Efficacy of BCG Vaccination Depends on Host Genetics.

We studied the effectiveness of anti-tuberculosis vaccination with BCG in mice of inbred strains and F1 hybrids (highly resistant to tuberculosis infection) that represent a wide range of genetically determined differences in susceptibility to infection with virulent Mycobacterium tuberculosis. The greatest relative effect was found in susceptible mice, with the exception of highly susceptible I/St mice that were practically not protected by vaccination. Despite significant effect of vaccination in inbred mice, their resistance to M. tuberculosis infection did not exceed that of non-vaccinated highly resistant F1 hybrids.

Host genetics in susceptibility to and severity of mycobacterial diseases.

The genetic analysis of susceptibility to infections has proven to be extremely useful for identification of key cells, molecules, pathways, and genes involved in the battle between two genomes - the essence of the infectious process. This is particularly true for tuberculosis and other mycobacterial infections which traditionally attracted much attention from both immunologists and geneticists. In this short review, we observe results of genetic studies performed in human populations and in animal models and compare relative input of forward and reverse genetic approaches in our knowledge about genetic control of and immune responses to mycobacterial infections.

"Suppressor Factor" of Neutrophils: A Short Story of a Long-Term Misconception.

A large body of evidence obtained during the last decade has demonstrated that neutrophils suppress T cell proliferation in different models of inflammation and cell interaction. The commonly used method for assessing cell proliferation and proliferation inhibition is measuring [3H]thymidine incorporation into cells. Earlier, we observed inhibition of [3H]thymidine uptake in experiments on neutrophil-mediated regulation of T cell response in tuberculosis immunity. Here, we used different types of proliferating cells to analyze the nature of the soluble "neutrophil factor" by a variety of methods (dialysis, HPLC, mass spectrometry, and NMR) and unambiguously demonstrated that neutrophils do not synthesize a specific factor inhibiting cell proliferation, but secrete high concentrations of extracellular thymidine that competitively inhibit [3H]thymidine incorporation. Although the physiological significance of thymidine secretion by neutrophils remains unknown, this phenomenon should be carefully considered when designing test systems for studying cell-cell interactions.

Formation of compact aggregates of B-lymphocytes in lung tissue during mycobacterial infection in mice depends on TNF production by these cells and is not an element of the host's immunological protection.

Tumor necrosis factor (TNF) plays a pivotal role in the early control of Mycobacterium tuberculosis and M. avium infections by a host. It was previously shown that both phagocyte-derived and T-cell-derived TNF productions are critical for protective immunity against M. tuberculosis, but the role of TNF produced by B-cells remained unclear. By comparing mice with B-cell-specific TNF deletion to littermate control mice, here we show that TNF production by B-lymphocytes is essential for the formation of infection-specific aggregates of B-cells in the lung. It is likely that these compact foci represent a pathogenic feature of inflammatory response rather than an element of protective immunity, since the capacity to form aggregates has no influence on the severity of M. tuberculosis- and M. avium-triggered diseases.

Mycobacterium tuberculosis Transcriptome Profiling in Mice with Genetically Different Susceptibility to Tuberculosis.

Whole transcriptome profiling is now almost routinely used in various fields of biology, including microbiology. In vivo transcriptome studies usually provide relevant information about the biological processes in the organism and thus are indispensable for the formulation of hypotheses, testing, and correcting. In this study, we describe the results of genome-wide transcriptional profiling of the major human bacterial pathogen M. tuberculosis during its persistence in lungs. Two mouse strains differing in their susceptibility to tuberculosis were used for experimental infection with M. tuberculosis. Mycobacterial transcriptomes obtained from the infected tissues of the mice at two different time points were analyzed by deep sequencing and compared. It was hypothesized that the changes in the M. tuberculosis transcriptome may attest to the activation of the metabolism of lipids and amino acids, transition to anaerobic respiration, and increased expression of the factors modulating the immune response. A total of 209 genes were determined whose expression increased with disease progression in both host strains (commonly upregulated genes, CUG). Among them, the genes related to the functional categories of lipid metabolism, cell wall, and cell processes are of great interest. It was assumed that the products of these genes are involved in M. tuberculosis adaptation to the host immune system defense, thus being potential targets for drug development.

[Production of mycobacterial antigenes merged with cellulose binding protein domain in order to produce subunit vaccines against tuberculosis].

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

[Prevention of tuberculosis: current approaches to development of vaccines].

This review is focused on recent advances in development of new vaccines for the prevention of tuberculosis. The main reasons for lack of BCG vaccine efficacy in different populations and geographic regions are presented. Design of new vaccines based on live modified strains of Mycobacterium bovis BCG, attenuated strains of Mycobacterium tuberculosis, recombinant proteins and viral vectors is considered in the specific examples. The usage of the heterologous "prime-boost" vaccination strategy against tuberculosis is discussed.

[A locus involved in tuberculosis infection control in mice locates in the proximal part of the H2 complex].

One of genetic loci involved in tuberculosis (TB) infection control in mice is located within the segment of Chr. 17 occupied by the H2 complex, the mouse MHC. As far as this region includes approximately 40 Mb and contains hundreds of genes affecting immune responses and host-parasite interactions, narrowing the interval by genetic recombination is pre-requisite for identification of particular gene(s). We have developed a panel of recombinant congenic strains bearing different parts of the H2 complex from TB-susceptible I/St mice on the genetic background of TB-resistant C57BL/6 mice. By superposing the phenotype "severe vs. mild infectious course" against the chart of alleles inherited by these new strains from the two parental strains, we have mapped a locus involved in TB control within the segment 33.305-34.479 Mb (-1.1 Mb) of the Chr. 17. Such a location indicates that allelic variants of the prominent pro-inflammatory factor TNF do not affect TB course in our experimental system. This result was confirmed by the assessment of the TNF level in the lung tissue of infected mice of different strains. The QTL (quantitative trait locus) mapped in our study influences several important parameters of TB infection: multiplication of mycobacteria in the lungs, severity of lung pathology and regulation of the early inflammatory response.

[Development of pulmonary chlamydia infection in inbred mice lines differentiated by genetically determinated sensitivity to tuberculosis infection].

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. The susceptibility does not depend on the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by little interacting QTL mapped to chromosomes 3, 9, 17. To find out whether the tuberculosis-susceptible I/St mice are susceptible to other intracellular bacteria taxonomically distant pathogen of Chlamydia pneumoniae was studied. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former were more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St, 9.2 +/- 1.2 days; A/Sn, 22.0 +/- 0 days (p < 0.001)). To estimate the degree of chlamydial multiplication in the lungs, we suggested a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite's genome equivalents in infected tissue from 1 to 16 days after challenge. The interstrain difference of chlamydia burden in lungs was observed only after 24 hours after infection. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection. The numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p < 0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-? and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p < 0.05). Because the prominent difference in survival time did not correlate with permanent difference in bacterial multiplication, we suggested that both infections trigger fatal pathological processes whose dynamics depends strongly upon the host genetics.

Adaptive Changes in Mycobacterium avium Gene Expression Profile Following Infection of Genetically Susceptible and Resistant Mice.

We performed a comparative analysis ofMycobacterium aviumtranscriptomes (strain 724R) in infected mice of two different strains- resistant and susceptible to infection. Sets of mycobacterial genes transcribed in lung tissue were defined, and differentially transcribed genes were revealed. Our results indicate thatM. aviumgenes coding for enzymes of the Krebs cycle, oxidative phosphorylation, NO reduction, fatty acid biosynthesis, replication, translation, and genome modification are expressed at high levels in the lungs of genetically susceptible mice. The expression of genes responsible for cell wall properties, anaerobic nitrate respiration, fatty acid degradation, synthesis of polycyclic fatty acid derivatives, and biosynthesis of mycobactin and other polyketides is increased in the resistant mice. In the resistant host environment,Mycobacterium aviumapparently transitions to a latent state caused by the deficiency in divalent cations and characterised by anaerobic respiration, degradation of fatty acids, and modification of cell wall properties.

[Immunomorphological characteristics of experimental granulomatous inflammation caused by Mycobacterium avium].

The results of the topical arrangement of T and B lymphocytes in the lung tissue granulomatous masses of the mice susceptible (C57BL/6Ycit) and resistant (I/StSnEgYCit) to aerosol inoculation with M. avium 724R in a dose of 2 x 10(3) CFU/mice are presented by means of labeled monoclonal antibodies. At week 8 of the inoculation, CD4+ lymphocytes were located both among the granuloma cells in the diffuse fashion and as small clusters and in the interalveolar and interlobular septa in the B6 mice and these were mainly in the granuloma in the I/St mice. At week 16, CD8+ lymphocytes were found in the peripheral granuloma portions in the B6 mice and these were diffusely throughout the area of granulomatous masses in the I/St mice. In the B6 mice, CD19+ lymphocytes formed cell aggregates from week 8 of the inoculation; and in the I/st mice, these were diffusely located among the granuloma cells and abundantly in the interalveolar and interlohular septa.

[Study of a role of B cells in the formation of antituberculosis immunity].

The authors have earlier demonstrated that CBA/N mice bearing nonsense mutation in the btk gene encoding B cell Bruton's thyrosine kinase after CG vaccination develop no protective response against subsequent M. tuberculosis H37Rv challenge. Adoptive transfer approaches are applied to show that this defect is determined by lymphoid cells and B lymphocytes play an essential role in its expression. In addition, experiments on CBA/N-xid mice with crude mixtures of soluble mycobacterial antigens suggest that the processing of live BCG could be impaired in the antigen-presenting cells of these mice.

[Tuberculosis: pathogenesis, immune responses and genetics of the host].

Tuberculosis is a chronic infectious disease predominantly affecting the lung. The hallmark of tuberculosis infection is the formation of granulomata in the vicinity of infectious foci. The tuberculous granuloma is a complex, cellulary and biochemically well-orchestrated structure, which development plays a dual role. Restricting dissemination of infection and forming a battlefield for protective immunity, granulomatous process may compromise lung function, threatinig the host health. Both the susceptibility to infection per se and the degree of lung failure and disease severity are under genetic control. Tuberculosis genetics is complex and far from being resolved, but the information available clearly indicates that the control of intracellular infections depends upon biochemical networks, which have not been appreciated with this regard until recently.

I/St mice hypersusceptible to Mycobacterium tuberculosis are resistant to M. avium.

We previously demonstrated that mice of the I/St strain are extremely susceptible to Mycobacterium tuberculosis, as well as to the taxonomically distant intracellular bacteria Chlamydia pneumoniae and Salmonella enterica. To broaden our knowledge about the control of susceptibility to intracellular pathogens, we studied the infection caused by Mycobacterium avium virulent strain 724 in a panel of inbred mouse strains and found that I/St mice are resistant to M. avium. By comparing I/St mice with B6 mice, we demonstrated that (i) B6 mice are much more susceptible to infection caused by M. avium in terms of bacterial multiplication in the lung tissue and severity of lung pathology; (ii) in B6 mice but not in I/St mice infection leads to prolonged leukocyte infiltration of the lung tissue, development of necrotic lung granulomata, and lethality; and (iii) the unfavorable infectious course in B6 mice is accompanied by elevated production of gamma interferon, tumor necrosis factor alpha, and especially interleukin-12 in the lungs. Importantly, M. avium-resistant I/St mice carry a functional r allele of the Slc11a1 (formerly Nramp1) gene, while B6 mice have the Slc11a1(s) genotype. Segregation genetic analysis of (I/St x B6) F2 hybrids demonstrated that susceptibility or resistance to infection caused by M. avium largely depended upon the Slc11a1 genotype and that other genetic traits had a relatively weak influence. This close-to-monogenic pattern differs sharply from the host control of many other intracellular bacterial infections, for which the involvement of numerous quantitative trait loci has been ubiquitously observed.

[Efficiency of anti-inflammatory therapy for experimental tuberculosis in genetically tuberculosis-sensitive mice].

The study assessed trends in death rates in genetically tuberculosis-sensitive I/St mice, mycobacterial seeding from their organs, and the magnitude of abnormal changes in the lung after intratracheal Mycobacterium tuberculosis infection of recipients. The experimental animals received two anti-inflammatory regimens: 1) a very small-dose intravenous injection of immature antigen-loaded bone marrow-derived syngeneic dendritic cells; 2) the nonsteroidal anti-inflammatory drug diclofenac injected intramuscularly in the course of disease development. Both anti-inflammatory regimens caused a significant increase in the animals' survival, a reduction in the amount of mycobacteria in the lung and the pulmonary infiltration with lymphoid cells as compared with untreated infected control animals. These findings indicate that suppression of an excessive inflammatory process in the lung may have a beneficial antituberculous effect in tuberculosis-sensitive animals.

Mycobacterium tuberculosis-susceptible I/St mice develop severe disease following infection with taxonomically distant bacteria, Salmonella enterica and Chlamydia pneumoniae.

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. To find out whether tuberculosis (TB)-susceptible I/St mice are susceptible to other intracellular bacteria, we investigated two different taxonomically distant pathogens, Chlamydia pneumoniae and Salmonella enterica serovar Typhimurium. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1(r)) demonstrated that the former are more susceptible to both salmonella and chlamydia, displaying a significantly shortened survival time following challenge. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia, despite their similar ability to control bacterial multiplication. Following infection with salmonella, substantial ( approximately 3 log) but very short (second day post-infection) interstrain differences in bacterial loads were observed, accompanied by higher levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in the peritoneal cavities of I/St mice. I/St macrophages were more permissive for salmonella growth during the first 24 h following infection in vitro. Because the prominent differences in survival time did not correlate with permanent differences in bacterial multiplication, we suggest that both infections trigger fatal pathological processes whose dynamics depend strongly upon the host genetics.

[Experimental approaches to designing vaccines against tuberculous infection reactivation].

Pulmonary tuberculosis (TB) in adults is considered to be a reactivation disease that develops after a long period of latent infection. The need for vaccines against TB reactivation is urgent; however, such vaccines are not available yet. The authors have developed an experimental model of TB reactivation in inbred mice of TB-susceptible strain I/St and attempted to vaccinate these animals in the preinfection (prophylactic) and postinfection (therapeutic) manner against the reactivation. Therapeutic vaccination with BCG resulted in an exacerbation of the disease, presumably, due to Koch's phenomenon. Prophylactic vaccination with proteins of the Rpf family induced IFN-gamma production by CD4+ T cells and slightly decreased mycobacterial multiplication in the organs. However, neither the vaccination protocol prevented infection reactivation, suggesting that heterologous prime-boost approaches should be further investigated in our model.

A new coding mutation in the Tnf-alpha leader sequence in tuberculosis-sensitive I/St mice causes higher secretion levels of soluble TNF-alpha.

I/St and A/Sn mice are polar extremes in terms of several parameters defining sensitivity to Mycobacterium tuberculosis. TNF-alpha, mainly produced by activated macrophages, can mediate both physiological and pathophysiological processes. Adequate TNF-alpha levels are essential for a forceful protective response to M. tuberculosis. We have functionally characterized a nonsynonymous substitution, Arg8 His, in the highly conserved cytoplasmic domain of the pro-TNF-alpha leader peptide from extremely M. tuberculosis-sensitive I/St mice. This was compared to the common pro-TNF-alpha variant found in A/Sn mice. Using cDNA constructs, both variants were constitutively expressed in HEK293A cells. A significantly higher secretion level of Arg8 His TNF-alpha was shown using flow cytometry and ELISA analysis (P=0.0063), while intracellular levels were similar for both protein variants. An even TNF-alpha distribution throughout the cells was seen using confocal microscopy. This suggests that the Arg8 His substitution affects pro-TNF-alpha processing. The I/St mouse may serve as a model to further explore the function of the well-conserved cytoplasmic region of TNF-alpha. However, other identified substitutions in the I/St promoter, introns and 3'UTR of Tnf-alpha, as well as the cellular environment in vivo may affect the balance between soluble and intracellular Arg8 His TNF-alpha before and during M. tuberculosis infection.

Direct comparison of low-dose and Cornell-like models of chronic and reactivation tuberculosis in genetically susceptible I/St and resistant B6 mice.

We applied the low-dose challenge (chronic) and reactivation following chemotherapy withdrawal (Cornell-like) TB models to mouse strains with genetically different susceptibility to and severity of Mycobacterium tuberculosis-triggered disease. Systemic infection caused by intravenous (i.v.) administration of approximately 70 cfus of M. tuberculosis H37Rv lead to chronic, persistent, non-lethal disease in genetically resistant B6 mice, but resulted in a fatal pathological process in the lungs of susceptible I/St animals. Thus, application of the identical experimental approach to genetically different murine hosts allows investigating both slowly progressive disease with the fatal outcome (I/St) and chronic life-span disease (B6). Under Cornell-like model conditions, both temporary eradication of cultivable bacilli from lungs and spleens due to chemotherapy and their re-appearance in organs following its withdrawal were demonstrated in mice of both strains. However, (i) reactivation occurred significantly earlier in I/St than in B6 mice; (ii) I/St mice survived not more than 6 month following chemotherapy withdrawal and demonstrated 100% TB relapse, whereas in B6 mice mortality did not exceed 50%, and no mycobacteria were recovered from some animals. I/St mice, with their genetically determined high TB severity, provide a more reliable tool for modeling TB relapse after chemotherapy withdrawal than mice of more resistant strains.

[Genomics and gene engineering: rationale to the development of new means of tuberculosis control]. / Genomika i gennaia inzheneriia: ratsional'nye podkhody dlia razrabotki novykh sredstv bor'by s tuberkulezom.

The review contains information on modern approaches to the development of antituberculosis vaccines and remedies. Data on the comparative effectiveness of different subunit and DNA vaccines against tuberculosis are presented. The use of comparative and structural genomics for the search and characterization of new Mycobacterium tuberculosis genes, whose products may prove to be important antigens for the development of vaccines or target proteins for remedies against tuberculosis, is considered.