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.

Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens.

Immunostimulating complexes (ISCOMs) are unique multimolecular structures formed by encapsulating antigens, lipids and triterpene saponins and are one of the most successful antigen delivery systems for microbial antigens. In the current study, both the route of administration and the antigen concentration of ISCOMs, containing Eimeria tenella antigens and saponins from native plants, were evaluated in their ability to stimulate humoral immunity and to protect chickens against a challenge infection with E. tenella. Broiler chickens were immunized with ISCOM preparations containing E. tenella antigens and the purified saponins Gg6, Ah6 and Gp7 isolated from Glycyrrhiza glabra, Aesculus hippocastanum and Gipsophila paniculata, respectively. The effects of the route of administration, dose of antigen and type of saponin used for construction of ISCOMs were evaluated for ability to stimulate serum IgG and IgM and to protect chickens against a homologous challenge. A single intranasal immunization was the most effective route for administering ISCOMs although the in ovo route was also quite effective. Dose titration experiments demonstrated efficacy after single immunization with various ISCOM doses but maximum effects were observed when ISCOMs contain 5-10mug antigen. Immunization of birds by any of the three routes with E. tenella antigens alone or antigens mixed with alum hydroxide adjuvant resulted in lower serum antibody and reduced protection to challenge relative to immunization with ISCOMs. Overall the results of this study confirm that significant immunostimulation and protection to challenge are achieved by immunization of chickens with ISCOMs containing purified saponins and native E. tenella antigens and suggest that ISCOMs may be successfully used to develop a safe and effective vaccine for prevention of avian coccidiosis.

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.

[Immunostimulating activity of a saponin-containing extract of Saponaria officinalis]. / Izuchenie immunostimuliruiushchei aktivnosti saponinsoderzhashchego ékstrakta Saponaria officinalis.

The immunostimulating activity of saponin-containing extract of Saponaria officinalis has been studied. Use of an S. officinalis extract in a concentration close to the critical concentration of saponin micella formation increased the immunogenicity of viral glycoproteins. The immunogenicity of glycoprotein complexes with S. officinalis was higher than the immunogenicity of intact virus and micellae of purified glycoproteins and was comparable to that of glycoprotein complexes with Quil A glycoside.

Assessment of dot-blot ELISA sensitivity on membrane sorbent using various peroxidase substrates.

The sensitivity of the peroxidase reaction in dot-blot ELISA significantly depends on the substrate. The highest sensitivity is observed using benzidine and diamine-phenol combinations as the substrates due to the reaction of the coupled oxidation (NADI).

[Interactions between Mycoplasma and human and animal viruses. Ultrastructural analysis]. / Vzaimodeistvie mikoplazm s virusami cheloveka i zhivotnykh. Ul'trastrukturnyi analiz.

Presented are the data on the ultrastructural analysis of interaction between mycoplasma and certain cancerogenic and infectious viruses in humans and animals. Revealed are spontaneous associations of mycoplasma with viruses of cattle leukemia, T-cell human leukemia and with a representative of Bunyaviruses. Immediate interaction of these agents is found possible. Simulated complexes of mycoplasma with infectious viruses are developed. Electron microscopy on supramolecular levels revealed immediate interaction of different agents in membranes. Some methodological procedures help to reveal that the interaction of M. pneumoniae and A. laidlawii with orthomyxo-, paramyxo and togavirus is of specific character and is realized as receptor ligand form due to the affinity in the receptor requirements of these pathogens. This property as well as a bequeath distribution and frequent association in respiratory infections enable one to suggest the possibility of their immediate interactions in a host body.

[Study of influenza virus hemagglutinin H3 avidity using monoclonal antibodies]. / Izuchenie avidnosti gemaggliutinina H3 virusa grippa s pomoshch'iu monoklonal'nykh antitel.

The results of a comparative study of the features of antigenic determinants of avid and non-avid variants of influenza virus hemagglutinin H3 and of the influence of the mode of antigen presentation on the degree of its avidity are presented. The avidity of influenza virus hemagglutinin was shown to be determined by the capacity of individual antigenic determinants to interaction with antibodies. The antigenic determinants of avid hemagglutinin possessing a high functional activity in interaction with antibodies may have the spatial configuration which does not change in different modes of the antigen presentation. Isolation of hemagglutinin from virions of non-avid virus variants may lead to increased functional activity of individual antigenic determinants (and the antigen molecule as a whole) probably due to an increased degree of exposure and/or complementarity of the determinants for active centres of antibody.

[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.

[A comparison of the spectra of antibodies to individual proteins of the human immunodeficiency virus in seropositive sera collected in the territories of the USSR and England]. / Sravnenie spektrov antitel k individual'nym belkam virusa immunodefitsita cheloveka v seropozitivnykh syvorotkakh, sobrannykh na territorii SSSR i Anglii.

Antibody spectra to individual proteins of human immunodeficiency virus (HIV) in 74 seropositive serum samples collected in the USSR and 65 serum samples collected in Britain were studied by immunoblotting techniques. Most of the sera belonged to clinically healthy persons, some of the sera collected in Britain contained specific IgM antibodies. The results were evaluated qualitatively and quantitatively. In the former case the study of samples collected both in the USSR and in Britain yielded similar results which also coincided with the data of literature regarding asymptomatic virus carriers: very high content of antibodies to protein gp41 and sufficiently high content of antibodies to protein p24 were registered in all sera. But the quantitative evaluation of the results of this investigation revealed differences between serum samples collected in these two countries. The main feature of sera collected in the USSR was their noticeably greater reactivity with respect to the products of HIV gene gag: proteins p24, p53 and p22. The explanations of this phenomenon are discussed.

Controlled organization of multimolecular complexes of enveloped virus glycoproteins: study of immunogenicity.

Some technological and immunological problems facing the preparation of subunit viral vaccines are discussed. Solubilization of enveloped virus glycoproteins with various detergents has been studied. It has been demonstrated that a novel non-ionic detergent, MESK, can be used to prepare the glycoproteins of enveloped viruses in defined supramolecular forms: monomers, micelles, liposomes and multimeric complexes. These preparations have been tested for immunogenicity. It has been shown that the immunogenicity of glycoproteins in micellar form or in liposomes is comparable with that of the whole virus. The immunogenicity of the glycoprotein complex with the glycoside Quil A appeared to be significantly higher in comparison with the whole virus and was similar to the immunogenicity of glycoproteins mixed with Freund's complete adjuvant.

[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.

[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.

[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.

[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.

[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.

[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.