As with drug discovery, there is a considerable vaccine pipeline, and in fact this has steadily developed over the past two decades. Despite this, the pyramid of ideas and candidates has become very narrow at the apex of this pyramid, and it seems to this observer that not only will many of the very innovative ideas proposed over this time never reach fruition, but the current dreadful funding environment will kill any new innovative ideas dead in their tracks (astonishingly, a very recent article on this topic [97
] described the field as ‘re-invigorated’). At the Global Forum on TB Vaccines conference held in September 2010 a questionnaire was circulated as a prelude to publishing a ‘Blueprint for TB Vaccines’ document, and it will be illuminating to see if other workers in the field share my viewpoint.
As this field grew, it became apparent that there were multiple types of vaccines that had some sort of protective response, and which could potentially replace BCG. This was necessary, most agreed at the time, because BCG simply did not work. There were some positive effects in children, but this was completely lost as the vaccinated individual approached adulthood. Now, a decade or so further on we appear to be back at square one, with an improved recombinant (r)BCG as the prime candidate.
The spectrum of candidates is in itself very impressive, and was initially driven by the ‘must replace BCG’ idea (an idea that might soon reappear, see below). Ideas, and subsequent candidates, ranged from using key secreted proteins, which soon transformed into the manufacture of fusion polypeptides, to DNA-based vaccines (an idea that died owing to the concern that these would not work in larger animals, including humans), to the use of viruses to deliver key antigens, all the way to taking the M. tuberculosis
organism itself and removing genes to make it less virulent or auxotrophic [38
To give a few examples, HyVac-4 is a fusion of Ag85B and TB10.4 and gives excellent results in mice using IC31 adjuvant [103
]. M72 (or Mtb72F) consists of a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)–Mtb39–Mtb32(N) [104
]. It was first identified in a murine screen conducted by the author under the NIH TB Vaccine Contract program at Colorado State University, CO, USA. Further studies in guinea pigs illustrated its potency further [104
], and it was shown to significantly prolong the survival of guinea pigs in a BCG prime boost model compared with BCG alone [105
]. In the latter case, in some animals the lung pathology even suggested disease reversal. More recently, it was tested in an AS02A adjuvant formulation in the cynomolgus monkey model [106
], where it was immunogenic and caused no adverse reactions. In a BCG prime boost protocol protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology and survival, and as in the previous guinea pig study evidence of disease reversal was observed. The immunological pro-file in these animals indicated IL-2, IFN-γ and TNF-α secretion by T cells, suggesting central memory generation.
A new generation vaccine, ID93, was recently described [107
]. It is a fusion protein using antigen targets implicated by human T-cell line recognition analysis [108
], in this case Rv1813, Rv3620 and Rv2608. When administered with the adjuvant GLA-SE, a stable oil-in-water nano-emulsion, this vaccine candidate was shown to be immunogenic in mice, guinea pigs and cyno-molgus monkeys. In mice, the ID93/GLA-SE combination induced polyfunctional CD4 Th1-cell responses and protected these animals from challenge with virulent strains including an MDR isolate [110
]. Similar results were observed in guinea pigs. What is particularly important is that this candidate was identified by screening cloned human T-cell lines and contains none of the ‘immunodominant’ antigens, yet it works. Avoiding these may be critical, as explained below.
A potential drawback to the efficacy of any nonliving vaccine is the need for adjuvant delivery vehicles; these can be complex to formulate (liposomes, for example) and also very expensive to make. Progress is now being made on less expensive materials including synthetic derivates of monophosphoryl lipid A; this material is a good Th1 adjuvant because it signals through TLR-4, and can be further formulated with other materials, including TLR9 agonists to drive a strong Th1-type immune response.
Using nonreplicating viruses to deliver antigens, either as prime or boost, continues to be very promising. The author has previously expressed his admiration [38
] for the team leading the development of the modified vaccinia Ankara (MVA)85 vaccine candidate, and their careful, sequential testing of this virus in animal models and then humans. However, as this line of research progresses, additional data regarding the immune response to this candidate is growing, not all of it positive. Recently, it was observed that following vaccination with MVA85A, antigen-specific IL-17A-producing T cells were induced in the peripheral blood of healthy volunteers [112
]. These T cells appeared later than IFN-γ+
cells. In individuals with pre-existing immune responses however, there were higher numbers of cells with a regulatory T-cell phenotype, and a concomitantly lower number of Th17 CD4 cells [112
]. A new strategy to possibly improve this further (in mice at least) used MVA expressing both the cytokine IL-15 (to boost memory T cells) plus an ESAT/Ag85 fusion in a prime boost study. This improved the number of ‘polyfunctional’ T cells, but otherwise was not any more protective than the BCG control [113
One would think that solid, reproducible animal efficacy data would be absolutely essential before any vaccine candidate would advance to clinical trials, let alone climb to the very top of the pipeline (of course it depends on who controls that pipeline). Certain candidates, notably MVA85, rBCG30, HyVac-4, and ΔureC hly+ rBCG, have undergone very considerable animal efficacy testing for all to see. While I am certain the lead Aeras candidates have also undergone appropriate testing, it is actually hard to find much data in the literature.
The lead rBCG (Aeras-403) consists of BCG expressing the (pH-independent) perfringolysin molecule, coupled with overexpression of the molecules Ag85A and Ag85B, and TB10.4. In a mouse study it was shown that control mice infected with the W-Beijing strain HN878 lived nearly 300 days [114
], this was improved if they were first given BCG, and this survival was improved further if they were given Aeras-403 instead.
If the astute reader finds this result strange, then he/she is not alone. When HN878 was first tested (by Kaplan) it was described as ‘hypervirulent’ [115
]. This description is apt; it is highly virulent both in mice and guinea pigs. In our hands, HN878 kills mice (infected by low dose aerosol) in approximately 75–90 days [29
]. This isolate is a very potent inducer of regulatory T cells [29
], and this completely interferes with BCG vaccine-induced immunity. While mice are protected at day 30, by day 60 this protection is completely lost [116
; ORDWAY DJ AND ORME IM, UNPUBLISHED DATA] . Thus, to claim that control mice live 300 days is baffling.
The insertion of a membrane perforation molecule in Aeras-403 is based on an earlier elegant study by Grode et al
]. They inserted the gene for listeriolysin into BCG; the idea here was that this lysin is known to drive the pathogenesis of listeriosis by perforating membranes, and thus could promote escape of antigens into the cytoplasm and thus into the Class-I pathway.
To their credit, this group compared the protective effect of the ΔureC hly+ rBCG against both the laboratory strain H37Rv and a W-Beijing strain. The authors stated that in the H37Rv challenge model the rBCG showed greater protection, but the results they present only show a minor difference between the rBCG and the control BCG at a very late time point; other than this there was no difference at all. In mice challenged with the W-Beijing strain the BCG control was initially protected but this was soon lost, whereas protection by the rBCG was extremely rapid and sustained. This is an important observation, with the caveat that the growth kinetics of the two challenge infections were identical (i.e., the clinical strain was not of any higher virulence than the laboratory strain, growing only to ~5.2-log despite deposition of >200 bacilli in the lungs).
Whereas Aeras has clearly made the decision that CD8 induction is a critical component of a new rBCG vaccine, and have thus engineered Aeras-403 to include a endosomal membrane disruption molecule, one can easily argue that any hard evidence that a CD8 response to a vaccine is essential is actually hard to find. In fact, is endosomal disruption actually needed? It seems that M. tuberculosis
at least can promote a perfectly good CD8 response without this, as I pointed a few years ago [38
], cross-talk between Class-II endosomal presentation and Class-I is actually a well-known phenomenon. One such mechanism involves Sec61, a protein translocation channel complex that is present on the phagosomal membrane where it can transport proteins from the phagosomal compartments across to proteosomes where peptides ready for Class-I presentation can be processed. Moreover, a recent study revisited the idea that virulent strains of M. tuberculosis
can actually progressively translocate from phagolysosomes into the cytosol in nonapoptotic cells [118
]. This event was dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. This resulted in significant cell death within a week. This is consistent with an earlier study showing that infection of macrophages with virulent strains causes necrosis rather than apoptosis [119
At this time, Aeras has pushed ahead with NHP and clinical evaluations of their vaccine. A recent study compared BCG Danish with the Aeras-403 (rBCG/AFRO-1) candidate, with boosting provided by two subsequent immunizations with Aeras-402 (a nonreplicating adeno-virus-35 expressing the same antigens), in rhesus macaques [120
]. The primary conclusions of this study were that Aeras-402 induced qualitatively and quantitatively different cellular immune responses as compared with BCG in the vaccinated monkeys. While this was so, the actual variation between monkeys was considerable; some responded and some did not in virtually every assay performed. However, two animals did show evidence for multifunctional T-cell generation, which was promising. However, since there was no data supporting a sustained memory CD4 T-cell response, this study instead emphasized responsiveness by CD8αα T cells, which the study regarded as encouraging. The reality though is that this is an unusual and poorly understood subset of CD8 T cells. There is some suggestion it is a memory T-cell precursor (in a study of acute viral infection in mice, and in a tumor study in humans) and one study showed these to be CCR7lo
, again consistent with an effector memory T-cell subset [121
]. More analytical studies have shown it recognizes nonclassical MHC class I molecules (TL) and that while CD8αα cells can bind presenting molecules at equal frequencies to CD8αβ, the β chain is essential to correct engagement of the MHC/peptide complex and to correct signaling [122
]. Further studies suggest that CD8αα represents a differentiation stage that leads to negative regulation of T-cell activation [123
]. If this is so, one could speculate that if these macaques in the above study were challenged, they would have no resistance at all.
In a more recent report, in humans, Aeras-402 was given alone to healthy volunteers. Here, the vaccine induced good CD4 and CD8 responses and evidence for multifunctional memory cells. The vaccine was safe and immunogenic, but even a single injection of this adenovirus induced seropositivity in 22% of the individuals [124
In newly ongoing trials, infants will receive BCG and then MVA85 4 months later. There are plans to compare this with Aeras-402 used as a booster vaccine, providing a head-to-head comparison of the two virus-based boosting approaches. Both boosts improve the numbers of ‘polyfunctional’ T cells, indicating that they generate some degree of memory immunity (hopefully, central memory). But why the timing of these studies? It is generally agreed that BCG protects children but the overall durability and protective longevity of the vaccine is highly variable. Therefore, is it likely that giving the boosting vaccine so soon after the BCG prime in these (barely immuno-competent) infants will have a desired or even noticeable effect? I have hypothesized that BCG causes a gradual expansion of effector memory T cells in children but this is gradually lost through attrition [125
]. If the viral vaccines expand central memory that should be a beneficial event, but what is the half-life of these cells? Would it not make more sense to wait maybe 6–8 years before boosting, when the memory response might be more amenable to effector memory to central memory transition, and the boosting effect potentially more likely to significantly extend protection?
Finally, a tremendous drawback in all these studies is the lack of an adequate biomarker of vaccine efficacy. Production of the cytokine IFN-γ was an initial approach, but this was soon questioned [126
]. More recently, it was shown that the frequency and cytokine pro-file of antigen-specific T cells, including CD4 IFN-γ responses, appeared to be poor correlates of protection [128
]. These ideas have now been replaced by the idea of poly-/multi-functional T cells, consistent with the knowledge that central memory T cells can often make IL-2 and TNF-α, as well as IFN-γ [129
]. Further bad news was obtained in a NHP animal model; this used a new aerosol model to look for immunological and clinical readouts that could be used in vaccine evaluation studies. However, neither animal survival, nor were the IFN-γ profiles induced following vaccination found to correlate with protection. Interestingly, the only data that did correlate was MRI of lungs combined with stereology [61
]. It should be noted, in this regard, that MRI seems a useful imaging technique both for small and large animal models [56
] because it can detect dead tissue containing necrosis, whereas the much more heavily promoted PET approach [69