного диабета, экстренность старта диализа. Пол, уровни натрия, калия, артериального давления не оказали влияния на выживаемость. Близкие по величине риски были преобразованы в баллы шкалы.Результаты. Шкала START продемонстрировала высокую предсказательную ценность в отношении риска смерти: AUC 0,82 (95% ДИ 0,79–0,85) для группы разработки шкалы и 0,79 (95% ДИ 0,74–0,84) для группы подтверждения (для обеих p < 0,001). Для точки отсечения 7–8 баллов чувствительность метода составила 81,1%, специфичность 69,9% для группы разработки и 78,9 и 67,9% соответственно – для группы подтверждения. Мы подтвердили близкие значения выживаемости пациентов в обеих группах для низких, средних и высоких значений шкалы START. Различия в выживаемости для подгрупп с тремя уровнями шкалы были близкими для групп разработки и подтверждения: критерий Wilcoxon = 8,78 (p = 0,02) vs 15,31 (p < 0,001) при сравнении выживаемости подгрупп низких и средних величин шкалы и 25,18 (p < 0,001) vs 39,21 (p < 0,001) при сравнении выживаемости подгрупп средних и высоких величин шкалы.Заключение. Разработанная шкала START, включающая модифицируемые факторы риска, продемонстрировала хорошую предсказательную ценность в отношении 5-летней летальности и может использоваться при принятии решения о времени старта диализа.

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Structural and phase transformations in single-crystal alloy ZhS36-VI [001] after holding in the range of 1050 - 1300°C

The special features of structure and phase transformations in alloy ZhS36-VI [001] after long high-temperature holds are considered. Formation of μ-phases with different morphology and of a (γ +γ′) structure of the nanosize level is discovered. The temperature-time domain of existence of the phases in alloy ZhS36-VI [001] is plotted. © 2012 Springer Science+Business Media, Inc