Effect of the method of preparation of bacille Calmette-Guérin (BCG) vaccine on the properties of four daughter strains

Samples of Bacille Calmette-Guérin (BCG) vaccines from four collaborating production laboratories, each of which had prepared vaccine from four different daughter strains of BCG, had previously been monitored for changes in colony morphology and the present study was undertaken to determine whether the changes observed were reflected in the patterns of protein secretion and lipid content. In the samples examined there was evidence for a correlation between colony morphology and the presence or absence of mycoside B. As the components of BCG that determine virulence and protective immunity are unknown, care must be taken to ensure constancy of the strains during the manufacture of vaccines.     

BCG vaccine: an investigation of colony morphology from four different strains after their introduction as seed for vaccine preparation in four production laboratories

Four collaborating BCG production laboratories had each prepared vaccine from four different BCG strains. In this laboratory the morphology of colonies cultured from samples of the 16 vaccine lots thus available was examined, and it was found that changes monitored in earlier small-scale experimental reconstructions had also occurred during some of the full-scale production procedures, in accordance with prediction. In particular, a minority population carried by the Danish BCG strain had replaced the original majority when this strain had been employed as seed for the production of vaccine by the British procedure. Similarly, a minority carried by the Japanese strain had replaced the original majority when this strain was introduced as seed into the production procedures in the United Kingdom and France. Serial subcultures made in this laboratory showed, in accordance with previous experience, that the changes that had occurred in the Japanese strain could be completely reversed by serial subculture as a surface pellicle on Sauton medium.     

The Efficacy of the BCG Vaccine against Newly Emerging Clinical Strains of Mycobacterium tuberculosis

To date, most new vaccines against Mycobacterium tuberculosis, including new recombinant versions of the current BCG vaccine, have usually been screened against the laboratory strains H37Rv or Erdman. In this study we took advantage of our recent work in characterizing an increasingly large panel of newly emerging clinical isolates [from the United States or from the Western Cape region of South Africa], to determine to what extent vaccines would protect against these [mostly high virulence] strains. We show here that both BCG Pasteur and recombinant BCG Aeras-422 [used here as a good example of the new generation BCG vaccines] protected well in both mouse and guinea pig low dose aerosol infection models against the majority of clinical isolates tested. However, Aeras-422 was not effective in a long term survival assay compared to BCG Pasteur. Protection was very strongly expressed against all of the Western Cape strains tested, reinforcing our viewpoint that any attempt at boosting BCG would be very difficult to achieve statistically. This observation is discussed in the context of the growing argument made by others that the failure of a recent vaccine trial disqualifies the further use of animal models to predict vaccine efficacy. This viewpoint is in our opinion completely erroneous, and that it is the fitness of prevalent strains in the trial site area that is the centrally important factor, an issue that is not being addressed by the field.     

Whole-genome sequences of four Mycobacterium bovis BCG vaccine strains

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is the only vaccine available against tuberculosis (TB). A number of BCG strains are in use, and they exhibit biochemical and genetic differences. We report the genome sequences of four BCG strains representing different lineages, which will help to design more effective TB vaccines.     

Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti

Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock-in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1-encoded proteins were localized in the cell wall, and two of them, the immunodominant T-cell antigens ESAT-6 and CFP-10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock-ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock-in, whereas BCG controls were cleared. Knocking-in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.     

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.     

Surface morphology of Mycobacterium bovis BCG: relation to mechanisms of cellular aggregation

Growing colonies of Mycobacterium bovis BCG, Tice and Glaxo substrains, and freshly ball milled and freeze-dried Tice BCG vaccines were examined by scanning and transmission electron microscopy (TEM) and by light microscopy after cytochemical staining. BCG organisms in colonies growing on agar were randomly oriented, despite colony morphology, and nearly completely covered by an amorphous material. Aggregates of organisms in vaccine suspensions were also covered with this material, but single cells were not covered. In TEM, the covering material was visualized between groups of cells as an electron-transparent area surrounded by a thin electron-dense layer. This material appeared to originate in the upper cell wall, between the cell wall skeleton and the outer dense layer. Staining of the covering material indicated the presence of protein, carbohydrate and acidic groups, but not exposed lipids. The covering material was absent from the ventral side of colonies, suggesting that its production is oxygen-dependent. These observations suggest that a mycobacterial exudate, previously observed and implicated as a virulence factor, may also bind the cells together, and accounts for the aggregative properties of the organisms in culture.     

Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP

Analysis of mycobacterial strains that have lost their ability to cause disease is a powerful approach to identify yet unknown virulence determinants and pathways involved in tuberculosis pathogenesis. Two of the most widely used attenuated strains in the history of tuberculosis research are Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (H37Ra), which both lost their virulence during in vitro serial passage. Whereas the attenuation of BCG is due mainly to loss of the ESAT-6 secretion system, ESX-1, the reason why H37Ra is attenuated remained unknown. However, here we show that a point mutation (S219L) in the predicted DNA binding region of the regulator PhoP is involved in the attenuation of H37Ra via a mechanism that impacts on the secretion of the major T cell antigen ESAT-6. Only H37Ra "knock-ins" that carried an integrated cosmid with the wild-type phoP gene from M. tuberculosis H37Rv showed changes in colony morphology, increased virulence, ESAT-6 secretion, and induction of specific T cell responses, whereas other H37Ra constructs did not. This finding established a link between the PhoP regulator and ESAT-6 secretion that opens exciting new perspectives for elucidating virulence regulation in M. tuberculosis.     

A point mutation in the mma3 gene is responsible for impaired methoxymycolic acid production in Mycobacterium bovis BCG strains obtained after 1927

BCG vaccines are substrains of Mycobacterium bovis derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the mma3 gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production and mma3 gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the mma3 sequence of the latter strains differs from that of Mycobacterium tuberculosis H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type mma3 (resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.     

Phage types of Mycobacterium bovis, substrains of BCG.

Nineteen substrains of Mycobacterium bovis, strain bacille Calmette-Guérin (BCG) used in laboratories throughout the world for the preparation of BCG vaccines were phage-typed with a battery of mycobacteriophages. The results revealed differences in their susceptibility to phage lysis that allow subdivision of these strains of BCG into two or more phage types.     

Relative persistence capacity of BCG substrains in mouse spleen. Computerized statistical analysis. Multiple comparison.

The relative persistence capacity in mouse spleen of 10 and 9 BCG substrains from liquid and dried vaccines, respectively, was evaluated in two studies. Recoverable BCG colony counts from mouse spleen were determined at given days on solid medium in the two studies during a period of 1-360 and 1-345 days, respectively, after the intravenous BCG vaccination, performed with two different viable units. From 36,000 (study 1) and 21,600 (study 2) recoverable BCG colony counts, 180 and 108 mean relative persistence capacity values were estimated to test the residual virulence during the follow-up time, using computerized statistical analysis. The early and late trends of mean relative persistence capacity of the BCG substrains in mouse spleen were tested by linear regression analysis and analysis of variance and covariance; then with ranked adjusted group mean relative persistence capacity, Gabriel's simultaneous test procedure was performed for multiple comparison to diminish type 1 error in statistical inference and in objective interpretation of the experimental results. The associations of the ranked mean relative persistence capacity of the BCG substrains at the different sacrifice days of mice were also analyzed by Kendall's test of concordance. The early, late, and overall relative persistence capacity reflects the residual virulence of the BCG substrains and provides information on the required protective efficacy (immunogenicity) and adverse reactions (reactogenicity), allowing the appropriate vaccination dose, expressed in viable units of the substrain used, to be determined.     

New tuberculosis vaccines based on attenuated strains of the Mycobacterium tuberculosis complex

The world urgently needs a better tuberculosis vaccine. Bacille Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, has been very widely used as a vaccine for many years but has had no major effect on reducing the incidence of tuberculosis. A number of alternative living and non-living vaccines are being investigated. Live vaccine candidates include genetically modified forms of BCG, genetically attenuated strains of the Mycobacterium tuberculosis complex and genetically engineered vaccinia virus and Salmonella strains. Non-living vaccine candidates include killed mycobacterial species, protein subunits and DNA vaccines. One requirement for acceptance of any new vaccine will be a favourable comparison of the protection it induces relative to BCG in a range of animal models, some of which may need further development. Molecular genetic techniques are now available that enable production of live attenuated strains of the M. tuberculosis complex with vaccine potential. In the first of two broadly different approaches that are being used, large numbers of mutants are produced by transposon mutagenesis or illegitimate recombination and are screened for properties that correlate with attenuation. In the second approach, putative genes that may be required for virulence are identified and subsequently inactivated by allelic exchange. In both approaches, mutants that are attenuated need to be identified and subsequently tested for their vaccine efficacy in animal models. Many mutants of the M. tuberculosis complex have now been produced and the vaccine properties of a substantial number will be assessed in the next 3 years.     

Immunogenicity of orally-delivered lipid-formulated BCG vaccines and protection against Mycobacterium tuberculosis infection

Lipid formulations containing BCG strains Danish 1331 or Moreau (Rio de Janeiro) were trialled as oral vaccines in rodent models. In mice, oral-delivery of either strain resulted in BCG colonisation of the alimentary tract lymphatics and induction of gamma-interferon responses. In guinea pigs, both strains provided pulmonary protection against Mycobacterium tuberculosis aerosol challenge, as shown by significantly reduced bacterial loads and lung:body weight ratios. Lipid-formulated BCG provided superior protection against M. tuberculosis over unformulated orally-delivered BCG (Moreau), and equivalent protection to sub-cutaneous BCG (Danish) immunisation. Oral-delivery of lipid-formulated BCG may offer a practical alternative to parenteral-route BCG vaccination.     

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.     

Improved protection by recombinant BCG

Mycobacterium bovis bacille Calmette Guérin (BCG) is one of the most widely used live vaccines. Technologic advancement in genome manipulation enables the construction of recombinant BCG (rBCG) strains, which can be employed as highly immunogenic vaccines against tuberculosis with improved safety profile.     

Genome Sequencing and Analysis of BCG Vaccine Strains

Background

Although the Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) has been available for more than 75 years, one third of the world''s population is still infected with Mycobacterium tuberculosis and approximately 2 million people die of TB every year. To reduce this immense TB burden, a clearer understanding of the functional genes underlying the action of BCG and the development of new vaccines are urgently needed.

Methods and Findings

Comparative genomic analysis of 19 M. tuberculosis complex strains showed that BCG strains underwent repeated human manipulation, had higher region of deletion rates than those of natural M. tuberculosis strains, and lost several essential components such as T-cell epitopes. A total of 188 BCG strain T-cell epitopes were lost to various degrees. The non-virulent BCG Tokyo strain, which has the largest number of T-cell epitopes (359), lost 124. Here we propose that BCG strain protection variability results from different epitopes. This study is the first to present BCG as a model organism for genetics research. BCG strains have a very well-documented history and now detailed genome information. Genome comparison revealed the selection process of BCG strains under human manipulation (1908–1966).

Conclusions

Our results revealed the cause of BCG vaccine strain protection variability at the genome level and supported the hypothesis that the restoration of lost BCG Tokyo epitopes is a useful future vaccine development strategy. Furthermore, these detailed BCG vaccine genome investigation results will be useful in microbial genetics, microbial engineering and other research fields.     

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18 kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.     

Biological attributes of colony-type variants of Candida albicans

Twenty 'commensal' oral or 'pathogenic' vaginal isolates of Candida albicans were examined for colony morphology on malt/yeast-extract and serum-based agar media. Diverse and variable colony morphology was seen on serum agar. In 17 strains, selective subculture of morphologically atypical colonies produced progeny which had reverted to the morphology of the majority of parental colonies. However, in one strain, a highly stable colony variant was isolated which did not revert on subculture. In two further strains, variants were isolated which could be maintained with at least 99% homogeneous colony type by selective colony subculture, but reversion to the parental type or switching to other morphologies occurred at rates of 10(-2) to 10(-4): a rapid switching phenomenon. The relative proportions of mycelial or yeast forms were the main determinants of colony morphology. The variants were biotyped using a selection of biochemical tests. The stable variant differed from its parent in several characters, including rate of production of a proteinase enzyme. The pathogenicity of variants was compared in mice, and both stable and switching variants differed in virulence from their parental strains. Colony-type variation on suitable media is thus a powerful tool in the isolation of mutants or variants of C. albicans which differ from 'isogenic' parents in significant biological properties. Such variants may aid identification and characterization at the molecular level of determinants of, for example, pathogenicity and morphogenesis.     

Variation in Colonial Morphology of Neisseria gonorrhoeae After Growth on Media Containing Antimicrobial Agents

Stable colonial types 1, 2, 3, and 4 were prepared from eight strains of Neisseria gonorrhoeae. Four of the strains, termed laboratory strains, had been transferred over 100 times; three strains, termed clinical strains, were transferred only three to five times after isolation from patients, and one stabilized clinical strain was transferred purposefully 30 times after isolation from a patient. Colonial types of the three categories were grown on four media containing the following agents at the level used in diagnostic media: (i) vancomycin, colistin, and nystatin; (ii) these antibiotics plus trimethoprim lactate; (iii) trimethoprim lactate alone; and (iv) a control with no antimicrobial agents. When grown on media containing the antimicrobial agents, colonial types 1, 2, and 3 of all strains showed specific and consistent changes that precluded accurate identification of the types. In general, the colonies were smaller, more dense to transmission of light, and more granular than colonies grown on control medium. More colonies showed these type changes in the clinical strains and on media containing trimethoprim lactate. Colonies of type 4 showed little or no change. The changes in colonial morphology of types 1, 2, and 3 were pronounced enough to make colony typing difficult if the antimicrobial agents, particularly trimethoprim lactate, were present in media.