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British Medical Bulletin 62:45-58 (2002)
© 2002 Oxford University Press

Bacterial pathogen genomics and vaccines

Richard Moxon and Rino Rappuoli

Molecular Infectious Diseases Group, Weatherall Institute of Molecular Medicine and University of Oxford Department of Paediatrics, John Radcliffe Hospital, Oxford UK
Chiron SpA, Siena, Italy

	Abstract

Top Footnotes Abstract Context and opportunities Facilitating the discovery of... Proof in principle of... Concluding comments References

 
Infectious diseases remain a major cause of deaths and disabilities in the world, the majority of which are caused by bacteria. Although immunisation is the most cost effective and efficient means to control microbial diseases, vaccines are not yet available to prevent many major bacterial infections. Examples include dysentery (shigellosis), gonorrhoea, trachoma, gastric ulcers and cancer (Helicobacter pylori). Improved vaccines are needed to combat some diseases for which current vaccines are inadequate. Tuberculosis, for example, remains rampant throughout most countries in the world and represents a global emergency heightened by the pandemic of HIV. The availability of complete genome sequences has dramatically changed the opportunities for developing novel and improved vaccines and facilitated the efficiency and rapidity of their development. Complete genomic databases provide an inclusive catalogue of all potential candidate vaccines for any bacterial pathogen. In conjunction with adjunct technologies, including bioinformatics, random mutagenesis, microarrays, and proteomics, a systematic and comprehensive approach to identifying vaccine discovery can be undertaken. Genomics must be used in conjunction with population biology to ensure that the vaccine can target all pathogenic strains of a species. A proof in principle of the utility of genomics is provided by the recent exploitation of the complete genome sequence of Neisseria meningitidis group B.

	Context and opportunities

Top Footnotes Abstract Context and opportunities Facilitating the discovery of... Proof in principle of... Concluding comments References

 
The availability of complete genome sequences of pathogenic microbes has revolutionised the possibilities for developing vaccines^1,2. Never before has there been a comprehensive catalogue of all vaccine candidate molecules from which to choose those most likely to be effective. This is the good news. On a more cautious note, the genomic revolution has brought to the fore the immense difficulties inherent in evaluating the large number of potential vaccine candidates emanating from complete genome sequence data. How to arrive at a judicious decision on which of many candidates to take forward into human trials is an immense challenge. The costs of vaccine development to the commercial sector are huge (millions to billions of dollars) and the process lengthy (5–10 years). Given that genomic screens may yield scores, even hundreds of candidates, there is a need to reduce to a handful those antigens that are taken forward into clinical testing. Here we review how genomics, in concert with a number of complementary methods, can improve the prospects of vaccine antigen discovery. Genomics now has a secure place in vaccinology and can facilitate the research and development process, placing it on a more rational footing and accelerating the rapidity with which novel candidates can be taken forward to clinical trials. Before considering the contribution of a complete bacterial genome sequence, some key principles relevant to vaccine development need to be mentioned. Although these principles are general ones and not specifically tied to the availability of whole bacterial genome sequences, they are an essential context in which to evaluate the utility of genomics.

Ideally, a vaccine should be effective against all pathogenic strains of a species. This can be achieved by identifying one or more epitopes that are conserved among all disease-causing strains of a pathogenic species^3,4. This is a tall order when one considers the diversity within natural populations of bacterial pathogens. The size of bacterial populations and their rapid replication generate extensive variation within populations through mutation and recombination and this genetic diversity is far greater for the pathogen than for its human host⁵. For this reason, there is a compelling need to consider population biology as an integral component of vaccine design³, an issue that is of no lesser importance for viral and parasitic, than it is for bacterial, vaccines. This amalgamation of population biology and genomics is critical since complete bacterial genome sequences are typically available for only one strain of a pathogen, although there are exceptions, for example Mycobacterium tuberculosis (two completed genome sequences) and Neisseria meningitidis (two completed genome sequences and a third in progress). Basing a genomic screen for vaccine antigen discovery on one representative strain of the species runs the risk of identifying candidate genes/epitopes that are absent in other genetically distinct pathogenic strains of the species. However, if a gene is present in some strains of the species but is absent from the genome of the sequenced strain, this is not a serious flaw since any robust vaccine candidate should be present in all strains. Most successful bacterial vaccines to date have targeted surface exposed B-cell epitopes. However, host immune responses to cell surface exposed epitopes, through natural selection, result in both intrastrain and interstrain genetic diversity (diversifying selection). However, if genetic drift or shift results in loss of fitness, including virulence, the selective drive towards antigenic variation is constrained. The importance of this functional conservation is that some antigenic structures are retained in a conserved conformation, while strains with variant surface antigens associated with reduced fitness are extinguished. For this reason, many successful vaccines target virulence factors (e.g. toxins and capsular polysaccharides) that are essential to the organism's potential to cause disease.

A second important characteristic for a successful vaccine is that the candidate antigen must be capable of eliciting a protective immune response in humans or the relevant animal for which the vaccine is intended. This raises several related strategic issues. Years of experimentation have shown clearly that one cannot assume any reciprocity between the safety, immunogenicity, and efficacy of a vaccine in humans compared to that observed in laboratory animal models. Most of the currently successful bacterial vaccines (Table 1) were developed through a process of rational empiricism, i.e. trial-and-error testing and where the target antigens are often major virulence factors. This approach has resulted in spectacular global progress in the control of infectious diseases, for example the vaccines against tetanus, meningitis, diphtheria, tuberculosis and whooping cough, all of which are now incorporated into the WHO's extended programme of immunisation. These successes must be tempered by the unmet challenges of preventing major bacterial diseases such as dysentery, pneumonia, urinary tract infections and gastric carcinoma. It can be argued that, to an extent, vaccines for the relatively 'easy' pathogens (none are that easy!) have already been discovered and those major bacterial pathogens for which improved or novel vaccines are needed require more sophisticated approaches (Table 1). There is some legitimacy to this claim. Some of the major bacterial diseases for which vaccines are needed lack the major target molecules such as toxins or capsules that have underpinned some of the existing successful vaccines. For example, the important diseases caused by Neisseria gonorrhoeae or non-typeable (capsule-deficient) Haemophilus influenzae pose diabolical challenges because to date no single antigen has been identified in these pathogens that can be targeted to provide comprehensive protective immunity in humans. A further problem is that these pathogens act predominantly at epithelial surfaces, so that a successful vaccine must be able to provide immunity that acts at the mucosal level. Thus, it may be justly argued that, for these pathogens, the bar has been raised with respect to developing effective vaccines and that rational empiricism is too blunt a tool to make efficient headway, both in the biological sense and, more pragmatically, in economic terms. These considerations strengthen the argument that innovations capable of decreasing the duration and cost of the development process are highly desirable.

View this table: [in this window] [in a new window]   Table 1 Bacterial vaccines (licenced) used in humans and the targeted antigen (if known)

 

	Facilitating the discovery of candidate antigens

Top Footnotes Abstract Context and opportunities Facilitating the discovery of... Proof in principle of... Concluding comments References

 
In early-phase vaccine research, a wide variety of methods can be used in conjunction with complete bacterial genome sequences to facilitate the discovery of promising candidate antigens. These methods include methods that have been developed to provide insights into the fundamentals of the host–microbial interaction in pathogenesis. Many were developed prior to, and independently of, the availability of whole genome sequences, but in conjunction with the latter, can be exploited to discover candidate vaccines. How can genomics facilitate efficient discovery of conserved, protective vaccine antigens in concert with state-of-the-art molecular methodologies? Prior to complete bacterial genome sequences, only a very few genes in any pathogen's genome were known. Classical genetics provided a powerful tool, but was limited in its scope. For example, mutagenesis could not identify genes where loss of their function was conditionally lethal. A major contribution of genomes to early phase vaccine discovery research programmes is that a complete bacterial genome sequence provides a comprehensive inventory of all potential candidate antigens. The utility of this is that one can achieve a sensitivity of 100% (i.e. initially every possible vaccine candidate is identified and its pros and cons can be considered); therefore, no candidate need slip through the net through ignorance of its existence. The only essential tool at this stage is a laptop computer! One of the major facts to emerge from the many completed bacterial genome sequences now available is that, on average, over a third of the genes in any pathogen genome have no known function and many of these have no existing data base matches.

Bioinformatics

The information from complete bacterial genome sequences allows those skilled in bioinformatics to identify genes of interest (genomic mining). However, the tally of candidates at this stage will be numerous and hence the need for adjunct methods to prioritise the most promising potential vaccines that can be realistically taken forward into detailed pre-clinical and clinical testing. The application of sophisticated bioinformatics is essential to unlock and make available the immense fund of information contained in whole genome sequences⁶. There exists a variety of software packages with which to assign functions to genes and predict key features such as cellular location (e.g. surface exposure), molecular weight, pI and solubility. However, even when these analyses have been expertly executed, they can be potentially misleading. For example, in assigning functions based on homology to genes, the function of which has been proved using appropriate assays, it may take only a few changes in a domain to alter its function⁷. On the other hand, there are examples where gene function can be complemented between species where the amino acid identity of the relevant protein is as low as 23%⁸. The message is that though much progress may be made in silico, this information must be supplemented by experimental biology.

Systematic, gene-by-gene analysis of bacterial pathogens

The potential for genomes to provide substantial contributions to vaccinology is reflected in recent publications on functional genomics^9–11. For the vast majority of important human bacterial pathogens, in addition to the availability of complete genome sequences, appropriate techniques for genetic transfer are in place and there are well-validated animal models. There is a wealth of observational data on the biology of infections, as well as comprehensive strain collections by clinical microbiologists. A starting point is the identification, through annotation of the pathogen's genome, of the coding sequences for all proteins and stable RNAs. Precise deletion of all genes, for example using inverse PCR with sequence tagging (bar-coding) and/or fluorescent labelling, can be attempted using a variety of directed strategies. These mutants can be investigated using high throughput screening techniques starting with growth in rich and minimal media in vitro and thence to tissue or organ culture, embryonated chicken eggs, small animals (rats, mice) and eventually natural hosts including, possibly, humans¹². For many of these experiments, pools of deletion mutants can be used in the first instance and aliquots sampled at various times during the growth of cultures or stages of infection. Tags can then be amplified and hybridised to microarrays (see below). Although apparently simple in outline, such ambitious, comprehensive analyses represent a formidable task. However, there are now a number of powerful methods for comparing global gene expression profile changes in pathogenic bacteria, as well as the cells of their natural hosts. These methods include oligonucleotide arrays¹³, serial analysis of gene expression (SAGE)¹⁴, differential cDNA screening¹⁵, expressed sequence tag data base comparison¹⁶, and two dimensional gel electrophoresis of cellular proteins. Many of these methodologies are labour-intensive and not yet suitable for high throughput use, such as SAGE. However, oligonucleotide arrays are a realistic high-throughput methodology, although many technological problems still need to be refined and validated. Nonetheless, there are already many published examples where microarrays have been used to provide identity and expression levels of selected genes simultaneously. The technological advances in the past decade have lead to miniaturisation of the appropriate synthesis and attachment chemistry; thousands of DNA or RNA molecules can be arrayed on a few square centimetres of a solid phase (e.g. glass slide)¹⁷. This allows monitoring of the expression level of genes. As a demonstration of the feasibility of this technology, RNA was extracted from S. pneumoniae cultures, with or without exposure to the competence stimulating peptide (CSP), and labelled with biotin. This allowed investigation of differences in the expression of genes by probing a microarray of S. pneumoniae genes. Whereas most of the studied genes showed no differences in expression, genes from the competence operon were induced¹⁸. There are now several other demonstrations of the feasibility of this approach to monitor and compare gene expression of organisms in different environmental conditions^19,20. However, convincing demonstrations of its application to pathogenic bacteria in vivo as an adjunct to identifying candidate vaccines are yet to come²¹, but we would expect this to be achieved to the point that, within a few years, such an approach is as routine a methodology as automated sequencing.

An elegant and novel example of the application of microarrays is their use to understand the basis of the current variations in the efficacy of Bacille Calmette Guerin (BCG) vaccines to protect against Mycobacterium tuberculosis. Comparative hybridisations between the genomes of M. tuberculosis, M. bovis and the various daughter BCG strains have been performed and 12 novel deletions were found in daughter lineages when these were compared with the progenitor strain²². The efficacy of BCG in different regions of the world is highly variable and both the temporal and geographical differences in its effects on populations are likely to be highly complex and multifactorial. However, at least one tractable reason for these differences may be through genetic differences in the live attenuated strains that have been used in mass immunisation programmes over the years.

Immune responses as an indicator of in vivo function

Using host immune responses to indicate genes expressed in vivo is both powerful and specific. There are numerous examples of using bacteria obtained from patients or infected animals followed by analysis using SDS-PAGE and immunoblotting with convalescent sera²³. However, an innovative recent approach is to capitalise upon the surprising finding that amplified PCR fragments can be rendered transcriptionally active when injected into animals, so-called DNA, nucleic acid²⁴ or genetic immunisation²⁵. Genetic immunisation can be used as an approach to identify vaccines. Since all of the candidate antigens are encoded in the genome, a systematic search for protective antigens can be realised through the construction of expression libraries. The principle is to immunise an animal with a representative library of pathogen DNA that expresses a range of antigens in vivo, challenge the animal and assay for protection. The expression library immunisation (ELI) protocol involves fusing digested whole genome DNA to the last exon of the gene for human growth hormone (hGH) such that the hGH-MP antigens are expressed and secreted. In a representative experiment, Mycoplasma pulmonis DNA was fused into 3 frames of hGH and 9 libraries of about 3000 members were prepared. Each of these would be expected to have about 5000 genes in frame. Following 4 immunisations of mice, the animals were challenged and some libraries were shown to induce protective immunity²⁶. With minor modifications, this approach can be used to carry out a systematic and comprehensive analysis in which the antibodies specific for in vivo expressed proteins are used to detect the microbial products expressed during infection. Such analysis could use fluorescence for in situ work or, more simply, an ELISA on ex vivo material. In the latter instance, plates could be coated with extracts of different infected tissues, normal tissues serving as controls, or the same tissues at different stages of infection to obtain a profile of in vivo expression during pathogenesis. The implication of this approach, published about the same time that whole genome bacterial sequences became available, is that libraries capable of inducing protection could be sub-fractionated to identify the relevant protective DNA immunogens. Only a few nucleotides of the insert DNA responsible for inducing protection need be sequenced using microarrays or anchor PCR and these can then be matched to the whole genome sequence to obtain the full DNA sequence of the gene. This approach would not of course be useful for identifying macromolecules such as capsule or lipopolysaccharide and would suffer from the potential loss of epitopes critical to antibody recognition through in vivo degradation or changes in conformation.

Gene expression in vivo

Signature tagged mutagenesis (STM), developed by David Holden, is an approach based on random mutagenesis to identify genes required for in vivo survival²⁷, and, thereby, a powerful tool for identifying potential virulence factors. A key feature of the technique is that every mutant carries an associated unique sequence of approximately 40 bp so that it is effectively bar coded and can be identified through hybridisation. The tags from a mixed population of bacterial mutants representing the inoculum (input) and bacteria recovered from infected hosts (output) are detected by amplification, radiolabelling and hybridisation analysis. This provides a method that can be used to screen pathogens for gene products whose expression and function in vivo suggests their candidacy as vaccine antigens. Although a whole bacterial genome sequence of the pathogen under study is not essential, its availability, in addition to traditional databases, is greatly facilitating. As an example, Sun et al used STM in conjunction with the two publicly available complete genome sequences of the meningococcus²⁸. Using an infant rat model of invasive infection, a library of 2850 insertional mutants of N. meningitidis was scored and 73 genes were identified that were essential for bacteraemia, many of which were of unknown function. In addition to 8 known virulence genes (providing a validation of the method), 65 novel genes were found, none of which had previously been identified as essential to infection in vivo. Among these, 16 represented surface-expressed candidate antigens that are now under intensive evaluation for their vaccine potential.

In vitro expression technology (IVET) is a genetic system, developed in the laboratory of John Mekalanos, that positively selects for bacterial genes that are specifically induced when bacteria infect their host²⁹. It is well known that the expression of many virulence genes is regulated by environmental conditions and these gene products are specifically required for different stages in the infection process³⁰. IVET sets out to distinguish genes that are active in vivo but not in vitro because essential signals present in the host, but not in culture media, induce their expression. A sub-class of these genes that meet these selection criteria will encode products required for the infection process, including previously unidentified microbial factors that may be candidate vaccine antigens. The IVET strategy begins with a bacterial strain carrying a mutation in a biosynthetic gene that greatly attenuates growth in vivo, for example a purA auxotroph. This mutant cannot grow in the host unless it is supplied with the mutated biosynthetic function. This function is provided by a promoterless purA gene, in which the missing transcription functions are provided by fragments obtained from a random library of whole genome DNA of the pathogen. This provide the basis for selecting in vivo expressed genes using a reporter system in which the purA gene, transcriptionally activated, has been placed in frame with lacZ. Prior to the availability of complete genome sequences, the positively selected (ß-galactosidase positive) fusions were then completely sequenced to identify the genes of interest. However, only a small number of nucleotides from the novel DNA fragment need to be sequenced to provide the information to identify the matching sequences in the whole pathogen genome sequence. Several versions of the IVET principle other than this universal rescue of purA auxotrophy have been developed, each offering different capabilities including detection of intracellular, use of antibiotic selection³¹, or stage-specific expression, use of genetic recombination³², of virulence factors in host tissues. A limitation of IVET is that it will not detect many relevant genes, for example, where there are subtle, but biologically significant, in vitro versus in vivo differences in gene activity. In IVET experiments using mice as an experimental model, a series of studies identified more than 100 virulence associated genes in the pathogen Salmonella typhimurium. Again, among these were many completely novel genes of unknown function. One of these genes was shown to control the expression of several pathogenicity-related functions, including DNA adenine methylase (dam) in S. typhimurium. Mutants deficient in dam were defective in replication in tissues such as spleen and liver and were, therefore, avirulent. These mutants were shown to be effective as live vaccines against murine typhoid fever³¹. Since dam is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality world-wide, Mahan has proposed that the dam gene product is a potentially excellent target for vaccines³³.

Valdivia and Falkow have devised an elegant strategy³⁴ that lends itself perfectly to an approach using genomics and high throughput screening, for example using FACS. The essence of this methodology is that, under particular conditions in vivo, transcriptionally active genes fused to green fluorescence protein (gfp) will fluoresce (differential fluorescence induction). FACS can then be used to identify either fluorescent bacteria or host cells containing fluorescent bacteria. The only genetic requirements are that the bacterial pathogen is able to maintain an episomal element and express the functional gfp. Although, as initially described, this system used random DNA fragments inserted upstream of a promoterless gfp gene, the system could be used for a site-specific strategy using a comprehensive approach based on whole genome sequences. The fluorescence intensity of individual bacteria grown in tissue culture can be compared with the same bacterial clone after release from infected cells. This allows selection of bacteria that are transcriptionally active only within the host cell (for example, a macrophage). In the original study, 14 insertion mutants with intracellular dependent activities were identified. This study was done prior to the availability of the relevant whole genome sequence of S. typhimurium, so the investigators were obliged to characterise these genes the hard way, i.e. by cloning, DNA sequencing and further characterisation. However, with the availability of the whole genome sequences of a pathogen under study, a high throughput, efficient and comprehensive approach should be feasible since, once the promoter is identified, the entire gene to which it corresponds can be easily isolated. Additionally, it will be possible to identify those promoter fusions that are unstable. An advantage of using fluorescence detection is that there is no dependence upon growth. Many pathogens enter viable, but non-culturable states, e.g. Vibrio cholerae. This is, therefore, a powerful strategy for studying environmentally triggered gene induction in bacteria obtained from infected animals or host cells that accurately reflect conditions of natural infections. As has been stressed, the activity of virulence genes is sensitive to precise combinations of environmental conditions. It is, therefore, useful to use such strategies to track differences in gene expression at different times and at different stages of pathogenesis.

	Proof in principle of the application of genomics to vaccine discovery

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An example of how genomics has already been used to identify vaccine candidates that are novel and currently being readied for clinical trials is afforded by research aimed at developing a vaccine against group B strains of the meningococcus (MenB)³⁵. This important pathogen is a major cause of meningitis and septicaemia and, although prevention of disease by most strains can be achieved by vaccines based on group specific capsular polysaccharides, this approach is not easily applicable to the development of vaccines against group B strains. This is for two reasons. First, the B capsular polysaccharide, an -2-8-linked polysialic acid, is an extremely poor immunogen even when conjugated to a carrier protein³⁶. Second, there are concerns about the safety of using the -2-8-linked polysialic acid as an immunogen since the identical structure is present on the surface of human cells and, therefore, the induction of autoantibodies could be harmful³⁷. As a result, there has been a substantial research effort over many years to identify alternative non-capsular antigens. In effect, antigens known to be surface expressed and of functional importance (therefore heightening the probability of broad conservation in virtually all relevant strains) have been put through their paces in numerous early phase research projects. After varying periods of this trial and error approach (rational empiricism), most of the proposed candidates have been found to be either insufficiently conserved or unable to elicit protective immunity and have, therefore, been discounted. Clearly, this has been an inefficient, frustrating, and costly process and sadly deficient in providing any convincing vaccine candidates. The availability of the complete genome sequence of MenB³⁸ provided an opportunity to take a different, genomic-based approach (Fig. 1). While the sequencing project was in progress, unassembled DNA fragments were analysed to identify open reading frames (ORFs) that potentially encoded novel surface-exposed or exported proteins. A primary screening for coding capacity was carried out on DNA segments or contigs using database and computer programmes to exclude proteins with cytoplasmic functions and identify surface exposed or secreted proteins. This search identified 570 such ORFs, and the sequences of these predicted genes were amplified using polymerase chain reaction and then cloned into E. coli to express each polypeptide. Successful expression was achieved in the majority (61%) and included lipoproteins, inner membrane, periplasmic, and outer membrane proteins. The recombinant proteins were used to immunise mice and the post-immunisation sera were analysed to verify the specific reactivity with, and surface localisation of, each polypeptide. This was carried out using both the immunising strain and a panel of genetically diverse MenB strains representative of the global diversity of the natural population of hypervirulent strain lineages³⁹. To further identify polypeptides capable of inducing functional antibodies, the post-immunisation sera and pre-immunisation sera were compared using bactericidal assays. Sera exhibiting killing activity provided a strong indication of proteins capable of inducing protective immunity⁴⁰. Out of 85 proteins that showed convincing evidence of surface localisation, 22 also showed activity using the bactericidal assay, some of which were comparable to the titres obtained using outer membrane vesicles, a vaccine that has been shown to confer protection in humans in clinical trials⁴¹. Most importantly, at least 5 proteins showed relatively complete conservation of their amino acid sequence across the panel of genetically distinct hypervirulent MenB strains. This represents an important finding since, prior to this genomic approach to vaccine discovery, the only Men B proteins that have been described and considered as vaccine candidates exhibited marked antigenic heterogeneity. The proteins inducing the most cross-reacting bactericidal response are now being studied alone or in combination in order to find a vaccine formulation that is effective against all strains. The combination of several antigens in the vaccine formulation should be able to avoid the generation of escape mutants which are to be expected for single antigens. One of the antigens identified by genome screening (GNA 33), although conserved in sequence, was found to induce immunity against only a subgroup of strains possessing the P1.2 sub-serotype porin A. It was found that this antigen contains a QTP peptide that induces strong antibodies against the loop 4 of porin A, where the QTP sequence is also present⁴². This example shows that the genome-based approach not only finds genuine antigens but can also identify mimetic antigens that induce protective immunity against other target epitopes present in the bacterium. Although the application of genome sequences to vaccine discovery has not yet resulted in a product of proven efficacy, several of the candidates identified through the complete MenB genome sequence are now under intensive investigation and a small number are being prepared for testing in phase 1 trials.

View larger version (70K): [in this window] [in a new window]   Fig. 1 Diagram depicting how complete microbial genome sequence data can accelerate vaccine development1.

 

	Concluding comments

Top Footnotes Abstract Context and opportunities Facilitating the discovery of... Proof in principle of... Concluding comments References

 
Infectious diseases remain the most important global cause of death and disability in mankind; pathogenic bacteria contribute about half of this burden. The use of whole genome sequences of bacteria has changed forever the approach to the investigation of bacterial pathogens and is being used to augment the discovery of novel therapeutics, diagnostics and vaccines. Other than improved water supplies, no public health measure to date can match the impact of vaccines in reducing the toll wrought by pathogens. Genomics is certain to be a facilitating component of the vaccine discovery process and can make unique contributions because the complete sequence database makes available every potential vaccine candidate. For most of the world's major bacterial pathogens, this information can be accessed through publicly available databases (http://www.sanger.ac.uk/pathogens/ or http://www.tigr.org/tdb/mdb/mdb.html).

	Footnotes

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Correspondence to: Prof. E Richard Moxon. Molecular Infectious Diseases Group, Weatherall Institute of Molecular Medicine and Department of Paediatrics, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK

	References

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