A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein.

Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection

Production & characterization of monoclonal antibodies to Mycobacterium tuberculosis

Background & objectives: Monoclonal antibodies (MAbs) against Mycobacterium tuberculosis H37Rv culture filtrate (CF) were raised by immunizing BALB/c mice and characterization was done. Attempts have been directed towards identifying mycobacterial antigens in biological fluids by employing polyclonal and monoclonal antibodies specific for M. tuberculosis. Immunohistologic studies, using MAbs for the localization of whole or fragmented bacilli in the biopsy specimens were also carried out. Methods: Intrasplenic IS and intraperitoneal IP routes of immunization, were compared. The MAbs were characterized for their isotype, binding specificity, nature of binding epitope, reactivity in immunoassays etc. Results: IS and IF’ routes of immunization, were compared and IP was found superior. Ten MAbs designated TRC l-10 were produced. Of these, 7 MAbs, TRC 1-7 reacted with the 30/31 kDa doublet (antigen 85 complex), TRC 8 with 12 kDa in addition to 30/31 kDa and TRC 9 and 10 with the 24 and 12 kDa antigens respectively. Six MAbs were classified as broadly cross reactive and 2 showed limited cross reactivity. TRC 8 and 10 showed species specificity. Employing TRC 8 in sandwich ELISA, antigen was detected in sera from 17 of 25 pulmonary tuberculosis patients and 3 of 20 controls. TRC 8 was found to be useful in detecting antigens specifically in M. tuberculosis and M. leprae infected tissues, by immunoperoxidase staining. Interpretation & conclusion: TRC 8 was found to be restricted in its reactivity to M. tuberculosis complex and M, leprae. TRC 8 may prove useful in immuno-diagnosis of tuberculosis

A Systematic Review of Immunological Studies of Erythema Nodosum Leprosum.

Erythema nodosum leprosum (ENL) is a painful inflammatory complication of leprosy occurring in 50% of lepromatous leprosy patients and 5-10% of borderline lepromatous patients. It is a significant cause of economic hardship, morbidity and mortality in leprosy patients. Our understanding of the causes of ENL is limited. We performed a systematic review of the published literature and critically evaluated the evidence for the role of neutrophils, immune complexes (ICs), T-cells, cytokines, and other immunological factors that could contribute to the development of ENL. Searches of the literature were performed in PubMed. Studies, independent of published date, using samples from patients with ENL were included. The search revealed more than 20,000 articles of which 146 eligible studies were included in this systematic review. The studies demonstrate that ENL may be associated with a neutrophilic infiltrate, but it is not clear whether it is an IC-mediated process or that the presence of ICs is an epiphenomenon. Increased levels of tumor necrosis factor-α and other pro-inflammatory cytokines support the role of this cytokine in the inflammatory phase of ENL but not necessarily the initiation. T-cell subsets appear to be important in ENL since multiple studies report an increased CD4+/CD8+ ratio in both skin and peripheral blood of patients with ENL. Microarray data have identified new molecules and whole pathophysiological pathways associated with ENL and provides new insights into the pathogenesis of ENL. Studies of ENL are often difficult to compare due to a lack of case definitions, treatment status, and timing of sampling as well as the use of different laboratory techniques. A standardized approach to some of these issues would be useful. ENL appears to be a complex interaction of various aspects of the immune system. Rigorous clinical descriptions of well-defined cohorts of patients and a systems biology approach using available technologies such as genomics, epigenomics, transcriptomics, and proteomics could yield greater understanding of the condition

Isolation and Evaluation of Diagnostic Value of Two Major Secreted Proteins of Mycobacterium Tuberculosis

Two secreted antigens of Mycobacterium tuberculosis, namely the antigen 85 complex (30/31) and 38kDa antigens, were purified from the whole culture filtrate by using two dimensional preparative electrophoresis and anion exchange chromatography, respectively. Individual components of the antigen 85 complex namely, antigen 85A, 85B and 85C, were separated using hydrophobic interaction chromatography. The humoral antibody activity to these antigens in sputum positive cases of active pulmonary tuberculosis and normal healthy volunteers was determined by enzyme linked immunosorbent assay (ELISA) and immunoblot. Recombinant 38kDa and antigen 6 were used as reference antigens for the assay. None of the healthy volunteers reacted with the 38kDa antigen, while 52% of the TB sera reacted with it. Of the three components of the antigen 85 complex, 85B gave the highest positivity of 40 per cent. The results of combination of 38kDa with antigen 6 offered better results with 76% positivity

Revealing the molecular signatures of host-pathogen interactions.

Advances in sequencing technology and genome-wide association studies are now revealing the complex interactions between hosts and pathogen through genomic variation signatures, which arise from evolutionary co-existence

Lipoproteins of Mycobacterium tuberculosis : an abundant and functionally diverse class of cell envelope components

Mycobacterium tuberculosis remains the predominant bacterial scourge of mankind. Understanding of its biology and pathogenicity has been greatly advanced by the determination of whole genome sequences for this organism. Bacterial lipoproteins are a functionally diverse class of membrane-anchored proteins. The signal peptides of these proteins direct their export and post-translational lipid modification. These signal peptides are amenable to bioinformatic analysis, allowing the lipoproteins encoded in whole genomes to be catalogued. This review applies bioinformatic methods to the identification and functional characterisation of the lipoproteins encoded in the M. tuberculosis genomes. Ninety nine putative lipoproteins were identified and so this family of proteins represents ca. 2.5% of the M. tuberculosis predicted proteome. Thus, lipoproteins represent an important class of cell envelope proteins that may contribute to the virulence of this major pathogen

Molecular biology and immunology of the 60-kDa stress protein (65-kDa antigen) of Mycobacterium paratuberculosis

M. paratuberculosis infection in sheep causes a chronic granulomatous enteritis, characterised by a massive cellular infiltration of macrophages, epithelioid cells and lymphocytes. Like other mycobacterial infections the predominant immune response is cell-mediated and this response centres on a complex interaction between T cells and macrophages infected with M. paratuberculosis organisms. T cells have been identified as the predominant inducers of the cell mediated immune response to mycobacteria. Stress proteins have been shown to be immunodominant antigens in immune responses to other pathogenic mycobacteria. T cells reactive to stress proteins have been isolated from infected animals. For these reasons, the 60-kDa stress protein of M. paratuberculosis was chosen for the investigation of the immune response to M. paratuberculosis infection in sheep.In this thesis the DNA encoding the 60-kDa stress protein of M. paratuberculosis was amplified by PCR, sequenced and the ORF of this protein expressed as a fusion protein with glutathione S-transferase. Recombinant M. paratuberculosis 60-kDa stress protein was obtained by cleavage of the fusion protein with the proteolytic enzyme thrombin.Recombinant M. paratuberculosis 60-kDa stress protein was used to generate polyclonal T and B cell responses in sheep which were assessed by in vitro proliferation and enzyme-linked immunosorbent assays, respectively. In addition, the recombinant protein was used to generate a monoclonal antibody. The assays and reagents generated in this thesis were subsequently used to investigate the immune response to M. paratuberculosis 60-kDa stress protein in ovine paratuberculosis.This investigation provides preliminary evidence that paratuberculosis-infected animals have T cells that recognise and are capable of responding by proliferation to M. paratuberculosis 60-kDa stress protein in vitro, and that antibodies to this protein could be detected in sera from these animals. Furthermore, M. paratuberculosis 60-kDa stress protein was detected in tissues from infected animals by immunocytochemical analysis. These preliminary findings support a role for M. paratuberculosis 60-kDa stress protein in the ovine immune response to M. paratuberculosis infection

Studies of immune response in human tuberculosis

TB (tuberculosis), caused by Mycobacterium tuberculosis (Mtb), continues to be a world-leading killer and a serious global health problem primarily affecting poor people in many developing countries. The difficult situation with TB/HIV co-infection is also a major key challenge to public health. Despite recent advances in TB research, the host- and pathogen-specific factors that lead to protective immunity, particularly in humans, remain unclear. While it is well-established that cell-mediated immunity is required to control TB infection, the role of humoral immunity including Th2 immune responses is debated. This thesis aimed to explore immune responses in human TB and the immunopathogenic mechanisms involved in the progression of clinical TB in both HIV-negative and HIV-positive individuals. To address the aims of this thesis work, well-defined study cohorts of active TB patients were obtained in close collaboration with the Black Lion University Hospital in Addis Ababa, Ethiopia. We used a novel immunodiagnostic test, Antibodies in Lymphocyte Supernatants (ALS), to demonstrate that IgG-secreting plasmablasts were significantly higher in the peripheral circulation of patients with active TB compared to latent TB cases and non-TB controls. Interestingly, BCG-specific IgG titers were particularly high in blood samples from TB/HIV co-infected patients with CD4 T cell counts <200 cells/ml who produced low levels of Mtb-specific IFN-γ in vitro. A technological platform including quantitative assessments of mRNA and protein expression in tissue (lymph nodes), fluids (bronchoalveolar lavage (BAL) and pleura fluid) and peripheral blood, was also used to investigate antimicrobial effector pathways and adverse immune responses in unique clinical samples obtained from the local site of Mtb infection. The results from these studies revealed that TB disease was associated with extensive tissue remodelling including an altered cellular composition, collagen deposition and granuloma formation. Here, the degree of necrotic granuloma formation was particularly prominent in TB/HIV-co-infected patients. Despite granuloma enrichment of activated CD68+ macrophages containing Mtb-antigens, mRNA levels of IFN-γ, TNF-α and IL-17 remained low in Mtb-infected lymph nodes. Accordingly, CD8+ T cells expressing cytolytic and antimicrobial effector molecules perforin and granulysin were low inside the TB lesions, while CD4+FoxP3+ regulatory T cells (Treg) and Th2/Treg cell cytokines IL-13 and TGF-β were up-regulated in the Mtb-infected tissues. The observed shift of the immune response from a Th1/Th17 towards an immunoregulatory phenotype was supported by our finding of a Th2 polarized response in the lung of patients with pulmonary TB. Here, multiplex protein analysis of BAL and plasma samples demonstrated low levels of Th1/Th17 cytokines and the T cell-chemoattractant CCL5, but significantly up-regulated levels of pro-inflammatory cytokines and the Th2 cytokine IL-4. The enhanced Th2 response was associated with increased levels of CCL4, suppressors of cytokine signaling-3 (SOCS3) and mycobacteria-specific IgG in BAL fluid from patients with active pulmonary TB. Contrary, IL-4, CCL4, SOCS3 and IgG-responses remained low in patients with less severe extrapulmonary pleural TB disease, who demonstrated up-regulated levels of both IFN-γ and CCL5. Taken together, our results provide evidence that human TB is associated with impaired Th1 immunity but elevated Th2/immunoregulatory responses and induction of antibody-mediated immunity. Importantly, enhanced Th2 and/or plasmablast responses may be used as relevant biomarkers or immune response signatures of active progressive TB disease that could be explored as potential targets for clinical TB management in the future

Effects of Mycobacterium vaccae NCTC 11659 (standard or recombinant) on cytokine production by human and murine cells

Killed M. vaccae is in clinical trials as an immunotherapeutic agent and adjuvant, but understanding of its mode of action is incomplete. To test its potential as a recombinant carrier organism and the nature of the responses evoked, recombinant strains were generated that expressed p27 of SFV. In mice these primed for specific production of IFNγ in the presence of p27, and induced serum IgG2a and, at higher doses, IgG1 responses to M. vaccae sonicate. Flow cytometry for intracellular cytokines after a single subcutaneous injection of 109 M. vaccae revealed accumulation of IFNγ-secreting CD8+ cells in lymph nodes. This finding, together with data generated simultaneously by another research group, implied an unusual adjuvant effect, not shared by other killed mycobacteria. In order to investigate this adjuvanticity the effects of killed M. vaccae on cytokine release from the human THP-1 monocyte line were investigated. M. vaccae, BCG and soluble bacterial preparations were all able to induce IL-12, IL-10 and TNFγ production in vitro to varying extents. Dose of mycobacterium and the addition of IFNγ influenced the balance of cytokines. Attempts to define active components in the IL-12 induction system were not successful, but it was noted that M. vaccae differed strikingly from the other killed mycobacteria in that its induction of IL-12 was greatly enhanced by exposure to lysozyme. The induction of IL-12 proved sufficiently reproducible to be used as a potency assay for material manufactured for clinical use. In conclusion, M. vaccae has been shown to be a potent Th1 inducer at appropriate doses, with the added ability to induce expansion of the CD8+ IFNγ+ population, perhaps via IL-12 release following exposure to lysozyme in vivo. CD8+ cells are strongly implicated in the clinical situations where M. vaccae has shown benefit

Host Responses to the Phenolic-Glycolipid-1 Antigen of Mycobacterium Leprae (Elisa, Leprosy, Epidemiology, Armadillo, Human).

Antibody responses to the apparently species specific phenolic-glycolipid-1 (Phen-Gl-1) antigen of Mycobacterium leprae were examined in humans and armadillos using enzyme-linked immunosorbent assays (ELISA). Statistical definitions for the interpretation of positive and negative reactions were derived. A retrospective serological survey of armadillos indicated that leprosy in the wild armadillo is a naturally acquired zoonosis. Presently 12.5% of the armadillos in 2 parishes in south central Louisiana have detectable IgM antibodies to Phen-Gl-1. Approximately 2.7% of these histologically exhibit clinical disease. Antibodies were not detected in Florida armadillo sera. Variations in prevalence rates were noted, and may be due to environmental conditions, population characteristics or some intricacies in the transmission of leprosy. Naturally acquired leprosy in the armadillo may be used as a model to study transmission and baseline data have been derived. The ELISA was shown to have application in the management of experimental leprosy infections in armadillos. Resistant armadillos were noted to have an irregular or absent antibody response to the Phen-Gl-1 antigen over the course of an experimental infection. Armadillos infected in the wild also had an irregular IgM response. Susceptible armadillos appeared to have a long-term IgM antibody response to Phen-Gl-1 becoming detectable some 186 days post-experimental infection. This antibody remained detectable for up to 1140 days post-infection. Antibody responses of susceptible armadillos correlated with the harvestable load of M. leprae in liver tissues and ELISA absorbances successfully predicted a harvest result 97% of the time. IgM antibodies to Phen-Gl-1 were earlier and more reliable than other indicators of infection previously applied. IgM, IgA, and IgG antibodies to Phen-Gl-1 were detected in leprosy patients and contacts. IgM appeared to be the predominate isotype detectable. Human patients showed no significant correlation of antibody relative their clinical status. IgM antibodies to Phen-Gl-1 were depressed as a result of therapy with thalidomide. Monitoring Phen-Gl-1 antibodies in human patients is not predictive of patient status or reaction and does not seem indicated for clinical management