METHODS FOR ASSESSMENT OF THE SPECIFIC ACTIVITY OF DRUGS FOR PREVENTION OF HEPATITIS B.

The immunologic activity (specific activity) is one of the main indicators of quality of vaccines for prophylaxis of hepatitis B, along with their safety. Retrospective analysis of the use of laboratory methods for assessment of specific (immunogenic) activity of modern vaccines against hepatitis B using indicators was carried out: in vitro method based on evaluation of HBsAg content and in vivo method based on evaluation of immunogenic activity in mice. Both methods are standardized and described in normative documents on the vaccines against hepatitis B of domestic production registered in the Russian Federation. Indicators of specific (immunogenic) activity of vaccines against hepatitis B were used to investigate more than 170 vaccine series using the ELISA method in the period from 2013 to 2015. The obtained control results confirmed the expediency and efficiency of enzyme immunoassay for determination of HBsAg content, as well as permissibility of use of ready sets of the Murex HBsAg Version 3 test systems for testing vaccines against hepatitis B by the ELISA method. Analysis of the results of laboratory control of series of vaccines against hepatitis B using a biological method for immunogenicity evaluation based on ED50 analysis confirms persistently high immunogenic activity of the Russian commercial vaccines intended for prophylaxis of hepatitis B. The confirmed comparability of methods allows the number of in vivo tests to be further reduced in favor of the enzyme immunoassay authentically characterizing the produced drug.

[Vitaherpavac is the first Russian herpes simplex virus vaccine obtained on the Vero B continuous cell line].

Vitaherpavac, a dry inactivated herpes simplex virus (HSV) culture vaccine, has been obtained, by using the Vero B continuous cell line as a substrate for accumulation of herpes simplex virus types 1 (US strain) and 2 (VN strain). Vitaherpavac and the similar vaccine Herpovax made by the Research Institute of Vaccines and Sera, Saint Petersburg (for which preparation a primary trypsinized chick embryo cell culture used as a substrate for accumulation of HSV types 1 and 2), underwent comparative clinical trials. The tolerability and therapeutic effectiveness of the vaccine were tested in patients diagnosed as having chronic frequently recurring herpes. The trials have yielded positive results that suggest that it is expedient to introduce of the new vaccine Vitaherpavac into practice to treat chronic recurrent herpetic infection of various localizations. Vitaherpavac has been registered in the Russian Federation and permitted for medical application.

[Diagnostic advantages of the test system "DS-EIA-HBsAg-0.01" for detection of HBV surface antigen].

The new highly sensitive test system "DS-EIA-HBsAg-0.01" (Priority Certificate No. 2006129019 of August 10, 2006) in detecting hepatitis B surface antigen (HBsAg) was assessed. The sensitivity of the test was estimated using the federal standards sample HBsAg 42-28-311-06, panels' samples Boston Biomedica Inc. (West Bridgewater, Mass, USA) and ZeptoMetrix Corp. (Buffalo, NY, USA). The findings have indicated that "DS-EIA-HBsAg-0.01" is equally effective in detecting different subtypes of HBsAg during a seroconversion period earlier than alternative assays. Along with its high analytical and diagnostic sensitivity, the system shows a high diagnostic specificity.

[The evaluation of the diagnostic value of PCR test systems according to the results of state trials].

The method for the diagnostic value evaluation of preparations, based on the calculation of the accuracy of test results and taking into account the spread of the diagnosed disease (the comparison of the PCR test systems for the diagnostics of hepatitis B and ureaplasmosis) is proposed. As shown in this work, evaluations obtained with the use of this method coincide with those obtained on the basis of prognostic value, but are more convenient in use and provide additional information.

[Evaluation of the reactogenicity and immunogenic potency of combined vaccine "Bubo-Kok" in the immunization of children against diphtheria, tetanus, pertussis and viral hepatitis B]. / Otsenka reaktogennosti i immunogennosti kombinirovannoi vaktsiny "Bubo-Kok" pri immunizatsii detei protiv difterii, stolbniaka, kokliusha i virusnogo gepatita B.

Combined vaccine "Bubo-Kok" is characterized by safety and high immunological activity. The number of postvaccinal reactions in children aged 1 and 2 years, immunized with vaccine "Bubo-Kok", was not statistically different from those in groups of children immunized with adsorbed DPT vaccine, as well with such vaccine in combination with vaccine against hepatitis B. After the completion of the primary course of immunization 100% of children had protective antibody titers against diphtheria, tetanus and hepatitis B. Antibody titers against pertussis, equal to or exceeding protective titers, were found in more than 70% of immunized children. The immunogenic potency of vaccine "Bubo-Kok" with respect to all its components was not inferior to that of adsorbed DPT vaccine and vaccine against hepatitis B, when introduced simultaneously in different areas of the body. Vaccine "Bubo-Kok" successfully passed state trials and was recommended for registration.

[Evaluation of the diagnostic efficacy of a test system for detecting hepatitis virus C RNA by polymerase chain reaction "AmpliSens(TM) HCV-240/BKO-440)"]. / Otsenka diagnosticheskoi éffektivnosti tests-sistemy dlia vyiavleniia RNK virusa gepatita C metodom polimeraznoi tsepnoi reaktsii "AmpliSens(TM) HCV-240/VKO-440".

The test system developed at the Central Research Institute of Epidemiology, Ministry of Health of the Russian Federation for identification of hepatitis C virus RNA was studied. The sensitivity of the test system which the rate of similar results was 100% with its 5-fold reproduction was evaluated. That was 5 x 103 genomic equivalents (or international units) per ml of a sample. A scheme for evaluation of the reproductibility of test systems based on the polymerase chain reaction (PCR) by using model samples is proposed. Whether it can be used for intra- and extra-laboratory assessment of the quality of PCR analyses is discussed.

[Stability of hepatitis C virus RNA in human blood plasma in a panel of control samples]. / Stabil'nost' RNK virusa gepatita C v plazme krovi cheloveka v paneli kontrol'nykh obraztsov.

The stability of hepatitis C virus (HPC) RNA concentration in 5 human plasma samples after storage at +22 degrees C for two months, at -20 degrees C, and +4 degrees C for six months after 10 freezing-unfreezing cycles was evaluated. In this study, the concentration of HCV RNA in the samples was stable after six months of storage at -20 degrees C. The concentration of HCV RNA decreased on the average of 92% after 2-month storage at +22 degrees C. After six months of storage at +4 degrees C and after 10 freezing-unfreezing cycles, that decreased by 28 and 42%, respectively. Based on their own findings, the authors developed a HCV-RNA panel containing 5 positive human plasma samples with RNA levels of 103-105 IU/ml. The panel may be recommended both for the standardization of PCR kits and for the intra- and interlaboratory quality control of PCR laboratories.

[Evaluation of reactogenicity and immunogenicity of Bubo-M, the Russian combined vaccine for immunization of adults against diphtheria, tetanus and viral hepatitis B]. / Otsenka reaktogennosti i immunogennosti otechestvennoi kombinirovannoi vaktsiny Bubo-M pri immunizatsii vzroslykh protiv difterii, stolbniaka i virusnogo gepatita B.

Bubo-M, the first Russian associated vaccine, was found to have low reactogenicity and high immunogenic potency. The frequency of postvaccinal reactions in the group of persons immunized with Bubo-M (20%) appeared to be considerably lower than among persons who received the combined injection of adsorbed DT toxoid with reduced antigen content and vaccine against hepatitis B (47.7%). Following the course of vaccination the level of anti-HBs considerably exceeded the protective level. Immune response to the diphtheria and tetanus components of Bubo-M exceeded that observed after immunization with absorbed DT toxoid with reduced antigen content (p < 0.05).

[Reactions to and immunogenic properties of the combined vaccine Bubo-M for immunization of children against diphtheria, tetanus and viral hepatitis B]. / Reaktogennost' i immunogennost' kombinirovannoi vaktsiny Bubo-M pri immunizatsii detei protiv difterii, stolbniaka i virusnogo gepatita B.

Bubo-M, the first Russian combined vaccine, was found to have low reactogenicity. The difference between the number of postvaccinal reactions in the group of children immunized with Bubo-M (25.9%) and those in the group of children who had been simultaneously injected into different sites of the body with ADS-M toxoid (adsorbed DT toxoid with reduced antigen content) and hepatitis B vaccine (26.7%) was not statistically significant. Following immunization a considerable increase in the level of diphtheria and tetanus antibodies (p < 0.005) was observed in all children (100%), the level of HBs antibodies in the group of children immunized with Bubo-M (the geometric mean titer: 13,721 IU/l) essentially exceeding that observed in the control group injected with ADS-M toxoid and hepatitis B vaccine (the geometric mean of the titer: 2,441 IU/l). Bubo-M was duly registered and allowed for industrial production and medical use.

[Construction of reference panel of immune serum against hepatitis C virus with standard level of IgG antibodies]. / Konstruirovanie referens-panelei syvorotok k virusu gepatita C s normirovannym urovnem IgG-antitel.

A panel of anti-HCV sera (lot 03HC) was prepared from human sera obtained at blood transfusion centers and infectious hospitals. Donor sera and high-titer sera from patients infected with HCV were used. For positive samples, specific sera reactive with the core and/or NS proteins of HCV 1b and 2 were selected. Positive sera were standardized by the concentrations of IgG with a pool of negative sera containing no HBsAg and antibodies to HIV, HCV, and syphilis. The sera for the panel were selected and titered in screening and specific tests. The anti-HCV panel includes negative and positive sera with low and high titers. The panel sera are stabilized and can be stored for a short time at room temperature. The anti-HCV panel of sera, lot 02HC, was certified at L. A. Tarasevich Institute for Standardization and Control as anti-HCV reference panel intended for sensitivity, specificity, and stability control of diagnostic systems for detection of antibodies to HCV in Russia.

[Development of sera reference panels for quality control of ELISA diagnostic kits in Russia]. / Razrabotka standartnykh panelei syvorotok dlia kontrolia kachestva immunofermenthykh test-sistem v Rossii.

In the past 5 years, the investigators of the "VECTOR" SRB VB and the L.A. Tarasevich State Institute of Standardization and Control of Medical Biological Preparations have jointly designed sera reference panels containing anti-HIV-1 IgG, anti-HCV IgG, and anti-HAV IgM which have been approved as national standard panels. The panels are intended for use in controlling the specificity and stability of the most widely used ELISA diagnostic kits and immunoblot test systems during production, control, and application stages. Some problems of development and production of these panels, including the representation of different sera in the panels and the selection of specific IgG concentrations in the different sera in the panel are described. The authors also attract attention to the stabilization of the specific characteristics of panel sera during storage and transportation.

[Constructing the reference panel of sera with standard level of IgG-antibodies]. / Konstruirivanie standartnykh panelei syvorotok c normirovannym urovnem IgG-antitel.

Fundamentals of designing reference panels of sera for effective control of commercial test systems and immunoblotting, intended for detecting antiviral antibodies, have been developed. Reference low-titer panels of anti-IgG antibodies to HIV-a and hepatitis C virus have been designed. A reference panel contains diluted reactive sera with a standard level of IgG antibodies and native sera with undetectable level of antibodies to the major viral antigens from risk group subjects. The reactive sera of a panel contain the whole spectrum of antibodies to all principal viral antigens.

[The use of Vero (B) cell line for medico-biological preparations]. / Liniia kletok Vero (B) dlia prigotovleniia mediko-biologicheskikh preparatov.

A variant of Vero cell line has been obtained, adapted to the Russian Eagle's medium with 8% fetal calf serum. The variant is characterized by intensive cell proliferation, possesses a different content of chromosomes, contains no oncogens and contaminants, specifically, Mycoplasma. The strain was characterized and certified in accordance with the WHO requirements. Inoculation (110 ampules) and working (100 ampules) stocks at the levels of the 170th and 178th passages were placed for storage in liquid nitrogen. Vero (B) cell line is sensitive to herpes simplex virus types 1 and 2, CMV, hepatitis A virus, recombinant variolovaccinia strain expressing HbS antigen, etc. The line retains its biological properties from passage 178 to passage 200 and is recommended for control and preparation of various biomedical agents, including antiviral vaccines.

[Isolation and characteristics of a new strain of diploid cells from human embryonic lung tissue]. / Poluchenie i kharakteristika novogo shtamma diploidnykh kletok iz émbrional'noi tkani legkogo cheloveka.

A new strain of the embryonic human fibroblasts L-68 was obtained and thoroughly characterized. It completely met all the requirements of the International Committee on the Cells Cultures. This strain can be recommended as a substrate for production of viral vaccines, diagnostic preparations and for research purposes.

[Comparative evaluation of methods for detecting HBsAg in sera]. / Sravnitel'naia otsenka metodov vyiavleniia HBsAg v syvorotkakh.

The results of the analysis of 1209 serum samples, made with a view to detecting those containing HBsAg, are presented. This analysis was made by the radioimmunoassay (RIA) on a polyethylene film, by the standard RIA technique with the use of a diagnostic kit obtained from Abbott Laboratories (USA) and by the passive hemagglutination (PHA) test. The RIA film technique was found to have the sensitivity of about 2 ng/ml HBsAg, which is similar to the sensitivity of the kit from Abbott Laboratories and exceeds the sensitivity of the PHA test approximately 50-fold. The percentage of detected HBsAg-positive sera, yielded by analysis with the use of the RIA film technique and the standard RIA technique, is the same. The RIA technique make it possible to detect more positive sera than the PHA test by about 2.5%.

[Use of a radioimmune method for determining HBsAg in preparations of human gamma-globulin]. / Ispol'zovanie radioimmunnogo metoda dlia opredeleniia HBsAg v preparatakh gamma-globulina cheloveka.

The work presents the results of studies made with a view to improve the method of testing gamma-globulin preparations for the presence of hepatitis B virus surface antigen (HBsAg) by means of radioimmunoassay (RIA). The work shows that this method requires the use of specially selected negative control samples made up of pooled gamma-globulin samples. Standard RIA techniques intended for detecting the presence of HBsAg in human plasma and blood serum is not suitable for the analysis of the preparations of human gamma-globulin.

[Use of a cocultivation method for detecting cytomegalovirus contamination of cell cultures of simian origin]. / Ispol'zovanie metoda sokul'tivirovaniia dlia vyiavleniia tsitomegalovirusnoi kontaminatsii kletochnykh kul'tur obez'ian'ego proiskhozhdeniia.

At present, nonanthropoid primates are widely used as sources of cell cultures for manufacture of live viral vaccines. Simian cell cultures, particularly kidney cell cultures are also known to be frequently contaminated with cytomegaloviruses. The isolation of the latter is rather difficult due to the late appearance of the cytopathic effect in cell cultures of natural hosts. In the present study, the sensitivity of 4 methods virus isolation from the test cells was compared: the method of long-term cultivation of cells; the method of long-term cultivation with one subpassage of the cells; the method of cocultivation of the test cells by mixing with sensitive cells; and the method of co-cultivation by overlaying the test cells on an incomplete monolayer of sensitive cells. The latter method shortened the observation period and yielded a higher percentage of isolation of contaminating viruses from African green monkey kidney cell cultures. This method is supposed to be used in future for the detection of viral contamination of African group monkey kidney cell cultures utilized in manufacture of live viral vaccines.