Tuberculosis vaccines: opportunities and challenges
Bingdong Zhu MD1, Hazel M. Dockrell BA, PhD 2, Tom H. M. Ottenhoff MD, PhD 3, Thomas G. Evans MD4, Ying Zhang MD, PhD 5
1, Lanzhou Center for Tuberculosis Research & Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University, Lanzhou, China, 730000.
2, Department of Immunology and Infection and Tuberculosis Centre, London School of Hygiene & Tropical Medicine, London WC1E 7HT, United Kingdom.
3, Department of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands
4, Vaccitech, Oxford, UK
5, Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA
AUTHORS’ CONTRIBUTIONS:
BZ wrote novel vaccine development and immune memory; HD wrote BCG vaccination and biomarkers for TB vaccine; TO and HD wrote immune-profiling correlates of protection; TE wrote clinical trials; HD and YZ wrote novel approaches to evaluate TB vaccines; YZ organized and revised whole manuscript. All authors contributed to writing status of TB vaccines and revising the manuscript.
ABSTRACT: (208 words)
Tuberculosis is a serious disease around the world. BCG is the only tuberculosis vaccine licensed for use in human beings, and is effective in protecting infants and children against severe miliary and meningeal tuberculosis. However, BCG’s protective efficacy is variable in adults and wanes with time since vaccination. Novel candidate TB vaccines being developed include whole cell vaccines (recombinant BCG, attenuated M. tuberculosis, killed M. tuberculosis or M. vaccae), adjuvanted protein subunit vaccines, viral vector-delivered subunit vaccines, plasmid DNA vaccines, RNA based vaccines etc. At least 14 novel TB vaccine candidates are now in clinical trials, including killed M. vaccae, recombinant BCG ΔureC::hly, adjuvanted fusion proteins M72 and H56, and viral vectored MVA85A.
Unfortunately, in TB there are no correlates of vaccine-induced protection although cell-mediated immune responses such as IFN-γ production are widely used to assess vaccine immunogenicity. Recent studies have suggested that central memory T cells and local secreted IgA correlate with protection against TB disease. Clinical TB vaccine efficacy trials should invest in identifying correlates of protection, and evaluate new TB biomarkers emerging from human and animal studies. Thus, accumulating new knowledge on M. tuberculosis antigens and immune profiles correlating with protection or disease risk will be of great help in designing next generation TB vaccines.
Short title: (40 characters maximum including spaces) Novel TB vaccines and evaluation
Keywords： Tuberculosis, Vaccines, Biomarkers, Immunity, Clinical Trials
1. Status of tuberculosis vaccines
BCG, the only tuberculosis (TB) vaccine licensed in human beings, is effective in protecting infants and children against severe miliary and meningeal tuberculosis, although its protection is variably lost in adults1. The reason for the low protective efficacy in adult might be that BCG induced effector memory T cells (TEM), which may protect humans for 10-15 years but gradually wane2. The protective effect of BCG varies geographically, possibly due to sensitization by environmental mycobacteria or to pre-exposure to Mycobacterium tuberculosis (M. tuberculosis) before vaccination3. A systematic review found a protective efficacy of 19% against infection among vaccinated children after exposure, and 58% protection against progression to disease among those infected as compared with unvaccinated children4. A retrospective population-based cohort study in Norwegian-born individuals showed that the BCG vaccine effectiveness against pulmonary tuberculosis up to 9 years was 67%, 10-19 years was 63%, 20-29 years was 50%, and could induce protection for as long as 50 years5, 6.
Understanding protective immunity against M. tuberculosis infection and disease is complex. It is still unclear which antigens and which immune cells and mechanisms provide the most effective immunity, preferably sterilizing immunity, against M.
tuberculosis. However, cell-medicated immune mechanisms, including Th1 type CD4
T cells, CD8 T cells, γδ T cells, and CD1 restricted and HLA-E restricted T cells7 have all been proven to have the capability to kill mycobacteria inside macrophages8. Humoral immunity, especially secretary IgA against cell wall components of M.
tuberculosis, may also contribute to blocking M. tuberculosis binding to epithelial
cells and macrophages, and thus take part in the protective immune response to M.
tuberculosis9, 10. Based on our limited knowledge on TB immunity, most novel TB vaccines are designed to induce cell-mediated immunity with desired additional humoral immunity. At least 14 novel TB vaccine candidates are now in clinical trials, as reported at July 2017 (see Figure 1).
2. Antigen screening and novel vaccine development 2.1 Protein antigen screening and subunit vaccines
M. tuberculosis expresses about 4000 proteins, many of which contain predicted
CD4/CD8 T-cell epitopes recognized by αβ type T cell receptors, and stimulate immune responses to infected macrophages11 (IE-DB data base, http://www.iedb.org).
The antigens found in current subunit vaccines were selected mainly according to their immunodominance in animal or human studies, as well as from differential expression between those latently infected without progression and those with active TB disease. Most are secreted proteins, including ESAT6, MPT64, Ag85B, and Ag85A, and cell wall proteins such as heat shock proteins (HSPs), etc.12. In recent years, Aagaard et.al reported that besides ESAT6 and CFP10, ESX or type VII secretion (T7S) dimer substrates EsxD-EsxC, EsxG-EsxH, and EsxW-EsxV were
highly immunogenic. Combining these in a fusion protein, H65, allowed induction of protection equivalent to BCG, with the advantage of high predicted population coverage without interfering with current ESAT-6 and CFP10-based diagnostics13. Besides secreted antigens, the cell wall-associated PE protein based subunit vaccine M72 protein 14 and the hemaglutinin HBHA15 have also had promising protective effect in mouse models. Moreover, HSPs showed both preventive and therapeutic effects against latent infection in mice16. Bi and colleagues expressed 90% of M.
tuberculosis proteins in yeast, made a proteome microarray and screened 20
candidates using an IFN-γ release assay17. Liu and colleagues also expressed and purified 1250 M. tuberculosis proteins in E. coli and evaluated humoral and cellular immune responses in human samples. They identified four protein candidates with high immunogenicity: Rv0232, Rv1031, Rv1198, and Rv201618.
In a different approach, elution of HLA-bound peptides from M. tuberculosis infected antigen presenting cells was pursued19, 20. This unbiased approach helped to identify new M. tuberculosis antigens presented during actual M. tuberculosis infection.
Furthermore, using unbiased transcriptomic analyses M. tuberculosis antigens expressed at the site of infection, the lung, were identified. These so-called in vivo expressed (IVE) M. tuberculosis antigens were highly expressed in the lungs of susceptible mice21. Using additional bioinformatics tools, many new antigens among these identified candidates were found that were able to stimulate both conventional and unconventional T-cells from latently infected individuals, including T cells
producing cytokines other than IFN-γ22.
The problem of antigen screening is complicated due to different phases of M.
tuberculosis infection, particularly its ability to enter a stage of chronic and
latent/persistent infection. During these phases of infection, ESAT6 is highly expressed, but Ag85B is expressed mainly in the early stage23. Other work has shown that some latency antigens are potent T cell stimulatory antigens for human T cells24, and have protective effects in animal models of TB. Moreover, the mycobacteria in the body consist of bacteria in different stages of metabolic activity with different antigen expression, including both replicating and dormant bacteria that maintain the ability to transition between these two states as in the Yin-Yang model25. For this reason, TB subunit vaccines should select antigens from different metabolic stages to provide broader immunity. The novel multi-stage subunit vaccines such as H56 (which contains latency antigen Rv2660c besides the replicating/secreted antigens Ag85B and ESAT-6 26)[17, 18], as well as ID9327, and LT7028, all consist of M.
tuberculosis antigens selected from different stages of bacteria (Table 1). In general,
they have induced higher protective efficacy than those only consisting of antigens highly expressed in replicating bacteria. H56, a first example of such a rationally designed TB subunit vaccine, has now entered multiple clinical trials and induced promising immune responses29.
2.2 Adjuvants for TB vaccination
As current adjuvant in use, aluminum based compounds, is driving humoral but not cellular immunity, novel adjuvants may be necessary for new TB protein-based subunit vaccines to induce cell-mediated Th1 type immune responses. Most adjuvants under investigation for this purpose are complex formulations consisting of vehicle and immunostimulator. Cationic liposomal dimethyldioctadecylammonium (DDA) and Quillaja saponaria fraction (QS21) have been used as vehicles to deliver and present antigens to induce Th1-type cell-mediated immune response. CAF01 adjuvant is composed of DDA and trehalose 6,6’-dibehenate (TDB), a synthetic analogue of the mycobacterial cell wall component trehalose 6,6'-dimycolate (TDM)34. Zhu and his team developed a new adjuvant DPC consisting of three components: DDA, Poly I:C, a ligand of TLR3 receptor, and cholesterol, which is used to enhance rigidity of lipid bilayer and make the adjuvant stable33. Some adjuvants used in clinical trials are listed in Table 1.
2.3 “Alternative antigens”: glycolipid components
The M. tuberculosis cell envelope contains a large abundance of lipids, especially glycolipids35. Mycobacterial lipid antigens can activate T cells via CD1-mediated antigen presentation. CD1d is conserved between mice and men, and activates invariant natural killer like T cells, whereas CD1a, b and c present antigens to
“adaptive” T cell subsets, including germline-encoded mycolyl-reactive (GEM) T cells36. Examples of such lipid molecules are mycolic acids, glucose monomycolate (GMM)37, diacylated sulfoglycolipids, or mannosyl-phosphatidylinositol-based
glycolipids, which were found to be presented by CD1b38. They were also reported to elicit cutaneous DTH responses39 and mediate protective immunity against mycobacterial infection. Although the immunogenicity of M. tuberculosis lipid components has been known for many years and some components have been identified, more efforts still need to be made to identify the most effective components, determine how to prepare in large quantities by purification or chemical synthesis.
2.4 Virus-vectored subunit vaccines
Live, attenuated non-replicating viruses have been genetically engineered to deliver foreign antigens. Viral vectors can deliver antigen encoding genes into host cells efficiently, trigger intracellular production of the antigen in vivo, and induce both CD4+ and CD8+ T cell-mediated immunity. Virus-vectored subunit vaccines have the potential to activating innate immunity and do not need additional adjuvants40. MVA85A, a modified vaccinia Ankara virus delivering Ag85A, has been a leading vectored vaccine entering clinical trials41. Other viruses such as adenovirus type 35, adenovirus type 5, Sendai virus, lentivirus, parainfluenza virus type 2, parainfluenza virus 5, influenza virus, vesicular stomatitis virus (VSV), chimpanzee adenovirus, and murine cytomegalovirus have also been exploited to construct TB subunit vaccines.
Most of the early generation vaccines used M. tuberculosis antigens Ag85A, Ag85B, ESAT6, and Mtb10.440. New virus-vectored vaccines also contain antigens from replicating bacteria and dormant bacteria, as discussed above (section 2.1), to induce
broader and multi-stage specific immunity. For example, recombinant VSV-846 presents the antigens Rv3615c, Mtb10.4, and Rv2660c42. Another advantage of virus- vectored vaccines is that some viral vectors, including influenza A virus, parainfluenza, Sendai virus and MVA, can be administrated by the intranasal route and induce mucosal antigen-specific Th1 type immune responses in the lung tissue40. Clinical trials showed that MVA85A and AERAS-402, an adenovirus type 35- vectored tuberculosis vaccine containing DNA coding for Ag85A, Ag85B, and TB10.4 were safe in BCG-vaccinated healthy adults43, 44, further documenting safety and acceptability of virus-vectored subunit vaccine application in human beings.
However, virus-vectored vaccines also face some challenges. First, pre-existing antibodies against the viral vehicle in the immune host might inhibit their activity. In addition, virus-vectored vaccines might induce less central memory T cells (TCM) than adjuvanted protein vaccines, given their extremely high immunogenicity and tendency to drive terminally differentiated T-cells. By comparing the immunogenicity of adjuvanted protein subunit vaccine vs. MVA vectored vaccine, both delivering the same fusion protein H28 which consisted of Ag85B-TB10.4-Rv2660c, immunization with H28 followed by a H28 booster vaccination induced stronger central memory T cell-mediated immune responses and a slightly more prominent reduction of disease and pathology in animals when compared to H28/MVA2845. Considering the lack of efficient MVA85A vaccination mediated boosting against clinical tuberculosis in infants vaccinated with BCG41, this character of different immune responses (central vs effector memory; lung homing vs circulation T cells) induced by virus-vectored
vaccines needs further investigation.
2.5 Whole cell TB vaccines
Live attenuated whole cell vaccines (WCVs) include recombinant BCG and attenuated M. tuberculosis and somewhat akin to killed whole-cell vaccines. Live attenuated WCVs have potential advantages over protein-adjuvant and viral-vectored subunit vaccines for their comprehensive antigen repertoire and their similarity to natural infection. The killed bacteria such as RUT1, detoxified and fragmented M.
tuberculosis cells, and heat-killed or irradiated M. vaccae have mainly been
investigated for therapeutic effects (Figure 1). Recombinant BCG strains or modified BCG delivery has thus been employed to improve and eventually replace BCG. A safer BCG for use in infants with HIV infection would also be an advantage.
Recombinant BCG approaches include the following two strategies:
(1) Overexpression of M. tuberculosis immunodominant antigens already present in BCG or antigens from M. tuberculosis absent from BCG. For example, rBCG30, highly expressing Ag85B, was shown to be more potent than BCG against a M. bovis challenge in animals46. Recombinant BCG expressing antigen Ag85B and Rv3425, the latter being absent from M.tuberculosis, improved the protection against M.tuberculosis challenge in mice47. A cocktail of recombinant BCG (rBCG) strains, which overexpress multistage antigens Ag85A, Ag85B, and HspX improved the protective effect of BCG compared to parental BCG48. Furthermore, recombinant BCG expressing RD1 antigens from M. tuberculosis or M. marinum are currently
being evaluated.
(2) BCG modifications for more effective antigen presentation and/or shorter persistence of BCG in the host, which aims to induce more long-lived memory T-cells with increased safety in the immunocompromised host49. One representative of this type is recombinant BCG ΔureC::hly, which expresses membrane-perforating listeriolysin (Hly) of Listeria monocytogenes and is devoid of urease C. This rBCG (VPM1002) elicits a profound type 17 cytokine response and abundant type 1 cytokines after antigens re-stimulation, and induced superior protection in mice over parental BCG, with about a 10-fold further reduced bacterial burden following M.
tuberculosis challenge50 at late time points, including against Beijing M. tuberculosis strains against which BCG itself had virtually no effect. More studies have shown that VPM1002 may induce more central memory T cells51 and induce autophagy52. rBCG VPM1002 has entered into Phase 2 clinical trials in both HIV-exposed infants, and as a method for preventing recurrence after initially successful TB treatment53 (see Figure 1).
With the accumulating knowledge on pathogenesis of M. tuberculosis, many M.
tuberculosis virulence genes have been identified. By deleting these bacterial
virulence-related genes, an attenuated M. tuberculosis vaccine can be developed, while maintaining the virtually full repertoire of M. tuberculosis antigens compared to native M. tuberculosis. MTBVAC, an attenuated M. tuberculosis with deleted phoP and fadD26, is the first live M. tuberculosis-based vaccine reaching clinical
assessment. A recent phase I trial showed that MTBVAC was at least as immunogenic as BCG and had similar safety profile as BCG54. Further trials are being planned.
3. Clinical trials of novel TB vaccines
The TB vaccines in clinical trials are shown in Figure 1. A Phase IIB trial in 3600 HIV-uninfected, adults latently infected with M. tuberculosis is under way in three African countries using the GSK M72 adjuvanted fusion protein vaccine. This study should further our understanding of the potential role of CD4+ TH-1 induced immunological responses in preventing the development of disease in latently infected individuals. In addition, a large 10,000 person study to prevent TB disease is under way in Guanxi province China using a killed non-tuberculous mycobacterial vaccine given as a series of multiple vaccinations to latently infected adults with PPD skin test induration greater than 15 mm.
In addition to these large-scale PoC trials using disease endpoints, a new set of human studies are under way, based on the use of innovative trials designs using more focused populations specifically selected to reduce sample size. The first of these trials is evaluating whether a novel vaccine (H4/IC31) or the use of BCG re- vaccination can prevent sustained infection by M. tuberculosis (as opposed to disease)55. The trial uses novel blood tests in which BCG vaccination does not interfere with the test result, and is enrolling adolescents in South Africa with a high rate of incident M. tuberculosis infection, thereby requiring only 330 subjects per arm
rather than the two thousand or more needed in the classic PoC trials. Results are expected mid-2017. The second innovative design is to evaluate a vaccine’s ability to prevent the 4-6% relapse and/or reinfection rate typically observed following successful treatment of active TB. A “Prevention of Recurrence” (POR) trial using the ID93 candidate will likely begin shortly and a similar POR study is being undertaken in India using the recombinant BCG candidate VPM1002.
Other innovative leads that will be aggressively pursued over the next 5 years will include the use of aerosolized candidates, either alone or in combination. The protection apparently afforded by latent tuberculosis infection of the lung has led to renewed interest in aerosolized whole cell TB vaccines, and two recent small studies in humans have shown that aerosolization of high dose BCG (105 CFU) is well tolerated (H McShane, personal communication). The combination of aerosolized or intramuscular adenoviral vectored vaccines followed by modified vaccinia virus Ankara has been especially promising in both preclinical models and in early human trials44, 56, 57. CMV-vectored candidates will likely move forward in both the TB and HIV area, as they induce prolonged and high levels of effector T cells in the mucosal location where the pathogen first encounters the human host, although a risk for overactivation is a potential detriment. The value of a number of novel BCG replacement strategies will also become clearer.
4. Immune memory and vaccine design
4.1 Memory T cell subsets
Following antigen stimulation, T cells are activated and develop into different subsets including effector T cells (TEFF), effector memory T cells, central memory T cells and tissue-resident memory T cells (TRM), etc. TEM broadly migrate between peripheral tissues, the blood, and the spleen, while TCM are restricted to the secondary lymphoid tissues and blood. Following antigen re-stimulation, TCM can produce interleukin (IL)- 2, proliferate extensively and develop into TEM, whereas TEM are less proliferative and develop into full blown effector cells, producing effector cytokines such as IFN-γ58. The purpose of vaccination is to induce long-term immunological memory that mediates protection from infection. Therefore, memory T cells would be expected to be an important correlate of immune protection against TB. According to the character of memory T cells, it is believed that TEM mediate immediate and short-term immune protection while TCM mediate long-term protection59. Lung TRM also play a role in immune protection against M. tuberculosis respiratory tract infection, which can be induced by mucosal immunization60.
4.2 TB vaccine-induced immune memory
Following BCG vaccination of human newborns, the antigen specific CD4+ T-cell response peaked 6–10 weeks after vaccination and gradually waned over the first year of life, displaying a predominant CD45RA–CCR7+ central memory phenotype but with a characteristic of effector memory cells on cytokine and cytotoxic marker expression2. Another study showed that the BCG Pasteur vaccine strain survived for
300 days following vaccination, and with the resulting persistent stimulation of immune cells, BCG vaccination induced more TEM than TCM61. TEM could respond quickly following pathogen infection but could not maintain persistent memory.
Recombinant BCG ΔureC::hly has a shorter persistence time in the host to 30 days, which may be beneficial to inducing higher proportions and numbers of antigen- specific central memory CD4+ T cells than BCG51. Adjuvanted protein subunit vaccines also persist only for a short time in host and were found to have the capability to activate TCM immune responses and to induce long-term protective efficacy62. Notwithstanding this data, there is discussion on the protective effect of memory T cells. Steigler used a murine memory T-cell depletion model and found that BCG-mediated protection was independent of memory T cells40. Therefore, more studies are needed to investigate the correlation between memory T cells and protection, especially in the human population.
5. Biomarkers for use in TB vaccine trials
There are currently no effective biomarkers that predict vaccine efficacy63. Most TB vaccine developers have used assays detecting IFN-γ to assess vaccine
immunogenicity. This is based on the assumption that memory T cells making IFN-γ are necessary (even though not sufficient) for protection. IFN-γ can be detected in stimulated supernatants of whole blood or peripheral blood mononuclear cells (PBMC), using ELISPOT assays or using intracellular cytokine staining followed by flow cytometry. More complex assays using multi-parameter bead arrays can be used
to assess a larger profile of secreted cytokines and chemokines, and have revealed changes in the bio-signature induced by BCG in different settings64. The recent move from intracellular cytokine staining analysed by flow cytometry (which has largely concentrated on IFN-γ, IL-2, TNF-α and IL-17 in combination with limited T cell or memory markers) to CyTOF65 enabling analysis of a larger number of cytokines (coupled with more phenotypic markers including more detailed analysis of the memory T cell subsets) will also yield greater information on the type of immune response induced by vaccines. For example, the simple ratio of
monocytes:lymphocytes splits both protected and not protected BCG vaccinated infants into 2 groups, indicating an additional source of heterogeneity in the immune responses to TB vaccines66.
In early clinical trials, vaccine developers may be reluctant to include more experimental assays that are not proven correlates of protection, and that provide a broader readout of the complex cellular changes following immunization. However the move towards “systems vaccinology” encourages analysis of antigen recognition, proposes integration of immune responses with metabolic changes67, and an in-depth analysis of the gene expression pathways, including epigenetic modifications that modulate immune responses68, not only determining the functional phenotype of cell subsets, but also in driving what is termed “trained immunity”69. BCG can induce such epigenetic changes in monocytes70 that may alter both cytokine responses and induce NK cell activation in BCG vaccinated infants71.
It is also worth noting that different biomarkers may be required for different types of vaccine. For example, live vaccines or those delivered using viral vectors may induce CD8 T cell responses as well as CD4 T cells, and assays to detect these responses must be used. Vaccines for different age groups and perhaps even different setting may also need to be “personalized”72, with associated fine tuning of any protective biomarker assays.
6. Immune-profiling based correlates of protection against TB disease
Vaccine development would be greatly facilitated if correlates of protection indicating protective efficacy were available. However, it is clear that at present we do not have assays that can identify the potential of a TB vaccine candidate to be protective in humans. The MVA85A vaccine given as a boosting vaccine to BCG vaccinated to South African infants did not significantly augment protective efficacy, despite impressive previous data showing boosting of T cell responses in volunteers and evidence that an IFN-γ ELISPOT response was induced in the infant vaccinees41. There are many possible explanations for this outcome including that BCG vaccination had not induced a significant primary immune response to Ag85A that could be boosted by MVA85A, or that Ag85A is regulated in its expression during later phase infection.
Besides correlates of risk, discussed in the accompanying chapter in this issue, the
cited large phase 2b MVA85A study in infants revealed two unexpected correlates of reduced risk of developing TB. In this study, higher levels of Ag85A IgG antibodies and higher frequencies of IFN--specific cells were associated with reduced risk of TB disease73. The association of BCG-specific IFN- secreting T cells at baseline (measured by ELISPOT, such that the source of IFN-γ could not be ascertained) with reduced risk of developing TB disease was somewhat counterintuitive initially, since Kagina et al74 in a similar population had shown a lack of correlation of BCG induced IFN-γ production by CD4 T cells with subsequent development of disease (using a short term ICS assay). However, the ICS and ELISPOT assays used are quite different, and the results are not necessarily in disagreement with each other, since much of the IFN-γ could be coming from other cells than T cells, such as NK cells or ILC. Further work should now identify the cellular source of this IFN-γ, since it may point to alternative cells involved in reducing TB risk in infants at least.
Other approaches for searching for correlates of protective immunity have focused on developing unbiased, functional measures of human protective responses. One example of such an assay is the in vitro mycobacterial growth inhibition assay which measures the ability of immune cells or whole blood to inhibit outgrowth of mycobacteria in in vitro systems75. Using this assay in BCG-vaccinated infants in the UK, there was an increased ability to control the growth of BCG following BCG vaccination, correlating with the previously recorded ability of BCG to induce significant protection against TB in the UK. Additional work showed that
polycytotoxic M. tuberculosis specific T cells recognizing M. tuberculosis lipoarabinomannan (LAM) also correlated with phenotypic protection in human TB76.
Thus, several new biomarkers of reduced TB risk are emerging from vaccine- and observational studies, which may fertilize further research to explore the underlying pathways, and inspire the development of practical tests to apply these markers to clinical studies, such as vaccine studies and other TB interventional approaches, including treatment studies. It will be important that these are tested in different settings and that the influence of co-infections and co-morbidities is considered.
7. Novel approaches and models to evaluate TB vaccines
As noted above the current TB vaccine pipeline contains vaccines of different types.
These vaccines are tested initially in mouse models and then usually in guinea pigs;
studies in non-human primates, rabbits and cattle are also used when available.
Different animal models have their advantages and limitations (Table 2). Recently, a new rabbit skin model was developed with features that reflect human lesions by producing liquefaction and has shown to be a visual and convenient model for effective evaluation of TB vaccines77. Further studies are needed to determine if the vaccine candidates identified by this rabbit skin model are more effective than those identified by other more commonly used mouse models in providing better protection or prediction of protection in humans. In addition, the route, dose, timing, schedule of vaccine immunization all will affect the development of T cell and humoral immune
responses. Furthermore, the dose, route, and timing of virulent bacteria challenging also affect the protective efficacy, which all add up to the complexity of vaccine evaluation. More studies are needed to identify the most relevant parameters for evaluating TB vaccines, in the context of systems approach to TB vaccine design and development63.
In some cases animal testing is performed comparing different vaccines head-to-head, but this is only possible through umbrella organizations such as the TuBerculosis Vaccine Initiative (TBVI; www.tbvi.eu), consortium grants such as the EU Horizon2020 funded grants TBVAC2020 (http://www.tbvi.eu/for-partners/tbvac2020/
), EMI-TB (http://emi-tb.org/) and Aeras. Such grants that can allocate funding for further vaccine development based on the outcome of such head-to-head testing can make strategic decisions about which vaccines should be supported but in general, different vaccines are developed by individual groups or laboratories.
Acknowledgements
We acknowledge funding by EC FP7 ADITEC (Grant Agreement No. 280873); EC HORIZON2020 TBVAC2020 (Grant Agreement No. 643381); EC ITN FP7
VACTRAIN project; EC FP7 EURIPRED (FP7-INFRA-2012 Grant Agreement No.
312661); The Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038); STW (Grant Agreement No. 13259). Research reported in this publication was supported by the National Institute of Allergy And Infectious
Diseases of the National Institutes of Health under Award Number R21AI127133.
B.Z. is supported by the National Natural Science Foundation of China (31470895) and National Major Science and Technology Projects of China (2012ZX10003-008- 006). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or any funder. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The Authors:
Bingdong Zhu MD, Professor and Director of Institute of Pathogen Biology, School of Basic Medical Sciences, Lanzhou University; major research interest in tuberculosis subunit vaccine, immunology of vaccine, and tuberculosis immunopathology.
Hazel M Dockrell, BA, PhD, Department of Immunology and Infection and Tuberculosis Centre, London School of Hygiene & Tropical Medicine, London WC1E 7HT, United Kingdom; major research interest in immunity to mycobacteria including correlates of protection for use in vaccine trials and biomarkers indicating the impact of TB treatment, including the impact of co-infections and co-morbidity.
Tom H. M. Ottenhoff, MD, PhD, Department of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands; Head Laboratory Infectious
Diseases; major research interest in immunology and immunogenetics of mycobacterial infectious diseases, including tuberculosis and leprosy. See https://www.lumc.nl/org/infectieziekten/medewerkers/tomottenhoff
Thomas Evans, MD, Vaccitech; major research interest is the product development of vaccines that will impact global health, and led Aeras, a TB vaccine Product Development Partnership, as CEO from 2013-2015
Ying Zhang, MD, PhD, Professor of Microbiology and Immunology, Johns Hopkins University and his research interests include tuberculosis pathogenesis, drug resistance, persistence and host responses to mycobacterial infections.
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Table 1 Subunit vaccine candidates with promising protective effects
Vaccines Antigens Adjuvant Effect Referenc
e
H56 Ag85B
ESAT-6 Rv2660c
IC31, combining a cationic peptide (KLKL(5)KLK) and a synthetic
oligodeoxynucleotide
(ODN1a), which is a Toll-like receptor 9 agonist
Preventive Post- exposure Therapeutic
26, 29
ID93 Rv2608
Rv3619 Rv3620 Rv1813
GLA-SE, a synthetic TLR4 agonist GLA formulated in the squalene-in-water stable emulsion (SE)
Preventive Post- exposure
30
M72 Mtb39A
Mtb32A
AS01E, containing
immunostimulants MPL and Quillaja saponaria fraction (QS21)
Preventive/
Post- exposure
31, 32
LT70 ESAT-6
Ag85B Mtb8.4 Rv2626c
DPC, consisting of DDA, Poly I:C, a ligand of TLR3 receptor, and cholesterol
Preventive Therapeutic
28, 33
Table 2. Animal models for TB vaccine evaluation
Animal Model
Advantages Disadvantages Referenc
e Mice Producing a strong cell-
mediated immunity (CMI) during M. tuberculosis infection
Producing weak delayed- type hypersensitivity (DTH), and inducing granuloma but not necrosis and cavity; In addition, their genetic susceptibility determines the pattern of lung pathology.
78-80
Guinea Pig
Producing a strong DTH response and inducing caseous necrosis
Producing too weak CMI responses to control the TB infection; generally being
unable to induce
liquefaction and cavity;
limitation of immunological reagents; higher cost compared with mice
78, 79, 81
Rabbits Generating strong levels of CMI and DTH, and inducing
caseous necrosis,
liquefaction and pulmonary cavity as in human beings; A
Containing two kinds:
resistance or susceptibility to TB, and the resistant rabbits can produce high level of immune response
81-83
new visual and convenient rabbit skin model was recently developed to evaluate new vaccine candidates.
and pathological features just like human beings; the susceptible rabbits are easy to form hematogenous spread and die. In addition, the use of rabbit model is limited due to the lack of the immunological reagents.
Cattle Natural host for M. bovis, which produce similar immune responses as in human beings
Large animal with high cost on containment facilities and experiment
84
Non- human primates
The genomes, physiology and immune systems are similar to those of human beings
High cost on containment facilities and experiment
85
Box1 Factors affecting the variability in protection and immunogenicity with BCG
Exposure to environmental mycobacteria or even to M. tuberculosis itself affects
Post-vaccination exposure to mycobacteria could reduce immunological memory
BCG mainly induces effector memory T cells, which may gradually wane and lose protection.
The role of latent TB infection in the mothers of vaccinated infants, and of BCG scars in the mothers had a temporary effect
The other factors: season, coinfections with CMV, HIV and helminths, nutrition, etc.
Figure 1. Global clinical pipeline of TB vaccine candidates
