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.     

Cloning of the gene encoding a protective Mycobacterium tuberculosis secreted protein detected in vivo during the initial phases of the infectious process

The existence of therapeutic agents and the bacille Calmette-Guérin (BCG) vaccine have not significantly affected the current tuberculosis pandemic. BCG vaccine protects against serious pediatric forms of tuberculosis but not against adult pulmonary tuberculosis, the most common and contagious form of the disease. Several vaccine candidates, including Mycobacterium tuberculosis recombinant proteins formulated in newer adjuvants or delivered in bacterial plasmid DNA have recently been described. An attractive source of vaccine candidates has been M. tuberculosis Ags present in culture supernatants of the initial phases of the bacterial growth in vitro. In this study we describe an Ag discovery approach to select for such Ags produced in vivo during the initial phases of the infection. We combined RP-HPLC and mass spectrometry to identify secreted or shed M. tuberculosis proteins eliminated in animal urine within 14 days after the infection. A peptide containing sequence homology with a hypothetical M. tuberculosis protein was identified and the recombinant protein produced in Escherichia coli. The protein was recognized by Ab (IgG2a and IgG1) and T cells (Th1) of mice infected with M. tuberculosis and by lymphoid cells from healthy donors who had a positive purified protein derivative skin test but not from tuberculosis patients. Moreover, this Ag induced protection in mice against M. tuberculosis at levels comparable to protection induced by BCG vaccine. These results validate the Ag discovery approach of M. tuberculosis proteins secreted or shed in vivo during the early phases of the infection and open new possibilities for the development of potential vaccine candidates or of markers of active mycobacterial multiplication and therefore active disease.     

Recombinant mycobacterial proteins future directions to improve protective efficacy

A large number of subunit vaccine candidates have recently been developed as alternatives to Mycobacterium bovis Bacillus Calmette-Guerin (BCG), which gives unpredictable and highly variable protection against tuberculosis. Immunological potential of various recombinant proteins against mycobacterial infections has been discussed. Further, strategies have been suggested, which include development of constructs coexpressing cytokines or regimens utilizing recombinant proteins for further improving the protective efficacy.     

Molecular cloning and sequencing of the merozoite surface antigen 2 gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in mycobacteria

Strain bacillus Calmette-Guerin (BCG) of Mycobacterium bovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis. In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-1/HN Plasmodium falciparum genome, sequenced, and expressed in M. bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis. The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa. This first report of expression of the full-length P. falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria.     

Vaccine platform for prevention of tuberculosis and mother-to-child transmission of human immunodeficiency virus type 1 through breastfeeding

Most children in Africa receive their vaccine against tuberculosis at birth. Those infants born to human immunodeficiency virus type 1 (HIV-1)-positive mothers are at high risk of acquiring HIV-1 infection through breastfeeding in the first weeks of their lives. Thus, the development of a vaccine which would protect newborns against both of these major global killers is a logical yet highly scientifically, ethically, and practically challenging aim. Here, a recombinant lysine auxotroph of Mycobacterium bovis bacillus Calmette-Guérin (BCG), a BCG strain that is safer than those currently used and expresses an African HIV-1 clade-derived immunogen, was generated and shown to be stable and to induce durable, high-quality HIV-1-specific CD4⁺- and CD8⁺-T-cell responses. Furthermore, when the recombinant BCG vaccine was used in a priming-boosting regimen with heterologous components, the HIV-1-specific responses provided protection against surrogate virus challenge, and the recombinant BCG vaccine alone protected against aerosol challenge with M. tuberculosis. Thus, inserting an HIV-1-derived immunogen into the scheduled BCG vaccine delivered at or soon after birth may prime HIV-1-specific responses, which can be boosted by natural exposure to HIV-1 in the breast milk and/or by a heterologous vaccine such as recombinant modified vaccinia virus Ankara delivering the same immunogen, and decrease mother-to-child transmission of HIV-1 during breastfeeding.     

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.     

Tuberculosis vaccines: beyond bacille Calmette-Guerin

Tuberculosis (TB) disease caused by Mycobacterium tuberculosis (M. tb) remains one of the leading infectious causes of death and disease throughout the world. The only licensed vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG) confers highly variable protection against pulmonary disease. An effective vaccination regimen would be the most efficient way to control the epidemic. However, BCG does confer consistent and reliable protection against disseminated disease in childhood, and most TB vaccine strategies being developed incorporate BCG to retain this protection. Cellular immunity is necessary for protection against TB and all the new vaccines in development are focused on inducing a strong and durable cellular immune response. There are two main strategies being pursued in TB vaccine development. The first is to replace BCG with an improved whole organism mycobacterial priming vaccine, which is either a recombinant BCG or an attenuated strain of M. tb. The second is to develop a subunit boosting vaccine, which is designed to be administered after BCG vaccination, and to enhance the protective efficacy of BCG. This article reviews the leading candidate vaccines in development and considers the current challenges in the field with regard to efficacy testing.     

Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis

Bacillus Calmette-Guerin (BCG) vaccine has failed to control the global tuberculosis (TB) epidemic, and there is a lack of safe and effective mucosal vaccines capable of potent protection against pulmonary TB. A recombinant replication-deficient adenoviral-based vaccine expressing an immunogenic Mycobacterium tuberculosis Ag Ag85A (AdAg85A) was engineered and evaluated for its potential to be used as a respiratory mucosal TB vaccine in a murine model of pulmonary TB. A single intranasal, but not i.m., immunization with AdAg85A provided potent protection against airway Mycobacterium tuberculosis challenge at an improved level over that by cutaneous BCG vaccination. Systemic priming with an Ag85A DNA vaccine and mucosal boosting with AdAg85A conferred a further enhanced immune protection which was remarkably better than BCG vaccination. Such superior protection triggered by AdAg85 mucosal immunization was correlated with much greater retention of Ag-specific T cells, particularly CD4 T cells, in the lung and was shown to be mediated by both CD4 and CD8 T cells. Thus, adenoviral TB vaccine represents a promising novel vaccine platform capable of potent mucosal immune protection against TB. Our study also lends strong evidence that respiratory mucosal vaccination is critically advantageous over systemic routes of vaccination against TB.     

Oral bacillus Calmette-Guérin vaccine against tuberculosis: why not?

The bacillus Calmette-Guérin (BCG) vaccine is the only licensed vaccine for human use against tuberculosis (TB). Although controversy exists about its efficacy, the BCG vaccine is able to protect newborns and children against disseminated forms of TB, but fails to protect adults against active forms of TB. In the last few years, interest in the mucosal delivery route for the vaccine has been increasing owing to its increased capacity to induce protective immune responses both in the mucosal and the systemic immune compartments. Here, we show the importance of this route of vaccination in newly developed vaccines, especially for vaccines against TB.     

Protective and therapeutic efficacy of Mycobacterium smegmatis expressing HBHA-hIL12 fusion protein against Mycobacterium tuberculosis in mice

Tuberculosis (TB) remains a major worldwide health problem. The only vaccine against TB, Mycobacterium bovis Bacille Calmette-Guerin (BCG), has demonstrated relatively low efficacy and does not provide satisfactory protection against the disease. More efficient vaccines and improved therapies are urgently needed to decrease the worldwide spread and burden of TB, and use of a viable, metabolizing mycobacteria vaccine may be a promising strategy against the disease. Here, we constructed a recombinant Mycobacterium smegmatis (rMS) strain expressing a fusion protein of heparin-binding hemagglutinin (HBHA) and human interleukin 12 (hIL-12). Immune responses induced by the rMS in mice and protection against Mycobacterium tuberculosis (MTB) were investigated. Administration of this novel rMS enhanced Th1-type cellular responses (IFN-γ and IL-2) in mice and reduced bacterial burden in lungs as well as that achieved by BCG vaccination. Meanwhile, the bacteria load in M. tuberculosis infected mice treated with the rMS vaccine also was significantly reduced. In conclusion, the rMS strain expressing the HBHA and human IL-12 fusion protein enhanced immunogencity by improving the Th1-type response against TB, and the protective effect was equivalent to that of the conventional BCG vaccine in mice. Furthermore, it could decrease bacterial load and alleviate histopathological damage in lungs of M. tuberculosis infected mice.     

Influence of bovine lactoferrin on expression of presentation molecules on BCG-infected bone marrow derived macrophages

The current vaccine for tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is an attenuated strain of Mycobacterium bovis bacillus Calmette-Guerin (BCG). BCG has proven to be effective in children, however, efficacy wanes in adulthood. Lactoferrin, a natural protein with immunomodulatory properties, is a potential adjuvant candidate to enhance efficacy of BCG. These studies define bovine lactoferrin as an enhancer of the BCG vaccine, functioning in part by modulating macrophage ability to present antigen and stimulate T-cells. BCG-infected bone marrow derived macrophages (BMMs) cultured with bovine lactoferrin increased the number of MHC II(+) expressing cells. Addition of IFN-gamma and lactoferrin to BCG-infected BMMs enhanced MHC II expressiona dna increased the ratio of CD86/CD80. Lactoferrin treated BCG-infected BMMs were able to stimulate an increase in IFN-gamma production from presensitized CD3(+) splenocytes. Together, these results demonstrate that bovine lactoferrin is capable of modulating BCG-infected macrophages to enhance T-cell stimulation through increased surface expression of antigen presentation and co-stimulatory molecules, which potentially explains the observed in vivo bovine lactoferrin enhancement of BCG vaccine efficacy to protect against virulent MTB infection.     

Contribution of L-alanine dehydrogenase to in vivo persistence and protective efficacy of the BCG vaccine

The tuberculosis (TB) vaccine strain Mycobacterium bovis BCG is unable to utilise alanine and this deficiency is thought to inhibit the growth of the vaccine in vivo and limit vaccine efficacy. In this report we demonstrate that L-alanine catabolism can be conferred on BCG by introduction of the gene encoding L-alanine dehydrogenase (Ald) of Mycobacterium tuberculosis. Restoration of Ald activity did not change the in vivo growth of BCG in macrophages or mice, and protection against aerosol M. tuberculosis infection was not altered by addition of ald to the BCG vaccine. These results demonstrate that the inability to utilise L-alanine is not a contributing factor to the attenuated phenotype of BCG and does not influence the protective efficacy of the vaccine against TB.     

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。     

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。     

Improved protection against disseminated tuberculosis by Mycobacterium bovis bacillus Calmette-Guerin secreting murine GM-CSF is associated with expansion and activation of APCs

Modulating the host-immune response by the use of recombinant vaccines is a potential strategy to improve protection against microbial pathogens. In this study, we sought to determine whether secretion of murine GM-CSF by the bacillus Calmette-Guérin (BCG) vaccine influenced protective immunity against Mycobacterium tuberculosis. BCG-derived GM-CSF stimulated the in vitro generation of functional APCs from murine bone marrow precursors, as demonstrated by the infection-induced secretion of IL-12 by differentiated APCs, and the ability of these cells to present Ag to mycobacterium-specific T cells. Mice vaccinated with BCG secreting [corrected] murine GM-CSF (BCG:GM-CSF) showed increased numbers of CD11c+MHCII+ and CD11c-CD11b+F480+ cells compared with those vaccinated with control BCG, and this effect was most apparent in the draining lymph nodes at 7 and 14 days postvaccination. Vaccination with BCG:GM-CSF also resulted in enhanced expression of costimulatory molecules on migratory dendritic cells in the draining lymph nodes. The increased APC number was associated with an increase in the frequency of anti-mycobacterial IFN-gamma-secreting T cells generated after BCG:GM-CSF vaccination compared with vaccination with control BCG, and this effect was sustained up to 17 wk in the spleens of immunized mice. Vaccination with BCG:GM-CSF resulted in an approximately 10-fold increase in protection against disseminated M. tuberculosis infection compared with control BCG. This study demonstrates the potential of BCG secreting [corrected] immunostimulatory molecules as vaccines to protect against tuberculosis and suggests BCG:GM-CSF merits further appraisal as a candidate to control M. tuberculosis infection in humans.     

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.     

A novel recombinant Mycobacterium bovis bacillus Calmette-Guerin strain expressing human granulocyte macrophage colony-stimulating factor and Mycobacterium tuberculosis early secretory antigenic target 6 complex augments Th1 immunity

Since Mycobacterium bovis bacillus Calmette-Guerin strain (BCG) fails to protect adults from pulmonary tuberculosis (TB), there is an urgent need for developing a new vaccine. In this study, we constructed a novel recombinant BCG strain (rBCG) expressing human granulocyte macrophage colony-stimulating factor (GM-CSF) and the 6 kDa early secretory antigenic target (ESAT6) of Mycobacterium tuberculosis, named rBCG:GE (expressing GMCSF-ESAT6 complex), and evaluated the immunogenicity of the construct in BALB/c mice. Our results indicated that the rBCG:GE was able to induce higher titer of antibody than the conventional BCG, the rBCG:G (expressing GM-CSF) and the rBCG:E (expressing ESAT6). Moreover, the rBCG:GE also elicited a longer-lasting and stronger Th1 cellular immune responses than the other groups, which was confirmed by the incremental proliferation of splenocytes, the increased percentages of CD4(+) and CD8(+) T cells of spleen, the elevated level of interferon-γ in splenocyte culture after tuberculin-purified protein derivative stimulation, and the increased concentration of GM-CSF in serum. The data presented here suggested the possibility that the recombinant BCG:GE might be a good vaccine candidate to TB.     

Preclinical development of an in vivo BCG challenge model for testing candidate TB vaccine efficacy

There is an urgent need for an immunological correlate of protection against tuberculosis (TB) with which to evaluate candidate TB vaccines in clinical trials. Development of a human challenge model of Mycobacterium tuberculosis (M.tb) could facilitate the detection of such correlate(s). Here we propose a novel in vivo Bacille Calmette-Guérin (BCG) challenge model using BCG immunization as a surrogate for M.tb infection. Culture and quantitative PCR methods have been developed to quantify BCG in the skin, using the mouse ear as a surrogate for human skin. Candidate TB vaccines have been evaluated for their ability to protect against a BCG skin challenge, using this model, and the results indicate that protection against a BCG skin challenge is predictive of BCG vaccine efficacy against aerosol M.tb challenge. Translation of these findings to a human BCG challenge model could enable more rapid assessment and down selection of candidate TB vaccines and ultimately the identification of an immune correlate of protection.     

Protection Induced by Simultaneous Subcutaneous and Endobronchial Vaccination with BCG/BCG and BCG/Adenovirus Expressing Antigen 85A against Mycobacterium bovis in Cattle

The incidence of bovine tuberculosis (bTB) in the GB has been increasing since the 1980s. Immunisation, alongside current control measures, has been proposed as a sustainable measure to control bTB. Immunisation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been shown to protect against bTB. Furthermore, much experimental data indicates that pulmonary local immunity is important for protection against respiratory infections including Mycobacterium tuberculosis and that pulmonary immunisation is highly effective. Here, we evaluated protection against M. bovis, the main causative agent of bTB, conferred by BCG delivered subcutaneously, endobronchially or by the new strategy of simultaneous immunisation by both routes. We also tested simultaneous subcutaneous immunisation with BCG and endobronchial delivery of a recombinant type 5 adenovirus expressing mycobacterial antigen 85A. There was significantly reduced visible pathology in animals receiving the simultaneous BCG/BCG or BCG/Ad85 treatment compared to naïve controls. Furthermore, there were significantly fewer advanced microscopic granulomata in animals receiving BCG/Ad85A compared to naive controls. Thus, combining local and systemic immunisation limits the development of pathology, which in turn could decrease bTB transmission.     

In vivo persistence and protective efficacy of the bacille Calmette Guerin vaccine overexpressing the HspX latency antigen

New strategies to control infection with Mycobacterium tuberculosis, the causative agent of tuberculosis, are urgently required, particularly in areas where acquired immunodeficiencies are prevalent. In this report we have determined if modification of the current tuberculosis vaccine, Mycobacterium bovis BCG, to constitutively express the mycobacterial HspX latency antigen altered its protective effect against challenge with virulent M. tuberculosis. Overexpression of M. tuberculosis HspX in BCG caused reduced growth in aerated cultures compared to control BCG, but growth under limited oxygen availability was not markedly altered. Upon infection of mice, BCG:HspX displayed tissue-specific attenuation compared to control BCG, with reduced growth within the lung and liver but not the spleen. Both BCG:HspX and control BCG protected mice against aerosol M. tuberculosis challenge to a similar extent, however, immunodeficient mice infected with BCG:HspX survived significantly longer than mice infected with the control BCG strain. Therefore, altering the in vivo persistence of BCG by overexpression of HspX may be one important step towards developing a new tuberculosis vaccine with an improved safety profile and suitable protective efficacy against M. tuberculosis infection.