[Rubella virus genetic determinant of attenuation].

Vaccination is the most effective and available way to prevent Rubella. Presently, 9 vaccine strains were registered. Nevertheless, the molecular mechanisms of the attenuation were poorly elucidated for the rubella virus. However, the study of these mechanisms identifying genotypic and phenotypic markers of attenuation, which together with sequence analysis could be used for the genetic stability control of vaccine strains, is still of current interest. Common trends of genetic changes in the process of adaptation to cold were found due to comparison of nucleic acid and amino acid sequences of the Russian strain C-77 with corresponding positions of the known rubella virus strains and its wild type progenitors, if available.

[Studying of molecular mechanisms of rubella virus attenuation evidence from Russian strain C-77].

Live attenuated rubella vaccine is used for vaccination. Temperature-sensitive (ts) phenotype was proved for almost all rubella vaccine strains, and the acquisition of the ts phenotype during cold adaptation was strongly correlated with the attenuation of the wild-type viruses. Nevertheless, the molecular mechanisms of the attenuation have been insufficiently understood for rubella virus. Study ofthese mechanisms, identifying genotypic markers of attenuation, which together with the sequence analyses could be used for genetic stability control of vaccine strains, is still of current interest. In this work, we determined nearly complete genome sequences of attenuated (ca) and the wildtype progenitor (wt) of the rubella virus strain C-77 isolated in Russia. Possible genetic determinants of attenuation were detected. Thus, 13 nucleotide differences leading to 6 amino acid substitutions were found. Four amino acid substitutions were found to be almost unique. Special consideration should be given to Tyr1042Cys substitution in the protease domain of C-77 strain, because it most probably plays the crucial role in acquisition of ts-phenotype.

[M-29 human diploid cells line--a substrate for the production of antiviral vaccines].

AIM: Study of morphologic and karyologic characteristics of 5 russian human diploid cell lines (HDC). MATERIALS AND METHODS: 5 HDC lines and HDC strain MRC-5 were studied; RK-13 and Vero continuous cell lines were used; viruses: rubella (RA27/3), measles (L-16), epidemic parotitis (L-3). Cytogenetic analysis of HDC was performed by using DAPI differential staining method. RESULTS: M-29 line has characteristics that are similar to those of MRC-5 diploid cell strain. M-29 cell culture is not contaminated with foreign viruses, mycoplasmata, does not have oncogenic potency. CONCLUSION: M-29 line has high virus-productive properties for accumulation of measles, rubella and epidemic parotitis vaccine viruses and may be recommended as a substrate for the production of antiviral vaccines.

[Quick test for measurement of rubella virus titer in virus-containing fluid using RT-PCR].

AIM: To develop method of rubella virus titer measurement in virus-containing fluid using real-time PCR (RT-PCR) with fluorescent detection. MATERIALS AND METHODS: Measurement of infectious titer of rubella virus (Wistar RA 27/3 strain) cultivated on Vero cells was performed simultaneously by RT-PCR and cytopathic effect assay (CEA) on PK-13 cell culture and then results obtained by each method were compared. RESULTS: Time interval after inoculation, in which difference between virus titer measured by both methods did not exceed 0.3 1gTCD50/ml (value acceptable by WHO), was 2 - 7 days. Pearson correlation coefficient between two values for the mentioned interval was close to 1, which point to good agreement of results. In control sample--international vaccine standard of rubella virus--difference in virus titer determined by RT-PCR and CEA was within 0.2 1gTCD50/ml that lower than value acceptable by WHO. CONCLUSION: Method for measurement of rubella virus titer in virus-containing fluid using RT-PCR was developed, which characterized by high specificity, sensitivity, standard performing, shorter time needed for procedure compared with classic methods and, at the same time, high correlation of its results with results obtained by the latter methods during defined time interval.

[Role of cytokines in immune response to T-independent antigens, type 2].

The influence of human and mouse cytokines on the induction of immune response to model T-independent (TI) antigens of type 2--DNP-dextran and DNP-ficoll--in the culture of mouse spleen cells was studied. For the first time this study revealed that the action of definite T-cell factors not only induced the polyclonal stimulation of B lymphocytes, but also the increased synthesis of specific antibodies, as well as switched over antibody isotypes from IgM to IgG. This confirmed the suggestion that T cells took part (directly or indirectly) in the regulation of immune response to so-called TI antigens. These results widened our knowledge on the mechanisms of the development of humoral immune response to TI antigens of type 2.

[Human monoclonal antibodies to synthetic viral peptides]. / Monoklonal'nye antitela cheloveka k sinteticheskim peptidam virusa.

Cells producing human antibodies to synthetic viral peptides MH-24 (corresponding to 302-325 residues of HIV-1 gp120) and p107 (main peptide of EBV antigen EBNA-1) were fused with human x mouse heteromyeloma B6B11. The efficacy of specific hybridization was 40%. Hybrid cells H1F2 (MN-specific) and HF4 (p107-specific) were cloned by the limiting dilutions method. Monoclonal antibodies (MAb) were tested in enzyme immunoassay on a panel of synthetic peptides. All resultant MAbs were polyspecific for tested synthetic peptides. The data permit a conclusion that previously detected polyspecificity of human cultural antibodies to virus peptides is due to the true polyspecificity of antigen-binding centers of MAb, but not to the presence of strange cells in the culture.

Role of different lymphocyte subpopulations in the formation of non-specific immunoglobulins induced by antigen injection.

The formation of antibody and non-specific immunoglobulin under the influence of T-dependent (TD) and type 2 T-independent (TI-2) antigens in mice of two congenic strains CBA (Lyb5-, Lyb5+) and CBA/N (Lyb5-) was studied. TD antigens induced in mice of both strains not only the appearance of antibody-forming cells (AFC), but also a great increase in the number of cells producing non-specific immunoglobulins (nIFC). TI-2 antigens induced the AFC and antigen-dependent nIFC formation in CBA mice only. It is concluded that during immune response to TI-2 antigens not only the AFC appearance but the increase in nIFC formation (polyclonal activation) is due mainly to the mature Lyb5+ B cells.

[Isolation of human B-cells, specific for viral peptides, using magnetic spheres]. / Vydelenie B-kletok cheloveka, spetsifichnykh k virusnym peptidam, s pomoshch'iu magnitnykh sharikov.

A method for isolation of human B cells specific to viral peptides using magnetic beads has been developed. The method was used for the isolation from human tonsils of B cells specific to HIV-1 gp 120 MN-24 peptide (residues 302-322) and to Epstein-Barr virus 107 EBNA-1 peptide. The cells selected with beads were transformed by Epstein-Barr virus. The resultant cell cultures produce human immunoglobulin antibodies to HIV-1 MN-24 and to Epstein-Barr EBNA-1 p107 peptides.

[The dynamics of Treponema-specific blood immunoglobulins in early forms of syphilis]. / Dinamika treponemospetsificheskoi immunoglobulinemii pri rannikh formakh sifilisa.

The study has involved 73 patients with syphilis, 31 female and 42 male ones, aged 18-42. Four of these suffered from primary seronegative, 14 from primary seropositive, 21 from secondary fresh, 22 from secondary recurrent, and 12 from early latent seropositive syphilis. Peripheral blood sera were under study. Treponema-specific antibodies of the IgM and IgG classes were titered by enzyme immunoassay. The detected changes in Treponema-specific immunoglobulinemia are in good correlation with clinical staged pattern of syphilis and antiinfectious immunity status.

Ir gene influence on DNP-specific B-memory-cell formation.

It is well known that Ir genes control the antibody production. To investigate whether they also influence B-memory-cell generation, CBA mice (nonresponders) were primed with dinitrophenyl-poly-(L-tyr,L-glu)-poly-(D,L-ala)-poly-(L-lys)-DNP-TGAL conjugate. At the same time animals were injected with ovalbumin (OVA) to activate OVA-specific T helpers. Two to four weeks later animals were challenged with DNP-OVA conjugate and the number of IgG-producing B cells was determined. The data presented indicate that carrier-specific MHC-restricted T helpers are not required for B-memory-cell generation. It is concluded that the defect of IgG response to DNP-TGAL in CBA mice is caused by a block in the maturation of memory cells to antibody-producing cells.