- Email: [email protected]
TB vaccine development: after the flood
COMMENT
TB vaccine development: after the flood Ian M. Orme and John T. Belisle
I
n this new era of DNA sequencing and associated bioinformatics, in which a relatively large number of genes can be rapidly sequenced, it is now quite possible that most of the leading bacterial pathogens will be fully sequenced within the next five to ten years1. From the perspective of those of us working in the mycobacteria field, the completion of the genome of Mycobacterium tuberculosis H37Rv is probably one of the most important advances in our field since the identification of the bacillus a century ago2. This achievement has unleashed a veritable flood of information that will undoubtedly bear fruit in both drug discovery and the rational design of new vaccines.
The H37Rv genome There are two principal approaches to the technical issue of DNA sequencing. The first is the wholegenome shotgun approach, in which the complete sequence is deduced from large numbers of small fragments3; the second is the use of cosmid or phagemid libraries, which are first ordered to produce a contiguous map of the genome4. The H37Rv genome sequencing project used elements of both approaches to produce the finished product2. Furthermore, because it used bacterial artificial chromosome clones containing large inserts5, the project had the additional benefit of isolating 68 clones, which, together, cover most of the genome and can be used in the future for comparative genome mapping and other uses. The H37Rv genome is 4411 kb in length, encoding ~4000 genes. The G1C content is very high (65%) and the genome is rich in repetitive DNA sequences, particularly insertion sequences, as well as containing new multigene families and copies of housekeeping genes2. There are 3924 open reading frames (ORFs), of which
~40% encode proteins that have been identified by database comparisons and there have been educated guesses about another 40%. The rest of the ORFs encode unknown proteins that might have specific mycobacterial functions. At least 10% of the ORFs encode two families of glycine-rich proteins and such genes often contain multiple polymorphic G1C-rich sequences or major polymorphic tandem repeats. The short peptide motifs that comprise common repetitive domains within these proteins could potentially be involved in a form of antigenic variation and thus these protein families could be potential vaccine targets. The bacterium contains genes encoding a full metabolic arsenal, as well as 13 sigma factors and at least 100 regulatory proteins. But perhaps the most striking aspect of the genome is the vast array of enzymes (.250) involved in the biosynthesis or catabolism of fatty acids; this is consistent with the myriad of lipid-containing structures involved in the structure of M. tuberculosis, particularly its cell wall6. Current efforts in TB vaccine development Although the technological advances in tuberculosis (TB) vaccine development have perhaps not been as dramatic as those in the sequencing technology there have, nevertheless, been a number of innovative approaches, including the use of transposon mutagenesis to develop auxotrophic mutants of the Mycobacterium bovis Bacillus Calmette–Guérin (BCG) strain and M. tuberculosis, the use of the existing BCG vaccine as a recombinant I.M. Orme* and J.T. Belisle are in the Dept of Microbiology, Colorado State University, Fort Collins, CO 80523, USA. *tel: +1 970 491 5777, fax: +1 970 491 5125, e-mail: [email protected]
0966-842X/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
394
vector, the realization that DNA itself is a potential vaccine and, last but not least, the proteomic analysis of the bacillus7,8. In addition to these approaches, a more simplistic approach has been to use subcellular fractionation of the bacillus to generate subunit vaccines. Some of these have been based on whole fractions, such as the culture filtrate, and others on various immunogenic proteins such as ESAT-6, the Ag85 complex and the heat shock protein Hsp70 (Refs 9–12). It has to be stressed, however, that these are very early days and the available information to date has been obtained only from mouse and guinea pig experimental models. In these models, the capacity of the vaccine candidate to confer protection (mainly against aerosol-challenge infection) and, in the guinea pig, to prevent the development of tissue necrosis in the lung, is used as a measure of efficacy10. What will clearly be needed in the future, however, are ways to answer more specific questions regarding the longevity of new vaccines – vaccine challenge interval issues – as well as an animal model to determine whether the vaccine would have any effect in people already inoculated with BCG or could be used specifically to target certain T-cell responses in patients already infected with TB. The impact of genomic information on new vaccine design The genome sequence potentially provides a road map of every drug target and every possible protein target for vaccines. The complex fatty acid pathways in which the bacillus has invested a large proportion of its genome will certainly be a gold mine for drug targeting directed at the biosynthesis of the mycolic acid layer of the cell wall, the mycocerocic acids that are unique to virulent M. tuberculosis isolates, as PII: S0966-842X(99)01591-7
VOL. 7 NO. 10 OCTOBER 1999
COMMENT
well as at the copious and ubiquitous lipoarabinomannan, which spans the entire cell envelope. However, despite the potential impact of genomic data on drug design and the current enthusiasm for ‘directly observed’ therapy, many people believe that the only long-term solution to the global TB pandemic is vaccination. Genomic data will help enormously in tracking down potential antigen targets identified from proteomic and microarray technologies and will also undoubtedly help in identifying new vaccine targets. One of the most interesting pieces of information from the genome sequence is the presence of the two extensive families of glycine-rich proteins (the PE and PPE families), which, given their abundance, might be immunogens. Now that the protein sequences are readily available, simple experiments can be performed to test their immunogenicity, such as recognition of synthetic or recombinant peptides by interferon-gsecreting T cells from infected mice13. This type of analysis will not only reveal if these protein families are potential immunogens, but will also
elucidate which family members are the most potent antigens, as well as indicating if they are differentially produced over the course of an infection. In addition, the genome information confirms earlier work14 suggesting a remarkable lack of genomic diversity at the nucleotide level. This implies that proteins encoded by different strains are likely to be the same, which is useful to know for rational vaccine design. There might also be a direct benefit in terms of understanding why the current BCG vaccine has such variable effects. A recent study by Behr and colleagues15, using comparative hybridization to a DNA microarray, has shown that BCG cultures kept in different laboratories around the world have undergone various gene deletions, resulting in distinct substrains. Although there is no evidence as yet that these deletions are the basis for differences in vaccine efficacy, if they can be related to changes in immunogenicity and the generation of protective and memory immunity to the vaccine, then this might provide a way to genetically manipulate the BCG vaccine to make it more effective.
Acknowledgements The authors are supported by NIH grant AI-40488 and NIH Contract AI-75320. References 1 Cole, S.T. and Barrell, B.G. (1998) Novartis Found. Symp. 217, 160–172 2 Cole, S.T. et al. (1998) Nature 393, 537–544 3 Fleischmann, R.D. et al. (1995) Science 269, 496–512 4 Blattner, F.R. et al. (1997) Science 277, 1453–1474 5 Brosch, R. et al. (1998) Infect. Immun. 66, 2221–2229 6 Brennan, P.J. and Besra, G.S. (1997) Biochem. Soc. Trans. 25, 188–194 7 Orme, I.M. (1999) Adv. Vet. Med. 41, 135–143 8 Orme, I.M. (1997) Int. J. Tuberc. Lung Dis. 1, 95–100 9 Andersen, P. (1994) Infect. Immun. 62, 2536–2544 10 Baldwin, S.L. et al. (1998) Infect. Immun. 66, 2951–2959 11 Horwitz, M.A. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1530–1534 12 Roberts, A.D. et al. (1995) Immunology 85, 502–508 13 Orme, I.M. et al. (1993) J. Immunol. 151, 518–525 14 Sreevatsan, S. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9869–9874 15 Behr, M.A. et al. (1999) Science 284, 1520–1523
Event
Mutation and adaption from the Great Lakes to the Rocky Mountains Caroline P.J. Ash
I
n their natural environments, microorganisms are assaulted by a variety of stimuli that require sensing and response, whether these are other species, pheromones, nutrients or stresses. Unlike colonies grown under laboratory conditions, a natural environment (e.g. the soil surface or an infected wound) can be extremely heterogeneous in space and time, and it is not surprising that the organisms that have been selected to thrive under such extraordinarily variable conditions should be extraordinarily genetically labile
The centenary meeting of the American Society for Microbiology was held in Chicago, IL, USA from 30 May to 3 June 1999 and the 43rd Wind River Conference on Prokaryotic Biology was held at Estes Park, CO, USA on 2–6 June 1999. C.P.J. Ash is Editor of Trends in Microbiology. e-mail: [email protected]
themselves. Both the American Society for Microbiology (ASM) and Wind River (WR) conferences
0966-842X/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
395
provided fascinating revelations on the ways in which microorganisms redesign themselves in the face of widely fluctuating conditions. Quorum sensing and social mobility In a natural environment, bacteria exist neither as isolated cells nor as clones, and often exhibit degrees of social coordination that require extracellular signalling and signal recognition. Bacteria alter gene expression in response to cell density, which is sensed by the recognition PII: S0966-842X(99)01571-1
VOL. 7 NO. 10 OCTOBER 1999
TB vaccine development: after the flood Ian M. Orme and John T. Belisle
I
n this new era of DNA sequencing and associated bioinformatics, in which a relatively large number of genes can be rapidly sequenced, it is now quite possible that most of the leading bacterial pathogens will be fully sequenced within the next five to ten years1. From the perspective of those of us working in the mycobacteria field, the completion of the genome of Mycobacterium tuberculosis H37Rv is probably one of the most important advances in our field since the identification of the bacillus a century ago2. This achievement has unleashed a veritable flood of information that will undoubtedly bear fruit in both drug discovery and the rational design of new vaccines.
The H37Rv genome There are two principal approaches to the technical issue of DNA sequencing. The first is the wholegenome shotgun approach, in which the complete sequence is deduced from large numbers of small fragments3; the second is the use of cosmid or phagemid libraries, which are first ordered to produce a contiguous map of the genome4. The H37Rv genome sequencing project used elements of both approaches to produce the finished product2. Furthermore, because it used bacterial artificial chromosome clones containing large inserts5, the project had the additional benefit of isolating 68 clones, which, together, cover most of the genome and can be used in the future for comparative genome mapping and other uses. The H37Rv genome is 4411 kb in length, encoding ~4000 genes. The G1C content is very high (65%) and the genome is rich in repetitive DNA sequences, particularly insertion sequences, as well as containing new multigene families and copies of housekeeping genes2. There are 3924 open reading frames (ORFs), of which
~40% encode proteins that have been identified by database comparisons and there have been educated guesses about another 40%. The rest of the ORFs encode unknown proteins that might have specific mycobacterial functions. At least 10% of the ORFs encode two families of glycine-rich proteins and such genes often contain multiple polymorphic G1C-rich sequences or major polymorphic tandem repeats. The short peptide motifs that comprise common repetitive domains within these proteins could potentially be involved in a form of antigenic variation and thus these protein families could be potential vaccine targets. The bacterium contains genes encoding a full metabolic arsenal, as well as 13 sigma factors and at least 100 regulatory proteins. But perhaps the most striking aspect of the genome is the vast array of enzymes (.250) involved in the biosynthesis or catabolism of fatty acids; this is consistent with the myriad of lipid-containing structures involved in the structure of M. tuberculosis, particularly its cell wall6. Current efforts in TB vaccine development Although the technological advances in tuberculosis (TB) vaccine development have perhaps not been as dramatic as those in the sequencing technology there have, nevertheless, been a number of innovative approaches, including the use of transposon mutagenesis to develop auxotrophic mutants of the Mycobacterium bovis Bacillus Calmette–Guérin (BCG) strain and M. tuberculosis, the use of the existing BCG vaccine as a recombinant I.M. Orme* and J.T. Belisle are in the Dept of Microbiology, Colorado State University, Fort Collins, CO 80523, USA. *tel: +1 970 491 5777, fax: +1 970 491 5125, e-mail: [email protected]
0966-842X/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
394
vector, the realization that DNA itself is a potential vaccine and, last but not least, the proteomic analysis of the bacillus7,8. In addition to these approaches, a more simplistic approach has been to use subcellular fractionation of the bacillus to generate subunit vaccines. Some of these have been based on whole fractions, such as the culture filtrate, and others on various immunogenic proteins such as ESAT-6, the Ag85 complex and the heat shock protein Hsp70 (Refs 9–12). It has to be stressed, however, that these are very early days and the available information to date has been obtained only from mouse and guinea pig experimental models. In these models, the capacity of the vaccine candidate to confer protection (mainly against aerosol-challenge infection) and, in the guinea pig, to prevent the development of tissue necrosis in the lung, is used as a measure of efficacy10. What will clearly be needed in the future, however, are ways to answer more specific questions regarding the longevity of new vaccines – vaccine challenge interval issues – as well as an animal model to determine whether the vaccine would have any effect in people already inoculated with BCG or could be used specifically to target certain T-cell responses in patients already infected with TB. The impact of genomic information on new vaccine design The genome sequence potentially provides a road map of every drug target and every possible protein target for vaccines. The complex fatty acid pathways in which the bacillus has invested a large proportion of its genome will certainly be a gold mine for drug targeting directed at the biosynthesis of the mycolic acid layer of the cell wall, the mycocerocic acids that are unique to virulent M. tuberculosis isolates, as PII: S0966-842X(99)01591-7
VOL. 7 NO. 10 OCTOBER 1999
COMMENT
well as at the copious and ubiquitous lipoarabinomannan, which spans the entire cell envelope. However, despite the potential impact of genomic data on drug design and the current enthusiasm for ‘directly observed’ therapy, many people believe that the only long-term solution to the global TB pandemic is vaccination. Genomic data will help enormously in tracking down potential antigen targets identified from proteomic and microarray technologies and will also undoubtedly help in identifying new vaccine targets. One of the most interesting pieces of information from the genome sequence is the presence of the two extensive families of glycine-rich proteins (the PE and PPE families), which, given their abundance, might be immunogens. Now that the protein sequences are readily available, simple experiments can be performed to test their immunogenicity, such as recognition of synthetic or recombinant peptides by interferon-gsecreting T cells from infected mice13. This type of analysis will not only reveal if these protein families are potential immunogens, but will also
elucidate which family members are the most potent antigens, as well as indicating if they are differentially produced over the course of an infection. In addition, the genome information confirms earlier work14 suggesting a remarkable lack of genomic diversity at the nucleotide level. This implies that proteins encoded by different strains are likely to be the same, which is useful to know for rational vaccine design. There might also be a direct benefit in terms of understanding why the current BCG vaccine has such variable effects. A recent study by Behr and colleagues15, using comparative hybridization to a DNA microarray, has shown that BCG cultures kept in different laboratories around the world have undergone various gene deletions, resulting in distinct substrains. Although there is no evidence as yet that these deletions are the basis for differences in vaccine efficacy, if they can be related to changes in immunogenicity and the generation of protective and memory immunity to the vaccine, then this might provide a way to genetically manipulate the BCG vaccine to make it more effective.
Acknowledgements The authors are supported by NIH grant AI-40488 and NIH Contract AI-75320. References 1 Cole, S.T. and Barrell, B.G. (1998) Novartis Found. Symp. 217, 160–172 2 Cole, S.T. et al. (1998) Nature 393, 537–544 3 Fleischmann, R.D. et al. (1995) Science 269, 496–512 4 Blattner, F.R. et al. (1997) Science 277, 1453–1474 5 Brosch, R. et al. (1998) Infect. Immun. 66, 2221–2229 6 Brennan, P.J. and Besra, G.S. (1997) Biochem. Soc. Trans. 25, 188–194 7 Orme, I.M. (1999) Adv. Vet. Med. 41, 135–143 8 Orme, I.M. (1997) Int. J. Tuberc. Lung Dis. 1, 95–100 9 Andersen, P. (1994) Infect. Immun. 62, 2536–2544 10 Baldwin, S.L. et al. (1998) Infect. Immun. 66, 2951–2959 11 Horwitz, M.A. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1530–1534 12 Roberts, A.D. et al. (1995) Immunology 85, 502–508 13 Orme, I.M. et al. (1993) J. Immunol. 151, 518–525 14 Sreevatsan, S. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9869–9874 15 Behr, M.A. et al. (1999) Science 284, 1520–1523
Event
Mutation and adaption from the Great Lakes to the Rocky Mountains Caroline P.J. Ash
I
n their natural environments, microorganisms are assaulted by a variety of stimuli that require sensing and response, whether these are other species, pheromones, nutrients or stresses. Unlike colonies grown under laboratory conditions, a natural environment (e.g. the soil surface or an infected wound) can be extremely heterogeneous in space and time, and it is not surprising that the organisms that have been selected to thrive under such extraordinarily variable conditions should be extraordinarily genetically labile
The centenary meeting of the American Society for Microbiology was held in Chicago, IL, USA from 30 May to 3 June 1999 and the 43rd Wind River Conference on Prokaryotic Biology was held at Estes Park, CO, USA on 2–6 June 1999. C.P.J. Ash is Editor of Trends in Microbiology. e-mail: [email protected]
themselves. Both the American Society for Microbiology (ASM) and Wind River (WR) conferences
0966-842X/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. TRENDS
IN
MICROBIOLOGY
395
provided fascinating revelations on the ways in which microorganisms redesign themselves in the face of widely fluctuating conditions. Quorum sensing and social mobility In a natural environment, bacteria exist neither as isolated cells nor as clones, and often exhibit degrees of social coordination that require extracellular signalling and signal recognition. Bacteria alter gene expression in response to cell density, which is sensed by the recognition PII: S0966-842X(99)01571-1
VOL. 7 NO. 10 OCTOBER 1999