Improving vaccines against tuberculosis

Tuberculosis remains a major cause of mortality and physical and economic deprivation worldwide. There have been significant recent advances in our understanding of the Mycobacterium tuberculosis genome, mycobacterial genetics and the host determinants of protective immunity. Nevertheless, the challenge is to harness this information to develop a more effective vaccine than BCG, the attenuated strain of Mycobacterium bovis derived by Calmette and Guérin nearly 90 years ago. Some of the limitations of BCG include the waning of the protective immunity with time, reduced effectiveness against pulmonary tuberculosis compared to disseminated disease, and the problems of a live vaccine in immuno-compromised subjects. Two broad approaches to vaccine development are being pursued. New live vaccines include either attenuated strains of Mycobacterium tuberculosis produced by random mutagenesis or targeted deletion of putative virulence factors, or by genetic manipulation of BCG to express new antigens or cytokines. The second approach utilizes non-viable subunit vaccines to deliver immunodominant mycobacterial antigens. Both protein and DNA vaccines induce partial protection against experimental tuberculosis infection in mice, however, their efficacy has generally been equivalent to or less than that of BCG. The comparative effects of cytokine adjuvants and vaccines targeting antigen presenting cells on enhancing protection will be discussed. Coimmunization with plasmid interleukin-12 and a DNA vaccine expressing Antigen 85B, a major secreted protein, was as protective as BCG. The combination of priming with DNA-85B and boosting with BCG was superior to BCG alone. Therefore it is possible to achieve a greater level of protection against tuberculosis than with BCG, and this highlights the potential for new tuberculosis vaccines in humans.     

全球结核病疫苗研究进展

本文旨在对全球结核病疫苗研究进展进行系统综述,描述国际上目前进入临床试验不同阶段的新型疫苗,包括重组卡介苗、亚单位疫苗、治疗性疫苗等,分析我国结核病疫苗研究现状,介绍国际研究发展趋势,如人类疫苗计划、全细胞疫苗、多阶段疫苗等,并对存在的问题和挑战进行讨论,展望未来发展趋势。     

Recombinant BCG vaccine candidates

Given the variable protective efficacy provided by Mycobacterium bovis BCG (Bacillus Calmette-Guérin), there is a concerted effort worldwide to develop better vaccines that could be used to reduce the burden of tuberculosis. Recombinant BCG (rBCG) are vaccine candidates that offer some potential in this area. In this paper, we will discuss the molecular methods used to generate rBCG, and the results obtained with some of these new vaccines as compared with the conventional BCG vaccine in diverse animal models. Tuberculosis vaccine candidates based on rBCG are promising candidates, and some of them are now being tested in clinical trials.     

A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis

With the advent of HIV and the widespread emergence of drug-resistant strains of Mycobacterium tuberculosis, newer control strategies in the form of a better vaccine could decrease the global incidence of tuberculosis. A desirable trait in an effective live attenuated vaccine strain is an ability to persist within the host in a limited fashion in order to produce important protective antigens in vivo. Attenuated M. tuberculosis vaccine candidates have been constructed by deleting genes required for growth in mice. These candidate vaccines did not elicit adequate protective immunity in animal models, due to their inability to persist sufficiently long within the host tissues. Here we report that an auxotrophic mutant of M. tuberculosis defective in the de novo biosynthesis of pantothenic acid (vitamin B5) is highly attenuated in immunocompromised SCID mice and in immunocompetent BALB/c mice. SCID mice infected with the pantothenate auxotroph survived significantly longer (250 days) than mice infected with either bacille Calmette-Guerin (BCG) vaccine or virulent M. tuberculosis (77 and 35 days, respectively). Subcutaneous immunization with this auxotroph conferred protection in C57BL/6J mice against an aerosol challenge with virulent M. tuberculosis, which was comparable with that afforded by BCG vaccination. Our findings highlight the importance of de novo pantothenate biosynthesis in limiting the intracellular survival and pathogenesis of M. tuberculosis without reducing its immunogenicity in vaccinated mice.     

结核病免疫机制及疫苗研究进展

结核病是一种棘手的重大传染病.虽然存在一些有一定疗效的治疗药物,亦有预防性疫苗--卡介苗(BCG);但结核病仍在世界范围流行,且发病率和病死率居高不下.结核病的免疫病理机制及疫苗研究近年来取得了一定的进展.结核分枝杆菌通过Toll样受体(TLR)等模式识别受体,激活巨噬细胞的天然免疫反应,清除细菌和调节获得性免疫反应....     

Recombinant BCG as a vaccine vehicle to protect against tuberculosis

Mycobacterium bovis Bacille Calmette Guérin (BCG) was first administered to humans in 1921 and has subsequently been delivered to an estimated 3 billion individuals, with a low incidence of serious complications. The vaccine is immunogenic and is stable and cheap to produce. Additionally, the vaccine can be engineered to express foreign molecules in a functional form, and this has driven the development of BCG as a recombinant vector to protect against infectious diseases and malignancies such as cancer. However, it is now clear that the existing BCG vaccine has proved insufficient to control the spread of tuberculosis, and a major focus of tuberculosis vaccine development programs is the construction and testing of modified forms of BCG. This review summarizes the strategies employed to develop recombinant forms of BCG and describes the potential of these vaccines to stimulate protective immunity and protect against Mycobacterium tuberculosis infection.     

对研发新型结核病疫苗的分析和展望

结核病是公共卫生当前面临的重要问题。由于BCG预防效果不佳,研究和开发新型结核病疫苗显得必须且急迫。新型结核病疫苗的研究开发路径和观念也经历了变迁,当前主流的研发路径有重组BCG或重组结核菌、重组痘病毒或重组腺病毒载体疫苗、蛋白质亚单位或重组融合蛋白质亚单位疫苗三类,它们在疫苗效力前景,抗原选型、配方、剂型,免疫应答,疫苗生产,疫苗质量控制,临床前研究动物试验,临床试验和使用,对结核病公共卫生政策的影响等方面各有优劣。新型结核病疫苗的成功研发,还需要病原学、发病机制、免疫学和疫苗研发科学的进一步努力。     

Toward novel vaccines against tuberculosis: current hopes and obstacles

Approximately 2 million people die of tuberculosis (TB) each year. The current vaccine, Bacille Calmette-Guérin (BCG), albeit widely employed, does not protect against adult pulmonary disease, and new vaccines are urgently needed to reduce the incidence of TB worldwide. New insights into the cellular and molecular mechanisms that underlie the interactions between Mycobacterium tuberculosis and its host have been exploited to develop novel vaccine candidates that recently have entered clinical trials. This review provides a brief overview of different approaches toward a new vaccination strategy and summarizes major challenges for the next decade.     

Development of vaccines to control bovine tuberculosis in cattle and relationship to vaccine development for other intracellular pathogens

Vaccination of cattle against bovine tuberculosis could be an important strategy for the control of disease either where there is a wildlife reservoir of Mycobacterium bovis infection or in developing countries where it is not economically feasible to implement a 'test and slaughter' control program. Advances in the understanding of protective immune responses to M. bovis infection in cattle and the advent of new molecular biological techniques, coupled with the sequencing of the M. bovis genome have provided opportunities for the rational development of improved tuberculosis vaccines. A number of new tuberculosis vaccines including attenuated M. bovis strains, killed mycobacteria, protein and DNA vaccines are under development and many are being assessed in cattle. Recent results have revealed several promising vaccine candidates and vaccination strategies. Ways of distinguishing between vaccinated and infected cattle are becoming available and the possibility of new approaches to the eradication of tuberculosis from domestic livestock is discussed. Similarities between the mechanisms of protective immunity against M. bovis and against other intracellular parasites continue to be found and discoveries from vaccine studies on bovine tuberculosis may provide helpful insights into requirements for vaccines against other intracellular pathogens.     

Influence of BCG vaccine strain on the immune response and protection against tuberculosis

The Bacille Calmette–Guérin (BCG) vaccine has been used for more than 80 years to protect against tuberculosis. Worldwide, over 90% of children are immunized with BCG, making it the most commonly administered vaccine, with more than 120 million doses used each year. Although new tuberculosis vaccines are under investigation, BCG will remain the cornerstone of the strategy to fight the worsening tuberculosis pandemic for the foreseeable future. The recent delineation of genetic differences between BCG vaccine strains has renewed interest in the influence of the vaccine strain on the protective efficacy against tuberculosis. This review critically examines the data from animal and human studies comparing BCG vaccine strains. Although there is good evidence to support the notion that the induced immune response and protection afforded against tuberculosis differs between BCG vaccine strains, currently, there are insufficient data to favour or recommend one particular strain. Identifying BCG strains with superior protection would have a dramatic effect on tuberculosis control at a population level: a small increment in protection provided by BCG immunization will prevent large numbers of cases of severe tuberculosis and deaths, particularly in children.     

Beyond BCG: the potential for a more effective TB vaccine.

The 'Bacille Calmette-Guerin' (BCG) vaccine has been used throughout most parts of the world for the majority of the century. It is safe to use and cheap to produce, but there have been increasing doubts about its effectiveness. These doubts could not come at a worse time, as tuberculosis (TB) rates continue to rise, compounded by the AIDS epidemic, and outbreaks of tuberculosis caused by multidrug-resistant strains are more common even in advanced countries. As a result, there is now a concerted research effort to produce new TB vaccine candidates. These include DNA vaccines, recombinant and auxotrophic vaccines and subunit vaccines, all of which hold promise. The real difficulty will probably arise at the clinical trial level, when a decision must be made either to replace BCG or to boost existing BCG-induced immunity using these new-generation vaccines.     

抗结核分枝杆菌疫苗的研究进展

结核病是由结核分枝杆菌感染引起的传染病,细胞免疫中的CD_4^+T细胞、CD_8^+T细胞、Th17细胞在对抗结核分枝杆菌感染中发挥重要作用,新近研究显示抗体特定的糖链修饰有助于清除病原体,提示体液免疫也可能参与免疫保护。目前使用的疫苗——卡介苗对婴幼儿重症结核病具有良好的保护力,但是对成人肺结核保护力欠佳,所以需要研发新的疫苗。目前已有数个新型疫苗进入临床试验。本文就结核分枝杆菌的免疫保护机制作一简要介绍,主要阐述现用疫苗——卡介苗及新型疫苗的研究现状,让读者对上述知识的进展有所了解。     

Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.     

Recent progress in the development and testing of vaccines against human tuberculosis

The growing pandemic of human tuberculosis has not been affected significantly by the widespread use of the only currently available vaccine, bacille Calmette Guerin. Bacille Calmette Guerin protects uniformly against serious paediatric forms of tuberculosis and against adult pulmonary tuberculosis in some parts of the world, but there are clearly populations in high-burden countries which do not benefit from the current vaccination regimen. New tuberculosis vaccines will be essential for the ultimate control of this ancient disease. Research over the past 10 years has produced literally hundreds of new tuberculosis vaccine candidates representing all of the major vaccine design strategies; protein/peptide vaccines in adjuvants, DNA vaccines, naturally and rationally attenuated strains of mycobacteria, recombinant mycobacteria and other living vaccine vectors expressing genes coding for immunodominant mycobacterial antigens, and non-peptide vaccines. Many of these vaccines have been tested for immunogenicity and protective efficacy in mouse and guinea pig models of low-dose pulmonary tuberculosis. In addition, alternative routes of tuberculosis vaccine delivery (e.g. oral, respiratory, gene gun) and various combinations of priming or boosting an experimental vaccine with bacille Calmette Guerin have been examined in relevant animal models. One of the most promising of these vaccines is currently in Phase I trials in human subjects, and others are expected to follow in the near future. This review will summarise the most recent progress made toward the development and preclinical evaluation of novel vaccines for human tuberculosis.     

Recombinant BCG expressing Mycobacterium tuberculosis major extracellular proteins

rBCG30, the first vaccine against tuberculosis demonstrated more potent than BCG in preclinical studies, is the prototype of a class of vaccines that utilize BCG as a host organism for expressing and secreting Mycobacterium tuberculosis major extracellular proteins. The vaccine is based on the concept that extracellular proteins of intracellular pathogens are key immunoprotective molecules. rBCG30, which expresses and secretes large amounts of the M. tuberculosis 30 kDa major secretory protein, is currently in human clinical trials.     

A Web-Based Platform for Designing Vaccines against Existing and Emerging Strains of Mycobacterium tuberculosis

Development of an effective vaccine against drug-resistant Mycobacterium tuberculosis (Mtb) is crucial for saving millions of premature deaths every year due to tuberculosis. This paper describes a web portal developed for assisting researchers in designing vaccines against emerging Mtb strains using traditional and modern approaches. Firstly, we annotated 59 genomes of Mycobacterium species to understand similarity/dissimilarity between tuberculoid, non-tuberculoid and vaccine strains at genome level. Secondly, antigen-based vaccine candidates have been predicted in each Mtb strain. Thirdly, epitopes-based vaccine candidates were predicted/discovered in above antigen-based vaccine candidates that can stimulate all arms of immune system. Finally, a database of predicted vaccine candidates at epitopes as well at antigen level has been developed for above strains. In order to design vaccine against a newly sequenced genome of Mtb strain, server integrates three modules for identification of strain-, antigen-, epitope-specific vaccine candidates. We observed that 103522 unique peptides (9mers) had the potential to induce an antibody response and/or promiscuous binder to MHC alleles and/or have the capability to stimulate T lymphocytes. In summary, this web-portal will be useful for researchers working on designing vaccines against Mtb including drug-resistant strains. Availability: The database is available freely at http://crdd.osdd.net/raghava/mtbveb/.     

Disease model: pulmonary tuberculosis

In spite of a massive effort to apply the tools currently available for tuberculosis (TB) control, both in this country and abroad, it is clear that complicating factors [for example, HIV co-infection, drug resistance, lack of patient compliance with chemotherapy, variable efficacy of Bacille Calmette-Guerin (BCG) vaccine] will prevent disease control unless new drugs, vaccines and diagnostic tests are developed (1). The publication of the complete genome sequence of Mycobacterium tuberculosis in 1998 (2) has facilitated a directed search for virulence genes, new drug targets, and vaccine antigens. This research effort has been made possible by the availability of highly biologically relevant animal models of pulmonary TB ((3)).     

Adjuvants in tuberculosis vaccine development

Tuberculosis remains a major public health problem around the world. Because the Mycobacterium bovis Bacilli–Calmette–Guerin (BCG) vaccine fails to protect adults from pulmonary tuberculosis, there is an urgent need for improved vaccine formulations. Unlike BCG, recombinant vaccines purified from bacterial expression vectors, as well as naked DNA, require an additional adjuvant. Recent improvements in our understanding of disease immunopathology, together with advances in biochemical and molecular techniques, have permitted the successful development of promising tuberculosis vaccine delivery and adjuvant combinations for human use. Here, we summarize the current state of adjuvant development and its impact on tuberculosis vaccine progress.     

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.