MORPHOLOGICAL ANALYSIS OF HEPATITIS B VIRUS WITH ESCAPE MUTATIONS IN S-gene G145R AND S143L.

BACKGROUND: In terms of serological properties and immunization, the wild type of HBsAg HBV and its G145R mutant behave as different antigens. This testifies to serious structural changes, which presumably could have a significant impact on the morphogenesis of virions and subviral particles. Nevertheless, morphological and ultrastructural investigations of HBV with G145R mutation have not been carried yet. OBJECTIVES: Research of structural and morphological organization of HBV in the presence of the G145R escape mutation. METHODS: Studies of sera, purified viruses and recombinant HBsAg were carried out by transmission electron microscopy by the method of negative staining and indirect reaction of immunelabeling using monoclonal antibodies of different specificity. Specimens of wild type HBV and HBV with S143L mutation obtained in an identical manner were used as the control. RESULTS: The presence of typical virus particles of HBV was shown in the specimens of wild strain and HBV with S143L mutation. Specimens of HBV with G145R mutation were characterized by expressed morphological heterogeneity. In the initial serum and in the specimen of purified virus containing G145R mutant, large oval particles 60-70 nm and up to 200 nm in size, respectively, were found. The presence of antigen structures of HBV in all heterogeneous forms was confirmed. It was shown that forming of subviral particles in the process of expression of the recombinant HBsAg with G145R mutation depends on conditions of expression and purification of the protein. They can vary from well-formed circular and oval particles to practically unstructured fine-grained masses. CONCLUSION: Direct data on the impact of G145R escape-mutation in S-gene, in contrast to S143L mutation, on the morphogenesis of virions and subviral particles of HBV were obtained.

[A comparative characteristic of antigenic properties of recombinant and native hbs-antigens with G145R mutation and evaluation of their immunogenicity].

BACKGROUND: One of the important reasons for spreading of hepatitis B virus (HBV) under conditions of vaccinepressure is emergence of escape mutations. Prevalent G145R mutation in S-gene leads to the most expressed changes of serological properties of HBV. Consequently, HBsAg is modifed so thoroughly that it cannot be recognized by the majority of anti-HBs. Mutant G145R also differs from a wild type HBsAg by its immunogenic properties. At present, the relevance of enhancement of hepatitis B vaccine in view of mutant virus variants has been recognized. OBJECTIVES: a comparative study of antigenic and immunogenic properties of native and recombinant G145R mutants and an estimation of possibility for developing antigenic component of hepatitis B vaccine with G145R mutation in HBsAg. METHODS: antigenic properties of recombinant HBsAg with G145R mutation were compared with each other and with native mutants by serological fngerprinting method. Then, BALB/c mice and sheep were immunized with selected recombinant antigen under different protocols. Titers of antibodies specifc to wild type or mutant G145R type of HBsAg in sera of immunized animals were measured. RESULTS: it was found that not all the recombinant HBsAg variants with G145R substitution have the same antigenic properties as native HBsAg with similar mutation. Recombinant HBsAg selected according to the principle of antigenic similarity possesses immunogenicity in mice and sheep causing the production of antibodies reacting with native wild and mutant type HBsAg. It was shown that mutant antigen is less immunogenic, requires larger doses and more time for the development of immune response; however, it is capable of causing an antibody level comparable with wild type antigen. CONCLUSIONS: preliminary selection of recombinant HBsAg containing G145R mutation with antigenic and immunogenic properties similar to the native analogue creates the basis for development of a specifc component of hepatitis B vaccine with escape mutation G145R in HBsAg.

[Anti-hepatitis B vaccination and postvaccinal immunity stimulation in patients with chronic obstructive pulmonary disease].

The problem of the anti-hepatitis B vaccination of patients with chronic obstructive lung disease (COPD) was discussed due to the lack of studies concerning the developing of the postvaccinal immunity, especially when vaccination is combined with the immunomodulating treatment. The data on the vaccination safety and its influence on the clinical course of COPD are also insufficient. Therefore, in this work we investigated the efficiency of the antihepatitis B vaccination in adults with chronic obstructive pulmonary disease under the treatment with the immunomodulating Affonoleikin drug. A total of 93 patients were tested including 59 patients with severe and moderate COPD (aged from 35 to 65 years). 34 of these 59 patients were vaccinated against hepatitis B (Kombioteh) according to 0-1-6 month scheme, and 25 of them were vaccinated against hepatitis B during the treatment with Affinoleikin. The control group, consisted of 34 healthy patients. Our study demonstrated good tolerance and high immune efficiency of the anti-hepatitis B vaccine. However, after the first vaccination the level of HBs-AT was below protective level in patients with COPD compared to healthy patients. Also, 64 to 70 % of patients with COPD were seronegative excluding the patients receiving the Affinoleikin treatment, whose antibody titer was protective after the first vaccine dose, but did not reach the level typical of healthy patients. After the second vaccination we detected low and medium protective antibody levels in 58.9% of patients from the 1st group, whereas 41% were seronegative. Introduction of the third vaccine-dose led to fast and significant increase in the antibody level mainly in high concentrations with 100% seroconversion in all patients. Combined antihepatitis B vaccination and Affinoleikin treatment in patients with COPD leads to faster biosynthesis of HBs- AT in protective concentrations and decrease of seronegative response, but it has no effect on frequency and type of general and local postvaccinal response.

[Hepatitis E: current concepts].

Hepatitis E belongs to the group of enetral hepatites. Its earlier cases caused by genotype I and II viruses were reported largely from epidemics affecting tropical and subtropical countries. Sporadic cases of hepatitis E recorded later in West Europe, North America, Southeast Asia, and Oceania were caused by genotype III and IV viruses. Until recently the disease has been supposed to be reversible and have positive outcome barring women at late stages of pregnancy in whom cases of fulminate clinical course and death were described. This review focuses on recent publications devoted to hepatitis E in immunodeficient patients, such (as recipients of solid organ transplants, HIV-infected subjects and those treated with chemotherapy. Immunosuppression was shown to turn the disease into the chronic form or liver cirrhosis. Also, the infection has extrahepatic, mostly autoimmune complications. Current approaches to antiviral therapy and prevention of hepatitis E are discussed.

[Immunization of patients with chronic obstructive pulmonary disease with vaccines of Bubo-M and hepatitis B].

AIM: To assess tolerability and immunological activity of Bubo-M vaccine and hepatitis B vaccine in patients with chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: Sixty-three patients with moderate and severe COPD aged 35-65 years were immunized against diphtheria, tetanus, and hepatitis B. Bubo-M vaccine as well as vaccine against hepatitis B were used for immunization. Immunologic effect of vaccination was assessed by measurement of serum antibody level to HBsAg as well as to diphtheria and tetanus toxoids. Assessment of antibody level to HBsAg was performed by ELISA, and levels of antibodies to diphtheria and tetanus toxoids--by micromethod in direct hemagglutination assay. Reactogenicity of Bubo-M vaccine was measured according to duration and intensity of local and systemic reactions. RESULTS: The local and systemic reactions were infrequent, serious adverse events after vaccination were not observed. Six months after vaccination, protective antibody titers to hepatitis B, diphtheria and tetanus were determined in all immunized persons--either healthy, or with COPD. During completion of vaccination schedule, significant reduction of acute respiratory infections rate and main disease exacerbations was noted in patients with COPD. CONCLUSION: Good tolerability and high immunogenicity of Bubo-M and hepatitis B vaccines were demonstrated in both groups of vacinees. These vaccines could be recommended for booster vaccination of adults with COPD.

Expression and characterization of hepatitis B surface antigen in transgenic potato plants.

Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.

[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.

[Analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis B virus]. / Analiz transgennykh rastenii tabaka, soderzhashchikh gen poverkhnostnogo antigena virusa gepatita B.

The plasmids carrying the gene encoding the hepatitis B surface antigen (HBsAg) under the control of 35S RNA single or dual promoters of the cauliflower mosaic virus CaMV 35S were constructed. These constructions were used for obtaining transgenic tobacco plants that synthesize the HBS antigen. The presence of HBsAg in tobacco plant extracts was confirmed by the enzyme-linked immunoassay using antibodies against the native HBs antigen. The antigen amount in plants carrying the HbsAg gene under a single 35 S promoter was 0.0001-0.001 of the total soluble protein whereas the use of a dual 35S promoter increased the antigen synthesis to 0.002-0.05% of the protein. The antigen-synthesizing ability was inherited by the offspring. In the F1 plants, the antigen expression varied in different lines comprising 0.001 to 0.03% of the total soluble protein, which corresponded to the antigen amount in the F0 plants.

[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.

[Russian recombinant vaccine against hepatitis B (results of controlled trials)]. / Otechestvennaia rekombinantnaia vaktsina protiv gepatita B (rezul'taty kontroliruemykh ispytanii).

Two yeast recombinant vaccines were compared: the Russian hepatitis B Combiotech vaccine in a dose of 20 mg and the Recombivax-HB vaccine in a dose of 10 mg manufactured in the USA (Merk, Sharp and Dohme Ltd.). Both agents were found safe and low reactogenic. After a course of 3 vaccinations with the Russian vaccine 92.5 to 97.5% subjects vaccinated after the 0-1-2 months and 0-1-6 months protocols had high levels of specific antibodies. In the group vaccinated with the American vaccine, antibodies to HBsAg were found in the sera of 95 to 100% subjects. These results demonstrate high immunological activity of both Russian and American vaccines.

Use of chromatography for the preparation of homologous tetanus antitoxin.

To obtain immunoglobulin of selected action with a higher titre, techniques for isolating antibodies from a donor's normal immunoglobulin by means of affinity chromatography were used. To obtain an affinity sorbent, tetanus toxoid was used as a ligand. Using an immunosorbent based on the purified ligand, tetanus antibodies were prepared with a specific activity of 65 IU/mg. According to gel-permeation chromatography, the tetanus antibodies obtained were represented mainly by IgG monomer (greater than 90%). The anticomplementary activity of the preparations obtained, being at least 11 mg of protein per 2CH50, does not rule out the possibility of intravenous administration of tetanus antibodies.

[The chromatography of yeast recombinant HBsAg in porous silicas]. / Khromatografiia drozhzhevogo rekombinantnogo HBsAg na poristykh kremnezemakh.

The results of the study of adsorption processes of recombinant HBsAg on various porous silicas are presented. The maximal yield of the recombinant antigen by absorption chromatography was observed on unmodified macroporous glass MPS-2000. A mean yield in this case was 85%. Silica C-80 modified with polyvinylpyrrolidone proved to be most acceptable for gel-penetrating chromatography. The adsorption and gel-penetrating chromatography on porous silicas proved to be an effective method for purification of recombinant HBsAg.

Eremomycin--new glycopeptide antibiotic: chemical properties and structure.

By a combination of chemical and spectroscopic (1H and 13C NMR) studies the structure of a glycopeptide antibiotic eremomycin has been elucidated. It is closely related to vancomycin, but differs in sugar and chlorine content. The eremomycin aglycone contains monodechlorovancomycinic acid; the only chlorine atom is situated in the second amino acid after the N-terminal amino acid residue of the peptide. The sugar part is composed of glucose and two residues of an amino sugar shown to be 2,3,6-trideoxy-3-amino-C-3-methyl-L-arabino-hexopyranose (4-epi-vancosamine). One of the amino sugar residues is a component of the disaccharide 2-O-(alpha-L-4-epi-vancosaminyl)-beta-D-glucopyranose, attached to a triphenyl ether moiety; the position of another one is at the serine oxygen in the C-terminal region of the aglycone.

[The antineoplastic antibiotic polypeptide 308 produced by Actinomadura recticatena. Its isolation and physicochemical properties]. / Protivoopukholevyi antibiotik-polipeptid 308, produtsiruemyi Actinomadura recticatena. Vydelenie i fiziko-khimicheskie svoistva.

A antitumor antibiotic belonging to the group of polypeptide antibiotics containing chromophore was isolated from the culture of Actinomadura recticatena Terekhova, Preobrazhenskaya et Galatenko, 1984, strain 308. Biosynthesis, isolation, physicochemical and biological properties of the antibiotic are described. The results of elemental analysis, the melting point, optical properties, UV, IR and NMR spectra and the data on acid hydrolysis showed that antibiotic 308 was most closely related to antibiotic BBM-928 A.

[Composition and structure of the antibiotic polypeptide 308-I from Actinomadura recticatena]. / Sostav i stroenie antibiotika-polipeptida 308-I iz Actinomadura recticatena.

Antibiotic 308-I isolated from Actinomadura recticatena and the products of its degradation were studied with the methods of electron impact mass spectrometry, chemical ionization with ammonia, field desorption and ion extraction from solution under atmospheric pressure. It was shown that by its composition and structure antibiotic 308-I was identical to antibiotic BBM-928 A.

[Physicochemical properties of octamycin]. / Izuchenie nekotorykh fiziko-khimicheskikh svoistv oktamitsina.

Octamycin, an original polyenic antibiotic was developed. The antibiotic is of an amphoteric character. The number and nature of the functional groups were determined by potentiometric titration. It was shown that the antibiotic contained the neutral sugar galactose. The equivalent weight of the antibiotic (990) was measured by potentiometric titration and the content of the sugar in the molecule. With high performance liquid chromatography it was demonstrated that the antibiotic consisted of 2 components at a ratio of 1:2. The molecular weights of the components were evaluated with mass spectrometry.

[The antibiotic varigomycin--a new representative of the carbonyl-conjugated pentaenes]. / Antibiotik varigomitsin--novyi predstavitel' karbonil-kon''iugiro-vannykh pentaenov.

In screening new antibiotics, varigomycin produced by a new fungal species Streptomyces variegatus Sveshnikova et Timuk was isolated. By physicochemical properties varigomycin belongs to carbonyl conjugated pentaens. It is a new representative of this antibiotic group. Varigomycin differs from the known representatives of this group by the presence of glucose, sugar in its molecule.