P.D. van Helden, E.G. Hoal/ Comparative Immunology, Microbiology and Infectious Diseases 36(2013)287-294 larger mammals have been made [17-26, 34 Some suc- Immune system stimulants, which directly affect cytokine cess in badgers, possums, deer and cattle has been claimed response for example 5. 9. 11, 12, 45, or alternatively, target but total protection has not been evident. The advantage the phagosome [9, 35, or interfere with the host cell cycle is that in these cases, a controlled challenge can be given The use of a simple antigen may seem counterintuitive. to directly assess protection. However, it can always be since it is widely believed that humoral immunity may not argued that the challenge is very unlikely to reflect the real, be important in mycobacterial infection or disease. How field condition and therefore the evaluation outcome of ever, we do not know this for certain and immune boosters vaccination may not reflect the true efficacy of the vaccine. or vaccination with antigens alone have shown promising Currently, TB in animals is either dealt with by a test-and- effects [17, 18, 36, 42]. Hence the rationale to vaccinate first slaughter policy or, as is generally the case with wildlife with a live vaccine and follow with a boost of antigen Tri- in many parts of the globe, left unmanaged and untreated. als currently underway in primates [46]and humans may These two approaches are both unsatisfactory for a variety show whether this approach suggests any advantage. of reasons which will not be the subject of discussion in this paper. At this time, it would seem that the best theo etical options for control would be test and slaughter, or 5. Possible complications? vaccination. Neither is simple nor clear-cut, but neverthe- less they are options to consider. Since M. bovis is generally Perhaps one of the most distressing problems is that n introduced oralien invader"pathogen, it may exe the preferred location of pathogenic mycobacteria is intra- serious negative effects on ecosystems, which does requir attention. This is particularly important in free-ranging ani normal macrophage function. It is thus difficult to imag- mals, where treatment is not possible, particularly in the ine how we may overpower these hidden organisms. Quite case of wildlife apart from this problem, there are also many other prob lems to consider. For example the Koch phenomenon 4. Choice of vaccine candidates suggests possible complications for a good vaccine uberculosis: in endemic areas, many individuals m For nearly a century, the vaccine given(bCg) has been a been infected and if they are exposed to a strong antigen, live one. There are good intellectual reasons to consider the ensuing host response may result in the exacerbation a live vaccine, not least of which is that we know that of occult disease leading to severe toxicities [47] he live vaccine bcg has some effect and that it reflects If we use a new live vaccine, what danger will this po the pathogen in that it is an intracellular organism. Newer live vaccine candidates may come in the form of mod- individuals? We already know BCG is problematic in this ified BCG (for example strongly expressing a particular regard. antigen)[11,35-37, or attenuated Mycobacterium tuber- Even if we overcome these problems, there is evidence culosis constructs(deletion mutants)[20, 38-40 In this that any protection will wane[48, 49], which then begs the case, it is thought to be necessary to knock out at least 2 question of when to vaccinate for maxim genes to negate any possibility that a pathogenic strain may [10 and whether a booster will be necessary or effective. regain virulence. However, live vaccines may also be the All these difficulties are compounded by the lack of com- source of adverse effects: in immunocompromised humans prehensive information on the effect of dosage [23 ,use for example, a live vaccine can cause disease(BCgosis i of adjuvants and timing of prime or boost vaccination. In seen frequently in HIV positive neonates)7. However, addition, it seems that there will be species-specific dif- the same situation may not be the case in immunocom- ferences, making extrapolation risky. Finally, it is possible promised individuals vaccinated with double knock-out that vaccination may interfere with some diagnostic tests live attenuated vaccines. It was shown that such a vac- for TB, complicating diagnosis [50]. This is particularly rel- cine candidate elicited good responses in FIV+ and FIv evant for control of TB in domestic and production animals cats, albeit weaker respons in those that were flv+ at present. with no adverse events noted. This work suggests that live vaccination may be possible in immunocompromised hosts [41. Despite this finding there are good reasons 6. where should we target our intervention step? o explore non-viable preparations for a vaccine. Much ttention has been given to this, ranging from individ Generally, vaccination is considered to be a preventa- ual antigens[11, 12, 18, 42. DNa or heat killed organisms tive activity. However, one may envisage the possibilities or crude preparations of mycobacterial extracts (24, 43. of a pre-exposure vaccine as is currently done, or a post which may also lead to adverse responses (tried by robert exposure vaccine a therapeutic vaccine or one that will Koch, where the adverse reaction has become known suppress reactivation disease. The use of Mycobacterium the Koch phenomenon). Most work has probably been vaccae as a post-exposure vaccine has been tried, but not devoted to protein antigens, perhaps because these can be been convincingly demonstrated to be of importance for loned, expressed and produced relatively easily. However general use [51], although there is some evidence to sug there is published evidence in animal models to suggest gest improved conversion during antibiotic treatment in that lipids or carbohydrates also hold potential as car humans 51. but no efficacy on its own. One might also didates [22, 42, 44 ]. Finally, a new-generation vaccine that envisage that if we find that all active cases of TB lack targets hostresponse can be an option. Such vaccines can be an adequate concentration of a bi ecule. such as aP.D. van Helden, E.G. Hoal / Comparative Immunology, Microbiology and Infectious Diseases 36 (2013) 287–294 289 larger mammals have been made [17–26,34]. Some suc￾cess in badgers, possums, deer and cattle has been claimed, but total protection has not been evident. The advantage is that in these cases, a controlled challenge can be given to directly assess protection. However, it can always be argued thatthe challenge is very unlikely to reflectthe real, or field condition and therefore the evaluation outcome of vaccination may not reflect the true efficacy of the vaccine. Currently, TB in animals is either dealt with by a test-and￾slaughter policy or, as is generally the case with wildlife in many parts of the globe, left unmanaged and untreated. These two approaches are both unsatisfactory for a variety of reasons which will not be the subject of discussion in this paper. At this time, it would seem that the best theo￾retical options for control would be test and slaughter, or vaccination. Neither is simple nor clear-cut, but neverthe￾less they are options to consider. Since M. bovis is generally an introduced or “alien invader” pathogen, it may exert serious negative effects on ecosystems, which does require attention. This is particularly importantinfree-ranging ani￾mals, where treatment is not possible, particularly in the case of wildlife. 4. Choice of vaccine candidates For nearly a century, the vaccine given (BCG) has been a live one. There are good intellectual reasons to consider a live vaccine, not least of which is that we know that the live vaccine BCG has some effect and that it reflects the pathogen in that it is an intracellular organism. Newer live vaccine candidates may come in the form of mod￾ified BCG (for example strongly expressing a particular antigen) [11,35–37], or attenuated Mycobacterium tuber￾culosis constructs (deletion mutants) [20,38–40]. In this case, it is thought to be necessary to knock out at least 2 genes to negate any possibility that a pathogenic strain may regain virulence. However, live vaccines may also be the source of adverse effects:in immunocompromised humans for example, a live vaccine can cause disease (BCGosis is seen frequently in HIV positive neonates) [7]. However, the same situation may not be the case in immunocom￾promised individuals vaccinated with double knock-out live attenuated vaccines. It was shown that such a vac￾cine candidate elicited good responses in FIV+ and FIV− cats, albeit weaker responses in those that were FIV+, with no adverse events noted. This work suggests that live vaccination may be possible in immunocompromised hosts [41]. Despite this finding there are good reasons to explore non-viable preparations for a vaccine. Much attention has been given to this, ranging from individ￾ual antigens [11,12,18,42], DNA or heat killed organisms or crude preparations of mycobacterial extracts [24,43], which may also lead to adverse responses (tried by Robert Koch, where the adverse reaction has become known as the Koch phenomenon). Most work has probably been devoted to protein antigens, perhaps because these can be cloned, expressed and produced relatively easily. However, there is published evidence in animal models to suggest that lipids or carbohydrates also hold potential as can￾didates [22,42,44]. Finally, a new-generation vaccine that targetshost response canbe anoption. Suchvaccines canbe immune system stimulants, which directly affect cytokine response for example [5,9,11,12,45], or alternatively,target the phagosome [9,35], or interfere with the host cell cycle. The use of a simple antigen may seem counterintuitive, since it is widely believed that humoral immunity may not be important in mycobacterial infection or disease. How￾ever, we do not know this for certain, and immune boosters or vaccination with antigens alone have shown promising effects [17,18,36,42]. Hence the rationale to vaccinate first with a live vaccine and follow with a boost of antigen. Tri￾als currently underway in primates [46] and humans may show whether this approach suggests any advantage. 5. Possible complications? Perhaps one of the most distressing problems is that the preferred location of pathogenic mycobacteria is intra￾cellular and pathogen survival is achieved by subverting normal macrophage function. It is thus difficult to imag￾ine how we may overpower these hidden organisms. Quite apart from this problem, there are also many other prob￾lems to consider. For example, the Koch phenomenon suggests possible complications for a good vaccine against tuberculosis: in endemic areas, many individuals may have been infected and if they are exposed to a strong antigen, the ensuing host response may result in the exacerbation of occult disease leading to severe toxicities [47]. If we use a new live vaccine, what danger will this pose to immunocompromised individuals, such as HIV positive individuals? We already know BCG is problematic in this regard. Even if we overcome these problems, there is evidence that any protection will wane [48,49], which then begs the question of when to vaccinate for maximum protection [10] and whether a booster will be necessary or effective. All these difficulties are compounded by the lack of com￾prehensive information on the effect of dosage [23], use of adjuvants and timing of prime or boost vaccination. In addition, it seems that there will be species-specific dif￾ferences, making extrapolation risky. Finally, it is possible that vaccination may interfere with some diagnostic tests for TB, complicating diagnosis [50]. This is particularly rel￾evant for control of TB in domestic and production animals at present. 6. Where should we target our intervention step? Generally, vaccination is considered to be a preventa￾tive activity. However, one may envisage the possibilities of a pre-exposure vaccine as is currently done, or a post￾exposure vaccine, a therapeutic vaccine, or one that will suppress reactivation disease. The use of Mycobacterium vaccae as a post-exposure vaccine has been tried, but not been convincingly demonstrated to be of importance for general use [51], although there is some evidence to sug￾gest improved conversion during antibiotic treatment in humans [51], but no efficacy on its own. One might also envisage that if we find that all active cases of TB lack an adequate concentration of a biomolecule, such as a