ovalbumin and Tuberculosis

The host immune response is generally sufficient to contain Mycobacterium tuberculosis infection. It does not, however, efficiently prevent subsequent infection with M. tuberculosis or provide sterilizing immunity. While the understanding of the immune response generated against this pathogen is incomplete, improvements have been achieved due to advances in immunological tools. In this study, we analyzed the multifunctional nature of primary and memory CD8 T-cell responses generated during murine M. tuberculosis infection. We generated a recombinant M. tuberculosis strain expressing ovalbumin (OVA) epitopes in order to expand the peptides for the detection of CD8 T cells during M. tuberculosis infection and enable us to use OVA-specific reagents. Our results indicate that the majority of M. tuberculosis-specific CD8 T cells are limited to either cytotoxicity or the secretion of gamma interferon (IFN-gamma), with cytotoxicity being far more prevalent than IFN-gamma secretion. Memory CD8 T cells responded earlier and reached higher levels in the lungs than naïve CD8 T cells, as was expected. They were, however, less cytotoxic and secreted less IFN-gamma than newly primed CD8 T cells, suggesting that one factor contributing to bacterial persistence and lack of sterilizing immunity may be the low quality of memory cells that are generated.

Topics: Animals; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Immunologic Memory; Interferon-gamma; Lung; Mice; Mycobacterium tuberculosis; Ovalbumin; T-Lymphocyte Subsets; Tuberculosis

Vaccine efficacy largely depends upon DC targeting and activation. The most potent TLR soluble ligands induce diffuse DC activation, which may be associated with marked pro-inflammatory responses and possibly adverse effects. This raises the concern that effective vaccine adjuvants may similarly rely on widespread DC activation. Using a promising candidate vaccine against tuberculosis (fusion protein of Ag85B and 6-kDa early secretory antigenic target (ESAT-6)) formulated in the potent IC31 adjuvant, DC targeting and activation was studied in vivo, following the fate of antigen and adjuvant in the draining lymph nodes, to define the magnitude of DC targeting/activation required in vivo to induce protective vaccine responses. Unexpectedly, protective IFN-gamma-mediated Ag85B-ESAT-6/IC31 responses were associated to the activation of a minute population (less than 0.3%) of CD11c(+) lymph node DC, without detectable systemic pro-inflammatory responses. This activated peripheral tissue-derived DC population, characterized by enhanced CD80, CD86, CD40 and IL-12p40 expression, was only identified when focusing on adjuvant- or antigen-labeled CD11c(+) DC, which were found to support T cell proliferation. Immunization with aluminum hydroxide adjuvant (Alum) resulted in a similar proportion of antigen-associated DC but without detectable enhancement of CD80, CD86, CD40 or IL-12p40 expression. Thus, potent protective IFN-gamma-producing responses may be elicited by the exquisite activation of a minute number of in vivo targeted DC.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigen Presentation; Antigens, Bacterial; Antigens, CD; Bacterial Proteins; CD11c Antigen; CD4-Positive T-Lymphocytes; Cell Proliferation; Dendritic Cells; Interferon-gamma; Interleukin-12 Subunit p40; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Oligodeoxyribonucleotides; Ovalbumin; Recombinant Fusion Proteins; Spleen; T-Lymphocytes; Tuberculosis; Vaccination

To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15(-/-)) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15(-/-) mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4(+) Th1 response capable of producing IFN-gamma was normally detected in spleen and lung of IL-15(-/-) mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-gamma and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15(-/-) mice. The degree of proliferation of Ag-specific CD8(+) T cells in IL-15(-/-) mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8(+) T cells significantly increased in IL-15(-/-) mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.

Topics: Animals; Antigens; Cell Division; Cell Movement; Cells, Cultured; Egg Proteins; Interleukin-15; Lung; Lymphocyte Count; Lymphocytes; Mice; Mice, Knockout; Mycobacterium bovis; Ovalbumin; Peptide Fragments; Spleen; Tuberculosis

During infection with lymphocytic choriomeningitis virus, CD8(+) T cells differentiate rapidly into effectors (CD62L(low)CD44(high)) that differentiate further into the central memory phenotype (CD62L(high)CD44(high)) gradually. To evaluate whether this CD8(+) T cell differentiation program operates in all infection models, we evaluated CD8(+) T cell differentiation during infection of mice with recombinant intracellular bacteria, Listeria monocytogenes (LM) and Mycobacterium bovis (BCG), expressing OVA. We report that CD8(+) T cells primed during infection with the attenuated pathogen BCG-OVA differentiated primarily into the central subset that correlated to reduced attrition of the primed cells subsequently. CD8(+) T cells induced by LM-OVA also differentiated into central phenotype cells first, but the cells rapidly converted into effectors in contrast to BCG-OVA. Memory CD8(+) T cells induced by both LM-OVA as well as BCG-OVA were functional in that they produced cytokines and proliferated extensively in response to antigenic stimulation after adoptive transfer. During LM-OVA infection, if CD8(+) T cells were guided to compete for access to APCs, then they received reduced stimulation that was associated with increased differentiation into the central subset and reduced attrition subsequently. Similar effect was observed when CD8(+) T cells encountered APCs selectively during the waning phase of LM-OVA infection. Taken together, our results indicate that the potency of the pathogen can influence the differentiation and fate of CD8(+) T cells enormously, and the extent of attrition of primed CD8(+) T cells correlates inversely to the early differentiation of CD8(+) T cells primarily into the central CD8(+) T cell subset.

Topics: Adoptive Transfer; Animals; Antigen-Presenting Cells; BCG Vaccine; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Survival; Cytokines; Egg Proteins; Female; Hyaluronan Receptors; Immunologic Memory; Immunophenotyping; Intracellular Fluid; L-Selectin; Listeriosis; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Peptide Fragments; T-Lymphocyte Subsets; Tuberculosis; Vaccines, Attenuated

Mice that were transgenic for a T-cell receptor (TCR) specific for ovalbumin peptide(323-339) (DO11.10) were able to survive an infection with Mycobacterium tuberculosis for approximately 80 days. This limited early control of infection was associated with gamma interferon production, inducible nitric oxide synthase expression within the lung, and an influx of clonotypic lymphocytes. The control of M. tuberculosis was lost in DO11.10 mice bred in a rag mutant background, demonstrating that the immune responsiveness was recombinase dependent and likely to be associated with the expression of an alternative alpha TCR by DO11.10 mice. A characterization of the antigen specificity in DO11.10 TCR transgenic mice demonstrated that the specificity was limited and dominated by the 26-kDa (Rv1411c) lipoprotein of M. tuberculosis. This study identifies this lipoprotein as an important and potent inducer of protective T cells within the lungs of mice infected with M. tuberculosis and therefore as a possible target for vaccination.

Topics: Animals; Antigens; CD4-Positive T-Lymphocytes; Cell Wall; Immunity, Cellular; Interferon-gamma; Lung; Mice; Mice, Transgenic; Mycobacterium tuberculosis; Ovalbumin; Receptors, Antigen, T-Cell; Tuberculosis

The use of cytokines during vaccination, particularly IL-15, is being considered due to the unique ability of IL-15 to enhance the proliferation of memory CD8(+) T cells. However, as homeostatic mechanisms limit excessive lymphocyte expansion, we addressed the consequences of this enhancement of T cell memory by IL-15. Infection of mice with either recombinant Mycobacterium bovis (BCG) expressing IL-15 (BCG-IL-15) or BCG and purified IL-15 resulted in an increased CD44, IL-2Rbeta expression and increased frequency of IFN-gamma-secreting CD8(+) T cells. Surprisingly, the enhancement of memory to concurrent infection by IL-15 exacerbated the attrition of pre-existing memory. Infection of mice with Listeria monocytogenes expressing OVA resulted in potent OVA(257-264)-specific CD8(+) T cell memory, and a challenge of these mice with either BCG-IL-15 or BCG and purified IL-15 resulted in an increased erosion of OVA(257-264)-specific CD8(+) T cell memory, relative to BCG. Enhancement in the erosion of OVA-specific CD8(+) T cell memory by BCG-IL-15 resulted in a consequently greater impairment in protection against a challenge with OVA-expressing tumor cells. We thus raise important questions regarding vaccinations that are aimed at maximizing T cell memory without considering the impact on pre-existing T cell memory.

Topics: Adjuvants, Immunologic; Animals; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Down-Regulation; Female; Immunity, Cellular; Immunization, Secondary; Immunologic Memory; Interleukin-15; Listeriosis; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mycobacterium bovis; Ovalbumin; Peptide Fragments; Plasmids; Tuberculosis; Up-Regulation

We evaluated CD8(+) T cell responses against the dominant CTL epitope, OVA(257-264), expressed by an acute (Listeria monocytogenes (LM) OVA) vs a chronic pathogen (Mycobacterium bovis bacillus Calmette-Guérin (BCG) OVA) to reveal the influence on CD8(+) T cell memory and consequent protection against a challenge with OVA-expressing tumor cells. Infection with lower doses of both pathogens resulted in stronger bacterial growth but weaker T cell memory indicating that memory correlates with pathogen dose but not with bacterial expansion. The CD8(+) T cell response induced by LM-OVA was helper T cell-independent and was characterized by a rapid effector response followed by a rapid, but massive, attrition. In contrast, BCG-OVA induced a delayed and weak response that was compensated for by a longer effector phase and reduced attrition. This response was partly dependent on CD4(+) T cells. CD8(+) T cell response induced by BCG-OVA, but not LM-OVA, was highly dependent on pathogen persistence to compensate for the weak initial CD8(+) T cell priming. Despite a stronger initial T cell response with LM-OVA, BCG-OVA provided more effective tumor (B16OVA) control at both local and distal sites due to the induction of a persistently activated acquired, and a more potent innate, immunity.

Topics: Animals; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Female; Immunologic Memory; Listeria monocytogenes; Listeriosis; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Neoplasms, Experimental; Ovalbumin; Peptide Fragments; Tuberculosis

Mice that had received adoptive transfer of DO11.10 TCR transgenic T cells polarized toward a Th1 or a Th2 phenotype were challenged with Ag-coated beads or with recombinant Mycobacterium tuberculosis expressing the OVA determinant. The resulting bead-induced pulmonary granulomas reflected the phenotype of the adoptively transferred T cells, with the Th2 cells promoting a fibrotic reaction. Mice receiving Th1 cells mounted an epitope-specific protective response to challenge with recombinant M. tuberculosis. Th2 recipients were characterized by enhanced weight loss and lung fibrosis during acute high-dose infection. The combination of TCR transgenic T cells and epitope-tagged mycobacteria provides a novel experimental model for investigation of the pathogenesis of tuberculosis.

Topics: Adoptive Transfer; Animals; Antigens; Cells, Cultured; Cytokines; Disease Models, Animal; Female; Granuloma, Respiratory Tract; Injections, Intravenous; Lymphocyte Transfusion; Mice; Mice, Inbred BALB C; Mice, Transgenic; Microspheres; Mycobacterium tuberculosis; Ovalbumin; Spleen; Th1 Cells; Th2 Cells; Tuberculosis

As the burden of infectious diseases becomes reduced in many countries, a remarkable increase in the incidence of allergies has occurred. The basis for the rise in atopic disorders as a correlate of the decline in infectious diseases has not been defined. In the present study, we tested experimentally whether prior systemic infection with Mycobacterium bovis bacillus Calmette Guérin (BCG) had any effect on ovalbumin (OVA) Al(OH)3 (alum)-induced immunoglobulin E (IgE) production, airway mucus production and eosinophilic inflammation. The data showed that allergen-specific IgE production and OVA-induced eosinophilia and goblet cell development were significantly inhibited by prior infection with BCG. Correspondingly, following immunization with OVA alum, BCG-infected mice exhibited significantly higher levels of allergen-driven interferon-gamma (IFN-gamma) production than the mice without infection. The ratio of IFN-gamma: interleukin (IL)-4 production was higher in OVA-sensitized mice with prior BCG infection than in those without infection. The abrogation of OVA-induced mucus production and pulmonary eosinophilia in BCG-infected mice correlated with significantly decreased IL-5 production and increased IFN-gamma and IL-12 production. These data provide direct evidence that intracellular bacterial infection (i.e. BCG) can inhibit antigen-specific IgE and airway reactivity induced by environmental allergen. Furthermore, the results suggest that changes in cytokine-producing patterns of T lymphocytes and other cells may be the mechanism by which infections influence allergies.

Topics: Allergens; Animals; Bronchi; Female; Goblet Cells; Immunoglobulin E; Immunoglobulin G; Interferon-gamma; Interleukin-12; Interleukin-4; Interleukin-5; Mice; Mice, Inbred C57BL; Mucus; Mycobacterium bovis; Ovalbumin; Pulmonary Eosinophilia; Rats; Rats, Sprague-Dawley; Tuberculosis

It has been proposed that the increase in prevalence and severity of atopic disorders inversely correlates with exposure to infectious diseases such as tuberculosis. We have investigated this issue by combining an intranasal Mycobacterium bovis-Bacillus Calmette-Guérin (BCG) infection with a murine model of allergen, (ovalbumin [OVA]) induced airway eosinophilia. BCG infection either 4 or 12 wk before allergen airway challenge resulted in a 90-95 and 60-70% reduction in eosinophilia within the lungs, respectively, compared to uninfected controls. The inhibition of airway eosinophilia correlated with a reduced level of IL-5 production by T cells from the lymph node draining the site of OVA challenge. Interestingly, BCG infection of the lung had no effect on IgG1 and IgE OVA-specific serum immunoglobulin or blood eosinophil levels. Furthermore, BCG-induced inhibition of airway eosinophilia was strongly reduced in interferon (IFN)-gamma receptor-deficient mice and could be partially reversed by intranasal IL-5 application. Intranasal BCG infections could also reduce the degree of lung eosinophilia and IL-5 produced by T cells after Nippostrongylus brasiliensis infection. Taken together, our data suggest that IFN-gamma produced during the T helper cell (Th)1 immune response against BCG suppresses the development of local inflammatory Th2 responses in the lung. Most importantly, this inhibition did not extend to the systemic immunoglobulin response against OVA. Our data support the view that mycobacterial infections have the potential to suppress the development of atopic disorders in humans.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Allergens; Animals; BCG Vaccine; Bronchial Hyperreactivity; Eosinophilia; Histocompatibility Antigens Class II; Immunoglobulin E; Immunoglobulin G; Interferon-gamma; Interleukin-5; Lung; Mice; Mice, Inbred C57BL; Nippostrongylus; Ovalbumin; Signal Transduction; Strongylida Infections; Th1 Cells; Th2 Cells; Tuberculosis

Cell-mediated immune responses are essential for protection against many intracellular pathogens. For Mycobacterium tuberculosis (MTB), protection requires the activity of T cells that recognize antigens presented in the context of both major histocompatibility complex (MHC) class II and I molecules. Since MHC class I presentation generally requires antigen to be localized to the cytoplasmic compartment of antigen-presenting cells, it remains unclear how pathogens that reside primarily within endocytic vesicles of infected macrophages, such as MTB, can elicit specific MHC class I-restricted T cells. A mechanism is described for virulent MTB that allows soluble antigens ordinarily unable to enter the cytoplasm, such as ovalbumin, to be presented through the MHC class I pathway to T cells. The mechanism is selective for MHC class I presentation, since MTB infection inhibited MHC class II presentation of ovalbumin. The MHC class I presentation requires the tubercle bacilli to be viable, and it is dependent upon the transporter associated with antigen processing (TAP), which translocates antigenic peptides from the cytoplasm into the endoplasmic reticulum. The process is mimicked by Listeria monocytogenes and soluble listeriolysin, a pore-forming hemolysin derived from it, suggesting that virulent MTB may have evolved a comparable mechanism that allows molecules in a vacuolar compartment to enter the cytoplasmic presentation pathway for the generation of protective MHC class I-restricted T cells.

Topics: Animals; Antigen-Presenting Cells; ATP-Binding Cassette Transporters; Cell Line; Escherichia coli; Hematopoietic Stem Cells; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Immunity, Cellular; Interleukin-2; Listeria monocytogenes; Macrophages; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Ovalbumin; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Tuberculosis

Malnutrition may be a predisposing host factor in the development of exogenous-reinfection tuberculosis. Outbred Hartley guinea pigs were given isocaloric diets containing either 30% ovalbumin (control animals) or 10% ovalbumin (low-protein-fed [LP] animals). Equal numbers of control and LP animals were assigned to one of three infection groups: (i) primary pulmonary infection with a low-virulence, streptomycin-resistant (LVsr) isolate of Mycobacterium tuberculosis and then reinfection 6 weeks later by the same route with a high-virulence (HV) isolate; (ii) only the primary infection (LVsr isolate); and (iii) only the secondary infection (HV isolate). Each infection resulted in the development of 4 to 12 pulmonary tubercles. Guinea pigs were skin tested with purified protein derivative and killed 6 weeks after the second infection. Protein deprivation suppressed the dermal responses to purified protein derivative in all infection groups. Primary infection of well-nourished animals with the LVsr isolate induced significant protection against infection with the HV isolate in the reinfected group, based upon the numbers of viable mycobacteria in the lung and spleen. Protein malnutrition did not exacerbate disease in the reinfected group beyond that observed in malnourished animals infected with the HV isolate only, but neither did the infection with the LVsr isolate protect the LP animals against reinfection with the HV isolate. We conclude that malnutrition interferes with the protection normally afforded by primary infection but does not result in more severe disease in reinfected individuals than would be observed in singly infected subjects.

Topics: Animals; Dietary Proteins; Female; Guinea Pigs; Mycobacterium tuberculosis; Ovalbumin; Protein Deficiency; Recurrence; Tuberculin; Tuberculosis; Virulence

Reaginic (IgE) antibodies are normally difficult to induce in laboratory rodents. The TB inbred mice constitute an exceptionally suitable model for the study of reaginic responses because of their ability to mount a strong and long lasting (over 4 months) IgE antibody response to hapten carrier conjugates, such as DNP3-OVO, administered in the presence of adjuvant. The maximum individual PCA titres determined for the sera of 8 animals after primary sensitization were in the range of 2,048 to 65,536 (geometric mean: 9,733) and after secondary sensitization the maximum individual PCA titres augmented to a level of 8,192 to 131,072 (geometric mean: 50,490).

Topics: Adjuvants, Immunologic; Administration, Oral; Animals; Antibody Formation; Dinitrobenzenes; Epitopes; Female; Immunologic Memory; Mice; Mice, Inbred Strains; Models, Biological; Ovalbumin; Passive Cutaneous Anaphylaxis; Reagins; Time Factors; Tuberculosis

Topics: Animals; Arthus Reaction; Coloring Agents; Exocrine Glands; Guinea Pigs; Inflammation; Injections; Lacrimal Apparatus; Mycobacterium bovis; Ovalbumin; Staining and Labeling; Staphylococcal Infections; Time Factors; Tuberculosis

Topics: Adult; Animals; Antibodies; Blood Proteins; Cattle; Erythrocytes; Hemagglutination Tests; Hot Temperature; Humans; Male; Ovalbumin; Serum Albumin; Sheep; Streptomycin; Tannins; Tuberculosis

Topics: Animals; Carbon; Connective Tissue; Electrons; Histiocytes; Lymph Nodes; Macrophages; Mice; Microscopy; Microscopy, Electron; Mononuclear Phagocyte System; Ovalbumin; Pharmacology; Rabbits; Research; Salmonella Infections; Salmonella Infections, Animal; Salmonella typhimurium; Spleen; Tuberculosis; Tuberculosis, Cutaneous; Typhoid-Paratyphoid Vaccines

Topics: Antibody Formation; Egg White; Mycobacterium; Mycobacterium tuberculosis; Ovalbumin; Tuberculosis