Real vaccines in the real world: tuberculosis vaccines move south

A successful vaccine must not only induce protection in an artificial laboratory environment and in genetically identical laboratory mice, but also be able to do the same in the real world. For new tuberculosis (TB) vaccines this poses a major challenge, as exposure to related mycobacterial species in the environment may both block and/or mask the immunity induced by a new vaccine. The environment may have additional, nonmycobacterial effects on how humans respond to TB vaccines, as well as to other vaccines. These effects, which are complex to both assess and understand, may include the results of nutrition, other infections and exposure to immune modulating agents, such as pesticides or toxins.

The pipeline of TB vaccine development

A number of new TB vaccines are currently being developed, including genetically modified Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccines, Mycobacterium tuberculosismutants, viral vectors expressing M. tuberculosis antigens and fusion proteins of M. tuberculosis antigens in adjuvant [1,2]. These vaccines are initially tested in animal models, where inbred mice can be used with group sizes that allow differences between vaccine formulations, routes of vaccination, adjuvants, immunogencity and protection against infection to be evaluated. TB vaccines showing promise in mice are usually evaluated in at least one other preclinical model, either guinea pigs and/or nonhuman primates. Vaccines that show sufficient promise, and for which there is sufficient financial backing, then move into early clinical testing in humans. The pipeline of Phase I, II and III trials moves the lead vaccine candidates from early clinical safety testing into ever-larger trials. However, as the lead candidates move through development, they are also moving increasingly into the real world. For vaccines that affect populations worldwide, or that predominantly affect those living in less-developed or tropical settings, this may mean that initial human trials are performed in Europe or the USA, while larger Phase II trials move south into Africa, Asia or South America. For new TB vaccines, that is a real challenge.

Exposure to environmental mycobacteria

The complexity that circulating strains and serotypes bring to vaccine development is well known for pneumococcal disease, meningococcal infections and influenza. For M. tuberculosis, genetic differences between clinical isolates are more common than was originally anticipated, and this genetic variation can modulate the interaction between the bacteria and the host macrophage [3] and, together with host genetics, may result in different clinical presentations [4]. However, genetic variation in the main antigens currently selected to be part of the TB vaccines under development, such as antigen 85 (Ag85), early-secreted antigenic target (ESAT)-6 or MT10.4, does not appear to be an issue. The main problem is the plethora of nontuberculous mycobacteria (NTM) to which we are exposed, either before or after receiving BCG or a new TB vaccine. There are the other pathogenic members of the mycobacterial genus, Mycobacterium leprae, M. bovis and Mycobacterium ulcerans, recently isolated from an aquatic Hemiptera [5], to which some of those living in areas of high TB incidence may also be exposed. Others are just known as NTM, mycobacteria other than TB (MOTT) or ‘environmental mycobacteria’, and we are all variously exposed to these members of the mycobacteria family. There are many of them – some more exotic than others – and most do not cause disease except in the immunocompromised [6]. Some are found in soil, others in water – with a limited number of studies performed in Africa [7,8]. Some are probably impossible to cultivate and completely undocumented. However, some are isolated from sputum or gastric washings of children or adults suspected of having TB [9–11].

On the one hand, immunologists and vaccinologists might be pleased that immunological memory induced by a new TB vaccine could be boosted by these closely related environmental species. Although only a few species, such as Mycobacterium kansasii, express the ESAT-6 gene [12], all have the secreted Ag85, and a recombinant BCG vaccine or an auxotropic mutant of M. tuberculosis will express predominantly crossreactive antigens. However, exposure to environmental mycobacteria may mask or block the immunity induced by BCG – and, perhaps, that induced by some of the new vaccine candidates. The concept of masking involves induction of antimycobacterial immunity that subsequent vaccination cannot improve, whereas blocking involves induction of antimycobacterial immunity that fails to provide protection and merely blocks induction of a protective immune response by subsequent vaccination [13]. In the USA, classical studies showed that skin test responses to purified protein derivatives (PPDs) from environmental mycobacteria increased from north to south, and that naval recruits with stronger skin-test reactivity to Mycobacterium intracellulare or Mycobacterium gause than to M. tuberculosis were less likely to develop TB [14,15]. Early studies in guinea pigs also produced evidence for protection induced by environmental mycobacteria [16]. More recent animal studies in mice and cattle have, however, provided evidence for both interference [17,18] and lack of interference [19] with subsequent BCG vaccination. In studies of BCG vaccination in adolescents and young adults in Malawi, where the BCG vaccine does not induce protection against pulmonary TB [20], most subjects had strong IFN-γ responses to mycobacterial antigens prior to BCG vaccination and did not show enhanced responses 12 months postvaccination [21]. Although not statistically significant, the greatest increases were seen in the small number of individuals with low initial IFN-γ responses to antigens from environmental mycobacteria [22]. In the UK, where most adolescents were nonresponsive to mycobacterial antigens prior to vaccination and where BCG vaccination does induce protective immunity [23], good increases in T-cell responses to mycobacterial antigens were detected following vaccination [21]. However, the subgroup of UK adolescents who did show pre-existing sensitization to mycobacterial antigens prior to vaccination still responded well to vaccination [24]. Thus, it may be the species of mycobacteria to which vaccinees are exposed that matters, or the dose or route of exposure.

In mice, the mycobacteria need to be live and multiply to interfere with the protection induced by BCG [17], and some strains of Mycobacterium avium induce a downregulation of IFN-γ and upregulation of antibody protection following BCG vaccination [25]. Another group studied the effect of M. avium when administered orally following BCG vaccination. Protection against a subsequent live M. tuberculosis challenge was reduced, perhaps owing to altered trafficking of the memory T cells to the brachial lymph nodes [26]. The literature on environmental mycobacteria and their effects is complex, with other studies failing to show such interference [19], but it is clear that exposure to such NTM has the potential to modulate, at least under some circumstances, the protection induced by an effective mycobacterial vaccine. More research is clearly needed in this area.

Nutrition & seasonal effects

As well as differences in exposure to environmental mycobacteria, which may prefer hotter and more humid environments, as a vaccine travels south along its development pipeline, population health and nutrition may change. Nutrition has a marked effect on resistance to disease, including TB, and nutritional deprivation in early life may have profound effects on lifetime health [27]. Deficiencies in micronutrients can also affect immune function, and low serum concentrations of vitamin D, resulting from either diet or a lack of sunshine, are associated with a higher risk of TB [28].

Seasonality is another factor that can affect the response to vaccination. A fascinating series of studies were performed in The Gambia that illustrate the ways in which immunity induced by vaccination can be affected by the month of vaccination, as well as the complexity of these associations and the difficulty of understanding their causes. In The Gambia, both pneumococcal and rabies vaccines showed significant associations between the month of vaccination in children and the antibody titer induced, but no such associations were seen for tetanus, diphtheria or typhoid vaccines in infants. In Pakistan, adult vaccinees showed associations of antibody titer with the month that the vaccine was administered for rabies and typhoid vaccines [29]. Another recent study assessed IFN-γ, IL-2 and CD154 (CD40L) expression in CD4 T cells stimulated with PPD following neonatal BCG vaccination in Gambian infants [30]. Infants born in the wet season had a significantly higher proportion of CD4 T cells expressing CD154 than those born in the dry season, with the main effects seen at 12 months of age. One explanation of such seasonal effects in low-income settings is that it reflects the nutritional status of the vaccinees, with subsequent effects on the immune system. Measurements of thymic index were lower in the wet/hungry season in The Gambia [31]. By 8 weeks of age, the thymic output of T cells was lower in infants born in the hungry season, and the IL-7 concentrations in the breast milk of their mothers were also lower [32].

Another study of immune responses induced by BCG vaccination of infants in Malawi identified the effect of birth month on IFN-γ production in diluted whole-blood cultures stimulated with PPD; a similar, albeit weaker, effect of seasonality was detected in UK infants (Lalor MK et al., Unpublished Data). In Malawi, this may reflect nutrition of the infant or the mother, as well as infectious diseases; in the UK, it is more likely to be a result of other infections.

Infectious diseases & immunity

It is often said that the burden of infectious diseases is higher in tropical or African settings. Certainly, malaria infections are common in many areas, as are intestinal helminth infections. Such infections may drive the immune system harder than in northern countries [33]. There are reports that the balance of naive and antigen-experienced T cells are shifted in Ethiopia compared with The Netherlands [34], and we have made similar observations in Malawian compared with UK adolescents and young adults (Ben-Smith A et al., Unpublished Data). In turn, this may impact on the maintenance of memory T cells. At younger ages, the impact of nutrition on thymic size and IL-7 concentrations in breast milk may also play a role [31,32].

Food toxins (and pesticides) can also influence immune responses. In humid climates, food is often stored under suboptimal conditions and, for example, aflatoxin from groundnuts has effects on growth and may influence both resistance to disease and immune function [35].

Genetics & environment

To find variations between different settings in responses to vaccines is not new. For example, for Haemophilus influenzae type b vaccination, there were differences between Chile and Belgium [36], and Alaska compared with Finland [37].

Some of this variation may be genetic. Studies of mono- and dizygotic twin pairs in The Gambia showed that genetic factors accounted for 51% of the considerable population variance in antibody levels to Hib, which still left 49% to be explained by environmental factors [38]. The impact of genetic determinants diminishes with time following vaccination, as with tetanus toxoid; the genetic influence seen in Gambian twins at 5 months of age was lost by 12 months of age, and environmental determinants had a greater influence on antibody persistence and affinity maturation [39]. For BCG, Gambian twin studies showed only 41% heritability in IFN-γ responses to M. tuberculosis PPD [40].

Southern pipeline

New vaccines for diseases such as TB, as well as those for malaria, HIV and other diseases prevalent in the less-developed areas of the world, therefore, have to travel not just along their individual development pipelines, but also to areas where the immune system is influenced by climate, by exposure to different infectious agents, by food restrictions that may impact on birth weight, growth and lifetime health and by exposure to toxins that may have a negative impact on the immune system. New vaccines must demonstrate efficacy and induce protective immunity in such challenging settings. We already know that considerable geographic variation has been observed in the protective efficacy of the BCG vaccine [23]. We cannot assume that Africa is the same as Asia, or even that different areas within one continent will be the same. Therefore, we need a far greater insight into the complexities of how the immune system develops and is maintained under these conditions. This will require comparative studies not just in north/south settings but within continents; but these should provide valuable insights into how the immune system develops and how immunity is maintained.

Acknowledgements

The author would like to thank H McShane, A Checkley and P Gorak-Stolinska for helpful comments.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.