Tuberculosis is caused by infection with Mycobacterium tuberculosis, a bacillus bacterium. It is spread by aerosol droplets and causes irreversible lung destruction. If it escapes the lung it may cause systemic disease affecting many organs including bones, joints, liver, spleen, gastrointestinal tract and brain. 50% of people exposed to M. tuberculosis are infected with the bacteria and 15% of those infected develop disease. Poverty, malnutrition and overpopulation contribute dramatically to the perseverance and wild spread of tuberculosis.
Past means of controlling TB have involved the use of combinations of antibiotics. Recently, because of complications due to multidrug-resistant strains, the number and combination of antibiotics administered must be individually tailored depending on the strain the patient is harboring. In extreme cases, surgical removal of the infected portion of the lung is required.
A new effective control strategy that is currently being implemented throughout the world is DOTS (Directly Observed Treatment, Short-course). This strategy has five main elements:
WHO Global Tuberculosis Programme
Current TB epidemic information
Initial growth of M. tuberculosis results in a delayed type hypersensitivity response which is characterized by the formation of small necrotic lesions with solid caseous centers. M. tuberculosis multiplication is probably restricted while encapsulated in these granulomatus lesions. Granulomas (lesions) consist of T lymphocytes and mononuclear phagocytes of different levels of maturation and activation. After initiation of delayed-type hypersensitivity (DTH) and tubercle formation, activation of macrophages by CD4+ TDTH cells enable the macrophages to destroy bacilli within the tubercle. Macrophage activation appears to be a central step of acquired resistance against M. tuberculosis.
Macrophage activation is achieved by T lymphocytes which are the principle mediator of cell mediated immune response against M. tuberculosis. CD4+ T cells are primarily helper T cells which secrete interleukins involved in the activation of macrophages. In the DTH response to M. tuberculosis, CD4+ T cells predominate over CD8+ T cells which are primarily cytolytic cells that lyse specific target cells.
Helper T cells are required to recruit and activate new monocytes and macrophages to the tubercle (lesion). CD4+ T cells are divided into TH1 and TH2 subsets depending on the type of cytokines produced. TH1 cells produce the cytokines interferon-gamma (IFN-gamma) and interleukin (IL-2), which are important for activation of antimycobacterial activities and essential for the DTH response. IFN-gamma specifically activates macrophages and stimulates them to ingest and kill mycobacteria more effectively.
Macrophage activation by cytokines provides only a partial explanation for immunity to M. tuberculosis. The presence of an MHC Class I-restricted response to mycobacterial infection has been shown in several T lymphocytes. CD8+ T lymphocytes contribute to macrophage activation by producing IFN-gamma. CD8+ T cells also may have cytolytic functions which enable them to recognize mycobacterial antigens presented by MHC Class I molecules on the surface of infected macrophages. CD8+ cytotoxic T lymphocytes (CTLs) are thought to be required to release intracellular M. tuberculosis residing in infected macrophages.
CTLs with specificity for mycobacterial antigens have been identified in the murine model of tuberculosis. This has shown that exogenous antigen can gain access to the class I processing and presentation pathway, and elicit a CD8+ T cell response in vivo. M. tuberculosis has the ability to survive within macrophages, providing metabolic antigens for processing and presentation with Class I molecules on the macrophage surface. It has been suggested that mycobacteria may be able to avoid processing within the phagolysosomal environment and in doing so antigen may enter the endogenous antigen-processing pathway and be presented to CD8+ T cells. (Turner & Dockrell, 1996)
It has been proposed that CD4+ T cells may also play a role in host defense against M. tuberculosis infection via cytolytic activity. CD4+ T cells may also be capable of lysing non-activated, infected macrophages and subsequently be taken up by activated macrophages which can destroy the mycobacteria. The presence of CD4+ T cells has been detected only in virtro human peripheral blood samples. (Mutis, Cornelisse, & Ottenhoff, 1993)
gamma/delta T cells are a minor population of T lymphocytes whose role in immune protection against M. tuberculosis remains unclear. They might have a role in the initial, innate immune response to M. tuberculosis because their population is expanded by mycobacteria and mycobacterial products in tissues. After subcutaneous and aerosol immunization of mice with mycobacteria, a significant proportion of the mycobacteria-reactive gamma/delta T cell population was stimulated. gamma/delta T cells also secrete IFN-gamma which may be an important trigger at initial stages of immune response.
Natural killer (NK) cells may also play an important part of the host response to M. tuberculosis. NK cells are capable of lysing host cells infected with mycobacterial pathogens indicating functional similarities with specific cytolytic T lymphocytes.
The cytokine interleukin-10 (IL-10), which is generally secreted by TH2 cells, affects macrophages by suppressing cytokine production, and thereby downregulating TH1 cell activity and proliferation. One study suggests that IL-10 plays a central role in enhancing the survival of M. tuberculosis within macrophages. As a result, it is imperative to avoid stimulation of IL-10 production when designing a vaccine against TB.
It has been shown in a variety of studies that both cytolytic T lymphocytes and IFN-g activated macrophages are necessary for conferring protective immunity against M. tuberculosis. As a result these are the immunological pathways that must be focused on in developing an efficacious vaccine to tuberculosis. The TH1 pathway of the immune response including CTL activation is best stimulated by antigens that have been synthesized and degraded inside the cell and presented on the cell surface with MHC Class I.
IFN-g is an important cytokine for immune protection, but in excess, it is also a key mediator of immunopathology. Massive activation of macrophages within tubercles by IFN-gamma results in concentrated release of lytic enzymes. These enzymes destroy neighboring healthy cells and create circular regions of necrotic tissue. CTL activity also causes tissue damage suffered as a result of M. tuberculosis infection. Care must be taken to ensure that vaccination induces protective immunity without damaging effects.
BCG vaccine is varied in its protective efficacy in different areas of the world. Average results polled from a range of studies indicate an overall reduction of risk of TB by 50%, with individual studies showing a range of efficacy from 0% to 80%. Variations throughout the world among strain and preparation of vaccines, exposure of recipients to environmental mycobacteria, strain of M. tuberculosis, and trial population are factors that may be responsible for the variability in the efficacy of the BCG vaccine. BCG seems to be less effective in populations exposed to environmental mycobacteria. (Fine, 1989)
The cytolytic response to purified protein antigens has been shown to be lower than to live BCG. It has been demonstrated that non-stimulated peripheral blood mononuclear cells (PBMCs) exhibit little or no cytotoxic activity towards target cells that have been loaded with soluble antigen purified protein derivatives (PPD) of M. tuberculosis. However, following stimulation of live M. bovis (BCG), PBMCs showed CTL activity towards several mycobacterial antigens. Such results indicate that live attenuated vaccines may be more effective than purified protein vaccines. (Turner & Dockrell, 1996)
The presence of a CD8+ T cell response demonstrates that antigens from mycobacteria can reach the MHC Class I processing and presentation pathway and be presented to Class I restricted cells. When a heat-killed preparation of M. tuberculosis was used, lower levels of MHC Class I antigen presentation were observed. It is thought that CD8+ T cell activation produced with live M. bovis BCG stimulation is altered if the mycobacteria is delivered in a non-viable form. A decrease in the degree of CD8+ T cell activation was seen when dead BCG was used. (Turner & Dockrell, 1996)
Mice inoculated with live attenuated M. tuberculosis were able to generate a protective immune response in adoptive transfer experiments, whereas dead M. bovis could not elicit immunity. This suggests that a live mycobacterial infection, with the generation of T-cell populations that respond to secreted antigens, is necessary for the induction of a protective immune response to M. tuberculosis. This may be due to activation of specific T cell populations by different antigens or by their presentation within a different pathway.
Live attenuated M. bovis BCG injection has been shown to activate CD4+ and CD8+ T cells in BCG vaccinated humans. Vaccination with a non-viable preparation of M. bovis BCG is comparable in CD4+ T cell activation, but it activates fewer CD8+ T cells. The TC cells behave cytotoxically towards both live and dead M. bovis BCG-pulsed macrophage targets, and to a lesser extent, PPD. These evidences suggest an important role for CD8+ T in protective immunity against M. tuberculosis infection. (Turner & Dockrell, 1996)
BCG Trials
Alternatively knocking out the genes in M. tuberculosis required for virulence or prolonged survival within macrophages is a strategy being used to produce a live attenuated M. tuberculosis vaccine. The development of a shuttle mutagenesis strategy to reproducibly obtain recombinants by allelic exchange in M. tuberculosis has recently been developed, enabling M. tuberculosis recombinants to be made. (Balasubramanian, et al., 1996)
Safety of the BCG vaccine is a growing concern due to the levels of HIV infection among recipients of the vaccine. In order to avoid potential adverse effects of BCG within immunocompromised individuals, BCG auxotrophs have been developed using the previously mentioned technique. Auxotrophs are mutants that require a specific nutrient or metabolite that is not required by the wild type. As a result, such mutants can only survive for a short period of time within a host, if the host lacks the specified nutrient. Five such strains were tested in mice with severe combined immunodeficiency disease (SCID) for safety, and in a susceptible strain of mice for protection. Results have shown that these stains are safe in SCID mice, and demonstrate the same amount of protective immunity as normal BCG in susceptible mice, suggesting that this could be a safer method of vaccination. (Guleria et al., 1996)
Mycobacterium tuberculosis is known to express numerous proteins, some of which are:
ESAT-6
The 10 kDa, 30 kDa, 38 kDa, and 65 kDa stress proteins were used as recombinant antigens and their capacity to elicit a human T cell proliferative response was studied. These four antigens were similar in inducing a protective Th1 response, thereby suggesting that none of them are immunodominant. Since human T cells were shown to be capable of responding to more than one mycobacterial antigen, then one approach for developing an effective vaccine is to incorporate multiple antigens. (Bloom, 1996)
ESAT-6 is a secreted protein of major importance. A mouse model was infected with Mycobacterium tuberculosis, treated and reinfected. Upon reinfection, this mouse elicited strong T cell responses to ESAT-6, therefore making this protein relevant as a means of providing protective immunity. ESAT-6 has also been demonstrated to be present only in M. tuberculosis and M. bovis. This protein could be of great value as a diagnostic reagent because the tuberculin purified protein derivative (PPD) is limited diagnostically because of its inability to differentiate between M. tuberculosis infection and sensitization after BCG vaccination. (Harboe, 1996)
The 30 kDa protein, also referred to as antigen 85 (Ag85), is comprised of three related protein components, 85A, 85B, and 85C. Ag85 is produced by Mycobacterium tuberculosis in abundance and can grow in human mononuclear phagocytes, as well as in broth culture. It has been shown that it provides immunogenicity in guinea pig models infected with tuberculosis. The importance and relevance of this protein is probably due to its involvement in the mycobacterium's cell wall synthesis. It has also been demonstrated that when human monocytes are infected with M. tuberculosis, they secrete the 30 kDa complex. This would be advantageous in developing a subunit vaccine, because the release of this protein is of significance for intracellular processing. (Horwitz, 1996)
The MPT64 is a classical protein, which is distinct and readily detectable not only in Mycobacterium tuberculosis, but also in some strains of Mycobacterium bovis BCG, and Mycobacterium bovis. A study was conducted by Haslov et al. demonstrating its possibility of use as a diagnostic skin test. (Andersen, 1995)
Even though these antigens have been demonstrated to be potential target molecules in animal models, whether or not they are apt to develop protective immunity in humans remains questionable.
Lipoarabinomannoside (LAM) is a long chain of polyarabinose capped with mannose which extends through the lipid layers. LAM is known to have many biological effects on host cells, especially macrophages. It is possible that LAM may act as a virulence factor. A somewhat different approach is to attempt developing a polysaccharide vaccine.
EpiMer and EpiMatrix is an approach that has recently been developed and will probably prove to be useful in the future for identifying T cell epitopes. EpiMer is an algorithm that was developed by the Brown TB/HIV Research Laboratory that searches and locates regions on a protein for clusters of MHC binding motifs. Specificity of binding is achieved because the MHC binding motifs describe anchor residues on the protein that are essential in binding to the MHC peptide binding groove. The prominence of EpiMer is its ability to predict universal or promiscuous epitopes which could be of significant value in developing subunit vaccines. (DeGroot, 1996)
EpiMatrix, also recently developed by the Brown TB/HIV Research Laboratory, is a computer driven algorithm. The presence of certain amino acids along a peptide may either aid or inhibit binding to the MHC. Using peptides that have previously been demonstrated to bind to MHC molecules will allow the determination of the relative occurrence of an amino acid at binding positions. Therefore, EpiMatrix compiles the information on the peptides that have shown to bind MHC molecules.
The choice of antigen for use in a DNA vaccine is primarily limited by the immunogenicity of the protein. Although the immunogenicity of many M. tuberculosis antigens has not yet been characterized, two candidate antigens currently being tested are heat shock protein 65 (hsp65) and antigen 85 (Ag85). Heat shock proteins are known antigens recognized most frequently in the immune responses to intracellular pathogens, including mycobacteria. (Silva, 1995) Ag85 is a protein complex composed of three proteins: Ag85A, Ag85B and Ag85C. The Ag85 complex belongs to a group of proteins secreted actively by dividing mycobacteria known to stimulate early and strong cellular immune responses in humans and mice infected with M. tuberculosis. Although a successful tuberculosis DNA vaccine will probably code for several antigens, preliminary experiments are being done with one antigen at a time. In fact, researchers are now applying the concept of DNA vaccines as a technique whereby expression libraries are used as vaccine material in mice in order to discover the best antigens for use in a tuberculosis vaccine.
One DNA vaccine experiment reporting positive results used the hsp65 gene into two vectors: one carrying the cytomegalovirus immediate-early gene promoter and the other carrying the p-hydroxymethylgluteral CoA reductase promoter. Results in the mouse model showed both DNA vaccines to be as effective as BCG in providing decreased bacterial counts after challenge with live M. tuberculosis. (Lowrie, 1994)
A more extensive study was done recently in which Ag85 DNA was used to immunize mice. Two antigens were tested: secreted Ag85A and the mature form of Ag85A. In both cases, immunization with the DNA resulted in reduced replication of M. tuberculosis by a factor of 20 to 40, a result similar to that obtained in BCG vaccinated mice. Results showed generation of protective immunity through induced humoral and cell-mediated immune responses. Ag85A gene vaccination resulted in high-titer antibodies reactive against Ag85, predominantly of the IgG1 and IgG2a types. Spleen cells from immunized mice were harvested and exhibited substantial lymphocyte proliferation when stimulated with BCG in vitro. The humoral and cell mediated responses induced by DNA vaccination in this study were indicative of a Th1-type of T-cell response, which is believed to be necessary for immunological protection from M. tuberculosis. (Huygen, 1996) However, in this case, care must be taken when extending data obtained from animal models to humans because of the discrepancies between humans and mice regarding the immunology of tuberculosis.
Balasubramanian, V. et al. Allelic Exchange in Mycobacterium tuberculosis with Long Linear Recombination Substrates. Journal of Bacteriology 1996; 178:273-279.
Bloom, B.R. et al. Immune Response to Recombinant Mycobacterial Proteins in Patients with Tuberculosis Infection and Disease. The Journal of Infectious Diseases 1996; 174:431-434.
DeGroot, AS, Meister GE, et al. A Novel Algorithm for the Efficient Identification of T-Cell Epitopes: Prediction and Testing of Candidate Tuberculosis Vaccine Peptides in Genetically Diverse Populations. Vaccines 96, Cold Spring Harbor Lab., Cold Spring Harbor NY 1996.
Fine, P.E.M. The BCG Story: Lessons from the Past and Implications for the Future. Reviews of Infectious Diseases 1996; 11:s353.
Fine, P.E.M. Variation in Protection by BCG: Implications of and for Heterologous Immunity. The Lancet 1995; 3346:1339.
Guleria, I. et al. Auxotrophic Vaccines for Tuberculosis. Nature Medicine 1996; 2:334-337.
Harboe, M. et al. Vaccines Against Tuberculosis. Vaccines 1996; 14:701-716.
Harboe, M. et al. Evidence for Occurrence of the ESAT-6 Protein in Mycobacterium tuberculosis and Viurlent Mycobacterium bovis and for its Absence in Mycobacterium bovis BCG. Infection and Immunity 1996; 64(1):16-22.
Horwitz, M.A. et al. Novel Insights into the Genetics, Biochemistry, and Immunocytochemistry of the 30-kilodalton Major Extracellular Protein of Mycobacterium tuberculosis. Infection and Immunity 1996; 64(8):3038-3047.
Huygen, K. et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nature Medicine 1996;2(8):893-898.
Kaufmann, Stefan, H.E., and Young, Douglas B. Vaccination Against Tuberculosis and Leprosy. Immunobiology 1992; 184:208.
Kemp, E.B., Belshe, R.B., Hoft, D.F. Immune Responses by Percutaneous and Intradermal Bacille Calmette-Guerin. Journal of Infectious Diseases 1996; 174:113-119.
Lowrie, D. et al. Towards a DNA vaccine against tuberculosis. Vaccine 1994;12:1537-1540.
Lowrie, D.B., Ricardo E.T., Celio L.S. Vaccination Against Tuberculosis. International Archives of Allergy and Immnunology 1995; 108:309-312.
Mendez-Samperio, Patricia. Antigen Presentation of Mycobacterial Peptides to Human T-Cell Clones can be Immunomodulated by Adding an MHC-Specific Inhibitor. Cellular Immunology 1993; 148:1-9.
Mustafa, Abu Salim, Fredrik Oftung. Cytokine Production and Cytotoxicity Mediated by CD4+ T-cells from Healthy Subjects Vaccinated with Mycobacterium bovis BCG and from Pulmonary Tuberculosis Patients. Nutrition 1995; 11:698.
Pisetsky, D. et al. Immunological Properties of Bacterial DNA. Annals of the New York Academy of Sciences 1995;772:152-163.
Pithie, A.D. et al. Generation of Cytolytic T-Cells in Individuals Infected by Mycobacterium tuberculosis Vaccinated with BCG. Thorax 1992; 57:695-701.
Roche, P.W., Triccas, J.A., Winter, N. BCG Vaccination Against Tuberculosis: PastDisappointments and Future Hopes. Trends in Microbiology 1995; 3:397-398.
Rosen, Hugh, Horn, David, Shaw, Alan. Perspectives for the Control of Tuberculosis. Therapeutic Immunology 1995; 2:105-113.
Silva, C. New vaccines against tuberculosis. Brazilian Journal of Medical and Biological Research 1995;28:843-851.
Turner, J. and Dockrell, H.M. Stimulation of Human Peripheral Blood Mononuclear Cells with Live Mycobacterium bovis BCG Activates Cytolytic CD8+ T-Cells In Vitro. Immunology 1996; 87:339.
Vordermeier, H.M. T-Cell Recognition of Mycobacterial Antigens. European Respiratory Journal Supplement 1995; 8(20):657s-667s.
Wallis, R. New Approaches to Identification of Antigens in Mycobacterium tuberculosis. Infectious Agents and Disease 1996; 5(2):119-125.
Young, D.B., Kenneth, Duncan. Prospectives for new Interventions in the Treatment and Prevention of Mycobacterial Disease. Annual Review of Microbiology 1995; 49:664-673.
Live Recombinant Vaccines
Efforts to modify BCG or M. tuberculosis by recombinant DNA technology to produce a new live attenuated vaccine against TB are in progress. One method is to express a variety of heterologous antigens in BCG. If the critical immune targets of M. tuberculosis were to be identified and expressed in BCG, this would provide a better live attenuated vaccine.
Peptide Vaccines
In order to develop an effective peptide vaccine for tuberculosis, the specific antigens must be identified, and their ability to induce protective immunity must be confirmed. It is insufficiently known whether certain proteins are particularly important in inducing protective immune responses. The most essential factor is probably getting proper antigen presentation, and this may occur most easily with certain proteins.
30/32 kDa (85 complex)
38 kDa (lipoprotein)
19 kDa (lipoprotein)
MPT64 (24 kDa)
Stress proteins (10 kDa, 65 kDa, 70 kDa, 90kDa)
DNA vaccines
An innovative vaccine approach currently being applied in the search for a BCG replacement is the nucleic acid, or DNA vaccine. Generally, the DNA vaccine involves the use of either antigen encoding naked DNA in buffer solution, which has been proven to transfect cells in vivo, or a viral vector coding for specific disease antigens. The possibility of using DNA vaccines is a promising alternative to BCG. It is constructed to code for very few antigens which can be selected so as not to interfere with skin sensitivity tests. This would provide the world with an easily produced and stable vaccine which would not interfere in skin sensitivity TB testing, allowing for a worldwide universal vaccination program.
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References
Andersen, B. et al. Mapping of the Delayed-Type Hypersenstivity-Inducing Epitope of Secreted Protein MPT64 from Mycobacterium tuberculosis. Infection and Immunity 1995; 63(12):4613-4618.