Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain

Purification and characterization of individual antigenic proteins are essential for the understanding of the pathogenic mechanisms of mycobacteria and the immune response against them. In the present study, we used anion-exchange chromatography to fractionate cell extracts and culture supernatant proteins from Mycobacterium bovis to identify T-cell-stimulating antigens. These fractions were incubated with peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle in lymphoproliferation assays. This procedure does not denature proteins and permits the testing of mixtures of potential antigens that could be later identified. We characterized protein fractions with high stimulation indices from both culture supernatants and cell extracts. Proteins were identified by two-dimensional gel electrophoresis followed by N-terminal sequencing or MALDI-TOF. Culture supernatant fractions containing low molecular weight proteins such as ESAT6 and CFP10 and other proteins (85B, MPB70), and the novel antigens TPX and TRB-B were associated with a high stimulation index. These results reinforce the concept that some low molecular weight proteins such as ESAT6 and CFP10 play an important role in immune responses. Also, Rv3747 and L7/L12 were identified in high stimulation index cell extract fractions. These data show that protein fractions with high lymphoproliferative activity for bovine PBMC can be characterized and antigens which have been already described and new protein antigens can also be identified in these fractions.

Humoral response to low molecular weight antigens of Mycobacterium tuberculosis by tuberculosis patients and contacts

Much effort has been devoted to the identification of immunologically important antigens of Mycobacterium tuberculosis and to the combination of target antigens to which antibodies from serum of tuberculous patients could react specifically. We searched for IgG antibodies specific for antigens of 45 to 6 kDa obtained after sonication of the well-characterized wild M. tuberculosis strain in order to detect differences in the antibody response to low molecular weight antigens from M. tuberculosis between patients with pulmonary tuberculosis and contacts. Specific IgG antibodies for these antigens were detected by Western blot analysis of 153 serum samples collected from 51 patients with confirmed pulmonary tuberculosis. Three samples were collected from each patient: before therapy, and after 2 and 6 months of treatment. We also analyzed 25 samples obtained from contacts, as well as 30 samples from healthy individuals with known tuberculin status, 50 samples from patients with other lung diseases and 200 samples from healthy blood donors. The positive predictive value for associated IgG reactivity against the 6-kDa and 16-kDa antigens, 6 and 38 kDa, and 16 and 38 kDa was 100% since simultaneous reactivity for these antigens was absent in healthy individuals and individuals with other lung diseases. This association was observed in 67% of the patients, but in only 8% of the contacts. The humoral response against antigens of 16 and 6 kDa seems to be important for the detection of latent tuberculosis since the associated reactivity to these antigens is mainly present in individuals with active disease.

Asynchronous expression of myeloid antigens in leukemic cells in a PML/RARalpha transgenic mouse model

Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor a (RARa) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARa fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARa develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARa TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 ± 16.68, 10.83 ± 8.11, 7.4 ± 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARa TM present a specific immunophenotype.

Responses of IgE, IgG1, and IgG4 to concanavalin A-binding Blomia tropicalis antigens in allergic patients

Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp) are the prevalent house dust mites in tropical countries and are associated with allergic diseases. Glycosylated antigens are highly immunogenic and involved in different pathologies. We evaluated the presence of IgE, IgG1, and IgG4 to concanavalin A-binding antigens (Bt-Con-A) isolated from Bt-total extract in sera of allergic and non-allergic subjects. Bt-total and Bt-Con-A extracts were evaluated by SDS-PAGE and ELISA for reacting with IgE, IgG1, and IgG4 in sera of 121 patients with allergic rhinitis and 36 non-allergic individuals. All subjects were skin prick tested with Bt-total extract and inhibition tests were performed for IgE, IgG1, and IgG4 using both extracts (Bt-total and Bt-Con-A). Skin prick test showed that 58% of the patients were sensitized to Bt (Bt+), with 52% reactive to both mites (Bt and Dp) and 6% to Bt only. A broad spectrum of proteins (14-152 kDa) was visualized in Bt-total and components >27 kDa for the Bt-Con-A extract. ELISA showed a similar profile of IgE, IgG1 and IgG4 levels in response to Bt-total and Bt-Con-A extracts in different groups, although Bt+ patients showed a lower IgG4 reactivity to Bt-Con-A extract. Specific IgG1 levels were higher in Bt+ patients than in control subjects, and IgG4 levels showed no significant difference among groups. ELISA inhibition showed a partial IgE and total IgG1 and IgG4 cross-reactivity with Dp extract for Bt-total and Bt-Con-A extracts. We conclude that Con-A-binding components isolated from Bt constitute major allergens and are involved in both allergen sensitization (IgE response) and homeostasis maintenance (IgG1 and IgG4 responses).

Interleukin-10-dependent down-regulation of interferon-gamma response to Leishmania by Mycobacterium leprae antigens during the clinical course of a coinfection

We have described a case of a patient with an intriguing association of mucocutaneous leishmaniasis with lepromatous leprosy, two opposite polar forms of these spectral diseases. In the present follow-up study, we investigated the effect of the addition of Mycobacterium leprae antigens on interferon-gamma (IFN-γ) production in Leishmania antigen-stimulated cultures of peripheral blood mononuclear cells (PBMC) from this patient. For this purpose, PBMC cultures were stimulated with crude L. braziliensis and/or M. leprae whole-cell antigen extracts or with concanavalin A. In some experiments, neutralizing anti-human interleukin (IL)-10 antibodies were added to the cultures. IFN-γ and IL-10 levels in culture supernatants were measured by ELISA. During active leprosy, M. leprae antigens induced 72.3% suppression of the IFN-γ response to L. braziliensis antigen, and this suppression was abolished by IL-10 neutralization. Interestingly, the suppressive effect of M. leprae antigen was lost after the cure of leprosy and the disappearance of this effect was accompanied by exacerbation of mucosal leishmaniasis. Considered together, these results provide evidence that the concomitant lepromatous leprosy induced an IL-10-mediated regulatory response that controlled the immunopathology of mucosal leishmaniasis, demonstrating that, in the context of this coinfection, the specific immune response to one pathogen can influence the immune response to the other pathogen and the clinical course of the disease caused by it. Our findings may contribute to a better understanding of the Leishmania/M. leprae coinfection and of the immunopathogenesis of mucosal leishmaniasis.

Recombinant vaccines and the development of new vaccine strategies

Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.