Vaccine strategies against tuberculosis

There has never been a more urgent need for a new vaccine against tuberculosis (TB). It is estimated that there are currently 8.8 million new cases per annum and 1.7 million deaths throughout the world [1]. The emergence of multiand now extensively drug-resistant strains ofMycobacterium tuberculosis (M.tb) has made control of this pathogen even more challenging. In addition, it is estimated that one third of the world (2 billion people) are latently infected with M.tb, and are at risk of reactivation of disease [2]. Globally, coinfection with HIV is the commonest cause of immunosuppression, and infection with HIV increases the risk of reactivation of latent M.tb infection from a 10% lifetime risk to a 10% annual risk [3]. In 2006, the Global Plan to Stop TB set out some detailed and ambitious targets for global TB control [1]. This plan explicitly recognises that in addition to the tools currently available, e.g. directly observed therapy, short-course (DOTS), new tools are needed in order for these targets to be achieved. Those new tools include new drugs, new diagnostic tests and new vaccines. Introduction


Helen McShane
The Jenner Institute, University of Oxford, United Kingdom There has never been amore urgent need for anew vaccine against tuberculosis (TB).It is estimated that there are currently 8.8 million new cases per annum and 1.7 million deaths throughout the world [1].The emergence of multi-and now extensively drug-resistant strains of Mycobacterium tuberculosis (M.tb) has made control of this pathogen even more challenging.In addition, it is estimated that one third of the world (2 billion people) are latently infected with M.tb,and are at risk of reactivation of disease [2].Globally,c oinfection with HIV is the commonest cause of immunosuppression, and infection with HIV increases the risk of reactivation of latent M.tb infection from a1 0% lifetime risk to a1 0% annual risk [3].
In 2006, the Global Plan to Stop TB set out some detailed and ambitious targets for global TB

Introduction
The only currently licenced vaccine against TB, bacilli Calmette-Guerin (BCG) is an attenuated strain of M. bovis.BCG was first used in humans in 1921, when it was administered per os [4].Since that time there have been many clinical trials throughout the world to evaluate the protective efficacy of this vaccine.When administered at birth, as it is throughout the developing world, BCG confers consistent and reliable protection against disseminated disease, particularly TB meningitis, in the first 10 years of life [5].However,t he protection conferred against pulmonary disease is much more variable [6].Ameta-analysis of 14 prospective trials and 12 case-control studies determined that overall the protective effect of BCG against pulmonary disease was 50%, but that latitude had as ignificant effect on this efficacy [6].Furthermore, ar ecent randomised controlled trial of BCG revaccination has shown that revaccination in adolescents does not improve protective efficacy [7].
Understanding the mechanism behind the variability in efficacy conferred by BCG is important in the development of better vaccines.Several factors have been cited to explain the variability in protective efficacy conferred by BCG throughout the world.It is clear that there are genetic differences between the different strains of BCG that have been used throughout the world [8], but the immunogenicity of different strains of BCG is comparable and it is not clear that these genetic differences confer any difference in pro-

Summary
The need for an improved vaccine against tuberculosis has never been more urgent.The HIV epidemic and the emergence of multi and extensively drug-resistant strains of Mycobacterium tuberculosis mean that global control of this pathogen remains inadequate.The existing vaccine, BCG, confers only variable protection against pulmonary disease.Exposure to environmental mycobacteria may contribute to this variability in protective efficacy.Protective immunity against Mycobacterium tuberculosis is dependant on acell-mediated immune response.Boosting BCG with as ubunit vaccine, and/or replacing BCG with an improved BCG are both strategies currently being investigated.Since 2002, there have been increasing numbers of TB vaccine candidates entering into clinical trials.The first of these candidates, MVA85A, is safe and highly immunogenic in all trials to date.In addition, the cellular immune response induced is highly polyfunctional.The protective efficacy of MVA85A will be evaluated in aPhase IIb trial commencing in early 2009 in South African infants.
tection [9].Nutritional differences in the populations in which the protective efficacy of BCG has been evaluated may also be ac ontributory factor [10].
The best explanation for the variable protective efficacy of BCG, however,lies in variability in exposure to environmental mycobacteria.Environmental mycobacteria are ubiquitous organisms which live in the soil, and exposure to these mycobacteria is universal but increases with increasing proximity to the equator.Itispostulated that these organisms interfere with BCG vaccination either by masking or by blocking.The masking hypothesis has been elegantly demonstrated by studies of BCG vaccination carried out in parallel in the UK (a population where BCG has been shown to be effective) and Malawi (a population where BCG is not effective) [11].In these studies, at baseline, BCG naïve adolescents in the UK had minimal anti-mycobacterial immune responses.In contrast, BCG naïve adolescents in Malawi already had high baseline responses to mycobacterial antigens.Most of the UK adolescents responded well to BCG vaccination, and there was as ignificant incremental rise in immune responses to mycobacterial antigens in this population.In contrast, in Malawi most responses did not significantly rise after BCG vaccination.This data fits with the masking hypothesis, whereby exposure to environmental mycobacteria induces some anti-mycobacterial immunity,a nd vaccination with BCG does not incrementally increase that response.Blocking is am ore active process, whereby it is postulated that pre-existing immunity induced by exposure to environmental mycobacteria inhibits the replication of BCG and hence vaccine 'take'.This mechanism has been demonstrated in animals [12].

Protective immunity to M.tb
In addition to an understanding of the mechanism behind the failure of BCG, an understanding of the nature of protective immunity to M.tb is important in the development of new vaccines.There is considerable interindividual variability in outcome after exposure to M.tb.A pproximately 70% of individuals exposed to M.tb successfully clear the infection [13].Only approximately 30% of people go on to become infected.This early response is attributable to an effective innate immune response, although the precise nature of this rapid response is not clearly defined [14].Of the 30% of individuals who become infected, 80-90% are able to contain the initial infection and the mycobacteria enter as tate of latency [14].Only 5-10% of adults develop primary disease.Once latently infected, individuals are at risk of reactivation of this latent infection should they become immunosuppressed for any reason.
M.tb is an intracellular pathogen and resides primarily inside macrophages.It is unlikely that antibodies have am ajor role to play in protective immunity to M.tb.Cellular immunity is essential.
It is clear from animal studies, and the increased susceptibility to TB disease seen in HIV infected subjects, that Class II restricted CD4 + Tc ells are essential for protective immunity to M.tb [3,15].Class Irestricted CD8 + Tcells probably also play ar ole in protective immunity,a lthough the precise mechanism by which they work is as yet not clearly elucidated.It may be that this Tcell subset is important in maintaining the latent state [16].
In addition to the importance of the different cell types, it is clear that ar obust Th-1 type immune response, with secretion of interferon gamma (IFN-g)f rom antigen specific Tc ells, is necessary for an effective immune response against M.tb.Animals and humans who have deficiencies in the IFN-g processing pathway are more susceptible to TB [17,18].However,IFN-g alone does not appear to be sufficient for protection.Other Th-1 cytokines such as IL-12 and TNF are also important.The importance of TNF has recently been highlighted by the reactivation of latent TB seen in patients commencing therapy with amonoclonal antibody against TNF [19].

Newvaccines in development
The two main current approaches in developing improved prophylactic TB vaccines are either to use modified BCG or M.tb to replace BCG; or to use selected immunodominant antigens in asubunit booster vaccine which is administered some time after BCG vaccination.For subunit vaccines, antigen delivery systems include recombinant viral vectors and protein/adjuvant combinations.BCG is likely to be included in any new TB vaccine regimen, at least in the short to medium term, because of the protection conferred against disseminated disease in childhood [5].The use of BCG in any new regimen, either alone as ar ecombinant BCG or when combined with asubunit boost, allows the retention of these protective effects of BCG in childhood.The two strategies can be combined and a booster vaccine could be used to boost an improved BCG.The leading new prophylactic TB vaccine candidates in development are summarised in table 1.
For the rest of this review paper the focus will be on MVA85A, the vaccine candidate developed by the University of Oxford.This candidate will be used to illustrate the pathway for development of a new TB vaccine.

MVA85A
MVA85A is ar ecombinant strain of modified vaccinia Ankara (MVA) expressing antigen 85A from M.tb.MVA is ahighly attenuated strain of vaccinia virus which has been passaged over 500 times through chick embryo fibroblasts.As aresult, host range and cytokine genes have been deleted and it cannot replicate in human cells [20].MVAwas used at the end of the smallpox eradication campaign to vaccinate over 100,000 people in southern Germany,and was found to have an excellent safety profile [21].Recombinant pox viruses have been demonstrated to be excellent at boosting previously primed cellular immune responses.The antigen selected for insertion into the MVAvector is antigen 85A.Antigen 85A is ah ighly immunodominant antigen in both preclinical and clinical studies which is present in all strains of mycobacteria sequenced to date [22].It is protective when administered alone in small animals [23].Importantly,the use of this antigen in anew vaccine does not interfere with the new diagnostic tests which are increasingly in clinical use [24].
MVA85A first entered into clinical development in September 2002 [25].These early clinical studies were designed primarily to demonstrate safety.The first studies were conducted in the UK in BCG naïve, tuberculin skin test (TST) negative adults.The aim with these studies was to identify subjects who were as mycobacterially naïve as possible in this first clinical trial, before going on to evaluate this vaccine sequentially in BCG vaccinated subjects and then M.tb latently infected subjects.In addition, each subject group was evaluated first in the UK before being evaluated in a TB endemic country.The reason for this caution was concern within the field of TB vaccine research about the induction of aK och phenome-non.The Koch phenomenon describes the induction of immunopathology at the site of infection in animals infected with M.tb.This phenomenon has been demonstrated in preclinical models and was also seen in humans, when Robert Koch developed his 'remedy' for TB at the end of the 19 th century [26,27].
The Gantt chart in figure 1outlines the clinical trials conducted with MVA85A since 2002.This is taken from the overall Gantt chart for the full product development of MVA85A.The outcome measures in all of these clinical trials have been safety and immunogenicity.The primary immunological readout in all of these clinical trials has been the ex-vivo IFN-g Elispot assay,using an 18-hour overnight incubation.In addition, amore detailed immunological analysis has been conducted using cryopreserved peripheral blood mononuclear cells, in order to fully characterise the vaccine induced immune responses.
To date in April 2008, 258 subjects have been vaccinated with MVA85A and there have been no vaccine related serious adverse events.MVA85A is administered intradermally,and mild local adverse events at the site of injection are common [28].Mild self-limiting systemic adverse events which typically occur in the first 12-24 hours after vaccination are also common [28].Importantly,there have been no signs of immunopathology in any of the trials to date, which include latently infected subjects in both the UK and South Africa (Sander et al., submitted; Ta meris, personal communication).
The immunogenicity results from the trials conducted to date are also encouraging.When administered to BCG naïve subjects in the UK, MVA85A induces as ignificant antigen specific Tcell response which reaches apeak one week after vaccination and then declines.Three weeks after vaccination, the response is not greater than baseline and boosting with asecond MVA85A vaccination at this timepoint does not amplify the response further [28].In contrast, administering MVA85A to subjects who had previously been vaccinated with BCG (median interval between BCG and MVA85A was 18 years) resulted in significantly higher antigen specific Tcell responses one week after vaccination when compared with the BCG alone or MVA85A alone group [28].Importantly for the induction of ac entral memory Tc ell response, these responses remained significantly higher than either vaccine alone up to 24 weeks after vaccination.
Amore detailed immunological analysis of the samples from the BCG-MVA85A vaccinated subjects has demonstrated that the antigen specific CD4 + Tc ells induced by vaccination with MVA85A are highly polyfunctional [29].One week after vaccination almost 50% of the antigen specific Tcells are positive for all four of the markers measured during this study: IFN-g,IL2,TNFa and MIP-1b.This polyfunctional profile persists, and six months after vaccination the responses remain considerably more polyfunctional than the baseline responses.Further analysis of the phenotype of these antigen specific CD4 + Tcells shows them to be relatively immature, with anon-terminally differentiated phenotype.In addition, proliferation studies confirm the proliferative potential of these cells [29].
Other trials with MVA85A have been conducted to investigate the effect of different intervals between BCG and MVA85A.Va ccination with MVA85A soon after BCG vaccination might be comparable to boosting in infancy when BCG has been given at birth.In contrast, vaccination with MVA85A many years after BCG might mimic a clinical scenario of boosting in adolescence.Studies comparing the magnitude of immune response after MVA85A vaccination soon (one month) and a long time (median 18 years) after BCG vaccination show that there is no difference in the magnitude of immune response seen in these two groups when comparing ex-vivo IFN-g Elispot responses [30].
When sufficient safety data had been accumulated in the early studies with MVA85A, astudy was conducted in M.tb latently infected subjects (Sander et al., submitted).Detailed clinical monitoring, including high resolution CT scans, was performed pre-and post-vaccination in this study in order to detect any clinical or sub-clinical immunopathology.R eassuringly,t he safety profile was very comparable to previous studies and there were no signs of any immunopathology.I mportantly,t he immunogenicity of MVA85A in this latently infected population was also very comparable to previous studies.
In 2005, aPhase IIa programme of clinical trials with MVA85A commenced in South Africa.These trials include aseries of studies in progressively younger populations: adults, adolescents, children and infants.To date the safety and immunogenicity data has been similar to that seen in the UK studies [31].Importantly,the responses in South African adults are as durable as the UK responses, with responses remaining significantly higher than baseline for at least one year after vaccination [31].In 2006 we commenced aPhase IIa non-interference study in Gambian infants.The purpose of this study was to assess whether co-administration of MVA85A together with the routine EPI schedule vaccines would lead to immunological interference; either with the humoral immune response to the EPI vaccines or with the cellular immune response to MVA85A (Ota, personal communication).
In summary,i na ll of the clinical trials conducted to date MVA85A is safe and immunogenic.The populations in whom it has been evaluated include M.tb infected adults in the UK and South Africa; HIV infected adults in the UK and South Africa; and children and infants in South and West Africa.The immune profile induced by MVA85A is consistent with what is understood about protective immunity against M.tb,inthat this vaccine induces high levels of highly polyfunctional CD4 + Tcells which proliferate and are not terminally differentiated.This vaccine also improves BCG induced protection in animal models.
The key question is, 'Does MVA85A enhance BCG induced protection in humans?'There are three main target populations most in need of an improved TB vaccine, and these are the groups in which one would wish to evaluate the protective efficacy of MVA85A: infants; adolescents; and HIV infected adults.In early 2009, aPhase IIb proof-ofconcept trial evaluating MVA85A in BCG vaccinated infants is scheduled to commence in South Africa, in collaboration with the South African TB Va ccine Initiative (SATVI) and Aeras Global TB Foundation.This important trial will provide insights into the potential immunological correlates of protection and increase our understanding of the relevance of the various animal models.