The aim of this investigation was a comparative analysis of isotypic structure of specific antiviral systemic humoral immune response in hospitalized patients with influenza caused by virus A(H3N2) or A(H1N1), including the A(H1N1)pdm09. Paired acute and convalescent phase sera from 109 adult patients aged 18 to 67 years with laboratoryconfirmed influenza A were analyzed by ELISA. Purified surface glycoproteins of influenza A viruses of different subtypes containing the hemagglutinin and neuraminidase were used as antigen for sensitization of plates in ELISA.The absence of type-specific conserved internal proteins in antigenic material allowed to carry out a subtype-specific differentiation of antibodies against influenza viruses in ELISA. Regardless of the subtype of influenza A viruses caused the disease, the most pronounced response was observed by subtype-specific IgG1 (70–90% of seroconversions). It has been shown for the first time that low activity of virus-induced IgG2 (6–9% of seroconversions) is a peculiarity of the immune response both to primary or recurrent infections with A(H1N1)pdm09. In patients repeatedly suffered by «seasonal» influenza A(H1N1) in 2007/2008 or influenza A(H3N2) in 2012–2014 IgG2 seroconversion’s rates were 40–59% (р < 0,05). Reaction virusspecific IgG3 was also weaker in patients with influenza A(H1N1)pdm09 (29–44% of seroconversions) than in subjects with influenza A(H1N1) or A(H3N2) (65% and 56% of seroconversions, respectively). Geometric mean titers of virus neutralizing antibodies identified during recovery phase in patients with primary and secondary influenza A(H1N1)pdm09 (1/28 and 1/103, respectively) were significantly lower than in patients recovered from influenza A(H1N1) or A(H3N2) (GMT were 1/594 and 1/378, respectively). It was shown that the surface glycoproteins of influenza A viruses may be an allergens. Virus-specific IgE seroconversion rates were comparable in all groups reaching 25–45%. The high activity of virus-induced serum IgA was detected in patients with influenza A(H3N2) or A(H1N1)pdm09 (60–79% of seroconversions). Thus, study of virus-specific activity of various immunoglobulin isotypes provides important information about the formation of adaptive antiviral immune response to influenza A viruses, and also estimate the contribution of its protective and immunopathogenic components to pathogenesis of the disease.

Diagnostic properties of new monoclonal antibodies (MAbs) to hexon adenovirus antigen (AB) monoclonal ELISA kit for early diagnosis of adenoviral infection were tested. Developed ELISA kit and FITC-conjugate of new monocional antibodies for immunofluorescent analysis were used for detection of different types of adenoviruses in clinical materials. The availability of their use in clinical and epidemiological practice was validated.

Diagnostic properties of new monoclonal antibodies (MAbs) to hexon adenovirus antigen (AB) monoclonal ELISA kit for early diagnosis of adenoviral infection were tested. Developed ELISA kit and FITC-conjugate of new monoclonal antibodies for immunofluorescent analysis were used for detection of different types of adenoviruses in clinical materials. The availability of their use in clinical and epidemiological practice was validated.

The article discusses the results of study that demonstrated the possibility of successful application of radial hemolysis reaction in analyzing the human inoculation immunity to new strains of influenza virus serotype A A (H5N1) andA (H5N2). The radial hemolysis reaction provides accurate results on introduction of erythrocytes of horse or sheep into hemolytic system instead of erythrocytes of hens applied previously. The technique combines high sensitivity (in comparison with reactions of hemagglutination-inhibition and micro-neutralization, correlation coefficient 0.84-0.85) and total absence of inhibitors impact on the reaction results. During the investigation of immune response of patients who had pandemic virus A (H1N1) pdm09, radial hemolysis reaction demonstrated not only primary detection of antibodies to virus-agent (67°%) during pandemic, but also elective heightened sensibility in the zone of low titer serums (1:20) in hemagglutination-inhibition reaction. These characteristics are very important in analysis of antibodies levels at early stages of disease. The radial hemolysis reaction continues to be a reliable instrument in evaluating qualitative and quantitative indicators of humoral immunity in ill patients and persons inoculated with new strains of human influenza virus.

The article deals with the study of characteristics of epitope-specific humoral immune response to respiratory syncytial viral infection depending on nature of disease and patients' age. The couple serums from 226 children and adults with respiratory syncytial viral infection were analyzed. To detect in enzyme immunoassay the epitope-specific IgG the synthetic peptides were applied imitating the structure of functionally depended epitopes of F-protein of respiratory syncytial virus with amino acid sequences 221-232 (F=SP12), 479-491 (F-SP13) and G-protein with amino acid sequences 152-164 (G-H13), 184-198 (G-T15). The respiratory syncytial viral infection neutralizing antibodies were detected using the microneutralization reaction. The rate of seroconversions of epitope-specific IgG consisted 21-25% in children 3-18 years old under primary respiratory syncytial viral infection and increased up to 42-50% in children 3-18 years old and adults under recurrent episodes of diseases. In these groups, the seroconversions of respiratory syncytial virus neutralizing antibodies were observed in 42%, 65% and 58% of cases correspondingly. Independently of age of patients with diagnosed respiratory syncytial viral infection, the absence of conversions of neutralizing antibodies was statistically significant associated with the absence of response from epitope-specific IgG. The presence among B-cell epitopes of immune dominance of surface glycoproteins of respiratory syncytial virus in patients with recurrent (but not primary) respiratory syncytial viral infection is detected. The hierarchy of epitope-specific immune response in case of complicated course of disease (response activity to epitopes: SP12=G-T15>G-H13) differed from the case of uncomplicated course of disease.

The article deals with the study of characteristics of epitope-specific humoral immune response to respiratory syncytial viral infection depending on nature of disease and patients' age. The couple serums from 226 children and adults with respiratory syncytial viral infection were analyzed. To detect in enzyme immunoassay the epitope-specific IgG the synthetic peptides were applied imitating the structure of functionally depended epitopes of F-protein of respiratory syncytial virus with amino acid sequences 221-232 (F = SP12), 479-491 (F-SP13) and G-protein with amino acid sequences 152-164 (G-H13), 184-198 (G-T15). The respiratory syncytial viral infection neutralizing antibodies were detected using the microneutralization reaction. The rate of seroconversions of epitope-specific IgG consisted 21-25% in children 3-18 years old under primary respiratory syncytial viral infection and increased up to 42-50% in children 3-18 years old and adults under recurrent episodes of diseases. In these groups, the seroconversions of respiratory syncytial virus neutralizing antibodies were observed in 42%, 65% and 58% of cases correspondingly. Independently of age of patients with diagnosed respiratory syncytial viral infection, the absence of conversions of neutralizing antibodies was statistically significant associated with the absence of response from epitope-specific IgG. The presence among B-cell epitopes of immune dominance of surface glycoproteins of respiratory syncytial virus in patients with recurrent (but not primary) respiratory syncytial viral infection is detected The hierarchy of epitope-specific immune response in case of complicated course of disease (response activity to epitopes: SP12 = G-T15 > G-H13) differed from the case of uncomplicated course of disease.

The article discusses the results of study that demonstrated the possibility of successful application of radial hemolysis reaction in analyzing the human inoculation immunity to new strains of influenza virus serotype A - A (H5N1) and A (H5N2). The radial hemolysis reaction provides accurate results on introduction of erythrocytes of horse or sheep into hemolytic system instead of erythrocytes of hens applied previously. The technique combines high sensitivity (in comparison with reactions of hemagglutination-inhibition and micro-neutralization, correlation coefficient 0.84-0.85) and total absence of inhibitors impact on the reaction results. During the investigation of immune response of patients who had pandemic virus A (H1N1) pdm09, radial hemolysis reaction demonstrated not only primary detection of antibodies to virus-agent (67%) during pandemic, but also elective heightened sensibility in the zone of low titer serums (1:20) in hemagglutination-inhibition reaction. These characteristics are very important in analysis of antibodies levels at early stages of disease. The radial hemolysis reaction continues to be a reliable instrument in evaluating qualitative and quantitative indicators of humoral immunity in ill patients and persons inoculated with new strains of human influenza virus.

AIM:Characterization of features of influenza pandemic development in Russia in relation to global process.MATERIALS AND METHODS:Pandemic monitoring was performed by using results of integrative analysis of laboratory diagnostic and population morbidity data from 49 supporting bases of Federal center of influenza from various cities in Russian Federation. Isolation of influenza virus was carried out in MDCK cells and chicken embryos under BSL-3 conditions. Reference virus A/California/07/09 obtained from CDC (Atlanta, USA) and antisera against this strain contained in WHO kit were used for antigenic analysis; rat antisera, new monoclonal antibodies against pandemic influenza virus developed by Research institute of influenza were also used.RESULTS:Based on PCR monitoring during epidemic peak, rate of pandemic influenza identification reached 45-49% of examined patients. About 53% of lethal cases of respiratory infections were caused by pandemic influenza virus, while predominately young people died from pneumonia and acute respiratory distress syndrome. Russian isolates generally were antigenically and genetically similar to the parent pandemic strain--influenza virusA/California/07/09, but contained S203T substitution in hemagglutinin. A number of strains contained D222G mutation that is responsible for the expansion of substrate specificity, as well as strain specific substitutions in hemagglutinin and neuraminidase molecules. The investigated isolates were resistant to remantadin, but sensitive to oseltamivir.CONCLUSION:Due to the formation of population immunity after the end of the first pandemic wave new drift variants of the virus capable of overcoming this formed immunity should be expected that apparently will require the correction of vaccine composition for the 2011 - 2012 season.

AIM:Assessment of reactogenicity, safety and immunogenicity after single intramuscular immunization of children with Grifor vaccine.MATERIALS AND METHODS:Reactogenicity, safety, and immunogenicity of Grifor vaccine compared with Vaxigrip vaccine was evaluated during phase III clinical trial in the Institute of Influenza. Thirty-six children aged 12 - 17 years, divided on 2 groups, participated in single blind comparative prospective randomized trial. Seroconversion factor, seroconversion and seroprotection levels were evaluated by hemagglutination inhibition assay.RESULTS:Results of study of systemic and local reactogenicity in children during first 7 days after immunization with Grifor and Vaxigrip vaccine showed good tolerability, areactogenicity and safety of both vaccines. Complete blood count, serum biochemistry and urinalysis results as well as serum IgE level did not change after vaccination. After immunization with Grifor vaccine, seroconversion rate to influenza virus subtypes A/H1N1, A/H3N2, and B was 70%, 50%, and 70% respectively, seroprotection rate--90%, 80%, and 85% respectively, and seroconversion factor--6.5, 2.7, and 4.0 respectively.CONCLUSION:This trial, which was performed in tightly controlled conditions, had demonstrated that Grifor vaccine is safe and highly immunogenic against influenza viruses A and B and satisfies criteria of both Federal Service for Surveillance for Protection of Consumers Rights and Human Welfare and CHMP of EMA. Obtained results allow to recommend the Grifor vaccine for use in pediatric practice according to national immunization schedule.

An indirect enzyme immunoassay (EIA) using the purified fraction of surface viral glycoproteins (GP) as an antigen for solid phase sensitization was not shown to be a specific method for the differential detection of influenza A(HS) (HS-Ab) virus antibodies (Abs) due to total conservative epitopes in the structure of GPs of influenza A(H5) and A(H1NI) viruses. The cross activity of some monoclonal Abs (MAbs) to influenza A(H5) and A(HIN1) viruses, which had been obtained at the Research Institute of Influenza, was proof of the presence of total immunodominant determinants in the structure of influenza H1 and H5 virus hemagglutinin (HA). In this connection, an EIA, which was based on the competition of influenza A(H5) H5-Ab virus HA-specific MAbs in the test sera for an association with influenza A(H5) virus, was proposed for the subtype-specific detection of H5 Ab. Comparison of the results of competitive EIA (cEIA), microneutralization (MN) test and HA inhibition test (HAIT) (using equine red blood cells) in the examination of sera obtained from 44 volunteers immunized with inactivated vaccine containing influenza A/Indonesia/5/2005 (H5N1) virus showed the high sensitivity and specificity of cEIA in detecting H5-specific Abs. The effectiveness of cEIA for the sera strictly positive for the content of H5 Abs was close to that of MN test and was 9-34% higher than HAIT (depending on those used in the analysis of H5 virus antigens). cEIA may be proposed to assess new influenza vaccines as an additional laboratory test. Since the infectious virus is not used during cEIA, it may be recommended for the serodiagnosis of influenza A(H5) at practical virological laboratories.

Conditions were developed for obtaining surface viral glycoprotein (GP) fraction intended for solid phase sensitization with the aim of constructing enzyme immunoassay test systems (EIATS) for detection of subtypical IgG and IgG to influenza A (H1N1) and A (H3N2) viruses. New variants of test systems were compared with the traditional methods for serological diagnosis of influenza. GP-based EIATS more often diagnosed influenza than EIATS based on purified whole-virion (WV) suspensions, hemagglutination inhibition and neutralization tests. Evaluation of conversions of subtypical IgG showed that the results coincided with the findings of neutralization and hemagglutination inhibition tests in 83-90% (EIATS-GP) and 50% (EIATS-WV). Cross-detection of antibodies to both virus subtypes in EIATS-GP and EIATS-WV was observed in 4 and 31% cases, respectively.

The antiviral drug arbidole reduces influenza and acute respiratory diseases (ARD) in children by 1.5-2 times when given in a dose of 0.1-0.2 g 2-3 or 5 times a week, by making influenza and ARD milder and decreasing their duration by 1.4-2.8 days. Unified regimens for prophylactic use of arbidole in influenza and ARD in children and adults are proposed.

Highly sensitive methods for the detection of viral antigens with the use of monoclonal or polyclonal enzyme immunoassay test systems, as well as virus-specific nucleic sequences (NS-gene) in polymerase chain reaction, made it possible to establish the fact of long-term asymptomatic virus carriership (AVC) in some of adolescent schoolchildren in a closed group. The occurrence of AVC varied at different phases of the epidemic process. The tendency to AVC was observed in individuals having blood group B (III) and HLA-A9 antigen of the major histocompatibility complex. The development of AVC occurred in the presence of a decreased level of humoral immunity. AVC was mainly registered among individuals, relatively resistant to relapsing clinically pronounced forms of respiratory virus infections.

Two serological tests--single radial hemolysis (RSH) and hemagglutination inhibition (HI) were used to evaluate the different techniques (intranasal, intradermal and combined methods) of application of inactivated influenza vaccines. When seroconversion to hemagglutinin (HA) was determined sensitivity of SRH proved to be higher as compared with HI by 6.7-41.4%. This test has also shown that the frequency of the seroconversion to HA was 2.1-5.6 times higher than that to neuraminidase (NA). It is important to standardize both HA and NA components in the influenza vaccine. It is interesting to study the local and cell immunity after intranasal inoculation of influenza vaccine because of the low postvaccinal level of serum antibodies and in connection with some publications concerning the protective role of this immunization method.