Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.

Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.

Crossprotection against nontuberculous mycobacterial infections by Mycobacterium tuberculosis memory immune T lymphocytes.

Adoptive immunization of T cell-deficient recipient mice with M. tuberculosis-specific memory immune T lymphocytes conferred upon these animals the ability to express significantly enhanced resistance both to the homologous infection, and to three strains of nontuberculous mycobacteria. These results support the hypothesis, therefore, that antigenic determinants possessed by the four mycobacterial strains that are relevant to the generation of protective cellular immunity are identical or closely crossreactive.

Protection against Mycobacterium tuberculosis infection by adoptive immunotherapy. Requirement for T cell-deficient recipients.

The results of this study demonstrate that spleen cells taken from mice at the height of the primary immune response to intravenous infection with Mycobacterium tuberculosis possess the capacity to transfer adoptive protection to M. tuberculosis-infected recipients, but only if these recipients are first rendered T cell-deficient, either by thymectomy and gamma irradiation, or by sublethal irradiation. A similar requirement was necessary to demonstrate the adoptive protection of the lungs after exposure to an acute aerosol-delivered M. tuberculosis infection. In both infectious models successful adoptive immunotherapy was shown to be mediated by T lymphocytes, which were acquired in the donor animals in response to the immunizing infection. It is proposed that the results of this study may serve as a basic model for the subsequent analysis of the nature of the T cell-mediated immune response to both systemic and aerogenic infections with M. tuberculosis.

Interleukin 12 (IL-12) is crucial to the development of protective immunity in mice intravenously infected with mycobacterium tuberculosis.

Immunity to Mycobacterium tuberculosis infection is associated with the emergence of protective CD4 T cells that secrete cytokines, resulting in activation of macrophages and the recruitment of monocytes to initiate granuloma formation. The cytokine-mediating macrophage activation is interferon-gamma (IFN-gamma), which is largely dependent on interleukin-12 (IL-12) for its induction. To address the role of IL-12 in immunity to tuberculosis, IL-12 p40(-/-) mice were infected with M. tuberculosis and their capacity to control bacterial growth and other characteristics of their immune response were determined. The IL-12 p40(-/-) mice were unable to control bacterial growth and this appeared to be linked to the absence of both innate and acquired sources of IFN-gamma. T cell activation as measured by delayed type hypersensitivity and lymphocyte accumulation at the site of infection were both markedly reduced in the IL-12 p40(-/-) mice. Therefore, IL-12 is essential to the generation of a protective immune response to M. tuberculosis, with its main functions being the induction of the expression of IFN-gamma and the activation of antigen-specific lymphocytes capable of creating a protective granuloma.

Disseminated tuberculosis in interferon gamma gene-disrupted mice.

The expression of protective immunity to Mycobacterium tuberculosis in mice is mediated by T lymphocytes that secrete cytokines. These molecules then mediate a variety of roles, including the activation of parasitized host macrophages, and the recruitment of other mononuclear phagocytes to the site of the infection in order to initiate granuloma formation. Among these cytokines, interferon gamma (IFN-gamma) is believed to play a key role is these events. In confirmation of this hypothesis, we show in this study that mice in which the IFN-gamma gene has been disrupted were unable to contain or control a normally sublethal dose of M. tuberculosis, delivered either intravenously or aerogenically. In such mice, a progressive and widespread tissue destruction and necrosis, associated with very high numbers of acid-fast bacilli, was observed. In contrast, despite the lack of protective immunity, some DTH-like reactivity could still be elicited. These data, therefore, indicate that although IFN-gamma may not be needed for DTH expression, it plays a pivotal and essential role in protective cellular immunity to tuberculosis infection.

Immunity to mycobacteria.

Recent progress in the field of immunity to mycobacteria has centered on T cell subset responses and the cytokines these cells secrete. In addition, there has been steady progress in identifying and characterizing several classes of major mycobacterial proteins; included amongst these are the secreted/export proteins of Mycobacterium tuberculosis, which several laboratories now believe may represent the key protective immunity-inducing antigens of the bacillus.

Prospects for new vaccines against tuberculosis.

The spectacular failure of the existing bacille Calmette-Guérin (BCG) vaccine in recent tuberculosis-control trials has prompted a search for potential replacements. Imaginative approaches currently under development include the modulation of BCG by the production of auxotrophic mutants or cytokine-secreting recombinants, and the development of subunit vaccines based on the major proteins of Mycobacterium tuberculosis.

Tuberculosis vaccine development: recent progress.

Recent years have seen a renewed effort to develop new vaccines against tuberculosis. As a result, several promising avenues of research have developed, including the production of recombinant vaccines, auxotrophic vaccines, DNA vaccines and subunit vaccines. In this article we briefly review this work, as well as consider the pros and cons of the animal models needed to test these new vaccines. Screening to date has been carried out in mouse and guinea pig models, which have been used to obtain basic information such as the effect of the vaccine on bacterial load, and whether the vaccine can prevent or reduce lung pathology. The results to date lead us to be optimistic that new candidate vaccines could soon be considered for evaluation in clinical trials.

The search for new vaccines against tuberculosis.

The failure of the BCG vaccine for tuberculosis in large, controlled clinical trials, coupled with the gradual consensus that it is mostly ineffective in preventing adult pulmonary disease in endemic areas, has led to a concerted effort to develop a new generation of vaccines. This work is ongoing in a variety of areas, including DNA vaccines, subunit vaccines, recombinant vaccines, and auxotrophic vaccines. Several such candidates are giving promising results in mouse and guinea pig, aerosol-challenge infection models and should move to clinical trials in the near future.

An age-related loss of the isogeneic barrier to the successful passive cell transfer of antimicrobial immunity in mice.

The results of this study show that an isogeneic barrier to the successful adoptive immunotherapy of Mycobacterium tuberculosis infected mice is progressively lost as these animals age. This was shown in this study by the demonstration that increasing levels of transferred acquired specific resistance could be conferred on normal old recipients of greater than 22 months of age, whereas in young recipients such transfers were unsuccessful without the prior exposure of recipients to sublethal levels of whole-body ionizing gamma irradiation. These data indicate that strategies of adoptive immunotherapy in the elderly may not have to overcome the age-related isogeneic physiological barrier to this procedure.

Changes in integrin/adhesion molecule expression, but not in the T-cell receptor repertoire, in old mice infected with tuberculosis.

The results of this study present data in support of the hypothesis that T lymphocytes in both young and old mice infected with virulent Mycobacterium tuberculosis undergo changes in expression of cell surface integrin/adhesion molecules as determined by flow cytometric analysis. These data thus further support the hypothesis that a reduced ability of T-cells in old mice to adequately and promptly accumulate at sites of inflammation induced by bacterial implantation is a central parameter underlying the increased susceptibility of these mice to this intracellular bacterial infection. In addition, however, no changes were observed in terms of the T-cell receptor expression (repertoire) of these animals, indicating that this facet of immunity is preserved in aging.

Similar responses by macrophages from young and old mice infected with Mycobacterium tuberculosis.

The growth of four isolates of Mycobacterium tuberculosis was compared in cultures of bone marrow-derived macrophages generated from young (3 months) and old (24 months) female C57BL/6 mice. In all four cases, no differences were seen in the course of the in vitro infection over a 10-day culture period. Macrophages from both young and old mice secreted similar levels of nitric oxide if treated with interferon gamma (IFN) 24 h prior to infection. Expression of mRNA encoding an array of early response genes in the two sets of cultures was also generally similar. These data indicate that the capacity of macrophages to respond to infection with a virulent intracellular bacterial infection does not seem to be influenced by the increasing age of the host.

Immunopathogenesis of Mycobacterium avium infection.

One of the most obvious problems one perceives when working with Mycobacterium avium isolates is the vast array of phenotypes expressed with regard to colonial morphotype, serovar and particularly virulence. Thus whenever experimental data derived from different MAC isolates is compared the variety of this group of mycobacteria must always be considered. Another issue of concern is the extrapolation of in vitro data to the in vivo disease. We have reported, in the past, that survival in murine macrophage culture does not always correlate with survival in vivo (23). It is plausible therefore, that the pathways outlined in section 5.2 and figure 3 play a crucial role in the initiation of the innate immune response in general and that there are components of this response which are not expressed by IFN-gamma activated macrophages but which are necessary for bacterial control. In conclusion, we suggest that the initial control of MAC infection requires a healthy lung (or gut) architecture and that control by unactivated macrophages includes respiratory burst activity and also the sequestration of free iron away from the mycobacterial phagosome. Acquired immunity is important in controlling bacteria which have overcome the innate response and this control is mediated by cytokine activation of infected macrophages. Finally, we have described an animal model of infection in which uncontrolled bacterial growth occurs and in which lesions similar to those seen in AIDS patients develop.

Immunology and vaccinology of tuberculosis: can lessons from the mouse be applied to the cow?

The nature of both innate and acquired immunity in the lungs are both still poorly understood, and how these two sets of mechanisms intersect with each other may have considerable bearing on the overall expression of host resistance. While the central role of CD4 T cells in the acquired phase is well established, cells bearing this marker may also contribute to innate immunity in the guise of both NK-positive and negative innate populations capable of secreting gamma interferon. In contrast, expansion of an antigen-specific memory T cell population is the purpose of current vaccine strategies, and several interesting and promising types of new candidates are briefly discussed.

The mouse as a useful model of tuberculosis.

Like other animal models of tuberculosis, the mouse has provided a large amount of information that can be applied to understanding the disease process in infected humans. The model is particularly useful in providing information about the immune response, given the huge database of reagents now available, including antibodies to lymphocyte markers and the growing number of available gene disrupted mice, and the model is validated by the fact that multiple mechanisms discovered in the mouse such as the TH1 pathway and the Toll-like receptor system are similarly important in humans. The model also has its limitations, particularly in terms of the immunopathologic response, in which similar elements occur but are expressed somewhat differently.

Dose of BCG does not influence the efficient generation of protective immunity in mice challenged with Mycobacterium tuberculosis.

It is generally agreed that BCG vaccination is relatively ineffective in adults exposed to tuberculosis infection. The reasons for this may well be multiple, and may include the possibility that higher doses of BCG may induce a mixed TH1 and TH2 response, which may lessen the protective effect of the vaccine. To test this hypothesis, mice were vaccinated with a range of doses of BCG and then challenged by the intravenous or aerogenic routes with virulent Mycobacterium tuberculosis. While the data support the hypothesis that a TH2 response is induced by higher doses of BCG, this was found to have no influence whatsoever on the capacity of the vaccinated mouse to express acquired specific resistance to the challenge infection.

Toll-like receptor 4 plays no role in susceptibility of mice to Mycobacterium tuberculosis infection.

Although various members of the pattern recognition Toll-like receptor (TLR) family have been implicated in host resistance to Mycobacterium tuberculosis infection, it remains unclear if the TLR4 receptor plays an important role. We demonstrate here that infection of TRL4-competent and TLR4-deficient mice on the C3H inbred mouse strain background had similar outcomes, measured in terms of the course of the disease, cell accumulation patterns in the lungs, and lung histopathology. These data argue against a significant role for TLR4 in immunity to tuberculosis in the mouse model.

Characterization of virulence, colony morphotype and the glycopeptidolipid of Mycobacterium avium strain 104.

SETTING: Members of the Mycobacterium avium complex (MAC) are responsible for mycobacterial disease in children, the aged and in immunocompromised individuals. The complex consists of different species, serovars and morphologic forms that vary in virulence. One isolate of the MAC is currently being sequenced (MAC 104) and was chosen based on its derivation from an AIDS patient and the fact that it could be genetically manipulated. OBJECTIVE: MAC 104 was therefore analyzed for virulence, colony morphotype and expression of the glycopeptidolipid (GPL) responsible for serotying differences and the rough to smooth morphological switch. RESULTS: The isolate was found to be virulent in the murine model of low-dose aerosol infection in that it could colonize the lung, proliferate within the tissue and disseminate to other organs. MAC 104 expressed a variety of colony morphotypes, the most prevalent of which were smooth opaque, smooth transparent and rough. All three morphotypes could persist in the lung; however, the transparent and rough morphotypes grew more rapidlyinvivo. The rough morphotype was unusual in that it expressed an atypical form of the GPL usually absent from rough morphotypes. CONCLUSION: This characterization complements the genome data and confirms that MAC 104 behaves similarly to other MAC isolates.

Activation marker expression on bovine peripheral blood gammadelta T cells during post-natal development and following vaccination with a commercial polyvalent viral vaccine.

Understanding the immunological function of bovine gammadelta T cells is essential for evaluating their role in the response to infectious agents and for determining the potential of targeting this population with vaccines. This study examined the age dependent changes of circulating CD2(+) and CD2(-) gammadelta T cells as well as differences in the expression of activation markers between these two populations. Changes in activation marker expression following vaccination with Vira Shield 5 are also discussed. CD62L was expressed on all CD2(-) gammadelta T cells but only a subset of CD2(+) gammadelta T cells and following vaccination there was a significant decrease in the percentage of CD2(-)/CD62L(+) gammadelta T cells but not CD2(+)/CD62L(+) gammadelta T cells. Both CD2(-) and CD2(+) gammadelta T cells consistently expressed high levels of CD44. The majority of both CD2(-) and CD2(+) gammadelta T cells also expressed CD45R, however, more of the CD2(-) cells were CD45R(neg/lo). Following vaccination there was a significant decrease in the percentage of CD2(-) and CD2(+) gammadelta T cells that expressed CD44 and CD45R. These data indicate significant differences in activation expression on CD2(-) and CD2(+) gammadelta T cells, which adds to the growing evidence that there may be functional as well as phenotypic differences between these two populations of bovine gammadelta T cells.

Tuberculosis in aged gammadelta T cell gene disrupted mice.

In young mice exposed to aerosol infection with Mycobacterium tuberculosis removal of the gammadelta T cell population by targeted gene disruption does not affect the expression of host resistance, but does influence the integrity of the early granulomatous response. The current study demonstrates that in aged gammadelta T cell gene disrupted mice similar immunopathologic changes ensued in both gene knockout and wild type control mice. Changes in cell surface marker expression, evident in other gene knockout models, was not observed in the aged gammadelta T cell knockout mice. These data imply that gammadelta T cell functions previously observed in young mice become much less important as the animal ages.