Current challenges and opportunities for bacillus Calmette‐Guérin replacement vaccine candidates

Bacillus Calmette‐Guérin (BCG) remains the only licensed vaccine against human tuberculosis (TB). BCG is a live‐attenuated strain of Mycobacterium bovis, with limitations in efficacy against respiratory TB, the most common form of the disease responsible for transmission. However, continues to be used in the immunization programmes of different countries in the absence of another alternative. In order to improve BCG efficacy against pulmonary TB, in the current clinical TB vaccine pipeline, there are live‐attenuated TB vaccines to replace BCG. This review discusses the current status of the development of live vaccine candidates designed to replace BCG from the rational strategies and immunological challenges to its clinical trial and identify key areas in the next years considered essential to confer improved safety and efficacy over BCG.     

Therapeutic host-directed strategies to improve outcome in tuberculosis

Bacille Calmette-Guérin (BCG) is the only licenced tuberculosis (TB) vaccine, but has limited efficacy against pulmonary TB disease development and modest protection against extrapulmonary TB. Preventative antibiotic treatment for Mycobacterium tuberculosis (Mtb) infections in high-prevalence settings is unfeasible due to unclear treatment durability, drug toxicity, logistical constraints related to directly observed treatment strategy (DOTS) and the lengthy treatment protocols. Together, these factors promote non-adherence, contributing to relapse and establishment of drug-resistant Mtb strains. Although antibiotic treatment of drug-susceptible Mtb is generally effective, drug-resistant TB has a treatment efficacy below 50% and can, in a proportion, develop into progressive, untreatable disease. Other immune compromising co-infections and/or co-morbidities require more complex prevention/treatment approaches, posing huge financial burdens to national health services. Novel TB treatment strategies, such as host-directed therapeutics, are required to complement pathogen-targeted approaches. Pre-clinical studies have highlighted promising candidates that enhance endogenous pathways and/or limit destructive host responses. This review discusses promising pre-clinical candidates and forerunning compounds at advanced stages of clinical investigation in TB host-directed therapeutic (HDT) efficacy trials. Such approaches are rationalized to improve outcome in TB and shorten treatment strategies.     

Inhibition of Mycobacterial Growth In Vitro following Primary but Not Secondary Vaccination with Mycobacterium bovis BCG

Despite the widespread use of the Mycobacterium bovis BCG vaccine, there are more than 9 million new cases of tuberculosis (TB) every year, and there is an urgent need for better TB vaccines. TB vaccine candidates are selected for evaluation based in part on the detection of an antigen-specific gamma interferon (IFN-γ) response. The measurement of mycobacterial growth in blood specimens obtained from subjects immunized with investigational TB vaccines may be a better in vitro correlate of in vivo vaccine efficacy. We performed a clinical study with 30 United Kingdom adults who were followed for 6 months to evaluate the abilities of both a whole-blood- and a novel peripheral blood mononuclear cell (PBMC)-based mycobacterial growth inhibition assay to measure a response to primary vaccination and revaccination with BCG. Using cryopreserved PBMCs, we observed a significant improvement in mycobacterial growth inhibition following primary vaccination but no improvement in growth inhibition following revaccination with BCG (P < 0.05). Mycobacterial growth inhibition following primary BCG vaccination was not correlated with purified protein derivative (PPD) antigen-specific IFN-γ enzyme-linked immunospot (ELISPOT) responses. We demonstrate that a mycobacterial growth inhibition assay can detect improved capacity to control growth following primary immunization, but not revaccination, with BCG. This is the first study to demonstrate that an in vitro growth inhibition assay can identify a difference in vaccine responses by comparing both primary and secondary BCG vaccinations, suggesting that in vitro growth inhibition assays may serve as better surrogates of clinical efficacy than the assays currently used for the assessment of candidate TB vaccines.     

Targeting multidrug-resistant tuberculosis (MDR-TB) by therapeutic vaccines

Tuberculosis (TB) has scourged humankind for millennia, and latent infection affects nearly one-third of today’s world population. The emergence of multidrug-resistant (MDR)-TB is a major global threat and reflects treatment failure of drug-sensitive disease. MDR-TB management is a burden for patients and society; success rates are unacceptably low with prolonged treatment duration. Mycobacterium tuberculosis (Mtb) possesses the ability to transform into a dormant state in which it can persist in the face of antimicrobial treatment and host defense. This sub-population of persisters is largely responsible for lengthy and difficult treatment. Targeting persistent bacilli could eventually improve the treatment success rate (currently 50–65 %) and shorten duration of treatment. A subset of therapies in the pipeline, termed therapeutic vaccines, use the host immune response to attack Mtb. The historical occurrence of an exacerbated host response has resulted in a negative perception of therapeutic vaccines. Thus, a renewed concept of immunotherapy is needed. We review current perspectives of immunotherapy in MDR-TB based on the knowledge of TB immunology and briefly discuss the profiles of several therapeutic vaccine products.     

Development of tuberculosis vaccines in clinical trials: Current status

Tuberculosis (TB ) is an important infectious disease worldwide. Currently, Bacillus Calmette‐Guérin (BCG ) remains the only TB vaccine licensed for human use. This TB vaccine is effective in protecting children against severe military TB but offers variable protective efficacy in adults. Therefore, new vaccines against TB are needed to overcome this serious disease. At present, around 14 TB vaccine candidates are in different phases of clinical trials. These TB vaccines in clinical evaluation can be classified into two groups including preventive pre‐ and post‐exposure vaccines: subunit vaccines (attenuated viral vectors or adjuvanted fusion proteins), and whole‐cell vaccines (genetically attenuated Mycobacterium tuberculosis (M. tb ), recombinant BCG , killed M. tb or M. vaccae ). Although, over the last two decades a great progress in the search for a more effective TB vaccine has been demonstrated there is still no replacement for the licensed BCG vaccine. This article summarizes the current status of TB vaccine development and identifies crucial gaps of research for the development of an effective TB vaccine in all age groups.     

Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis

Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by “dormant” M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ⁺/TNF⁺) and IFN-γ⁺ CD4⁺ T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.     

Selective delipidation of Mycobacterium bovis BCG enables direct pulmonary vaccination and enhances protection against Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is the leading killer due to an infectious organism. Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved against TB, however, its efficacy against pulmonary TB is poor. While BCG is currently inoculated intradermally, the natural route of M.tb infection is through the lung. Excessive lung pathology caused by pulmonary inoculation of BCG has prevented the use of this immunization route. Here, we show that selective chemical treatment of BCG with petroleum ether removes inflammatory lipids from the bacterial surface while keeping BCG viable. Pulmonary vaccination using this modified BCG attenuated inflammatory responses, prevented immunopathology of the lung, and significantly increased protection against M.tb infection in mice. We further directly linked IL-17A as the responsible contributor of improved immunity against M.tb infection. These results provide evidence that selective removal of cytotoxic lipids from the BCG surface attenuates inflammation and offers a safer and superior vaccine against TB causing less damage post-infectious challenge with M.tb.     

A murine model of tuberculosis/type 2 diabetes comorbidity for investigating the microbiome,metabolome and associated immune parameters

Tuberculosis (TB) is one of the deadliest infectious diseases in the world. The metabolic disease type 2 diabetes (T2D) significantly increases the risk of developing active TB. Effective new TB vaccine candidates and novel therapeutic interventions are required to meet the challenges of global TB eradication. Recent evidence suggests that the microbiota plays a significant role in how the host responds to infection, injury and neoplastic changes. Animal models that closely reflect human physiology are crucial in assessing new treatments and to decipher the underlying immunological defects responsible for increased TB susceptibility in comorbid patients. In this study, using a diet‐induced murine T2D model that reflects the etiopathogenesis of clinical T2D and increased TB susceptibility, we investigated how the intestinal microbiota may impact the development of T2D, and how the gut microbial composition changes following a very low‐dose aerosol infection with Mycobacterium tuberculosis (Mtb). Our data revealed a substantial intestinal microbiota dysbiosis in T2D mice compared to non‐diabetic animals. The observed differences were comparable to previous clinical reports in TB patients, in which it was shown that Mtb infection causes rapid loss of microbial diversity. Furthermore, diversity index and principle component analyses demonstrated distinct clustering of Mtb‐infected non‐diabetic mice vs. Mtb‐infected T2D mice. Our findings support a broad applicability of T2D mice as a tractable small animal model for studying distinct immune parameters, microbiota and the immune‐metabolome of TB/T2D comorbidity. This model may also enable answers to be found to critical outstanding questions about targeted interventions of the gut microbiota and the gut‐lung axis.     

Vaccination against tuberculosis: How can we better BCG?

Tuberculosis remains one of the most significant human diseases of the developing world, accounting for 3800 worldwide deaths per day. Although we currently have a vaccine for tuberculosis, BCG, this is insufficient at protecting from adult pulmonary tuberculosis in the parts of the world where a good vaccine is most needed. This has prompted the search for new vaccination strategies that can protect better than BCG, or can boost BCG-induced immunity. We discuss these subjects in line with what is known of the immune responses to BCG and Mycobacterium tuberculosis – the etiological agent of the disease, as well as the particular difficulties facing development of new vaccines against tuberculosis. A greater understanding of the factors constituting optimal protection against Mycobacterium tuberculosis infection, as well as which pathogenic factors facilitate active disease, will accelerate the delivery of safe vaccines able to restrict active tuberculosis and thus impede contagion.     

Pulmonary Mycobacterium bovis BCG Vaccination Confers Dose-Dependent Superior Protection Compared to That of Subcutaneous Vaccination

Worldwide, the Mycobacterium bovis BCG vaccine is one of the most widely used vaccines. However, it appears to be ineffective in preventing pulmonary tuberculosis. Here, we show that pulmonary BCG vaccination of mice with a broad dose range provides superior protection against Mycobacterium tuberculosis challenge compared to that of subcutaneous vaccination.     

Evaluation of specific humoral immune response and cross reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigens induced in mice immunized with liposomes composed of total lipids extracted from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

The development of a new tuberculosis (TB) vaccine has become one of the main objectives of the scientific community. Protein antigens have been widely explored as subunit TB vaccines, however lipid antigens could be equally important to be used or included in such a vaccine. The aim of this study was to demonstrate the potential of a liposome formulation composed of an extract of lipids from Mycobacterium smegmatis (Ms) as a TB vaccine candidate. We evaluated the immunogenicity of this formulation as well as the cross reactive response against antigens from Mycobacterium tuberculosis (MTb) in BALB/c mice. We determined the anti-liposome IgG response in sera from TB patients and from healthy subjects who displayed a positive (PPD+) or negative (PPD-) tuberculin skin test. A significant increase in anti-liposome IgG (p<0.05) was detected in animals immunized with Bacille Calmette-Guérin (BCG) compared with all groups, and in the group immunized with liposomes from Ms (LMs) compared to animals immunized with either LMs adjuvanted with aluminium (LMs-A) or the negative control group (phosphate buffered saline, PBS) respectively. With respect to the cross reactive response against a cocktail of cell wall antigens (CWA) from MTb, significantly higher IgG levels were observed in animals immunized with BCG and LMs compared to negative controls and either, aluminium-adjuvanted liposomes (LMs-A) or montanide (LMs-M) (p<0.05). Furthermore, the anti-liposome IgG response was significantly superior in sera from pulmonary TB patients compared to PPD+ and PPD- healthy subjects (p<0.001) suggesting the expression of these antigens in vivo during active MTb infection. The results obtained provide some evidence for the potential use of liposomes containing total lipid extracts of Ms as a TB vaccine candidate.     

Manipulation of BCG vaccine: a double-edged sword

Mycobacterium bovis Bacillus Calmette–Guérin (BCG), an attenuated vaccine derived from M. bovis, is the only licensed vaccine against tuberculosis (TB). Despite its protection against TB in children, the protective efficacy in pulmonary TB is variable in adolescents and adults. In spite of the current knowledge of molecular biology, immunology and cell biology, infectious diseases such as TB and HIV/AIDS are still challenges for the scientific community. Genetic manipulation facilitates the construction of recombinant BCG (rBCG) vaccine that can be used as a highly immunogenic vaccine against TB with an improved safety profile, but, still, the manipulation of BCG vaccine to improve efficacy should be carefully considered, as it can bring in both favourable and unfavourable effects. The purpose of this review is not to comprehensively review the interaction between microorganisms and host cells in order to use rBCG expressing M. tuberculosis (Mtb) immunodominant antigens that are available in the public domain, but, rather, to also discuss the limitations of rBCG vaccine, expressing heterologous antigens, during manipulation that pave the way for a promising new vaccine approach.     

Chemokines in tuberculosis: The good,the bad and the ugly

Mycobacterium tuberculosis (Mtb) infects about one-third of the world's population, with a majority of infected individuals exhibiting latent asymptomatic infection, while 5–10% of infected individuals progress to active pulmonary disease. Research in the past two decades has elucidated critical host immune mechanisms that mediate Mtb control. Among these, chemokines have been associated with numerous key processes that lead to Mtb containment, from recruitment of myeloid cells into the lung to activation of adaptive immunity, formation of protective granulomas and vaccine recall responses. However, imbalances in several key chemokine mediators can alter the delicate balance of cytokines and cellular responses that promote mycobacterial containment, instead precipitating terminal tissue destruction and spread of Mtb infection. In this review, we will describe recent insights in the involvement of chemokines in host responses to Mtb infection and Mtb containment (the good), chemokines contributing to inflammation during TB (the bad), and the role of chemokines in driving cavitation and lung pathology (the ugly).     

A review of the literature on the economics of vaccination against TB

The BCG vaccine was introduced in 1921 and remains the only licensed vaccine for the prevention of TB worldwide. Despite its extensive use, the BCG vaccine lacks the ability to fully control the TB-endemic and -pandemic situations. The BCG vaccine is most effective in preventing pediatric TB, in particular, miliary TB and tuberculous meningitis. However, it has a limited effect in preventing pulmonary TB, which occurs more frequently in adults. BCG vaccination has now been implemented in more than 157 countries worldwide. For various countries, the benefits of vaccination are only limited and potentially not cost effective. The International Union Against Tuberculosis and Lung Diseases had set the criteria for discontinuation of BCG vaccination in 1994. This decision, however, was not based on economic considerations. Many developed countries have met the criteria set by the International Union Against Tuberculosis and Lung Disease and stopped universal BCG vaccination. For developing countries, the BCG vaccine is still an effective intervention in protecting young children from TB infection. A lot of effort has been spent on R&D of new TB vaccines, the first of which are expected to be available within 5-7 years from now. Novel TB vaccines are expected to be better and more effective than the current BCG vaccine and should provide a viable strategy in controlling TB morbidity and mortality. In this review, the aim is to explore economic evaluations that have been carried out for vaccination against TB worldwide. In addition to epidemiological evidence, economic evidence can play a crucial role in supporting the governments of countries in making proper public health decisions on BCG vaccination policies, in particular, to implement, continue, or discontinue.     

Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection

Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen‐specific IFN‐γ and IL‐2‐expressing CD4⁺ and CD8⁺ T cells. In exosome‐vaccinated mice, there was a similar T_H1 immune response but a more limited T_H2 response compared to BCG‐vaccinated mice. Using a low‐dose M. tuberculosis mouse aerosol infection model, exosomes from CFP‐treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell‐free vaccine against an M. tuberculosis infection.     

Immune requirements for protective Th17 recall responses to Mycobacterium tuberculosis challenge

Tuberculosis (TB) vaccine development has focused largely on targeting T helper type 1 (Th1) cells. However, despite inducing Th1 cells, the recombinant TB vaccine MVA85A failed to enhance protection against TB disease in humans. In recent years, Th17 cells have emerged as key players in vaccine-induced protection against TB. However, the exact cytokine and immune requirements that enable Th17-induced recall protection remain unclear. In this study, we have investigated the requirements for Th17 cell-induced recall protection against Mycobacterium tuberculosis (Mtb) challenge by utilizing a tractable adoptive transfer model in mice. We demonstrate that adoptive transfer of Mtb-specific Th17 cells into naive hosts, and upon Mtb challenge, results in Th17 recall responses that confer protection at levels similar to vaccination strategies. Importantly, although interleukin (IL)-23 is critical, IL-12 and IL-21 are dispensable for protective Th17 recall responses. Unexpectedly, we demonstrate that interferon-γ (IFN-γ) produced by adoptively transferred Th17 cells impairs long-lasting protective recall immunity against Mtb challenge. In contrast, CXCR5 expression is crucial for localization of Th17 cells near macrophages within well-formed B-cell follicles to mediate Mtb control. Thus, our data identify new immune characteristics that can be harnessed to improve Th17 recall responses for enhancing vaccine design against TB.     

Tuberculosis: vaccines in the pipeline

TB is presenting new challenges as a global health problem, especially with new threats of HIV coinfection and multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis. The current TB vaccine, Mycobacterium bovis bacillus Calmette-Guerin (BCG), is the most widely used vaccine worldwide but its efficacy against pulmonary TB in adults in many high-burden countries is limited. Different vaccine strategies will probably be required for the various needs that exist within a population in which some individuals have been previously immunized with BCG, coinfected with HIV and/or latently infected with M. tuberculosis. In the last 15 years, new strategies to improve or replace BCG in the laboratory have led to several promising vaccine candidates that are actively being evaluated in human clinical trials. Some of these new vaccines may eventually be recommended for travelers to TB high-burden countries. This paper summarizes the progress of vaccine candidates in animal models to improve, replace or augment BCG vaccination.     

DAP10 contributes to CD8(+) T cell-mediated cytotoxic effector mechanisms during Mycobacterium tuberculosis infection

The activating C-type lectin-like receptor NKG2D, which is expressed by mouse NK cells and activated CD8 T cells, was previously demonstrated to be involved in tumor rejection and as a defense mechanism against viral and bacterial infections. Because CD8 T cells are important for protective immune responses during chronic Mycobacterium tuberculosis (Mtb) infection and represent a promising target for new vaccine strategies to prevent human pulmonary tuberculosis (TB), we studied the immune response in mice deficient for the NKG2D adapter molecule DAP10 during experimental TB. After aerosol infection, DAP10-defcient mice displayed an unimpaired recruitment, activation and development of antigen-specific CD8 T cells. Whereas the frequency of interferon-gamma-producing CD8 T cells from Mtb-infected DAP10-defcient mice was not affected, CD8 T cell-mediated cytotoxicity was significantly reduced in the absence of DAP10. The loss of cytotoxic activity in DAP10-deficient CD8 T cells was associated with an impaired release of cytotoxic granules. Together, our results suggest that during Mtb infection DAP10 is required for maximal cytolytic activity of CD8 T cells.     

Interleukin-17-dependent CXCL13 mediates mucosal vaccine–induced immunity against tuberculosis

The variable efficacy of tuberculosis (TB) vaccines and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) emphasize the urgency for not only generating new and more effective vaccines against TB but also understanding the underlying mechanisms that mediate vaccine-induced protection. We demonstrate that mucosal adjuvants, such as type II heat labile enterotoxin (LT-IIb), delivered through the mucosal route induce pulmonary Mtb-specific T helper type 17 (Th17) responses and provide vaccine-induced protection against Mtb infection. Importantly, protection is interferon-γ (IFNγ)-independent but interleukin-17 (IL-17)-dependent. Our data show that IL-17 mediates C-X-C motif chemokine ligand 13 (CXCL13) induction in the lung for strategic localization of proinflammatory cytokine-producing CXCR5⁺ (C-X-C motif chemokine receptor 5-positive) T cells within lymphoid structures, thereby promoting early and efficient macrophage activation and the control of Mtb. Our studies highlight the potential value of targeting the IL-17–CXCL13 pathway rather than the IFNγ pathway as a new strategy to improve mucosal vaccines against TB.