The therapeutic use of Abs in cancer, autoimmunity, transplantation, and other fields is among the major biopharmaceutical advances of the 20th century. Broader use of Ab-based drugs is constrained because of their high production costs and frequent side effects. One promising approach to overcome these limitations is the use of highly diluted Abs, which are produced by gradual reduction of an Ab concentration to an extremely low level. This technology was used to create a group of drugs for the treatment of various diseases, depending on the specificity of the used Abs. Highly diluted Abs to IFN-g (hd-anti-IFN-g) have been demonstrated to be efficacious against influenza and other respiratory infections in a variety of preclinical and clinical studies. In the current study, we provide evidence for a possible mechanism of action of hd-anti-IFN-g. Using high-resolution solution nuclear magnetic resonance spectroscopy, we show that the drug induced conformational changes in the IFN-g molecule. Chemical shift changes occurred in the amino acids located primarily at the dimer interface and at the C-terminal region of IFN-g. These molecular changes could be crucial for the function of the protein, as evidenced by an observed hd-anti-IFN-g-induced increase in the specific binding of IFN-g to its receptor in U937 cells, enhanced induced production of IFN-g in human PBMC culture, and increased survival of influenza A-infected mice.
In preliminary ELISA studies where released-active forms (RAF) of antibodies (Abs) to interferon-gamma (IFNg) were added to the antigen-antibody system, a statistically significant difference in absorbance signals obtained in their presence in comparison to placebo was observed. A piezoelectric immunosensor assay was developed to support these data and investigate the effects of RAF Abs to IFNg on the specific interaction between Abs to IFNg and IFNg. The experimental conditions were designed and optimal electrode coating, detection circumstances and suitable chaotropic agents for electrode regeneration were selected. The developed technique was found to provide high repeatability, intermediate precision and specificity. The difference between the analytical signals of RAF Ab samples and those of the placebo was up to 50.8%, whereas the difference between non-specific controls and the placebo was within 5%–6%. Thus, the piezoelectric immunosensor as well as ELISA has the potential to be used for detecting the effects of RAF Abs to IFNg on the antigen-antibody interaction, which might be the result of RAF’s ability to modify the affinity of IFNg to specific/related Abs.
Цель исследования - изучение возможного прямого влияния препарата «Анаферон детский» на жизненный цикл вируса гриппа А в процессе развития инфекции, а также дозозависимости противовирусного эффекта in vitro . Методика. Исследование противовирусной активности препарата «Анаферон детский» in vitro было проведено с использованием культуры клеток MDCK (Madin Darby canine kidney) и эталонных штаммов вируса гриппа A (H1N1) pdm09: A/California/07/09 и А/California/04/09, полученных от ВОЗ. Использовались методы оценки подавления Анафероном детским вирусной репликации (по результатам иммуноферментного анализа по определению экспрессии внутренних белков NP и M1 вируса гриппа А) и его влияние на ультраструктурные особенности морфогенеза вируса гриппа методом электронной микроскопии. В качестве положительного контроля был использован Озельтамивир карбоксилат в концентрации 10 мкМ. Для мониторинга валидности экспериментальной модели в работе использовали клетки, зараженные вирусом без добавления экспериментальных образцов (контроль вируса), а также интактные клетки (клеточный контроль). Результаты. В ходе исследования показан дозозависимый противовирусный эффект препарата «Анаферон детский» для 3 тестируемых разведений - 1/8, 1/12, 1/16. Методом электронной микроскопии показано, что применение препарата «Анаферон детский» при сравнении с контрольным образцом влияло на процесс почкования вирионов. Заключение. Впервые показана дозозависимость противовирусного действия препарата «Анаферон детский», а также подтверждена его эффективность в отношении двух штаммов вируса пандемического гриппа А/H1N1. Документировано, что применение препарата «Анаферон детский» нарушает жизненный цикл вируса гриппа А. Механизмы развития такого эффекта требуют дополнительного изучения, однако можно предположить их связь с ИФН-индуцирующими свойствами препарата «Анаферон детский», так как было показано, что в начале лечения вирусной инфекции препарат вызывает индукцию синтеза белков системы интерферонов.
The aim of this study was to evaluate a possible direct effect of Anaferon for Children on the life cycle of influenza A virus during infection development and a dose response of the antiviral effect in vitro. Methods. The in vitro antiviral activity of Anaferon for Children was studied on cultured MDCK cells and reference strains of influenza virus A (H1N1) pdm09: A/California/07/09 and A/California/04/09, both from the WHO. Inhibition of viral replication by Anaferon for Children and its effect on ultrastructural features of the influenza morphogenesis were evaluated using electron microscopy. Results. The study demonstrated a dose dependence of Anaferon for Children antiviral activity for three dilutions - 1/8, 1/12, and 1/16. Anaferon for Children affected the process of virion budding as compared to placebo. Conclusion. The study showed that the anti-influenza action of Anaferon for Children was dose-dependent and confirmed that this drug was effective against two strains of pandemic A/H1N1 influenza. Furthermore, Anaferon for children disrupted one or several stages of the virus life cycle.
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