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Applications of molecular microbiology to vaccinology
THE LANCET
Vaccine series
Applications of molecular microbiology to vaccinology E Richard Moxon Genetics, cell biology, and whole-genome sequencing of pathogens have changed dramatically the opportunities to investigate the epidemiology, pathogenesis, diagnosis, and control of microbial diseases. For example, recombinant DNA and PCR are powerful tools used to isolate genes whose role in pathogenicity can be investigated in biologically relevant virulence assays. Vaccines that target one or more of these genes can then be developed. Complete genome sequences of microbes provide an inventory of the genes encoding every virulence factor and potential immunogen. Candidate vaccines can be selected and developed using various approaches, including the recent innovation of immunisation with nucleic acids. Although many successful vaccines have been and will continue to be developed through empirical approaches, molecular microbiology provides a rational basis for discovery, development, and implementation of safer, more effective and, potentially cheaper vaccines. In the Golden Age of microbiology (the latter part of the 19th and early part of the 20th Century), it was discovered that particular microbes were responsible for specific diseases. Allied with the concept of host immunity, this eventually led to the development of vaccines which have controlled several major microbial diseases (yellow fever, diphtheria, tetanus, pertussis, poliomyelitis, measles, mumps, and rubella) and eradicated small pox. Starting with sulphonamides and penicillin, the decades of 1930, 1940, 1950, and 1960 witnessed the discovery of most of the important classes of antimicrobial drugs. By the middle of the 1990s, improved socioeconomic circumstances, immunisation, and antibiotics had transformed our capacity to treat, control, and even prevent the ravages of many of the most common and serious microbial diseases, at least in relatively affluent societies. In retrospect, this seems to have caused an inappropriate complacency concerning the impact of infections. Towards the end of this century, there has been appropriate recognition of the continuing public-health challenges posed by microbes, including the emergence of new pathogens, or old foes with novel attributes, antibiotic resistance, and of the vast unmet challenges of many infections for which vaccines are not available. Over the past three decades, the Golden Age of molecular biology, genetics, and cell biology have been used to dissect the details of host microbial interactions. This has brought about a revolution which has transformed our knowledge of epidemiology, pathogenesis, diagnosis, and prevention of microbial diseases. This article attempts to place in perspective some of the opportunities and the challenges, especially those emanating from the impact of molecular microbiology, in the field of vaccinology. From birth until death, man is besieged by a myriad of viruses, bacteria, fungi, and parasites competing to stay alive and to perpetuate their genes. Starting at birth, a variety of microbes establish themselves on the skin,
Lancet 1997; 350: 1240–44 Molecular Infectious Diseases Group, Institute of Molecular Medicine and Oxford University Department of Paediatrics, John Radcliffe Hospital, Oxford OX3 9DU, UK (Prof E R Moxon FRCP)
1240
mucous membranes, or gastrointestinal tract in the process of colonisation. In most instances, microbes and man coexist in a mutually benign or even beneficial partnership since these commensal microflora stimulate host immunity to protect against other invading organisms and may contribute micronutrients or growth factors. Rarely, colonisation by these living invasive organisms results in infectious disease and the impairment of health. This potential to injure or kill hosts (pathogenicity) is a general characteristic of all microbes and, directly or indirectly, is a constant challenge to human health. Pathogenicity is dependent on both the state of the host and a variety of microbial factors, and this mutuality and the implicit co-evolutionary implications are key concepts.
Molecular approaches to vaccinology The application of molecular biology to the identification of virulence genes has opened the door to understanding the fundamental basis of the pathogenic personality of virulent microbes. Starting from the classical version of Koch’s postulates, the new genetics has given us the molecular tools to help our understanding of virulence, as follows:1 identify a gene, or group of genes, responsible for virulence; isolate the gene through recombinant DNA technology (cloning) or PCR; make many copies of these genes in vitro; and show the essential role of this gene in pathogenicity by showing that a mutation of a specific gene results in attenuation of virulence in an appropriate model or that a monoclonal antibody specific to this gene product can protect against disease. The major functions of virulence genes include: tropism for the host species, or even for specific cells of individual hosts; mechanisms for the microbe to survive host clearance and multiply in the host; and the capacity to cause tissue damage or cytoxicity. For each of these, there are examples of vaccines which are effective because they target one or more of the virulence molecules responsible for these virulence functions (table). The molecular genetic definition of virulence has enormous implications for vaccinology. Although the concept of inducing immunity to essential virulence determinants is not new, the use of molecular techniques has helped in the discovery of many more ways of
Vol 350 • October 25, 1997
THE LANCET
Bacterial species
Microbial determinant
Biological function
Contribution to virulence
Vaccine
Bordetella pertussis
Filamentous haemagglutinin (FHA)
Adhesin (promotes attachment to respiratory epithelium)
Facilitates damage to respiratory epithelium
Component of acellular pertussis vaccines
Neisseria meningitidis
Transferrin-binding protein
Acquisition of iron
Increases survival and replication in vivo
Sub-unit vaccine (at preclinical trial stage)
Haemophilus influenzae
Type b capsular polysaccharide
Inhibits phagocytosis and complement mediated lysis
Enhances survival and dissemination via blood-stream
Conjugate vaccine
Clostridium tetani
Toxin
Cytotoxicity
Blocks synaptic transmission and causes tetanus
Toxoid
Some examples of biological, virulence, and vaccine potential of pathogenic bacteria
identifying and characterising virulence genes of pathogens and the molecules that they encode. Furthermore, using the techniques of recombinant DNA or PCR, virtually unlimited amounts of purified antigen can then be made. This is very different from the more empirical approach to vaccine development in which whole organisms are inactivated, for example with formalin, or attenuated by growth in an unnatural host thereby preventing it from causing disease in its natural host. Although this classic approach to immunisation has been remarkably successful and will undoubtedly continue to contribute to the provision of many more new and improved vaccines, it has some obvious disadvantages which can be addressed by the new genetics. These include inadvertent retention of some live organisms, reversion to virulence (eg, Sabin poliomyelitis vaccine occasionally causes paralytic disease) and inclusion of components not contributory to immunity but harmful to the recipient of the vaccine (reactogenicity). There are also difficulties in maintaining the biological activity of attenuated vaccines in transit and in storage. In contrast, modern molecular techniques offer improved characterisation of, and therefore control over, biological properties, including elimination or reduction of side effects. Subunit virus vaccines illustrate particularly well the application of the reductionist approach to making a candidate protein vaccine. In many instances, protective immune responses are elicited by surface polypeptides which coat the virus (eg, hepatitis B)2 or protrude from the viral cell surface projecting through the lipid envelope (eg, influenza).3 It is now relatively straight forward to identify the gene encoding a candidate immunogen. The nucleotide sequence (DNA or RNA) can be cloned into a suitable vector, replicated, and made to produce virtually unlimited amounts of the desired protein even when the virus, as is the case for hepatitis B, cannot be. Another elegant example of the application of this molecular approach to vaccine development is the genetic modification of the bacterial Bordetella pertussis toxin (PTX). PTX is thought to be an essential component of all vaccines that are effective in preventing whooping cough. The development of vaccines that cause fewer reactions than the whole-cell preparations of killed pertussis organisms has been achieved with chemically inactivated PTX alone or in combination with other antigens. By cloning the relevant genes, it was possible to change aminoacids of one of the subunits of PTX so as to inactivate the enzymatic properties responsible for toxicity. When used in an acellular whooping cough vaccine, this mutated PTX was found to be highly immunogenic, safe, and effective.4 A further very important use of these molecular techniques is in epidemiology, diagnosis, and in the establishment of the impact of a microbe as a public-
Vol 350 • October 25, 1997
health problem. Increasingly, molecular epidemiology is an essential tool used in clinical trials and surveillance of the impact of vaccines. Not only are genes passed on to progeny through the normal replicative processes of chromosomal or extra-chromosomal nucleic acids (vertical transmission), but these genes may also be spread to other organisms (horizontal spread) by a variety of mechanisms, including transformation by DNA released from a donor cell, transduction involving phage (bacterial viruses), and transposition of mobile genetic elements. These are highly efficient mechanisms by which extant genetic material is exchanged, added, or deleted. These mechanisms are also pomiscuous in that the transfer of genetic material may occur between closely related or unrelated organisms. Genetics can be used to track the prevalence and spread of particular genes or sequences relevant to virulence across time and geographical boundaries, while monitoring associations with disease. In 1982, for example, distinctive clones of Escherichia coli (O157:H7) were found to harbour a toxin (verotoxin) that causes haemorrhagic colitis and haemolytic-uraemic syndrome.5 With PCR and assays specific for the toxin, the prevalence and spread of this virulence factor could be tracked. This application of molecular techniques to facilitate epidemiological surveillance focuses on the detection of the virulence gene rather than the strain. This has lead to the discovery that verotoxin is not confined to O157:H7 strains since the identical gene has been found in association with other, epidemiologically distinct E coli strains (eg, serotype O103:H3).6 A vexing problem in public health is the unexpected manner in which epidemics or outbreaks of microbial diseases occur. Despite efforts to understand the dynamics of host microbial interactions so as to inform health policies for individuals and populations, the imperfections of our knowledge are such that the occurrence of epidemics is often unanticipated. The mutuality of host and microbe is central to understanding their co-evolution and the potential for disease, yet genetically determined variations of microbes and their hosts (the latter occurring at a slower rates than in microbes), followed by natural selection, complicate these interactions. As a consequence, it has been difficult to disentangle the relative roles of microbial virulence and host-immune responses responsible for epidemics. Antigenic variation is a striking feature of the biology of many viruses and free-living pathogenic parasites, fungi and bacteria.7 Examples include the drift and shift of key molecules (haemagglutinins and neuraminidase) on the surface of the influenza virus,8 or the glycoproteins of HIV-1.9 Among macroparasites, antigenic variation is seen in the var genes of Plasmodium falciparum,10 and the variant surface glycoproteins of trypanosomes.11 In bacteria, antigenic variation is exemplified by adhesins 1241
THE LANCET
Current status of whole genome sequences of major bacterial pathogens and parasites Completed and published
Completed but unpublished
In progress
Haemophilus influenzae Mycoplasma genitalium Mycoplasma pneumoniae Escherichia coli K-12 Helicobacter pylori
Borrelia burgdorferi Streptococcus pneumoniae Treponema pallidum
Bacillus subtilis Chlamydia trachomatis Enterococcus faecalis Legionella pneumophila Leishmania major Mycobacterium leprae
(fimbriae or pili)12 and in fungi, in the global switching of Candida albicans.13 Antigenic variations underlie much of the biological complexity of host-microbe interactions— eg, malaria with its variable patterns of disease, the occurrence of mini-epidemics, and the slowly acquired, imperfect nature of host immunity. Appreciation of the extent of variations in cell-surface molecules is critically important to the choice of vaccine candidates and their design and development because these changes impart the potential to evade the immunity induced by immunisation. Typically, phenotypic switching (eg, phase variation) occurs in individual organisms at frequencies of about one per 100 to one per 10 000 per generation. But a crucial factor, in addition to the frequency with which variation occurs, is its unpredictability. When and which of the variant phenotypes occurs is by chance and not by a programmed event. Indeed, the very fitness of microbes to adapt and survive through natural selection, attributes which include commensal or virulence behaviour, is a dynamic trade-off between variation and stability—an evolving agenda which underpins the interactions between microbe and host14. Another poorly understood but important issue is whether microbial virulence and transmissibility are coupled. Central to this is the concept that pathogens tend to evolve a level of virulence which maximises their reproductive rate (R0), the average number of new infections caused by a single infectious host in a population of susceptible individuals.15 The relation between transmissibility and microbe-induced morbidity and mortality varies among pathogens. For many pathogens, virulence and transmission are closely associated, but this is by no means always so. Although an increased rate of replication or its potential to injure host tissues might be expected to increase the transmission of a microbe, in at least two circumstances this is not the case. First, the host organ or tissue in which the organisms normally multiply and produce transmissible progeny may be different from that in which the organisms caused disease. Second, in many infections, host immuneresponses may be the major determinant of disease, which is a situation which will not increase transmissibility and may even decrease it. A detailed understanding of the links between transmission and virulence for a particular organism has important implications for public-health measures to control microbial diseases, including the policies informing the use of vaccines and antibiotics.16
Whole-genome sequencing The complete nucleotide sequences of several viral genomes have been available for several years but, in the past 2 years, technology has advanced to the point in which this is possible for other pathogens (panel). In the near future, the whole nucleotide sequences of the genomes of Mycobacterium tuberculosis and the malaria 1242
Mycobacterium tuberculosis Neisseria gonorrhoeae Neisseria meningitidis Plasmodium falciparum Pseudomonas aeruginosa Pyrobaculum aerophilum
Rickettsia prowazekii Staphylococcus aureus Staphylococcus epidermitis Streptococcus pyogenes Trypanosoma cruzi Vibrio cholerae
parasite Plasmodium falciparum will be known. The era of genomic sequences affords extraordinary opportunities for the use of immunisation since the complete DNA sequence of an organism provides a catalogue of the genes for every virulence factor and potential immunogen from which to select potential vaccine candidates.17 A particularly exciting fact revealed by the availability of whole genome sequences is that a large proportion of genes are of unknown function and will probably include many that would not have been revealed by classic genetic approaches such as random mutagenesis. In the future, therefore, the key issue will be much more one of selecting which of the many potential candidates most warrant investment as potential vaccine candidates and what constitutes the optimum methods of antigen presentation. An example is the use of the complete genome sequence of H influenzae to identify and characterise the multiple genes involved in lipopolysaccharide biosynthesis. Lipopolysaccharide is a critical structural and functional component of the cell envelope of a whole range of bacterial genera. In H influenzae, lipopolysaccharide is critical to the pathogenesis of infection. It modulates interactions with respiratory epithelial cells, plays a part in the invasive process whereby bacteria translocate from the respiratory tract to the blood, prevents clearance of bacteria from the blood, and is a major factor in the modulation of inflammation and tissue injury. It is a highly complex glycolipid whose biosynthesis requires a large number of genes (more than 30) for synthesis of the precursor units which include fatty acids, sugars, and side-chain substituents such as phosphoryl choline. These are synthesised and assembled initially in the cytoplasm, transported outwards, and assembled on the cell surface. Only a few of the relevant genes had been identified before the availability of the whole genome sequence, but once this was available, more than 25 additional genes were identified.18 A powerful method of extracting the information from the genome database was the use of previously published sequences deposited in general databases, particularly those known to be involved in lipopolysaccharide synthesis of other organisms. These database probes were used to search the H influenzae genome for homologous sequences. When matches were found within open reading frames, these candidate lipopolysaccharide genes were cloned and characterised. By making mutations in each of these genes, it was possible to determine whether they were involved in lipopolysaccharide biosynthesis because mutations would alter the lipopolysaccharide molecule. This change could be detected by reactivity with monoclonal antibodies, gel fractionation patterns, and electrospray mass-spectrometry (figure).23,24 Studies in an experimental animal infection model provided an estimate of the minimal lipopolysaccharide structure required for virulence. The extent of conservation of
Vol 350 • October 25, 1997
THE LANCET
How a whole-genome sequence of a pathogenic microbe can be used to develop a candidate vaccine 1: representation of lipopolysaccharide, lipid A (endotoxin) is inserted into the cell envelope of the bacterium; inner and outer core saccharides, projecting outwards from the cell envelope, are important in mediation or modulation of interactions with host cells.23 2: whole-genome sequence of H influenzae Rd, includes all of the genes involved in the biosynthesis of H influenzae [reproduced with permission from Fleischmann et al24]. 3: nucleic acid sequences of genes or their deduced aminoacid sequences involved in lipopolysaccharide biosynthesis of other bacteria, obtained from general database searches, are used as database probes to search the H influenzae genome for homologues that are candidate lipopolysaccharide genes. 4: when a match is found the sequence can be inspected in the H influenzae genome database and is available as the basis for further experiments. 5: oligonucleotide primers can be constructed to obtain multiple copies of the desired candidate lipopolysaccharide gene using PCR. 6: these candidate lipopolysaccharide genes are cloned into a suitable plasmid vector; a mutation of this gene can then be constructed. In this example the gene has been disrupted by an insertion of a cassette of DNA containing a gene for kanamycin resistance that acts also as a selectable marker. 7: the cloned and mutagenised gene is introduced into H influenzae by transformation to achieve allelic replacement; the phenotype of the parent strain and its mutant can then be compared to determine whether there are differences in lipopolysaccharide. 8: with colony immunoblotting, the reactivity of the parent and mutant strain are compared; the mutant lipopolysaccharide has lost its capacity to bind to monoclonal antibody, whereas colonies of the parent strain bind this antibody. This provides strong evidence in support of a function for this gene in lipopolysaccharide biosynthesis. 9: further tests of biological function on parent and mutant can be done to further characterise the role of lipopolysaccharide in virulence. 10: information on the many genes involved in lipopolysaccharide biosynthesis provides detailed information on structure and its potential for use in vaccine development.18
Vol 350 • October 25, 1997
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THE LANCET
lipopolysaccharide-related genes in other strains can now be investigated to provide a rational basis for the evaluation of the candidacy of lipopolysaccharide for broad range vaccine development. Indeed, the information provided by whole-genome sequencing opens up a wealth of possibilities in vaccine research and development. Attenuation can be approached at the genome level by identifying and deleting the minimal set of genes required for virulence so as to achieve optimal attenuation and safety as vaccine candidates in their own right or as vectors for delivery of foreign antigens.
Nucleic acids as immunogens In 1990, Wolff and colleagues reported that direct intramuscular injection of mice with DNA, a plasmid containing the complete sequence of a gene, resulted in persistent expression of the encoded protein.19 These astonishing observations have been confirmed and expanded in animals to show durable and reproducible protective immunity for several important pathogens including influenza,20 malaria,21 and HIV-1.22 Although many issues, including theoretical safety considerations associated with polynucleotide vaccination, will need to be carefully examined, there could be some substantial advantages to this approach. These include the induction of immunity in the absence of the infectious agent, a considerable advantage for vaccines against organisms such as HIV or those with a propensity for long-term persistence in the body—eg, cytomegalovirus. Another advantage is that individual immune responses are dictated by particular MHC gene variations (alleles). In outbred populations, naked DNA immunisation would allow a selection of nucleic acids corresponding to a range of polypeptides to overcome this problem of T-cell restriction, thereby increasing the number of responders. Finally, stability and ease of production would make naked DNA an attractive proposition for global immunisation strategies.
Future prospects As our understanding of microbes deepens, new approaches to the control of infections, especially vaccines, will be feasible. In particular, there is a need to consider more broadly the ways in which novel vaccines may be used to control or modulate microbial behaviour without the aggressive disruption of their ecology which is characteristic of many antimicrobials. The difficulty of nosocomial infections caused by bacteria and fungi in immune compromised individuals—trauma, cancer, transplantation, prematurely born infants—is one of microbial opportunism. The blunderbuss use of antibiotics has proved a two-edged sword, successful sometimes in treating life-threatening infections but at the expense of drastic decimation of normal commensal flora and the selection of antibiotic resistance (eg, enterococci and staphylococci). The complex orchestration and organisation of genes permissive for, or directly involved in, commensal or virulence behaviour are beginning to be unravelled. There are signalling systems which allow microbes to sense changes in their environment—eg, as an organism translocates from an extracellular to an intracellular location in the host. The transcription of genes which results in the translation of proteins can be
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monitored by global regulators of the microbe so that several gene activities can be coordinately and appropriately switched on or off. An enormous body of information about the structural details of molecular interactions of microbial ligands and host receptors is now available that provides a rational basis for the interruption, in a selective manner, those events that presage pathogenicity and offer novel strategies for the control of microbial diseases, including vaccines. I thank H Masoud and JR Brisson for providing the structural and conformational representations of H influenzae lipopolysaccharide used in the figure, D Hood and JC Richards for helpful discussions, and D Watson for her help in the preparation of the typescript and the figure. I received support from the Medical Research Council Programme Grant.
References 1 2 3 4 5
6
7
8 9 10
11 12 13
14 15 16 17 18
19 20
21
22
23
24
Falkow S. Molecular Koch’s postulates applied to microbial pathogenicity. Rev Infect Dis 1988; 10: S274–276. Jilg W, Schmidt M, Deinhardt F. Four year experiences with a recombinant hepatitis B vaccine. Infection 1989; 17: 70–76. Murphy BR, Webster RG. Influenza viruses. In: Fields BN, ed. Virology. New York: Raven Press, 1985. 1179–239. Pizza MG, Covacci A, Bartoloni A, et al. Mutants of pertussis toxins suitable for vaccine development. Science 1989; 246: 497–500. Riley LW, Remis RS, Helgerson SD, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Eng J Med 1983; 308: 681–85. Tarr PI, Fouser LS, Stapleton AE, et al. Hemolytic-uremic syndrome in a six year old girl after a urinary tract infection with Shiga-toxinproducing Escherichia coli O103:H2. N Engl J Med 1996; 335: 635–38. Deitsch KW, Moxon ER, Wellems TE. Shared themes of antigenic variation and virulence in bacterial, protozoal and fungal infections. Microbiol Molec Biol Rev 1997; 61: 281–93. Kendall AP. Epidemiological implications of changes in the influenza virus genome. Am J Med 1987; 82: S4–14 Nowak MA, Bangham CR. Population dynamics of immune responses to persistent viruses. Science 1996; 272: 74–79. Baruch DI, Pasloske BL, Singh B, et al. Cloning the P falciparum gene encoding PfEMP1, a malaria variant antigen and adhesion receptor on the surface of parasitised human erythrocytes. Cell 1995; 82: 77–87. Borst P, Rudenko G. Antigenic variation in African triponosomes. Science 1994; 264: 1872–873. Heckels JE. Structure and function of pili of pathogenic neisseria species. Clin Microbiol Rev 1989; 2: S66–73. Kennedy MJ, Rogers AL, Hanselman LR, et al. Variation in adhesion and cell surface hydrophobicity in Candida albicans white and opaque phenotypes. Mycopathologia 1988; 102: 149–56. Moxon ER, Thaler DS. The tinkerer’s evolving toolbox. Nature 1997; 387: 659–62. Anderson RM, May RM. Coevolution of hosts and parasites. Parasitology 1982; 85: 411–26. Lipsitch M, Moxon ER. Virulence and transmissibility of pathogens: what is the relationship? Trends Microbiol 1997; 5: 31–37. Moxon ER. Whole genome sequencing of pathogens: a new era in microbiology. Trends Microbiol 1995; 3: 335–37. Hood DW, Deadman ME, Allen T, et al. Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis. Molec Microbiol 1996; 22: 951–65. Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science 1990; 247: 1465–68. Ulmer J, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993; 259: 1745–49. Hoffman SL, Doolan DL, Sedagah M, et al. Nucleic acid malaria vaccines. Current status and potential. Ann N Y Acad Sci 1995; 772: 88–94. Wang B, Ugen KE, Srikantan V, et al. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proc Natl Acad Sci 1993; 90: 4156–60. Masoud H, Moxon ER, Martin A, Krajcarski D and Richards JC. Structure of the variable and conserved lipopolysaccharide oligosaccharide epitopes expressed by Haemophilus influenzae serotype b strain Eagan. Biochemistry 1997; 36: 2091–103. Fleischmann RD, Adams MD, White O, et al. The genome of Haemophilus influenzae Rd. Science 1995; 269: 496–512.
Vol 350 • October 25, 1997
Vaccine series
Applications of molecular microbiology to vaccinology E Richard Moxon Genetics, cell biology, and whole-genome sequencing of pathogens have changed dramatically the opportunities to investigate the epidemiology, pathogenesis, diagnosis, and control of microbial diseases. For example, recombinant DNA and PCR are powerful tools used to isolate genes whose role in pathogenicity can be investigated in biologically relevant virulence assays. Vaccines that target one or more of these genes can then be developed. Complete genome sequences of microbes provide an inventory of the genes encoding every virulence factor and potential immunogen. Candidate vaccines can be selected and developed using various approaches, including the recent innovation of immunisation with nucleic acids. Although many successful vaccines have been and will continue to be developed through empirical approaches, molecular microbiology provides a rational basis for discovery, development, and implementation of safer, more effective and, potentially cheaper vaccines. In the Golden Age of microbiology (the latter part of the 19th and early part of the 20th Century), it was discovered that particular microbes were responsible for specific diseases. Allied with the concept of host immunity, this eventually led to the development of vaccines which have controlled several major microbial diseases (yellow fever, diphtheria, tetanus, pertussis, poliomyelitis, measles, mumps, and rubella) and eradicated small pox. Starting with sulphonamides and penicillin, the decades of 1930, 1940, 1950, and 1960 witnessed the discovery of most of the important classes of antimicrobial drugs. By the middle of the 1990s, improved socioeconomic circumstances, immunisation, and antibiotics had transformed our capacity to treat, control, and even prevent the ravages of many of the most common and serious microbial diseases, at least in relatively affluent societies. In retrospect, this seems to have caused an inappropriate complacency concerning the impact of infections. Towards the end of this century, there has been appropriate recognition of the continuing public-health challenges posed by microbes, including the emergence of new pathogens, or old foes with novel attributes, antibiotic resistance, and of the vast unmet challenges of many infections for which vaccines are not available. Over the past three decades, the Golden Age of molecular biology, genetics, and cell biology have been used to dissect the details of host microbial interactions. This has brought about a revolution which has transformed our knowledge of epidemiology, pathogenesis, diagnosis, and prevention of microbial diseases. This article attempts to place in perspective some of the opportunities and the challenges, especially those emanating from the impact of molecular microbiology, in the field of vaccinology. From birth until death, man is besieged by a myriad of viruses, bacteria, fungi, and parasites competing to stay alive and to perpetuate their genes. Starting at birth, a variety of microbes establish themselves on the skin,
Lancet 1997; 350: 1240–44 Molecular Infectious Diseases Group, Institute of Molecular Medicine and Oxford University Department of Paediatrics, John Radcliffe Hospital, Oxford OX3 9DU, UK (Prof E R Moxon FRCP)
1240
mucous membranes, or gastrointestinal tract in the process of colonisation. In most instances, microbes and man coexist in a mutually benign or even beneficial partnership since these commensal microflora stimulate host immunity to protect against other invading organisms and may contribute micronutrients or growth factors. Rarely, colonisation by these living invasive organisms results in infectious disease and the impairment of health. This potential to injure or kill hosts (pathogenicity) is a general characteristic of all microbes and, directly or indirectly, is a constant challenge to human health. Pathogenicity is dependent on both the state of the host and a variety of microbial factors, and this mutuality and the implicit co-evolutionary implications are key concepts.
Molecular approaches to vaccinology The application of molecular biology to the identification of virulence genes has opened the door to understanding the fundamental basis of the pathogenic personality of virulent microbes. Starting from the classical version of Koch’s postulates, the new genetics has given us the molecular tools to help our understanding of virulence, as follows:1 identify a gene, or group of genes, responsible for virulence; isolate the gene through recombinant DNA technology (cloning) or PCR; make many copies of these genes in vitro; and show the essential role of this gene in pathogenicity by showing that a mutation of a specific gene results in attenuation of virulence in an appropriate model or that a monoclonal antibody specific to this gene product can protect against disease. The major functions of virulence genes include: tropism for the host species, or even for specific cells of individual hosts; mechanisms for the microbe to survive host clearance and multiply in the host; and the capacity to cause tissue damage or cytoxicity. For each of these, there are examples of vaccines which are effective because they target one or more of the virulence molecules responsible for these virulence functions (table). The molecular genetic definition of virulence has enormous implications for vaccinology. Although the concept of inducing immunity to essential virulence determinants is not new, the use of molecular techniques has helped in the discovery of many more ways of
Vol 350 • October 25, 1997
THE LANCET
Bacterial species
Microbial determinant
Biological function
Contribution to virulence
Vaccine
Bordetella pertussis
Filamentous haemagglutinin (FHA)
Adhesin (promotes attachment to respiratory epithelium)
Facilitates damage to respiratory epithelium
Component of acellular pertussis vaccines
Neisseria meningitidis
Transferrin-binding protein
Acquisition of iron
Increases survival and replication in vivo
Sub-unit vaccine (at preclinical trial stage)
Haemophilus influenzae
Type b capsular polysaccharide
Inhibits phagocytosis and complement mediated lysis
Enhances survival and dissemination via blood-stream
Conjugate vaccine
Clostridium tetani
Toxin
Cytotoxicity
Blocks synaptic transmission and causes tetanus
Toxoid
Some examples of biological, virulence, and vaccine potential of pathogenic bacteria
identifying and characterising virulence genes of pathogens and the molecules that they encode. Furthermore, using the techniques of recombinant DNA or PCR, virtually unlimited amounts of purified antigen can then be made. This is very different from the more empirical approach to vaccine development in which whole organisms are inactivated, for example with formalin, or attenuated by growth in an unnatural host thereby preventing it from causing disease in its natural host. Although this classic approach to immunisation has been remarkably successful and will undoubtedly continue to contribute to the provision of many more new and improved vaccines, it has some obvious disadvantages which can be addressed by the new genetics. These include inadvertent retention of some live organisms, reversion to virulence (eg, Sabin poliomyelitis vaccine occasionally causes paralytic disease) and inclusion of components not contributory to immunity but harmful to the recipient of the vaccine (reactogenicity). There are also difficulties in maintaining the biological activity of attenuated vaccines in transit and in storage. In contrast, modern molecular techniques offer improved characterisation of, and therefore control over, biological properties, including elimination or reduction of side effects. Subunit virus vaccines illustrate particularly well the application of the reductionist approach to making a candidate protein vaccine. In many instances, protective immune responses are elicited by surface polypeptides which coat the virus (eg, hepatitis B)2 or protrude from the viral cell surface projecting through the lipid envelope (eg, influenza).3 It is now relatively straight forward to identify the gene encoding a candidate immunogen. The nucleotide sequence (DNA or RNA) can be cloned into a suitable vector, replicated, and made to produce virtually unlimited amounts of the desired protein even when the virus, as is the case for hepatitis B, cannot be. Another elegant example of the application of this molecular approach to vaccine development is the genetic modification of the bacterial Bordetella pertussis toxin (PTX). PTX is thought to be an essential component of all vaccines that are effective in preventing whooping cough. The development of vaccines that cause fewer reactions than the whole-cell preparations of killed pertussis organisms has been achieved with chemically inactivated PTX alone or in combination with other antigens. By cloning the relevant genes, it was possible to change aminoacids of one of the subunits of PTX so as to inactivate the enzymatic properties responsible for toxicity. When used in an acellular whooping cough vaccine, this mutated PTX was found to be highly immunogenic, safe, and effective.4 A further very important use of these molecular techniques is in epidemiology, diagnosis, and in the establishment of the impact of a microbe as a public-
Vol 350 • October 25, 1997
health problem. Increasingly, molecular epidemiology is an essential tool used in clinical trials and surveillance of the impact of vaccines. Not only are genes passed on to progeny through the normal replicative processes of chromosomal or extra-chromosomal nucleic acids (vertical transmission), but these genes may also be spread to other organisms (horizontal spread) by a variety of mechanisms, including transformation by DNA released from a donor cell, transduction involving phage (bacterial viruses), and transposition of mobile genetic elements. These are highly efficient mechanisms by which extant genetic material is exchanged, added, or deleted. These mechanisms are also pomiscuous in that the transfer of genetic material may occur between closely related or unrelated organisms. Genetics can be used to track the prevalence and spread of particular genes or sequences relevant to virulence across time and geographical boundaries, while monitoring associations with disease. In 1982, for example, distinctive clones of Escherichia coli (O157:H7) were found to harbour a toxin (verotoxin) that causes haemorrhagic colitis and haemolytic-uraemic syndrome.5 With PCR and assays specific for the toxin, the prevalence and spread of this virulence factor could be tracked. This application of molecular techniques to facilitate epidemiological surveillance focuses on the detection of the virulence gene rather than the strain. This has lead to the discovery that verotoxin is not confined to O157:H7 strains since the identical gene has been found in association with other, epidemiologically distinct E coli strains (eg, serotype O103:H3).6 A vexing problem in public health is the unexpected manner in which epidemics or outbreaks of microbial diseases occur. Despite efforts to understand the dynamics of host microbial interactions so as to inform health policies for individuals and populations, the imperfections of our knowledge are such that the occurrence of epidemics is often unanticipated. The mutuality of host and microbe is central to understanding their co-evolution and the potential for disease, yet genetically determined variations of microbes and their hosts (the latter occurring at a slower rates than in microbes), followed by natural selection, complicate these interactions. As a consequence, it has been difficult to disentangle the relative roles of microbial virulence and host-immune responses responsible for epidemics. Antigenic variation is a striking feature of the biology of many viruses and free-living pathogenic parasites, fungi and bacteria.7 Examples include the drift and shift of key molecules (haemagglutinins and neuraminidase) on the surface of the influenza virus,8 or the glycoproteins of HIV-1.9 Among macroparasites, antigenic variation is seen in the var genes of Plasmodium falciparum,10 and the variant surface glycoproteins of trypanosomes.11 In bacteria, antigenic variation is exemplified by adhesins 1241
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Current status of whole genome sequences of major bacterial pathogens and parasites Completed and published
Completed but unpublished
In progress
Haemophilus influenzae Mycoplasma genitalium Mycoplasma pneumoniae Escherichia coli K-12 Helicobacter pylori
Borrelia burgdorferi Streptococcus pneumoniae Treponema pallidum
Bacillus subtilis Chlamydia trachomatis Enterococcus faecalis Legionella pneumophila Leishmania major Mycobacterium leprae
(fimbriae or pili)12 and in fungi, in the global switching of Candida albicans.13 Antigenic variations underlie much of the biological complexity of host-microbe interactions— eg, malaria with its variable patterns of disease, the occurrence of mini-epidemics, and the slowly acquired, imperfect nature of host immunity. Appreciation of the extent of variations in cell-surface molecules is critically important to the choice of vaccine candidates and their design and development because these changes impart the potential to evade the immunity induced by immunisation. Typically, phenotypic switching (eg, phase variation) occurs in individual organisms at frequencies of about one per 100 to one per 10 000 per generation. But a crucial factor, in addition to the frequency with which variation occurs, is its unpredictability. When and which of the variant phenotypes occurs is by chance and not by a programmed event. Indeed, the very fitness of microbes to adapt and survive through natural selection, attributes which include commensal or virulence behaviour, is a dynamic trade-off between variation and stability—an evolving agenda which underpins the interactions between microbe and host14. Another poorly understood but important issue is whether microbial virulence and transmissibility are coupled. Central to this is the concept that pathogens tend to evolve a level of virulence which maximises their reproductive rate (R0), the average number of new infections caused by a single infectious host in a population of susceptible individuals.15 The relation between transmissibility and microbe-induced morbidity and mortality varies among pathogens. For many pathogens, virulence and transmission are closely associated, but this is by no means always so. Although an increased rate of replication or its potential to injure host tissues might be expected to increase the transmission of a microbe, in at least two circumstances this is not the case. First, the host organ or tissue in which the organisms normally multiply and produce transmissible progeny may be different from that in which the organisms caused disease. Second, in many infections, host immuneresponses may be the major determinant of disease, which is a situation which will not increase transmissibility and may even decrease it. A detailed understanding of the links between transmission and virulence for a particular organism has important implications for public-health measures to control microbial diseases, including the policies informing the use of vaccines and antibiotics.16
Whole-genome sequencing The complete nucleotide sequences of several viral genomes have been available for several years but, in the past 2 years, technology has advanced to the point in which this is possible for other pathogens (panel). In the near future, the whole nucleotide sequences of the genomes of Mycobacterium tuberculosis and the malaria 1242
Mycobacterium tuberculosis Neisseria gonorrhoeae Neisseria meningitidis Plasmodium falciparum Pseudomonas aeruginosa Pyrobaculum aerophilum
Rickettsia prowazekii Staphylococcus aureus Staphylococcus epidermitis Streptococcus pyogenes Trypanosoma cruzi Vibrio cholerae
parasite Plasmodium falciparum will be known. The era of genomic sequences affords extraordinary opportunities for the use of immunisation since the complete DNA sequence of an organism provides a catalogue of the genes for every virulence factor and potential immunogen from which to select potential vaccine candidates.17 A particularly exciting fact revealed by the availability of whole genome sequences is that a large proportion of genes are of unknown function and will probably include many that would not have been revealed by classic genetic approaches such as random mutagenesis. In the future, therefore, the key issue will be much more one of selecting which of the many potential candidates most warrant investment as potential vaccine candidates and what constitutes the optimum methods of antigen presentation. An example is the use of the complete genome sequence of H influenzae to identify and characterise the multiple genes involved in lipopolysaccharide biosynthesis. Lipopolysaccharide is a critical structural and functional component of the cell envelope of a whole range of bacterial genera. In H influenzae, lipopolysaccharide is critical to the pathogenesis of infection. It modulates interactions with respiratory epithelial cells, plays a part in the invasive process whereby bacteria translocate from the respiratory tract to the blood, prevents clearance of bacteria from the blood, and is a major factor in the modulation of inflammation and tissue injury. It is a highly complex glycolipid whose biosynthesis requires a large number of genes (more than 30) for synthesis of the precursor units which include fatty acids, sugars, and side-chain substituents such as phosphoryl choline. These are synthesised and assembled initially in the cytoplasm, transported outwards, and assembled on the cell surface. Only a few of the relevant genes had been identified before the availability of the whole genome sequence, but once this was available, more than 25 additional genes were identified.18 A powerful method of extracting the information from the genome database was the use of previously published sequences deposited in general databases, particularly those known to be involved in lipopolysaccharide synthesis of other organisms. These database probes were used to search the H influenzae genome for homologous sequences. When matches were found within open reading frames, these candidate lipopolysaccharide genes were cloned and characterised. By making mutations in each of these genes, it was possible to determine whether they were involved in lipopolysaccharide biosynthesis because mutations would alter the lipopolysaccharide molecule. This change could be detected by reactivity with monoclonal antibodies, gel fractionation patterns, and electrospray mass-spectrometry (figure).23,24 Studies in an experimental animal infection model provided an estimate of the minimal lipopolysaccharide structure required for virulence. The extent of conservation of
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How a whole-genome sequence of a pathogenic microbe can be used to develop a candidate vaccine 1: representation of lipopolysaccharide, lipid A (endotoxin) is inserted into the cell envelope of the bacterium; inner and outer core saccharides, projecting outwards from the cell envelope, are important in mediation or modulation of interactions with host cells.23 2: whole-genome sequence of H influenzae Rd, includes all of the genes involved in the biosynthesis of H influenzae [reproduced with permission from Fleischmann et al24]. 3: nucleic acid sequences of genes or their deduced aminoacid sequences involved in lipopolysaccharide biosynthesis of other bacteria, obtained from general database searches, are used as database probes to search the H influenzae genome for homologues that are candidate lipopolysaccharide genes. 4: when a match is found the sequence can be inspected in the H influenzae genome database and is available as the basis for further experiments. 5: oligonucleotide primers can be constructed to obtain multiple copies of the desired candidate lipopolysaccharide gene using PCR. 6: these candidate lipopolysaccharide genes are cloned into a suitable plasmid vector; a mutation of this gene can then be constructed. In this example the gene has been disrupted by an insertion of a cassette of DNA containing a gene for kanamycin resistance that acts also as a selectable marker. 7: the cloned and mutagenised gene is introduced into H influenzae by transformation to achieve allelic replacement; the phenotype of the parent strain and its mutant can then be compared to determine whether there are differences in lipopolysaccharide. 8: with colony immunoblotting, the reactivity of the parent and mutant strain are compared; the mutant lipopolysaccharide has lost its capacity to bind to monoclonal antibody, whereas colonies of the parent strain bind this antibody. This provides strong evidence in support of a function for this gene in lipopolysaccharide biosynthesis. 9: further tests of biological function on parent and mutant can be done to further characterise the role of lipopolysaccharide in virulence. 10: information on the many genes involved in lipopolysaccharide biosynthesis provides detailed information on structure and its potential for use in vaccine development.18
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lipopolysaccharide-related genes in other strains can now be investigated to provide a rational basis for the evaluation of the candidacy of lipopolysaccharide for broad range vaccine development. Indeed, the information provided by whole-genome sequencing opens up a wealth of possibilities in vaccine research and development. Attenuation can be approached at the genome level by identifying and deleting the minimal set of genes required for virulence so as to achieve optimal attenuation and safety as vaccine candidates in their own right or as vectors for delivery of foreign antigens.
Nucleic acids as immunogens In 1990, Wolff and colleagues reported that direct intramuscular injection of mice with DNA, a plasmid containing the complete sequence of a gene, resulted in persistent expression of the encoded protein.19 These astonishing observations have been confirmed and expanded in animals to show durable and reproducible protective immunity for several important pathogens including influenza,20 malaria,21 and HIV-1.22 Although many issues, including theoretical safety considerations associated with polynucleotide vaccination, will need to be carefully examined, there could be some substantial advantages to this approach. These include the induction of immunity in the absence of the infectious agent, a considerable advantage for vaccines against organisms such as HIV or those with a propensity for long-term persistence in the body—eg, cytomegalovirus. Another advantage is that individual immune responses are dictated by particular MHC gene variations (alleles). In outbred populations, naked DNA immunisation would allow a selection of nucleic acids corresponding to a range of polypeptides to overcome this problem of T-cell restriction, thereby increasing the number of responders. Finally, stability and ease of production would make naked DNA an attractive proposition for global immunisation strategies.
Future prospects As our understanding of microbes deepens, new approaches to the control of infections, especially vaccines, will be feasible. In particular, there is a need to consider more broadly the ways in which novel vaccines may be used to control or modulate microbial behaviour without the aggressive disruption of their ecology which is characteristic of many antimicrobials. The difficulty of nosocomial infections caused by bacteria and fungi in immune compromised individuals—trauma, cancer, transplantation, prematurely born infants—is one of microbial opportunism. The blunderbuss use of antibiotics has proved a two-edged sword, successful sometimes in treating life-threatening infections but at the expense of drastic decimation of normal commensal flora and the selection of antibiotic resistance (eg, enterococci and staphylococci). The complex orchestration and organisation of genes permissive for, or directly involved in, commensal or virulence behaviour are beginning to be unravelled. There are signalling systems which allow microbes to sense changes in their environment—eg, as an organism translocates from an extracellular to an intracellular location in the host. The transcription of genes which results in the translation of proteins can be
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monitored by global regulators of the microbe so that several gene activities can be coordinately and appropriately switched on or off. An enormous body of information about the structural details of molecular interactions of microbial ligands and host receptors is now available that provides a rational basis for the interruption, in a selective manner, those events that presage pathogenicity and offer novel strategies for the control of microbial diseases, including vaccines. I thank H Masoud and JR Brisson for providing the structural and conformational representations of H influenzae lipopolysaccharide used in the figure, D Hood and JC Richards for helpful discussions, and D Watson for her help in the preparation of the typescript and the figure. I received support from the Medical Research Council Programme Grant.
References 1 2 3 4 5
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