Adjuvant activity of multimolecular complexes based on Glycyrrhiza glabra saponins, lipids, and influenza virus glycoproteins.

Numerous studies have shown that immunostimulatory complexes containing Quil-A saponin and various antigens are effective in stimulating the immune response and can be used as vaccine preparations for animals and humans. However, Quil-A saponin possesses toxicity and haemolytic activity. In the present work, a saponin-containing preparation named "Glabilox" was isolated from the roots of a Glycyrrhiza glabra L. plant by high-performance liquid chromatography (HPLC). The results showed that Glabilox has no toxicity or haemolytic activity and can form stable immunostimulatory complexes. Subcutaneous immunization of mice with an immunostimulating complex containing Glabilox and H7N1 influenza virus antigens stimulated high levels of humoral and cellular immunity. Vaccination of chickens with the same immunostimulating complex protected 100% of the animals after experimental infection with a homologous virus. Comparative studies showed that the immunogenic and protective activity of immunostimulatory complexes containing Quil-A and immunostimulatory complexes containing Glabilox are comparable to each other. The results of these studies indicated that Glycyrrhiza glabra saponins show great promise as safe and effective adjuvants.

Adjuvant activity of saponins from Kazakhstani plants on the immune responses to subunit influenza vaccine.

In recent years, there have been a number of reports on the successful use of immunostimulatory complexes with saponins and viral glycoproteins as veterinary vaccines and in clinical trials for human medicine. The saponins Algiox, Sapanox and Pangisan were isolated and purified by HPLC from Allochrusa gypsophiloides, Saponaria officinalis and Gypsophila paniculata plants in Kazakhstan and they proved to have low toxicity in experiments with mice, chickens and chicken embryos. Algiox, Sapanox and Pangisan can be used to create immunostimulatory complexes (ISCOMs) similar to saponin-Quil-A-containing ISCOMs both in structure and in immunostimulatory efficiency. The adjuvant effect of the obtained saponins was studied by subcutaneous injection of mice with ISCOMs containing these herbal saponins and lipids and glycoproteins of H7N1 influenza virus. Sapanox, Pangisan and Algiox from Kazakhstani plants of the family Caryophyllaceae could be considered an additional source of highly effective adjuvants not only for veterinary vaccines but also for human medicine.

[Molecular genetic characteristics of the newcastle disease virus velogenic strains isolated in Russia, Ukraine, Kazakhstan, and Kirghizia].

The F gene fragment of 79 Newcastle disease virus (NDV) strains isolated from domestic and synanthropic birds in Kazakhstan, Kirghizia, Ukraine, and Russia in 1993 to 2007 was comparatively analyzed. Phylogenetic analysis of test isolates and reference NDV strains obtained from the GenBank was carried out by polymerase chain reaction with subsequent sequencing and comparative analysis of 154-bp nucleotide sequences in the main functional region of the F gene. All newly characterized isolates belong to three NDV genotype VII subgroups: VIIa, VIIb, VIId. The results show it necessary to monitor of NDV strains isolated in the CIS countries since the spread of NDV among migratory and synanthropic birds (pigeons, crows, and jackdaws) poses a serious threat to commercial poultry industry.

Immunostimulating complexes incorporating Eimeria tenella antigens and plant saponins as effective delivery system for coccidia vaccine immunization.

Immunostimulating complexes (ISCOMs) are unique, multimolecular structures formed by encapsulating antigens, lipids, and triterpene saponins of plant origin, and are an effective delivery system for various kinds of antigens. The uses of ISCOMs formulated with saponins from plants collected in Kazakhstan, with antigens from the poultry coccidian parasite Eimeria tenella, were evaluated for their potential use in developing a vaccine for control of avian coccidiosis. Saponins isolated from the plants Aesculus hippocastanum and Glycyrrhiza glabra were partially purified by HPLC. The saponin fractions obtained from HPLC were evaluated for toxicity in chickens and chicken embryos. The HPLC saponin fractions with the least toxicity, compared to a commercial saponin Quil A, were used to assemble ISCOMs. When chicks were immunized with ISCOMs prepared with saponins from Kazakhstan plants and E. tenella antigens, and then challenged with E. tenella oocysts, significant protection was conveyed compared to immunization with antigen alone. The results of this study indicate that ISCOMs formulated with saponins isolated from plants indigenous to Kazakhstan are an effective antigen delivery system which may be successfully used, with low toxicity, for preparation of highly immunogenic coccidia vaccine.

[Preparative isolation of myxovirus glycoproteins and the study of their immunogenic properties]. / Preparativnoe poluchenie glikoproteinov miksovirusov i izuchenie ikh immunogennykh svoistv.

Preparative isolation of glycoproteins from ortho- and paramyxoviruses is described. The purified concentrated virus has been treated with nonionic detergent MESK with subsequent removal of viral cores by centrifugation. Supernatant was sterilized by filtration through the nuclear filters and cleared from detergent by dialysis. Glycoproteins obtained have not contained contaminating cellular or core viral proteins or viral shell lipids. In the absence of detergent, glycoproteins have formed the peculiar mycelial complexes. Biological activity of glycoproteins was kept at high level. Glycoproteins output at isolation from different strains of influenza viruses A, B and Sendai virus varied from 75 to 98%. Immunogenetic study of the preparations obtained has demonstrated their capability to stimulate the formation of antibodies against both viral glycoproteins comparable with the capability of intact virus. The obtained level of immunity was enough to protect organism against homologous infection. Samples of glycoproteins obtained are up to standards for subunit vaccines, and the technique of their preparation is perspective as far as the production of vaccine preparations is concerned.

[Role of carbohydrates in the manifestation of the hemagglutinating and neuraminidase activities of the influenza virus]. / Rol' uglevodov v proiavlenii gemaggliutiniruiushchei i neiraminidaznoi aktivnosti virusa grippa.

Suspensions of influenza virions (MRC-11, X-7, and A/PR8/34 strains) were treated with a mixture of glycosidases including alpha-D-mannosidase, alpha-L-fucosidase, beta-D-galactosidase, and beta-D-N--acetylglucosaminidase for the study of the influence of carbohydrate part of influenza virus surface glycoproteins on the hemagglutinating and neuraminidase activity. The availability of oligosaccharide chains for the effect of glycosidases was found to change in relation to the functional status of the glycoprotein molecule. When carbohydrate residues were cleaved from neuraminidase in virus particle, its enzymatic activity did not change, whereas the hemagglutinating activity of virions after treatment with glycosidases decreased more than 50-fold.

[Use of the radioimmunoprecipitation method for testing a monospecific serum to an isolated influenza virus hemagglutinin]. / Primenenie metoda radiommunopretsipitatsii dlia testirovaniia monospetsificheskoi syvorotki k izolirovannomu gemaggliutininu virusa grippa.

Influenza virus hemagglutinin of H3 type was isolated using the proteolytic enzyme bromlaine. The isolated hemagglutinin was shown to have no neuraminidase activity and to contain no admixtures of extraneous proteins. This antigen was used to immunize rabbits and the resulting antiserum had high antibody titers in HI tests. The method of precipitation with the serum of labeled viral proteins showed the serum to be monospecific and to contain antibody to the hemagglutinin alone.

[Conditions for efficient antibody binding with proteins of the human immunodeficiency virus studied by immunoblotting]. / Izuchenie uslovii éffektivnogo sviazyvaniia antitel s belkami virusa immunodefitsita cheloveka v immunoblote.

The authors have investigated the antibody binding with individual HIV protein by "western" blotting method. The optimal condition for binding was incubation of nitrocellulose strips at 37 degrees C for 2 hrs followed by incubation at 4 degrees C overnight. Differences in the serologic activities of a number of sera in "western" blotting were shown by using two different antigens. It was concluded that mixing of different antigens before electrophoresis may be useful for more effective "western" blotting analysis.

[Preparative isolation of purified antigens of the herpes simplex virus type I and a study of their immunogenic properties]. / Preparativnoe poluchenie ochishchennykh antigenov virusa prostogo gerpesa tipa I i izuchenie ikh immunogennykh svoistv.

The HSV-I-containing material prepared in chick embryo fibroblast cultures was concentrated on nuclear filters followed by chromatography on a column with large-pore silica. The MESK detergent was used for isolation of glycoproteins. The glycoprotein preparation was highly immunogenic in experimental animals.

[Inhibition of the infectivity of enveloped viruses while retaining the specific biological activity of the viral proteins]. / Podavlenie infektsionnosti obolochechnykh virusov s sokhraneniem spetsificheskoi biologicheskoi aktivnosti virusnykh belkov.

A method of inactivation of enveloped viruses by treatment of virus-containing material with a nonionic detergent MESK was studied. Treatment with the detergent of allantoic or culture fluids containing influenza, parainfluenza, herpes simplex, and Venezuelan equine encephalomyelitis viruses was shown to result in significant (to 10 lg ID50) decrease of the infectivity of the viruses. At that, the specific biological activity of viral proteins: the hemagglutinating and neuraminidase activities in the case of influenza and parainfluenza viruses, and the hemagglutinating activity in the case of Venezuelan equine encephalomyelitis virus, remained unchanged. Treatment of the viruses with the detergent in the presence of 2% BSA, 5% gamma-globulin, or 50% blood serum also inactivated the infectivity of virions. The inactivating effect of the MESK detergent is associated with solubilization of external glycoproteins of the virus envelope. The method of mild nondenaturating inactivation of enveloped viruses with the MESK detergent may prove to be useful in laboratory practice.

[Binding of the surface antigen of the hepatitis B virus with antibodies studied with different denaturation exposures and with different analytical methods]. / Izuchenie sviazyvaniia poverkhnostnogo antigena virusa gepatita B s antitelami pri razlichnykh denaturiruiushchikh vozdeistviiakh i raznykh metodakh analiza.

The effect of delipidizing agents and a reducing agent on the antigenicity of 20 nm particles of hepatitis B virus surface antigen (HBsAg) was studied. The antigenicity was determined from the capacity of binding with antibody in passive hemagglutination test (PHAT) and dot-blot immune binding (DBIB). Delipidation was found to lead to an apparent decrease of antigenicity caused by aggregation. Subsequent destruction of the aggregates by treatment with sodium dodecylsulphate (SDS) reduced the antigenicity. Treatment with the reducing agent in the presence of SDS results in the loss of antigenicity detectable by PHAT, the DBIB titres remaining unchanged. Some examples demonstrate a high sensitivity of DBIB in detection of HBsAg. The advantages of the latter test over other methods of immune diagnosis are discussed.

[Immunogenicity of a subunit influenza vaccine in experiments on animals]. / Izuchenie immunogennosti sub"edinichnoi grippoznoi vaktsiny v opytakh na zhivotnykh.

The data on isolation of purified influenza virus glycoproteins and their reconstruction into liposomes by means of a new nonionic detergent, MESK, are presented. Sedimentational and flotational distribution of glycoprotein and liposome preparations was studied. In the presence of the detergent, the isolated glycoproteins were shown to occur mostly in a monomeric form. Removal of the detergent by dialysis resulted in formation of protein micelles, and the presence of exogenously introduced lipids in reconstruction of liposomes heterogeneous in composition. The resulting preparations had a high degree of biochemical purity and biological activity meeting the requirements for subunit vaccine properties. The immunogenic potency of a subunit influenza vaccine produced with the use of the MESK detergent was studied. The glycoproteins isolated with the use of MESK were shown to be comparable in their immunogenic potency with glycoproteins comprising viral particles. The influence of the form of glycoproteins presentation on their immunogenic potency was studied. Glycoproteins in the form of micelles and as components of liposomes were found to have a good immunogenic potency and to be able to induce protective immunity preventing experimental influenza infection in mice. Monomeric forms of glycoproteins had no such properties.

[Use of microfiltration and gel filtration technics for purifying and concentrating the Venezuelan equine encephalomyelitis virus]. / Primenenie mikrofil'tratsionnoi i gel'-fil'tratsionnoi tekhniki dlia ochistki i kontsentrirovaniia virusa venesuél'skogo éntsefalomielita loshadei.

A system for purification and concentration of Venezuelan equine encephalomyelitis (VEE) virus omitting large-scale ultracentrifugation was developed. The first step consists in prefiltration through large pore nuclear filters (NF) to remove large particle admixtures from the virus-containing fluid. In the second step, the resulting filtrage undergoes concentrating microfiltration through small pore NF. In this way the virus, but not total protein, is concentrated approximately 30-fold without any loss of biological activity. For VEE virus purification the next step uses gel filtration chromatography in a column with chemically modified macropore silica. In this stage, the purification factor by protein is approximately 100-fold and virus yield reaches 40%. It is suggested that the procedure used be applied for purification and concentration of a wide range of enveloped viruses.

[Isolation of glycoproteins of the Venezuelan equine encephalomyelitis virus and an evaluation of their immunogenic activity]. / Vydelenie glikoproteidov virusa venesuél'skogo éntsefalomielita loshadei i otsenka ikh immunogennoi aktivnosti.

Isolation of purified Venezuelan equine encephalomyelitis virus glycoproteins by means of a new Soviet nonionic detergent, MESK, is described. The MESK detergent was shown to permit isolation of approximately 70% of virus particle glycoproteins. The resulting preparation had a high hemagglutinating activity, contained no admixtures of foreign proteins and was not infectious. The study of the immunogenicity of purified glycoproteins in experimental mice and rabbits showed them to be capable of inducing high levels of serum antibodies. The immunogenicity of the isolated glycoproteins was comparable to their immunogenicity as components of virus particles. Treatment of the virus with MESK detergent also yielded preparations with predominant content of capsid protein. The described procedure for disintegration and purification of viral proteins is technologically simple and may be the basis for manufacture of a subunit vaccine. The resulting material may also be used for preparation of diagnosticums and in laboratory studies.

[Expert diagnosis of infectivity with the AIDS virus using immune blotting: the development of a system and assessment of its parameters]. / Ekspertnaia diagnostika infitsirovannosti virusom SPID s pomoshch'iu immunnogo blotinga: razrabotka sistemy i otsenka ee paranetrov.

A system for serodiagnosis of infection with AIDS viruses by means of immune ("western") blotting has been developed. The system has been found to be highly sensitive and suitable for expert serodiagnosis of AIDS disease and infection with human AIDS viruses.

[Masking of HIV antigen by specific antibodies in a model experiment]. / Maskirovanie antigena virusa immunodefitsita cheloveka spetsificheskimi antitelami v model'nom éksperimente.

An immuno-diagnostic test system of competitive EIA detecting HIV antigen in a concentration up to 1 ng/ml has been developed. Using this system, a phenomenon of binding of HIV antigen by antibody in sera from infected persons consisting in masking of antigenic determinants was demonstrated. The "undetectability" of HIV antigen in the system of competitive EIA caused by this phenomenon is considered to be a model of clearance of antigen at the excess of antibody in vitro. The experimental results are in agreement with the suggestion that repeated HIV antigenemia occurs as a result of exhaustion of specific immune responses.

[Chromatographic behavior of the hepatitis B virus surface antigen in donor plasma]. / Khromatograficheskoe povedenie poverkhnostnogo antigena virusa gepatita B v donorskoi krovi.

Behavior of HBsAg in adsorption chromatography of the antigen-containing plasma in a column with unmodified macropore silica was studied. Under the employed conditions of overlaying and elution, five maximum yields of plasma proper proteins from the column (peaks 1-5) were observed. HBsAg was determined mainly in the material of peaks 4 and 5 in which the dominating host protein was lipoprotein HDL. No HBsAg was detected in albumin-enriched fraction. Similarities of chromatographic behavior of all the three classes of particles whose surface is presented by HBsAg, 20 nm particles, filamentous forms, and Dane particles, were observed.

[The low frequency of false positive results in the serological detection of infectivity with the human immunodeficiency virus in collectives of healthy young people]. / Nizkaia chastota lozhnopolozhitel'nykh rezul'tatov pri serologicheskom vyiavlenii infitsirovannosti virusom immunodefitsita cheloveka v kollektivakh zdorovykh molodykh liudei.

Among 1002 foreign students examined in Odessa 11 subjects with antibody to HIV, i.e. infected with HIV, were detected. A complete agreement of the results of the enzyme immunoassay and immune blotting test was observed. The reasons of this are discussed. A specific regional distribution of seropositive subjects by their permanent residence places was revealed.

[Isolation of polymeric glycoprotein complexes of enveloped viruses using the Quill A glycoside and a study of their immunogenic activity]. / Poluchenie mul'timernykh kompleksov glikoproteidov obolochechnykh virusov s glikozidom Kvil A i izuchenie ikh immunogennoi aktivnosti.

Specific multimer complexes of glycoproteins of enveloped viruses were prepared by treatment of suspensions of purified concentrated virus with a non-ionic detergent MECK mixed with 0.2% glycoside Quil A. Mixed complexes of glycoproteins with glycoside Quil A were formed upon removal of the detergent from the mixture of solubilized glycoproteins and glycoside Quil A. According to the results of electron microscopy, the formed complexes differed morphologically from the conventional micelles of glycoproteins and presented spherical reticular structures 20-30 nm in diameter. The immunogenic potency of the complexes was much higher than that of virus particles and micelles of purified glycoproteins and was comparable to the immunogenic potency of glycoproteins mixed with complete Freund adjuvant. The protective activity of the complex of protein G of rabies virus with glycoside Quil A was higher than that of the subunit rabies vaccine adsorbed on aluminium hydroxide.

[Use of an immune blotting method (western) for studying viral antigens and for detecting antibodies to viral proteins]. / Primenenie metoda immunnogo ("vestern") blotinga dlia izucheniia virusnykh antigenov i vyiavleniia antitel k virusnym belkam.

The technique of the procedure and the results of the use of immune ("western") blotting method for studies on viral antigens and detection of antibodies to individual proteins of viral particles are described. The possibility of detection and study of individual viral antigens in the whole plasma of patients or carriers is demonstrated by the example of HBs antigen of human hepatitis B virus. The method of immune blotting was used for screening of human sera for the detection of antibodies to the AIDS virus proteins. The sera under study positive for antibodies to AIDS virus by preliminary solid-phase enzyme-immunoassay were shown to contain actually the antibodies to different proteins of AIDS virus. The antibody levels to individual AIDS virus proteins varied in different sera. Some sera positive for antibodies to AIDS virus by the solid-phase enzyme-immunoassay contained no antibody to AIDS virus proteins but reacted with cellular proteins present in the antigen. The immune blotting method was also used for determinations of the spectrum of antibodies in animal sera produced by immunization with different viral antigens.