Tuberculosis Vaccine Research Research Articles

Immunopathogenesis of tuberculosis and novel mechanisms of vaccine activity

The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brought together a wide and diverse community involved in tuberculosis vaccine research and development to discuss the current status of, and future directions for this critical effort. This paper summarizes the sessions on Immunopathogenesis of Tuberculosis, and Immunopathogenesis and Novel Mechanisms of Vaccine Activity. Summaries of all sessions from the 4th Global Forum are compiled in a special supplement of Tuberculosis.

Novel approaches to preclinical research and TB vaccine development

The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brought together a wide and diverse community involved in tuberculosis vaccine research and development to discuss the current status of, and future directions for this critical effort. This paper summarizes the sessions on Low-Dose NHP Challenge Models, Novel Approaches to Animal Models for TB Vaccine R&D, Novel Antigen Delivery Strategies, and Next Generation TB Vaccines and Vaccine Concepts. Summaries of all sessions from the 4th Global Forum are compiled in a special supplement of Tuberculosis. [August 2016, Vol 99, Supp S1, S1-S30].

Advocacy, partnership and political commitment for TB vaccine research

The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brought together a wide and diverse community involved in tuberculosis vaccine research and development to discuss the current status of, and future directions for this critical effort. This paper summarizes the sessions on Advancing the Pipeline: A Vision for the Next Decade, Engaging the BRICS: Basic Research to Manufacturing, and Regulatory and Access Issues for New TB Vaccines. Summaries of all sessions from the 4th Global Forum are compiled in a special supplement of Tuberculosis. [August 2016, Vol 99, Supp S1, S1-S30].

Epidemiologic studies and novel clinical research approaches that impact TB vaccine development

The 4th Global Forum on TB Vaccines, convened in Shanghai, China, from 21 - 24 April 2015, brought together a wide and diverse community involved in tuberculosis vaccine research and development to discuss the current status of, and future directions for this critical effort. This paper summarizes the sessions on Developing TB Vaccines for Prevention of Disease, Prevention of Infection, and Immunotherapy Indications; Concepts and Approaches in Clinical Research & Development; and Epidemiological Research. Summaries of all sessions from the 4th Global Forum are compiled in a special supplement of Tuberculosis.

Engaging adolescents in tuberculosis and clinical trial research through drama.

BackgroundThe South African Tuberculosis Vaccine Initiative is based in Worcester where tuberculosis (TB) is endemic, and incidence rates are amongst the highest nationally. In high TB burden settings after an early childhood peak, incidence rates start to rise again in adolescents, therefore they are an important target group for tuberculosis vaccine research. In 2012, learners from a local school developed a one-off theatrical production out of an educational comic book Carina’s Choice, developed by the South African Tuberculosis Vaccine Initiative in 2010. A Wellcome Trust International Engagement grant allowed for this one-off production to be further developed, with input from university students and staff, and rolled out to schools in the Worcester area as an engagement and education intervention.MethodsFocus group feedback was used to identify key messages and to develop the play’s script. Qualitative methods were used to collect and analyse relevant data. Interviews were conducted with learner-actors, pre- and post-focus group feedback was obtained from a sample of school-going adolescents, and pre- and post-questionnaires were administered to adolescent audience members.ResultsFrom the pre-drama focus group discussions, topics such as TB symptoms, stigma and transmission were identified as areas that needed attention. After the performances, adolescents showed improved knowledge on the identified topics and they discussed TB prevention measures. They highlighted transmission of TB during pregnancy as a further topic to be addressed in future iterations of the drama. Although stigma is a difficult phenomenon to interpret, post-drama participants understood that TB transmission could occur in all individuals. Learner-actors agreed with focus group participants that the play could impact the wider community if it were rolled out. Feedback from the South African Tuberculosis Vaccine Initiative staff verified that recruitment for an upcoming trial was facilitated by the preparedness that the play provided in recruitment areas. The study showed that before and after evaluations provide data on the usefulness of the play as an education tool.ConclusionsTheatre, presented and motivated by adolescent peers, can raise awareness of TB, and assist clinical trial preparedness and further engagement between trial staff and their trial community.

Performance qualification of a 13-color intracellular cytokine staining assay for the assessment of T cell responses in human PBMCs (TECH3P.932)

Abstract One challenge to tuberculosis vaccine research is the lack of a correlate of immunity, which researchers are attempting to address through use of more high-throughput, high-data assays for assessing trial specimens. Multiparameter intracellular cytokine staining (ICS) assays have proven to be one of the most cost-effective tools available. Previously, we reported the development of a 13-color ICS panel for use in assessing T cell responses from human PBMCs. In an effort to better understand the metrics of the assay performance, a qualification study under GCLP conditions was executed. The parameters assessed in this performance qualification included specificity, sensitivity, selectivity, linearity, and intermediate precision. The lower limits of quantification for cytokine detection were shown to be under 0.03%. Using Fisher’s exact test to determine positivity, the false positive and false negative rates were shown to be 0% in this performance qualification. Furthermore, assessment of intermediate precision suggested that the CD3, CD4, CD8, CCR7, and CD45RO markers each showed a percent coefficient of variation (%CV) of 25% or under, while intracellular and functional markers were generally under a 30% CV as measured via responses to Staphylococcal enterotoxin B. The results of this performance qualification study illustrate the capabilities of this panel for assessing clinical trial responses and suggest it is a reliable assay for the assessment of T cell responses.

Tonsils of the soft palate do not mediate the response of pigs to oral vaccination with heat-inactivated Mycobacterium bovis.

Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research.

Homologous prime boosting based on intranasal delivery of non-pathogenic invasive Escherichia coli expressing MPT64, decreases Mycobacterium tuberculosis dissemination

Protein-subunit vaccines as boosting strategies against tuberculosis (TB) infection are currently in the pipeline of TB vaccine research. Their main limitation is represented by their poor immunogenicity, which makes it necessary to couple protein-subunits with adjuvant molecules. In this study, we employed replication-deficient invasive Escherichia coli strains to deliver Mycobacterium tuberculosis proteins to the cytoplasm of non-phagocytic eukaryotic cells using various priming and prime-boosting vaccination protocols. Our results demonstrate that intranasal administration of invasive E. coli expressing the M. tuberculosis protective antigen MPT64 to mice primed with a recombinant BCG strain over-expressing MPT64 on its surface, decrease bacterial burden in mice spleens. Our data suggest that replication-deficient invasive E. coli may represent a suitable platform for BCG/rBCG priming followed by homologous-boosting immunization strategies.

Novel vaccination strategies against tuberculosis.

The tuberculosis (TB) pandemic continues to rampage despite widespread use of the BCG (Bacillus Calmette-Guérin) vaccine. Novel vaccination strategies are urgently needed to arrest global transmission and prevent the uncontrolled development of multidrug-resistant forms of Mycobacterium tuberculosis. Over the last two decades, considerable progress has been made in the field of vaccine development with numerous innovative preclinical candidates and more than a dozen vaccines in clinical trials. These vaccines are developed either as boosters of the current BCG vaccine or as novel prime vaccines to replace BCG. Given the enormous prevalence of latent TB infection, vaccines that are protective on top of an already established infection remain a high priority and a significant scientific challenge. Here we discuss the current state of TB vaccine research and development, our understanding of the underlying immunology, and the requirements for an efficient TB vaccine.

TB Vaccines: The (Human) Challenge Ahead.

Tuberculosis (TB) remains a global health threat, with 8.6 million new cases and 1.3 million deaths (including 0.3 million in HIV-positive people) in 2012 [1]. An estimated one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb) [2]. TB is one of the leading causes of death by an infectious disease worldwide, second only to HIV (1.6 million deaths in 2012), and the increase in drug resistant strains of Mtb is alarming [1,3]. An effective vaccine to prevent infection and/or pulmonary TB disease is desperately needed. However, there are several hurdles that have made TB vaccine research and development painfully slow, including the slow growth rate of Mtb, the lack of a predictive animal model, and the lack of an immune correlate [3]. In addition, animal challenge models are very expensive due to the requirement of an ABSL-3 facility, and clinical efficacy studies are long and require large numbers of patients. Funding continues to be an issue in the TB vaccine field (Figure 1) [1,4-6], particularly given the difficulties and the expense of preclinical and clinical studies. New tools are needed to evaluate candidate vaccines that produce a more rapid and accurate assessment of the potential for vaccine efficacy and, therefore, help to increase the speed, and reduce the cost, of vaccine candidate selection.

An adult zebrafish model for preclinical tuberculosis vaccine development

Tuberculosis remains a major global health challenge despite extensive vaccination schemes with the current live vaccine, Bacillus Calmette–Guérin. Tuberculosis vaccine research has been hampered by a scarcity of animal models which replicate human disease and are suitable for large-scale studies. We have shown recently that Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, causes an infection resembling human tuberculosis in adult zebrafish (Danio rerio). In the present study we use this model to show that BCG vaccination as well as DNA vaccination with selected mycobacterial antigens (Ag85B, CFP-10 and ESAT-6) protects adult zebrafish from mycobacterial infection. Using a low-dose (∼20–30 bacteria) intraperitoneal M. marinum infection, both the number of granulomas and the amount of infected organs were reduced in the DNA vaccinated fish. Likewise, when infecting with a lethal infection dose (∼20,000–27,000 bacteria), vaccination significantly reduced both mortality and bacterial counts in a manner dependent on the adaptive immune response. Protective effects of vaccination were associated with enhanced expression of interferon gamma. Our results indicate that the zebrafish is a promising new model for preclinical tuberculosis vaccine research.

Interview: An Insight Into Cutting-Edge Tuberculosis Vaccine Research

Else Marie Agger speaks to Katie Lockwood, Assistant Commissioning Editor Else Marie Agger was appointed Director of the Department of Infectious Disease Immunology, Statens Serum Institut in Denmark in 2011. Prior to this, she had been heading two different research groups focusing on tuberculosis (TB) vaccine research and adjuvant development, respectively. She holds a postdoctoral degree within immunology from the University of Copenhagen (Denmark). Agger has extensive experience within adjuvant research, TB vaccine research and preclinical development of vaccines. In addition, she has previously been involved in antigen discovery, primarily with a focus on developing novel diagnostic tests within TB. She has been involved in the coordination of vaccine delivery optimization within the EU-funded TBVAC and NEWTBVAC consortia and also has previous experience as a work-package leader within an EU-funded project, focusing on adjuvantation of influenza vaccines. Agger is the coinventor of several adjuvant patents and has published more than 60 papers within TB, vaccines and adjuvant research.

In vitro infection of human cells with Mycobacterium tuberculosis

It has been estimated that approximately 50% of individuals exposed to Mycobacterium tuberculosis never become tuberculin skin test positive, which may indicate that successful human immunological responses are able not only to inhibit mycobacterial growth, but also to kill the bacteria. Nevertheless, it has been extremely difficult to reproduce this effect invitro and the ability of human phagocytes to eliminate the bacteria is controversial. This is one of the reasons why we do not fully understand either tuberculosis resistance or susceptibility. Nowadays there is a pressing need in tuberculosis vaccine research to find biomarkers of successful responses to tuberculosis and the use of invitro models may allow the identification of the immunological mechanisms responsible for the mycobacterial killing that could be tested in clinical settings. Besides biosafety concerns, the manipulation of mycobacteria is technically very demanding, and the optimization of invitro infection protocols have been difficult. As a result, there are a large number of variations in the methodology that make arduous the comparison of results obtained in different research groups. In this review an overview of the mycobacterial and human cellular models most frequently studied are presented, together with a description of several of the modifications tried in infection protocols, from the preparation of the inoculum to the quantification of surviving mycobacteria after infection. A comment about statistical methods that may improve the detection of relevant biological effects is also included.

Vaccines against Tuberculosis: Where Are We and Where Do We Need to Go?

In this review we discuss recent progress in the development, testing, and clinical evaluation of new vaccines against tuberculosis (TB). Over the last 20 years, tremendous progress has been made in TB vaccine research and development: from a pipeline virtually empty of new TB candidate vaccines in the early 1990s, to an era in which a dozen novel TB vaccine candidates have been and are being evaluated in human clinical trials. In addition, innovative approaches are being pursued to further improve existing vaccines, as well as discover new ones. Thus, there is good reason for optimism in the field of TB vaccines that it will be possible to develop better vaccines than BCG, which is still the only vaccine available against TB.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG <em>lux</em>/M.Tb <em>lux</em>)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays. Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date. Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model. Note: Definitions/Abbreviations BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. CFU = Colony Forming Unit (a measure of mycobacterial viability).

Sensitive profiling of chemically diverse bioactive lipids

Here, we present an improved method for sensitive profiling of lipids in a single high-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry experiment. The approach consists of i) sensitive isocratic elution, which takes advantage of C18 column material that is resistant to increased pH values induced by piperidine, ii) chemometric alignment of mass spectra followed by differential analysis of ion intensities, and iii) semiquantitative analysis of extracted ion chromatograms of interest. A key advantage of this method is its wide applicability to extracts that harbor lipids of considerable chemical complexity. The method allows qualitative and semiquantitative analysis of fatty acyls, glycerophospholipids (such as glycerophosphatidylinositols, glycerophosphatidylserines, and glycerophosphatidylcholines in brain extracts), phosphatidylinositol mannosides, acylated glycerophospholipids, sphingolipids (including ceramides and gangliosides in brain extracts), and, for the first time with ESI, prenols and mycolic acids (MAs). MAs are targets in antimycobacterial therapy, and they play an important immunomodulatory role during host-pathogen interactions. We compared high-resolution mass spectra of MAs derived from Mycobacterium bovis Bacille Camette-Guérin during entry into nonreplicative conditions induced by oxygen deprivation (hypoxic dormancy). Although the overall composition is not drastically altered, there are pronounced differences in individual MAs. alpha-MAs accumulate during entry into dormancy, whereas a subpopulation of keto-MAs is almost entirely eliminated. This effect is reversed upon resuscitation of dormant mycobacteria. These results provide detailed chemical information with relevance to drug development and immunobiology of mycobacteria.

Development of New Tuberculosis Vaccines: A Global Perspective on Regulatory Issues

In May 2005, the TB Vaccine Initiative of the World Health Organization (WHO) Initiative for Vaccine Research convened a working meeting of regulators, investigators, and clinicians from developing and developed countries involved in tuberculosis (TB) vaccine regulation and research (see Text S1 for a list of participants). The purpose of the meeting was to specifically discuss the regulatory challenges for testing and introducing investigative TB vaccines into countries where the disease is endemic.

Heterologous boost vaccines for bacillus Calmette–Guérin prime immunization against tuberculosis

The current tuberculosis (TB) epidemic continues to call for the development of effective vaccination strategies. The initial TB vaccine research effort mostly focused on the search for a vaccine that might be better than, and thus could replace, the current bacillus Calmette–Guérin (BCG) vaccine. It has increasingly been realized that BCG or an improved BCG will continue to be used as a prime TB vaccine and there is a need to develop effective boost vaccines that could enhance and prolong the protective immunity of BCG prime immunization. Mounting experimental evidence suggests that recombinant vaccines, including both recombinant protein and genetic vector vaccines, are effective in boosting immune activation and protection by BCG vaccination. This review will discuss recent advances and the authors’ views in the development of there boost vaccines.

Bacillus Calmette-Guérin vaccination and infant mortality

When the bacillus Calmette-Guérin (BCG) vaccine was introduced in the 1920s, it was suggested that BCG occasionally had nonspecific beneficial effects on mortality beyond the specific protection against tuberculosis. Considering that BCG has since then become the most used vaccine in the world, surprisingly few studies have been undertaken into the effect of BCG on general mortality and morbidity. Recent studies suggest that BCG has beneficial nontargeted effects on general infant morbidity and mortality in low-income countries, often with the most pronounced effect among girls. These observational findings are supported by early trials in which children were randomized or alternated to BCG vaccination. Furthermore, a BCG scar and a positive tuberculin reaction are related to better survival among BCG-vaccinated children in low-income countries, especially for girls. The findings are not explained by frailty bias, in other words, that healthy children are more likely to receive BCG vaccination. A nonspecific, gender-differential effect of BCG on general infant mortality may have large implications for tuberculosis vaccine research and routine vaccination policy.