British Medical Bulletin

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British Medical Bulletin 62:73-86 (2002)
© 2002 The British Council

Tuberculosis vaccines

Douglas B Young and Graham R Stewart

Centre for Molecular Microbiology and Infection, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, UK

	Abstract

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
The increasing incidence of disease associated with HIV infection highlights the crucial role of the immune response in susceptibility to tuberculosis and has stimulated renewed efforts to develop improved vaccines. Vaccine targets include prevention of infection in naive individuals, prevention of re-activation in individuals harbouring latent infection, and prevention of relapse by immunotherapy in tuberculosis patients. Advances in mycobacterial molecular genetics have facilitated development of a range of live attenuated and subunit vaccine candidates that have been screened in experimental models of infection. Evaluation of the immunogenicity of selected candidate vaccines in clinical trials should be combined with a continuation of fundamental research on the immune response to mycobacterial infection and persistence.

	The natural history of tuberculosis

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
Mycobacterium tuberculosis is one of mankind's most successful pathogens, causing 8 million new cases of disease and around 2 million deaths every year¹. The bacteria are capable of causing disease in most organs of the body, but the predominant pulmonary form of tuberculosis is central to spread of the infection. Mycobacterial replication together with a tissue-damaging immune response converts the lung into a highly efficient aerosol-generating chamber that provides the source for continued transmission. As a result of this, it is estimated that one-third of the global population is currently infected with M. tuberculosis. In the vast majority of individuals, infection is apparent only in the form of an immune response that is sufficient to restrict bacterial multiplication, but often insufficient to completely eliminate the infection. There are two pathways by which this initial infection can progress to clinical disease. Around 2–5% of individuals fail to mount an appropriate containment response and go on to develop primary tuberculosis. In children, this is often manifest as extrapulmonary disease, including the frequently fatal tuberculous meningitis. Individuals who are successful in controlling initial infection remain susceptible to development of secondary tuberculosis, as a result of re-infection or re-activation of the initial infection. This is most commonly pulmonary tuberculosis and has a peak incidence in young adult age groups in endemic countries. The incidence of both forms of tuberculosis is markedly enhanced in individuals infected with the human immunodeficiency virus (HIV). Infection with M. tuberculosis is generally associated with a 10% life-time risk of clinical disease; in HIV-co-infected individuals this becomes a 10% annual risk. The HIV pandemic has reversed earlier progress in treatment-based tuberculosis control programmes, triggering alarming increases in disease incidence in sub-Saharan Africa. Together with the emergence of multidrug-resistant tuberculosis, the worsening disease situation has stimulated a renewal of efforts to develop immunological tools for tuberculosis control.

	BCG vaccination

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
Attempts to control tuberculosis by vaccination were initiated shortly after identification of the tubercle bacillus by Robert Koch at the end of the 19th century. Koch himself experimented with a therapeutic vaccine based on culture filtrate components, but it was the live attenuated vaccine of Albert Calmette and Camille Guerin that dominated the next century of tuberculosis vaccination². The bacillus of Calmette and Guerin (BCG) was prepared by serial passage of an initially virulent isolate of Mycobacterium bovis in a bile-containing laboratory medium. From modern molecular genomics, we now know that deletion of a 9.5 kb fragment of DNA – the RD1 region – was one important element of this initial attenuation process³. Further deletions occurred over the subsequent decades of laboratory passage prior to the introduction of freeze-drying technology, generating a series of genetically distinct BCG substrains possibly differing in their vaccine efficacy⁴. The original BCG strain was shown to be attenuated in a series of animal models and was introduced as a human vaccine in 1921.

BCG was designed to function according to the principles established by Edward Jenner and Louis Pasteur, priming the natural immune response to make an accelerated response on subsequent exposure to the virulent pathogen. It has been shown to achieve this goal in a wide range of experimental animals, from mice, guinea pigs and rabbits, to cattle, deer and non-human primates^5–8. Infection of naive animals is associated with an initial period of relatively unrestricted mycobacterial growth prior to the onset of a mycobacteria-specific cell-mediated immune response; this period is reduced in BCG-vaccinated animals, thereby restricting the extent of initial disease (Fig. 1). The subsequent course of the infection depends on the host species and on the dose and route of the challenge. Typically, bacterial numbers are maintained at a stable level, with accumulating pathological damage leading to impaired lung function and eventual death. This phase of the infection is similar in naive and vaccinated animals; the protective efficacy of BCG is associated with its ability to limit the initial extent of the infection rather than to modulate the subsequent chronic or persistent phase of the disease.

View larger version (12K): [in this window] [in a new window]   Fig. 1 Schematic representation of infection profiles in naive and BCG-vaccinated hosts. The initial phase of bacterial expansion is reduced by the more rapid onset of a cell-mediated immune response in the BCG-primed host. This is generally followed by a partial clearance of the infection leaving a persistent population of bacteria that contributes to chronic disease or to later re-activation. Four conceptual rationales are envisaged for novel tuberculosis vaccines: (1) pre-exposure vaccination inducing an immune response that can be recalled to the site of infection more rapidly than the BCG response; (2) pre-exposure vaccination inducing an immune response capable of more effective bacterial clearance (sterilising immunity); (3) postexposure vaccination capable of reducing the persistent bacterial population; and (4) postexposure vaccination which protects against re-activation.

 
Analysis of the effect of BCG vaccination in human populations has been dogged by confusion and controversy. Clinical trials that have examined the ability of neonatal BCG vaccination to protect against primary tuberculosis in children have produced consistently positive results, demonstrating efficacy in the range of 50–70% protection⁹. A more complex pattern is seen in trials involving older age groups². Vaccination of 12–14-year-olds in an MRC trial in the UK demonstrated 75% protective efficacy, but trials in other countries – most notably in the southern US and a large World Health Organization (WHO) trial in South India – failed to show any protection. While there may be multiple factors contributing to these discordant results (including the chronological changes in BCG strains discussed above⁴), geographical differences in exposure to environmental mycobacteria are thought to be a key influence¹⁰. Although M. tuberculosis itself is restricted to infected humans, other mycobacterial species are commonly found in soil and water supplies, particularly in tropical climates. These bacteria share antigenic determinants with M. tuberculosis and are capable of inducing a protective immune response in animal models¹¹. There are two possible mechanisms by which exposure to environmental mycobacteria might 'neutralise' the effect of BCG vaccination. First, they may confer a degree of protection that is equal to that achieved by BCG. Alternatively, they may induce a level of antimycobacterial immunity that is sufficient to prevent replication of the BCG vaccine, but that remains suboptimal in terms of protection against virulent M. tuberculosis. If this second hypothesis is correct, there may be an important role for a non-replicating vaccine in enhancement of immunity in exposed adult populations. Currently, the predominant use of BCG vaccination is for global prevention of primary tuberculosis in children. In the UK, BCG continues to be given to the school-age group shown to be protected in the MRC trials, in addition to use as a neonatal vaccine in communities at particular risk of exposure to infection.

	Prospects for new vaccines

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
Given the limitations of BCG in protection against adult pulmonary tuberculosis, there is clearly considerable scope for improved vaccination strategies. Two approaches can be considered. The first would involve replacement of BCG by a more potent pre-exposure vaccination inducing an immune response capable either of complete elimination of all of the infecting organisms, or of reliable containment of persistent infection. In practice, since BCG has proven efficacy against childhood tuberculosis (and against leprosy¹², and perhaps other causes of childhood mortality¹³), it is likely that any new pre-exposure vaccine would have to be introduced alongside existing BCG vaccination. The second approach would entail postexposure vaccination to boost immunity in individuals whose natural immune response has already been primed by infection or by BCG vaccination. In either case, enhancement of BCG – or more ambitiously, enhancement of natural immunity – would be the target of new tuberculosis vaccines. Over the last decade, two interlinked research programmes have moved towards this goal. On the one hand, there has been a concerted effort to dissect the fundamental mechanisms by which the immune system responds to infection with M. tuberculosis. In parallel, a wide range of potential vaccine candidates have been generated and subjected to empirical testing for protective efficacy in experimental models of infection.

	Immunity to tuberculosis

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
The initial response

The ability to survive and multiply inside phagocytic cells is central to the pathogenesis of M. tuberculosis. This ability is mediated in part by their relatively impermeable lipid-rich cell wall, but also by an ability to interfere with the normal processes of phagocytic digestion. In cell culture models, live tubercle bacilli are able to arrest phagosome maturation, allowing survival in a relatively congenial non-acidified intracellular vacuole¹⁴. The molecular mechanisms underlying phagosomal arrest are unknown, but the effect can be counteracted in macrophages that have been activated by T-cell stimulation¹⁵. The major arm of mycobacterial immunity is, therefore, the delivery of a macrophage activation signal – predominantly IFN- – by antigen-specific T lymphocytes¹⁶. The timing of the T-cell response is important. As the infection progresses, M. tuberculosis further alters macrophage physiology, reducing T-cell recognition and the response to IFN- stimulation¹⁷. During disease, continued macrophage activation in the presence of large numbers of bacteria becomes a major cause of pathology rather than protection. Although IFN- is clearly an essential element of the protective response, the total amount of IFN- produced does not necessarily reflect the effectiveness of the immune response^8,18.

HIV-induced susceptibility to tuberculosis, together with experiments in knockout mice, identifies a central role for CD4⁺ T-cells in antimycobacterial immunity^16,19. This is consistent with the expected presentation of mycobacterial antigens by MHC class II molecules on the surface of infected macrophages. BCG vaccination provides a very effective stimulus for priming of Th1, IFN- producing, CD4⁺ T-cells. Other immune mechanisms complement this central CD4 pathway and represent possible targets for vaccines designed to enhance BCG. CD8⁺ T-cells are able to recognise mycobacteria-infected cells and make an important contribution to protection against M. tuberculosis in mice^16,19. They may act as a supplementary source of IFN-, or perhaps as a means of destroying infected macrophages that are no longer responsive to cytokine activation. Triggering of apoptotic pathways can promote killing of intracellular mycobacteria, and some human CD8⁺ T-cells have been shown to secrete antibacterial peptides into target cells. T-cells that recognise non-protein antigens presented by CD1 molecules may also make an important contribution to the human immune response to mycobacterial infection; glycolipids are able traffic out of the mycobacterial phagosome and have been shown to co-localise with CD1 molecules in infected dendritic cells. T-cells with an antigen-specific receptor comprising and chains are also prominent in the immune response to mycobacterial infection and may play some supplementary role in protection. Finally, although humoral immunity is ineffective against mycobacteria, it is possible that antibodies to surface components could influence the initial interaction between mycobacteria and macrophages with some consequent effect on cell-mediated immune pathways.

Is there an appropriate combination of these different T-cell subsets that will guarantee elimination of all infecting mycobacteria? Animal models are discouraging. The most promising of the new vaccine candidates resemble BCG in triggering an early response that restricts, but fails to eliminate, the infection²⁰. However, given that the immune system does have the means to kill M. tuberculosis, sterilising immunity may simply require early recruitment of a sufficient number of T-cells. Novel vaccine strategies may evolve from an improved understanding of the precise kinetics by which different presentation pathways make antigens available on the surface of infected cells, and of the signals involved in trafficking and recruitment of lymphocytes. It is conceivable that some people naturally mount a sterilising immune response. Our current strategy to monitor infection is based on detection of an immune response rather than detection of bacteria. If we could measure bacterial load we might find a great diversity amongst the one-third of the world we currently deem to be 'infected'.

Persistence and re-activation

Why should individuals who have mounted an effective immune response to initial infection succumb to subsequent disease? It may be that containment of the infection requires constant immune surveillance (Fig. 2). A drop in T-cell numbers, as a result of HIV infection or perhaps more transiently due to stress or malnutrition, might then be sufficient to allow renewed growth of the pathogen. The kinetics of T-cell turnover, and the number of lymphocytes that the thymus is prepared to commit to recognition of different infectious agents, are additional factors likely to influence the long-term balance of the antimycobacterial response. An alternative hypothesis is that containment is independent of the immune response. The human immune response to mycobacteria involves isolation of the infectious focus within an organised granuloma. The bacteria are contained in the anoxic central region of the granuloma within a caseating mass of necrotic cells enclosed by a fibrotic capsule. Such caseous lesions have the potential to liquefy, establishing growth conditions suitable for extensive mycobacterial multiplication. Rupture of liquefied lesions releases large numbers of mycobacteria into the pulmonary cavity during active disease, overwhelming attempts at immune containment.

View larger version (19K): [in this window] [in a new window]   Fig. 2 Two possible pathways for re-activation. In model A, bacterial multiplication is prevented by active immune surveillance. A reduction in T-cell numbers (due to HIV, for example) triggers re-activation of the infection. In model B, persistent bacteria are sequestered from the immune response within a caseous necrotic granuloma. Physiological changes in the granuloma (liquefaction) allow re-activation of bacterial growth, exposing the immune system to a localised high dose challenge. It is possible that both pathways contribute to the phenomena of latent tuberculosis and re-activation disease.

 
The role of vaccination in prevention of re-activation will depend on the extent to which the bacterial population is accessible to the immune system. Analysis of the location and physiology of persisting mycobacteria has frustrated generations of tuberculosis researchers²¹. The most direct experimental approach involves dissection of autopsy material from individuals dying from causes other than tuberculosis. Mycobacteria can often be identified within well-defined granulomatous lesions in such samples, but additional viable organisms have been observed in parts of the lung free of any obvious lesions²². While most such studies date back to the 1920s, a recent PCR-based revival of this approach also detected disseminated infection in multiple cell types in the lung²³. There is a pressing need for methods that would allow us to measure the bacterial load, and also the bacterial location, in individuals with latent tuberculosis infection.

In the absence of a genuine understanding of the immune mechanisms involved in persistence, vaccination strategies targeted towards prevention of re-activation focus on augmenting the lymphocyte subsets identified as beneficial in controlling the acute stage of the infection.

	New vaccine candidates

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
The strategies that have been used to generate novel tuberculosis vaccine candidates are summarised in Table 1.

View this table: [in this window] [in a new window]   Table 1 Strategies for generation of novel tuberculosis vaccine candidates

 
Live attenuated mycobacterial vaccines

The development of techniques for genetic manipulation of mycobacteria has stimulated a variety of attempts to enhance the existing BCG vaccine. Auxotrophic mutants generated by inactivation of genes involved in essential biosynthetic pathways offer potential advantages over wild-type BCG in terms of safety and lack of interference with skin test assays^24,25. The immunogenicity of BCG can be modified by introduction of genes encoding mammalian cytokines, such as IFN-²⁶, or cytokine antagonists such as the latency activating peptide (LAP) that regulates transforming growth factor-ß (TGF-ß)²⁷. A potential drawback to this strategy is that the enhanced Th1 response may cause accelerated clearance of the vaccine, with a detrimental impact on the resultant memory response. Increased expression of protective antigens represents an alternative approach to enhancement of BCG immunogenicity. A recombinant BCG strain carrying multiple copies of the gene encoding the antigen 85B secreted protein showed increased protection in a guinea pig model²⁸, and enhanced immunogenicity was observed when expression of the 70 kDa heat shock protein was increased by deletion of a regulatory gene²⁹. The ability of BCG to prime MHC class I-restricted CD8⁺ T-cells was increased by incorporation of the gene encoding a haemolytic enzyme from Listeria monocytogenes³⁰.

Attenuation of M. tuberculosis by alternative mutations may generate strains with vaccine efficacy superior to BCG. Encouraging results have been obtained with auxotrophic mutants, for example³¹, and there are a growing number of reports of attenuation of M. tuberculosis as a result of alteration of surface properties or disruption of regulatory genes^32–35. By selecting mutations that interfere with different stages of the infectious process, it may be possible to optimise immunogenicity and induction of memory whilst eliminating pathological sequelae. It is attractive to speculate that mutations which alter the ability of M. tuberculosis to interfere with events in the infected cell might generate strains that are more immunogenic than the natural infection. Safety, particularly in immunosuppressed individuals, is a major concern with any vaccine development strategy based on live attenuated M. tuberculosis. While contemporary safety regulations are much more rigorous than those required at the time of introduction of BCG, the ability to engineer additional mutations provides scope for a robust approach to attenuation. Clearly, multiple mutations would be required to eliminate any possibility of reversion to virulence and extensive testing would have to include studies in immunosuppressed animal models.

Subunit vaccines

An important development over the last decade has been the identification of subunit vaccines with efficacy in animal models comparable to that previously achieved only with live mycobacteria³⁶. Subunit vaccine strategies have focused in particular on proteins present in filtrates prepared from in vitro cultures of M. tuberculosis, though non-secreted antigens have also been shown to induce protective responses when delivered in appropriately immunogenic form^37,38. The most extensively studied antigens are members of the antigen 85 complex – a family of mycolyl transferase enzymes involved in cell wall biosynthesis that comprise the major protein fraction in culture filtrates³⁹. A second important family of antigens are low molecular weight proteins related to ESAT6^36,40, a highly immunogenic culture filtrate protein with unknown physiological function that is encoded by a gene in the RD1 region and, therefore, absent from BCG³. These, and other mycobacterial proteins, have been shown to induce a protective immune response in experimental models when delivered as purified proteins combined with adjuvant^37,41, as DNA vaccines^42,43, or as recombinant viral vaccines⁴⁴. The latter strategies are attractive in allowing induction of both CD4⁺ and CD8⁺ T-cell subsets.

Selection of antigens for evaluation as subunit vaccines is generally based on their prominence as targets of the natural immune response. In designing vaccines to enhance pre-existing responses, it may be useful to screen for antigens that are presented on the surface of infected cells but that may be poorly immunogenic during the natural infection. Protection associated with a naturally sub-dominant epitope of ESAT6 provides an interesting example in this regard⁴⁵.

Novel candidate vaccines are generally screened initially in relatively inexpensive murine challenge models²⁰. Promising candidates are then more fully examined in guinea pigs. Guinea pigs develop a granulomatous pathology resembling that of human infection⁶ and provide the opportunity to study the effects of vaccines on chronic pathology as well as their effect on the initial acute phase of infection. More recently, there has been a move to screen vaccine candidates in non-human primate models as a final stage of preclinical development⁸. Current favourites include: (i) recombinant fusion constructs involving antigen 85, ESAT6, and other prominent protein immunogens⁴¹; (ii) modified vaccinia Ankara (MVA) expressing antigen 85⁴⁴; and (iii) a DNA vaccine encoding an M. leprae heat shock protein that has been shown to have therapeutic activity in a mouse model⁴⁶.

	Clinical trials

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
Clinical trials of BCG vaccination in the 1990s focused primarily on its effect on leprosy. An interesting observation was that a repeat BCG vaccination in a Malawi population had no effect on the susceptibility to tuberculosis, but did confer additional protection against leprosy¹². To date, this is the only trial that has assessed the use of a booster BCG, but an extensive evaluation of this strategy is currently underway in Brazilian schoolchildren⁴⁷. A neonatal vaccination trial comparing different BCG substrains and different delivery systems has recently been initiated in South Africa⁴⁸. This trial will test whether the high level of childhood tuberculosis in this community is a consequence of suboptimal BCG vaccination. An attractive strategy to test whether different substrains of BCG confer different levels of protection would involve alternating vaccines from different manufacturers in different years in national BCG vaccination programmes⁴⁹. In addition to addressing an important scientific hypothesis, this trial design may assist in co-ordinating global supplies of the vaccine.

The only new tuberculosis vaccine subjected to clinical trial in recent years is Mycobacterium vaccae, an environmental bacterium delivered as a killed formulation. This has been tested as a therapeutic vaccine. The vaccine had no effect on the rate of clearance of viable M. tuberculosis from the sputum of patients who were also receiving standard chemotherapy⁵⁰. While it seems unlikely that mycobacteria released into the lung during acute disease will be susceptible to immune control, it is attractive to suggest that immune stimulation might assist in removal of persistent organisms. Testing of this hypothesis would require monitoring of relapse in shortened treatment schedules or efficacy against drug-resistant disease.

Clinical trials represent the next important stage in development of new tuberculosis vaccine candidates. These will involve phase I safety trials initially in individuals with no prior exposure to tuberculosis, and later in healthy exposed controls. Given the gaps in our understanding of the nature of latent tuberculosis, it is important to keep in mind the possibility that immune modulation could have an adverse effect on individuals harbouring subclinical infection. Subsequent phase II clinical trials providing information on the immunogenicity of new vaccine candidates can be expected to make a major contribution to further vaccine development strategies.

Phase III protection trials will be prolonged and expensive. The on-going BCG trials discussed above will be important in developing appropriate infrastructure for future trials. Two types of trial can be anticipated. The first would compare BCG with some form of enhanced BCG in neonatal populations. Possible versions of 'enhanced BCG' could include genetically manipulated strains, or conventional BCG delivered alongside supplementary vaccination with protein formulations, recombinant vaccinia virus, or perhaps DNA constructs. While the incidence of childhood tuberculosis would provide a relatively early outcome measure in such trials, the key criterion for success will be a subsequent reduction of pulmonary disease in young adults.

A second type of trial would focus on vaccination of the young adult population most at risk of disease. This has the advantage of a more immediate impact on the main disease target, but would require vaccines effective in individuals in whom an antimycobacterial immune response has already been established as a result of M. tuberculosis infection, BCG vaccination, or environmental exposure. Immunogenicity trials involving the various subunit vaccine candidates described above will be important in assessing the feasibility of such an approach.

	Concluding comments

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
Tuberculosis continues to present a formidable challenge to vaccinologists. Progress has been achieved over the last decade by combining fundamental research on the immune response to mycobacterial infection with empirical testing of novel candidates in animal models. Investigation of the immunogenicity of selected candidates in human trials represents the next important stage in vaccine development.

	Acknowledgements

Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 
The authors gratefully acknowledge Programme Grant support from the Wellcome Trust.

	Footnotes

 
Correspondence to: Prof. Douglas Young, Centre for Molecular Microbiology and Infection, Flowers Building, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AZ, UK

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Top Abstract The natural history of... BCG vaccination Prospects for new vaccines Immunity to tuberculosis New vaccine candidates Clinical trials Concluding comments Acknowledgements References

 

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