Interaction of plum pox virus with specific colloidal gold-labeled antibodies and development of immunochromatographic assay of the virus.

Two monoclonal antibodies (mABs) raised against plum pox virus (PPV) were shown to recognize its D, M, and C strains. Conjugates of the antibodies with colloidal gold (CG) nanoparticles averaging 26 nm in diameter were synthesized. The binding constants of PPV with both the native and conjugated mABs were determined using a Biacore X device. The complexes between the CG-mAB conjugates and plum pox virions were examined by means of transmission electron and atomic force microscopy. Using the conjugates with optimal component ratio, an express immunochromatographic assay of PPV was developed with a detection limit of 3 ng/ml and duration of 10 min. The assay was tested for PPV detection in samples of stone fruit tree leaves and demonstrated a good compatibility with the data obtained by "sandwich"-ELISA. The developed assay can be used in the field and applied for monitoring viral infection and for quarantine purposes.

[Role of specific immunoglobulins of classes G, E and subclasses G1, G4 in the diagnosis of tuberculosis in children with hyperergic tuberculin sensitivity].

The study covered 56 patients (aged 6-14 years) having hyperergic tuberculin sensitivity, including 28 patients with pulmonary tuberculosis (Group 1) and 28 patients infected with Mycobacterium tuberculosis (MBT) (Group 2). All tuberculous processes were asymptomatic and limited. Minor forms of tuberculosis, diagnosed by computed tomography, were found in 71.4% of cases. The signs of incomplete calcification were detectable in 93% of cases. In the past year, tuberculin sensitivity has progressed to hyperergic one in 75% of Group 2 patients. The patients with tuberculosis were treated with 3 drugs; those infected with M BT received 2 agents. In the patients with minor forms of tuberculosis, the level of specific IgG antibodies was equal to those in healthy MBT-infected individuals with hyperergic tuberculin sensitivity, the levels of immunoglobulins of subclasses G1 and G4 were lower, that of IgE antibodies were higher than in the MTB-infected. These data may suggest the stability of the immune system in patients with minor forms of tuberculosis, detected in the phase of calcification, and its instability in the MBT-infected with tuberculin sensitivity increasing up to hyperergic one.

[Effect of use of bone marrow cells in therapy for experimental tuberculosis].

The authors studied the efficiency of use of bone marrow cells in the treatment of experimental tuberculosis. Bone marrow cell transplantation to H37Rv tuberculosis-infected H37Rv mice was shown to prolong the life span in the animals as compared to untreated animals. Examination of humoral immunity indicated that administration of allogenic bone marrow cells resulted in the nonspecific polyisotypic stimulation of antituberculosis antibodies, which is essential in producing the protective humoral background. A more significant generation of IgG2a antibodies than that of IgG1 antibodies was also found in therapy with bone marrow cells, which pointed to the fact that there was a Th1 response that is obviously protective in tuberculosis. The high level of IgG2a antibodies correlated with the high specific cellular immune response estimated by the delayed hypersensitivity reaction.

[Tuberculous IgE antibodies. Part II. Study of its concentrations in different forms of tuberculosis]. / Protivotuberkuleznye IgE-antitela. Chast' II. Issledovanie kontsentratsii pri razlichnykh formakh tuberkuleza.

Tuberculosis-afflicted lung are infiltrated by two functionally types of lymphocytes, which presumably counteract with each other by producing proinflammatory (type 1) and anti-inflammatory (type 2) cytokines. It is held that irregular sequestration of antigen into different compartments of the lung may lead to preferential activation of T-helper 1 or T-helper 2 lymphocytes. Unlike IgE antibodies, specific tuberculosis IgE antibodies are seen only in tuberculosis infection. The mean values of IgE antibodies in tuberculosis (7.661 +/- 0.849 IU/ml) are significantly greater than those in other pulmonary diseases (1.768 +/- 0.116 IU/ml). Low concentrations of tuberculosis IgE antibodies in persons with a marked hyperergic response to tuberculin (1.808 +/- 0.097 IU/ml) are of importance. Significant concentrations of mycobacterial IgE antibodies are mainly detected in fibrocavernous (14.56 +/- 1.11 IU/ml), infiltrative (10.10 +/- 1.08 IU/ml), peripheral lymph nodal (10.53 +/- 1.09 IU/ml) tuberculosis rather than intrathoracic lymph nodal tuberculosis (4.555 +/- 0.340 IU/ml). There is a particularly considerable increase in specific IgE antibodies in a phase of decay (15.98 +/- 1.64 IU/ml) and infiltration (12.66 +/- 1.08 IU/ml). These groups also show a concurrent rise in tuberculosis IgG antibodies, which nevertheless disagree with the increase of IgE (the correlation coefficient is 0.599).

[Anti-tuberculous IgE antibodies. I. Immunodominant antigens]. / Protivotuberkuleznye IgE-antitela. I chast'. Immunodominantnye antigeny.

It is widely accepted that protection against tuberculosis is provided by the formation of type 1 immune response, which is characterized by the production of IFN-gamma and IL-2. However, type 2 antimycobacterial immune response is also present: specific IgE antibodies that are IL-4 dependent, are usually found in tuberculosis patients. There is elevated production of type 2 cytokines in some cases. Thus, both types of an immune response can simultaneously develop, probably counteracting with each other. It is unknown which of mycobacterial antigens are capable of inducing a preferential type 2 response. To detect these antigens, the authors studied tuberculosis IgE antibodies in the sera of 500 tuberculosis patients by using the ELISA assay with ultrasonic disintegrated M. Tuberculosis H37Rv (sonicate). Antigens recognized by IgE antibodies were found to be localized in the cell wall of mycobacteria. The IgE-response was specific since the sera did not react with the antigens of atypical mycobacteria and other bacterial species.

Deletion of the 19kDa antigen does not alter the protective efficacy of BCG.

Expression of the Mycobacterium tuberculosis 19kDa lipoprotein in saprophytic mycobacteria has been found to reduce their ability to prime a protective response to subsequent virulent challenge in the mouse model. The present study was designed to test whether 19kDa expression has an analogous detrimental effect on the efficacy of BCG vaccination. In contrast to the results in saprophytes, neither overexpression of the 19kDa antigen, nor deletion of the endogenous 19kDa gene altered the ability of BCG to protect against M. tuberculosis challenge in a mouse model.

[Improving the efficacy of tuberculosis immunodiagnosis using anti-human IgG monoclonal antibodies]. / Povyshenie éffektivnosti immunodiagnostiki tuberkuleza putem primeneniia monoklonal'nykh antitel protiv IgG cheloveka.

Monoclonal antibodies (MAb) F5 to human IgG were used for creating immunoperoxidase conjugate. MAb dissociation constant was 10(-9)M-1 and the number of binding sites 1. Enzyme immunoassay (EIA) and immunoblotting showed that MAb F5 specifically recognize conformation epitope on intact human IgG molecule but not on other human immunoglobulins or denatured IgG. The resultant peroxidase conjugate with MAb F5 was used for EIA titration of antibacterial antibodies in sera from 30 patients with pulmonary tuberculosis, 28 patients with nonspecific pulmonary diseases (bronchitis and/or asthma, pneumonia), and 12 donors. For comparison similar studies were carried out with reference commercial immunoperoxidase conjugate to human IgG(H + L) manufactured at N. F. Gamaleya Institute of Epidemiology and Microbiology. Mycobacterium tuberculosis monoantigen (mol. weight 15-18 kD), affinity isolated by antibacterial MAb S4C1G4 (alpha S4C1G4), and PPD (Batch RT 45, Stattens Seruminstitut, Denmark) were used. Sensitivity and specificity of serum antibacterial antibodies were compared. The specificity of conjugate based on MAb F5 with monoantigen alpha S4C1G4 was 78.21%, sensitivity 94.50%, while those of conjugate to human IgG(H + L) were 53.30 and 76.89%, respectively (p < 0.001). For PPD the specificity and sensitivity were 56.75 and 72.33%, respectively (conjugate with MAb F5) versus 47.67 and 62.38% for conjugate against IgG(H + L), p < 0.001. Similar values were obtained in assessment of the concentrations of antibodies to alpha S4C1G4 for MAb F5 conjugate: specificity and sensitivity 47.16 and 71.56%, respectively, versus 23.67 and 95.16% for PPD (p < 0.05). No statistically significant differences between the experimental groups were detected with IgG(H + L) conjugate. We believe that specific MAb-based conjugate to human IgG will improve the efficacy of EIA as a method for the diagnosis of pulmonary tuberculosis.

[Comparative analysis of two mycobacterium tuberculosis antigens and two methodological approaches to determining serum antimycobacterial antibodies]. / Sravnitel'nyi analiz dvukh antigenov mycobacterium tuberculosis i dvukh metodicheskikh podkhodov pri opredelenii protivotuberkuleznykh antitel syvorotki v immunofermentnom analize.

The glycoprotein (15-18 kDa) antigen of Mycobacterium tuberculosis H37Rv was affinity isolated on the immunosorbent with monoclonal antibodies S4C1G4 (specific to M. tuberculosis H37Rv; Avdiyenko V. G., Kondrashov S.Yu, Lyashenko S.M.@Probl. Tuberk. 1996, v. 1, p. 6-8 (in Russian). This antigen and PPD (Batch RT 45, Stattens Seruminstitute, Denmark) that was a standard antigen were used for enzyme-linked immunosorbent assay (ELISA), by detecting serum IgG antibodies in patients with pulmonary tuberculosis, and control groups of patients with lung diseases other than tuberculosis (bronchitis and/or asthma, pneumonia) as well as healthy volunteers. The diagnostic parameters of specificity and sensitivity for titers and the same parameters for optic density (OD) (in serum dilution with maximum differences for groups of patients and donors) were compared. The new monoantigen method provided 86.11% specificity and 87.87% sensitivity, which were higher those obtained for optic density (63.89 and 80%, respectively). With PPD, the specificity and sensitivity were 58.04 and 78.78 (for the new titer method) versus 50 and 78.78% (OD data). The method error for titer determination was 10% and for standard OD determination was 27%. The new approach offers additional possibilities of enhancing the quality of ELISA for diagnosis of tuberculosis.

[Serological and immunochemical properties of carbohydrate-containing fraction from mycobacterium tuberculosis H37Rv]. / Serologicheskie i immunokhimicheskie svoistva uglevodosoderzhashchei fraktsii iz mycobacterium tuberculosis H37Rv.

Using affinity chromatography on concanavalin A (Con A) sepharose CL 6B, a carbohydrate-containing fraction was derived from Mycobacterium tuberculosis H37Rv sonicate. Surprisingly, the main component of Con A fraction was a protein having a molecular weight of a 30-kD range which is generally absent in the Con A-adsorbed fraction from the culture filtrate. ELISA by means of an antimycobacterial monoclonal antibody panel showed that the 30-kD range of Con A fraction contained antigen 85. It is suggested that the derived components antigen 85 are a glycosylated form of the proteins associated with the mycobacterial cell wall. The Con A fraction, antigen 85 (Department of Virology, Pasteur Institute, Brussels, Belgium), and PPD (Batch RT 45, Stattens Seruminstitute, Denmark) were used for ELISA determination of antimycobacterial antibody titers in the sera of 30 patients with pulmonary tuberculosis, 28 patients with nonspecific lung diseases (bronchitis and/or asthma, pneumonia), as well as in the sera of 12 healthy volunteers. The sensitivities were 46.42, 57.34, and 72.33% and the specificities were 36.97, 26.73, and 56.75% for Con A fraction, antigen 85, and PPD, respectively. The authors suggest that serodiagnostic properties of Con A fraction are extremely limited.

[Systematic approach to detection of anti-idiotypic antibodies to antimycobacterial idiotype in man and animals]. / Metodicheskii podkhod k opredeleniiu antiidiotipicheskikh antitel k antimikrobakterial'nomy idiotipu cheloveka i zhivotnykh.

ELISA in the sandwich modification was developed to detect anti-idiotypic antibodies to the antituberculosis idiotype of monoclonal antibodies (MAb) S4C1G4 (anti 15-18 kDa H37Rv). Sera immunoglobulins were determined by Staphylococcus protein A capture. F[ab+]2, the MAb fragments S4C1G4 (IgG2a, kappa) and 1.3.3.B5 (IgG2a, kappa), were used as a negative control, which were not recognized mycobacterial antigens and human immunoglobulins. The anti-idiotypic antibody titers were determined in the murine hyperimmune sera during the period of immunization with the MSAb SaC1G4. The method detected anti-idiotypic antibodies in the sera of infected mice and in patients with pulmonary tuberculosis. It is suggested that this method is applicable while using diagnostic tools.

Influence of the mouse Bcg, Tbc-1 and xid genes on resistance and immune responses to tuberculosis infection and efficacy of bacille Calmette-Guérin (BCG) vaccination.

We have studied the role of three mouse distinct non-H-2 genes (Bcg, Tbc-1, xid) in several phenomena of antituberculosis immunity and resistance. On the basis of median survival time (MST) of mice following infection with virulent Mycobacterium tuberculosis H37Rv, Bcg gene did not control resistance to the lethal dose of H37Rv infection in non-vaccinated and Myco. bovis (BCG)-vaccinated mice. However, Bcgr allele, in comparison with Bcgs allele, determined more effective suppression of an early multiplication in spleens of H37Rv mycobacteria after a low dose (5x10(4) colony-forming units (CFU)) injection. CBA/N mice, which are not protected efficiently against tuberculous challenge by BCG vaccination, were characterized by a decreased in vitro proliferation of immune lymph node cells, both spontaneous and stimulated with mycobacterial antigens. The decreased proliferation was due to immunosuppression caused by interactions between responding T cells and CBA/N antigen-presenting cells (APC). We have confirmed that the defective response to BCG-vaccination in CBA/N mice is linked with the X-chromosome and thus is presumably determined by the xid gene itself. I/St mice (Tbc-1s), supersusceptible to H37Rv infection, were not able to restrict the growth of H37Rv mycobacteria in spleens, even following infection with a low dose (5x10(4)), but restricted the growth of Myco.bovis BCG more effectively than Bcgs mice.

[An analysis of the specificity of anti-Mycobacterium monoclonal antibodies and of the chemical nature of the antigenic determinants with which they react]. / Analiz spetsifichnosti antimikobakterial'nykh monoklonal'nykh antitel i khimicheskoi prirody antigennykh determinant, s kotorymi oni reagiruiut.

Seven hybridomas of BALB/c mice producing monoclonal antibodies (mAb) against M. tuberculosis H37Rv have been obtained. The mAb acted against M. tuberculosis H36Rv with molecular mass 14, 17-15, 25 27 30 kD excluding mAb S5B3B8 and S3H5D7 which acted against the main antigen with 54 kD mass and 5-6 bands of antigens. Immunoglobulin isotypes of mAb were IgG2a and IgG2b. mAb can be divided into 2 groups by interaction with mycobacterial antigens in EIA: specific to M. tuberculosis complex antigens, cross-reactive to antigens of different mycobacteria. Chemical nature of antigenic determinants recognizable by a panel of mAb was investigated. Mild sodium periodate oxidation and protease digestion of mycobacterial antigens showed that mAb recognize both carbohydrate-containing epitopes (S2H6G4) and protein epitopes (S5B11E10, S5B3B8, S4C1G4) or protein and carbohydrate-containing antigenic determinants (S2F5C10, S2F2E2).

[An experimental analysis of Mycobacterium smegmatis as a possible vector for the design of a new tuberculosis vaccine]. / Eksperimental'nyi analiz Mycobacterium smegmatis v kachestve vozmozhnogo vektora dlia konstruirovaniia novoi protivotuberkuleznoi vaktsiny.

This study was inspired by WHO program for vaccine development. Our experimental model of lethal tuberculosis in mice was used to test whether rapidly growing avirulent M. smegmatis could be used as a live vaccination vector for screening rationalized M. tuberculosis cosmid library. The results suggest that M. smegmatis is not likely to possess protective activity against tuberculosis in C57B1/6 TB-susceptible mice. This negative result is satisfactory in that the immunity induced by M. smegmatis itself could not interfere with expected protection caused by the expression of M. tuberculosis genes in this vector. Our model makes it possible to find out which gene(s) of M. tuberculosis provide an ability to multiply in the host and whether virulence and persistence are controlled by the same genetic traits(s).

[Modulation of the course of experimental tuberculosis in mice by in vivo administration of monoclonal antibodies to interleukin-2, gamma-interferon and interleukin-4]. / Modulatsiia techeniia éksperimental'nogo tuberkuleza u myshei vvedeniem in vivo monoklonal'nykh antitel k interleikinu-2, gamma-interferonu i interleikinu-4.

The impact of the in vivo administration of monoclonal antibodies against cytokines on the development of a pathological process in mice infected with Mycobacterium tuberculosis H37Rv-infected mice. The susceptibility to fatal experimental tuberculosis, the level of DTH to mycobacterial antigens, the production of specific antimycobacterial IgG and IgM, T-cell proliferative responses to M. tuberculosis H37Rv antigens. Anti-gamma-interferon and anti-IL-2 treatment was found to provide virtually no effects on the course of experimental tuberculosis, the intravenous administration of anti-IL-4-monoclonal antibodies on days 2, 4, and 6 after inoculation with a lethal dose of M. tuberculosis significantly increases the median survival time (MST). This increase in MST is accompanied by a more prominent DTH response and lymphocytic proliferative reaction on the one hand, and by lower concentrations of specific IgG on the other. The therapeutical prospects of anti-IL-4 treatment in tuberculosis are discussed.

[Development of protective antituberculosis immunity after BCG vaccination in mice with silicon deficiency in water and feed]. / Formirovanie protektivnogo protivotuberkuleznogo immuniteta posle vaktsinatsii BTsZh u myshei pri nedostatke kremniia v vode i kormakh.

An experimental mouse model of normal and deficient content of silicon in the diet in CBA strain has been developed. It is shown that after BCG vaccination silicon deficiency brought about a dramatic decrease of in vitro proliferation of splenic lymphocytes in the presence of mycobacterial antigens, ConA or in the absence of the stimuli. DTH response to tuberculin in silicon deficient mice was also reduced. The level of specific serum IgG did not depend upon silicon content in the diet. The addition of silicon into the diet of silicon-deficient mice beginning from BCG vaccination restored the level of immune reactions.

Distinct H-2 complex control of mortality, and immune responses to tuberculosis infection in virgin and BCG-vaccinated mice.

We have studied the impact of distinct haplotypes and of different alleles at specific H-2 loci on: (i) the susceptibility to lethal form of experimental tuberculosis; (ii) the level of DTH to mycobacterial antigens; (iii) the efficacy of vaccination with bacille Calmette-Guérin (BCG); and (iv) the IgG production and T cell proliferative response to H37Rv antigens. On the basis of median survival time (MST) following primary inoculation with lethal dose of Mycobacterium tuberculosis, susceptibility to infection associated with I-Ab and Db alleles, host resistance associated with I-Ak and Dd alleles. Mice bearing a disease-resistant phenotype also developed a vigorous DTH response. Vaccination with BCG before H37Rv infection significantly prolonged the survival time of both resistant and susceptible animals, except in B10.M (H-2f) mice. The latter exhibited intermediate resistance to infection before but slight decrease in the MST following a high-dose BCG vaccination. Distinct H-2 regulation of susceptibility to lethal infection and of BCG vaccination efficacy was confirmed in another relatively resistant H-2f-bearing strain A.CA, in which mortality occurred more rapidly in vaccinated compared with primarily infected animals. The expression of the H-2f haplotype was associated with a low DTH response to tuberculin following vaccination and subsequent lethal infection. The lack of BCG protection against Myco, tuberculosis challenge in B10.M mice associated with the high titre of specific IgG. In addition, these mice exhibited a unique ability to respond to 65-kD antigen by both IgG synthesis and T cell proliferation.

[The isolation and testing of syngeneic anti-idiotypic antibodies against antimycobacterial monoclonal antibodies]. / Poluchenie i ispytanie singennykh antiidiotipicheskikh antitel protiv antimikobakterial'nykh monoklonal'nykh antitel.

Fifteen monoclonal antibodies (MAb) were obtained to cell walls (CW) of M. bovinus-8, two of them were limited specific against human and bovine mycobacteria. One MAb reacted in immunoblotting with a protein having molecular mass of 19.1 kDa. It was also found that all MAb bind with the antigen determinants of mycobacterial proteins. The competitive enzyme immunoassay (EIA) helped reveal that the antigenic determinants "recognized" by two MAb are located in the same area despite the fact that one reacted in immunoblotting with a denatured protein and the other "recognized" only a native antigen in EIA. The syngeneic antiidiotypic (anti-ID) immune response was induced by these MAb in BALB/c mice. The EIA showed the binding of anti-ID-antibodies isolated from mice serum both MAb inducing their synthesis and to antimycobacterial serum antibodies of caws with tuberculosis. Data suggesting a similarity existing between the mycobacterial antigen and anti-ID-antibodies were also obtained in the blast transformation reaction: in M. bovinus antigen stimulation of 8 mice lymph node cells sensitized by anti-ID-antibodies and in the reverse situation when the cells sensitive to KC M. bovinus-8 proliferated in response to stimulation by anti-ID-antibodies.

[Experimental influenzal and tuberculous infections under conditions of specific and nonspecific immunosuppression]. / Eksperimental'naia grippoznaia i tuberkuleznaia infektsii v usloviiakh spetsificheskoi i nespetsificheskoi immunosupressii.

The course of influenza and tuberculosis infections under the conditions of disturbances in the immune response of experimental animals has been studied. As revealed in the survival test, the induction of secondary T- and B-cell-mediated immunodeficiency in mice leads to an increase in the sensitivity of the body to influenza virus, especially in cases of T-cell-mediated immunodeficiency. The injection of BCG in combination with cyclophosphamide into mice induces tolerance to this antigen in the animals; this tolerance has a "split" character, i.e. it affects only T-cell-mediated, but not humoral immunity. The induction of T-cell-mediated immunodeficiency or tolerance to BCG in mice has been shown (in the survival test) to lead to the development of the sensitivity of the animals to experimental tuberculosis infection. B-cell-mediated immunodeficiency did not influence the animal survival rate.