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Evaluate reactogenicity, safety and immunogenicity in phase 2 clinical trials of 2 immunization schedules with Ultragrivac--an allantoic intranasal life influenza vaccine based on A/17/ duck/Potsdam/86/92 [17/H5] reassortant strain.4 groups of volunteers participated in the study: group 1--40 individuals were vaccinated twice with a 10 day interval; group 2--40 individuals were vaccinated twice with a 21 day interval; group 3 (control)--10 individuals received placebo twice with a 10 day interval; group 4 (control)--10 individuals received placebo twice with a 21 day interval. Local (secretory IgA), cellular and humoral immune response were evaluated. Humoral immunity was evaluated by the intensity of increase of geometric mean antibody titers against 2 influenza virus strains A/17/duck/Potsdam/86/92 [17/H5] and A/chicken/Suzdalka/Nov-1 1/2005 (H5N1), and by the level of significant (4 times or more) antibody seroconversions after the vaccination.After the use of Ultragrivac the level of secretory IgA in the nasal cavity of vaccinated volunteers in the groups with revaccination intervals of 10 and 21 days increased significantly. The second immunization with 10 or 21 day intervals significantly increased postvaccinal humoral immune response. Humoral immune response induction after 2 vaccinations with 10 day interval was no less effective than with 21 day interval.Ultragrivac allantoic intranasal live influenza vaccine is areactogenic, harmless for vaccinated individuals, safe for those around, and has immunogenic properties against not only homologous virus A(H5N2), but also against influenza strain A(H5N1).

To obtain human recombinant 70 kDa heat shock protein (Hsp70) in baculovirus expression system and to study its antiviral activity.Baculovirus expression system was used to obtain recombinant HSP70. Plasmid pFastBacHTb-Hsp70 containing sequence coding HSP70 gene with insertion of 6 histidine residues in protein reading frame was constructed. Competent cells MAX Efficiency DH 10 Bac were transfected with pFastBacHTb-Hsp70 plasmid with following extraction of recombinant bacmid Bac-Hsp70. In order to obtain baculovirus expressing HSP70, Sf-9 cells were transfected with Bac-Hsp70 bacmid. Hsp70 extraction and purification was performed with column metal-chelating affinity chromatography using Ni2+ ions. Protective efficacy of recombinant human HSP70 was estimated using model of Venezuelan equine encephalitis (VEE) in mice.Recombinant bacmid Bac-Hsp70 was constructed based on Bac-to-Bac expression system. Baculovirus expressing human HSP70 have been produced after transfection of Sf-9 cells with Bac-Hsp70 bacmid. Cultivation of recombinant baculovirus in Sf-9 cells and application of metal-chelating affinity chromatography allowed to extract purified fraction of HSP70. Experiments on mice infected with VEE virus demonstrated significant protection from death after administration of HSP70 in dose 15 mcg/mice.Application of baculovirus expression system and insect cell line for accumulation of recombinant baculoviruses in combination with Ni(2+)-mediated metal-chelating affinity chromatography allowed to obtain highly purified human recombinant HSP70 with marked antiviral activity.

To study tolerability and safety of pandemic vaccines against influenza A/H1N1 "INFLUVIR" and "PANDEFLU" on limited group of volunteers during phase I clinical trial.Thirty healthy volunteers were participated in phase I clinical trial. Clinical and laboratory tests of volunteers randomized to 2 groups (20 persons received vaccine and 10 persons - placebo) were performed. "INFLUVIR" vaccine was administered by intranasal route. Volunteers were hospitalized and followed up for development of local and systemic adverse events during 7 days after vaccination. "PANDEFLU" was administered intramuscularly; vaccinees were followed for 7 days in outpatient settings. RESULTS. Phase I clinical trial showed good tolerability and low reactogenicity of "INFLUVIR" and "PANDEFLU" vaccines. There were no moderate and severe local and systemic adverse events registered.On the basis of performed phase I clinical trial, phase II trial was recommended to perform in order to assess the reactogenicity, safety and immunogenicity of studied vaccines.

To perform preclinical study of subunit monovalent adsorbed inactivated influenza vaccine "PANDEFLU" [strains A/California/7/2009 (HIN1)v].Preclinical study of acute toxicity on experimental animals (assessment of vaccine's toxic effects on organs and body systems; pathomorphologic study of organs and tissues after administration of the vaccine; assessment of its influence on hematologic indicators).It was shown that administration of the vaccine did not lead to death of animals as well as to decrease of body mass or development of pathologic, focal sclerotic changes in parenchymal cells and visceral stroma; the vaccine did not negatively change hematologic and biochemical indicators of blood. Results of necropsy and histological study after acute administration of the vaccine in standard dose did not lead to irritation, inflammation or destruction of tissues in the place of inoculation. The vaccine was apyrogenic and did not have local irritating and allergenic effects. Status of animals after acute inoculation of the vaccine demonstrated its good tolerability and safety in doses exceeding standard human doses more than tenfold. CONCLUSION. Performed research demonstrated absence of contraindications for conduction of clinical trials of "PANDEFLU" vaccine on limited contingent of volunteers.

To perform preclinical assessment of new live monovalent vaccine Influvir against pandemic influenza virus A/H1N1 [strainA/17/California/2009/38 (H1N1)].Preclinical studies of acute toxicity and effect of Influvir vaccine on systems and organs of laboratory animals (rats and outbred white mice) was performed according to modern requirements of Institute of Toxicology.According to results of toxicometry and necroscopy, live monovalent influenza vaccine Influvir during intransal application was safe and had good tolerability during 14 days of observation for experimental animals after acute application. Performed preclinical studies allow to label the studied vaccine as class V virtually nontoxic drugs.According to results of preclinical studies, clinical trials of live monovalent intranasal influenza vaccine Influvir can be permitted.

Perspectives of using reverse genetics methods for constructing of recombinant influenza virus strains acceptable for use as live attenuated vaccines are discussed. Using of attenuated NS-vectors of influenza virus opens possibilities for the development of recombinant vaccines with optimal ratio of immunogenicity and safety. Reverse genetics is applicable for development of effective vaccines against new pathogens such as highly pathogenic avian influenza A/H5N1.

To confirm and prove on the extended contingent of volunteers the non-reactogenicity, safety and immunogenicity of "Grifor" vaccine in comparative trial with registered in Russia commercial vaccine "Vaxigrip".Phase II clinical trial was performed on the research bases of Mechnikov Institute of Vaccines and Sera and Institute of Influenza. In single-blind comparative prospective randomized trial 300 adult volunteers (150 volunteers on each base) aged 18 - 60 y.o. were divided on 3 equivalent groups. Assessment of antigenic characteristics of "Grifor" vaccine was performed using hemagglutination inhibition assay (HAI) with chicken erythrocytes measuring geometric mean titer (GMT), seroconversion factor as well as level of seroconversion and seroprotection.Previously performed studies proved non-reactogenicity, safety and high immunogenicity of "Grifor", whereas this comparative trial performed with commercial vaccine "Vaxigrip" did not reveal significant advantage in any of studied vaccine."Grifor" vaccine meet the requirements of both EMEA CPMP and methodic guidelines MY 3.3.2. 1758-03 for inactivated influenza vaccines, which allows to register vaccine "Grifor" in Russian Federation.

Phase I clinical trial of inactivated virosomal split influenza vaccine "Grifor" was conducted in the Mechnikov Research Institute of Vaccines and Sera as accredited base for such trials. Forty healthy volunteers (males and females) aged 18 - 50 years consented to participate in the trial. Reactogenicity, safety, and immunogenicity of new Russian influenza vaccine were assessed. Analysis of obtained results showed that there was evidence of safety and low reactogenicity of the vaccine as well as of its high immunogenic characteristics, which satisfied both the EMEA's Committee for Proprietal Medicinal Products criteria and requirements of Federal Service for Surveillance for Protection of Consumers Rights and Human Welfare (MU 3.3.2.1758-03) for inactivated influenza vaccines.

New Russian virosomal split vaccine against influenza "Grifor" was developed. The vaccine is represented by mix of highly purified protective external and internal antigens of influenza A (H1N1 and H3N2) and B viruses. Developed technology of manufacture allowed to provide presentation of external antigens of influenza virus in the form of virosomes, and presentation of internal antigens in the form of micelles with maximal preservation of their antigenic activity. Using electron microscopy, electrophoresis in 10% polyacrilamide gel with sodium dodecyl sulfate, and polymerase chain reaction, morphologic and biochemical properties of the vaccine were studied. Preclinical study, including assessment of antigenic characteristics of "Grifor" vaccine compared to vaccine "Vaxigrip" (France), was performed. It was established that administration of the vaccine did not result in death of experimental animals, decrease of body mass, development of pathologic (including inflammatory, dystrophic and necrobiotic) changes in viscera or render adverse effects on blood hematologic and biochemical parameters and on the immune system. The vaccine was not pyrogenic and allergenic, did not have local irritating effects. Obtained results supported the appropriateness of conducting the clinical trials of "Grifor" vaccine on limited number of volunteers.

According to opinion of WHO's experts, development and use of tetravaccine, which contains both interdemic and pandemic (H5N1) serotypes of influenza viruses, is one of the most promising approaches to control possible influenza pandemic. Results of recently obtained data from clinical trials allowed experts from WHO to make a conclusion that protective immunity against avian influenza virus can be achieved after 2-doses immunization, when the immune system will be primed to hemagglutinin after the 1st dose and sufficient protective immunity level will be formed after the 2nd dose. However, in case of real threat of pandemic, the time for immunization with 2 doses of the vaccine will be absent. In order to provide protection for population of Russia in a limited time frame it is reasonable to vaccinate them with H5 hemagglutinin beforehand. In that case, when real threat of pandemic will arise, not two but one injection with monovalent vaccine against avian influenza will be sufficient. This idea formed the basis for concept of development of tetravaccine. The essence of the concept is vaccination of population with tetravaccine, consisting of antigens of influenza virus serotypes H3N2, H1N1, B, and H5, before the influenza pandemic caused by H5N1 virus will begin. Such vaccination will induce immunologic memory to hemagglutinin of avian influenza virus serotype H5 and, when the real threat of the pandemic will occur, only single immunization with monovaccine against avian influenza instead of 2 doses will be required. In 2006 Scientific-Production Association "Microgen" conducted extended preclinical study of immunogenic and protective characteristics of candidate vaccines against avian influenza prepared from vaccine strains of H5N1 and H5N2 serotypes. It has been shown that candidate vaccines prepared from both strains have high protective ability against Russian epidemic isolate A/chicken/Kurgan/Russia/2/2005(H5N1). To this time Scientific-Production Association "Microgen" has produced monovalent bulk of H3N2, H1N1, and B serotypes, which are included in interdemic influenza vaccines, as well as monovalent bulk of H5N1 and H5N2 serotypes. This intermediate products are ready to be produced into tetravaccine for conducting extended preclinical studies of its safety, reactogenicity, immunogenicity, and protective properties. If results of such studies will be positive then it is possible to begin clinical trials of the tetravaccine in 2007 and to discuss the questions about its dosage, methods of challenge and schedule.

Scientic-production association "Microgen" has finished 1st phase of clinical trials of candidate vaccines against avian influenza in order to assess their reactogenicity, safety, and immunogenicity. Two vaccines constructed from NIBRG-14 vaccine strain [A/Vietnam/1 194/2004 (H5N1)], obtained from World Health Organization, were studied: "OrniFlu" (inactivated subunit influenza vaccine adsorbed on aluminium hydroxide) and inactivated polymer-subunit influenza vaccine with polyoxydonium (IPSIV). Clinical trial of the vaccines with different quantity of antigen (15, 30, and 45 mcg of H5N1 virus hemagglutinin) was carried out in Influenza Research Institute (St. Petersburg) and in Mechnikov Research Institute of Vaccines and Sera (Moscow). Analysis of results allowed to conclude that both vaccines were safe, well tolerated and characterized by low reactogenicity. Two-doses vaccination schedule was needed to meet required seroconversion and seroprotection rates (or =1:40 inor =70% of vaccinated volunteers). "Orni-Flu" vaccine containing 15 mcg of hemagglutinin and optimal quantity of aluminium hydroxide (0.5 mg) in one dose as well as IPSIV containing 45 mcg of hemagglutinin and 0.75 mg of polyoxydonium in one dose were most immunogenic after 2 doses - seroprotection rates in microneutralization assay were 72.2% and 77.0% respectively. Marked influence of aluminium hydroxide content on immunogenicity of the "OrniFlu" vaccine was confirmed in the study. Optimal quantity of adjuvant was 0.5 mg per dose. According to basic concept of vaccine development, preference is given to vaccine that under minimal quantity of antigen induces sufficient specific immune response and is safe in volunteers. "OrniFlu" vaccine containing 15 mcg of H5N1 virus hemagglutinin and optimal quantity of aluminium hydroxide (0.5 mg) corresponded to these requirements that allowed researchers to recommend it for clinical trials of 2nd phase.

The results of the molecular biological detection of the etiologic agent of hemorrhagic fever in Rostov Province are presented. The role of the causative agents of Astrakhan rickettsial fever, hemorrhagic fever with the renal syndrome, Q fever, leptospirosis and listeriosis has been excluded by means of such immunochemical reactions as the direct and indirect immunofluorescent tests, the solid-phase immunoenzyme assay, the complement fixation test and the agglutination test. The relationship between the cases of hemorrhagic fever in the focus of the outbreak and Crimean-Congo hemorrhagic fever virus has been demonstrated due to the use of the polymerase chain reaction with preliminary reverse transcription.