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Prospects of new vaccines for resurgent and emergent diseases Paul Everest and Gordon Dougan Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK Changing lifestyles around the world are contributing to the phenomenon of emergent and resurgent infections. At the same time the application of molecular techniques to the study of infection has given rise to new opportunities for the development of novel vaccines. The trend in vaccinology is towards more defined and safer vaccine formulations. Here we discuss recent developments in the field and how these might impact on resurgent and emergent infection control. Correspondence to Prof Gordon Dougan, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK The phenomenon of emergent and resurgent infectious diseases is a result of a combination of factors affecting the human population together with our improved ability to identify the aetiological agents of diseases. Emergent diseases are caused by a wide variety of organisms1"6, hi some cases we suspect that a novel agent may have moved into the human population from an animal reservoir, as with HTV or new influenza virus strains. In other cases, improved technology has allowed the identification of previously undetected pathogens, such as Helicobacter pylori. In addition, microorganisms themselves are undergoing rapid evolution as a consequence of changing lifestyles and environments. These factors have all contributed to the emergence of multidrug resistant pathogens and new variants of existing microorganisms, such as enterohaemorrhagic Eschertchia coli. As a consequence of the large variety of novel emerging/resurging agents it is unlikely that a simple approach can be used to tackle this complex problem. For example, novel vaccines developed against different emerging disease targets are likely to be of a variety of types. In this short review, we will summarise some of the advances in modern vaccinology and discuss, using examples, how this technology might be applied to the problem of emergent infections. We will also try to illustrate some of the special problems that might be encountered in this rapidly evolving area. Emergent disease targets Different pathogens associated with the phenomenon of emergent diseases are discussed in detail throughout this issue. A simple survey of British Medical Bulletin 1998,54 (No 3) 557-568 C The British Council 1998 Resurgent/emergent infectious diseases the titles will serve to illustrate the wide variety of disease agents. For many of these microorganisms no vaccines are currently available, for example malaria and HIV. Here we have to consider how to generate completely novel vaccines. However, for some of the resurgent infectious agents, well established vaccines have been used throughout the world, as m the case of diphtheria. Here we might consider how to improve the existing vaccine to adapt it to either a changing disease pattern (e.g. adults rather than children) or use in a socially unstable environment. It is also clear that the pathogens associated with the various emerging diseases exploit widely differing mechanisms of pathogenesis or colonise different niches in the host, such as mucosal surfaces or intracellular environments. Others have evolved the ability to undergo antigenic variation, as with HIV and Borrelia, presenting particularly severe challenges for vaccine design. Thus, for many of these agents vaccine development is unlikely to be a trivial challenge. The demands of modern vaccinology Many of the existing commercially available vaccines would be unlikely to receive a licence if they were relaunched for use today. This is because the standards set by control agencies in different countries have improved beyond recognition. This is, in part, a consequence of improvements in modern molecular biology taken together with hard lessons learned from mistakes made in the past. Any modern, non-living, human vaccine would probably have to be exceptionally well defined in terms of its antigenic composition. Any live vaccine would probably have to be constructed using well-defined attenuating mutations that could not revert to generate virulent derivatives under any circumstances. As we move away from whole organism vaccines to defined antigens, we encounter problems with poor immunogenicity; adjuvants (immune potentiators) have to be added to vaccine formulations in order to overcome this problem. As more vaccines are developed, there is an increasing requirement to combine different vaccines into a single or a few vaccine shots7. At this point it is possible to encounter antigenic competition between different vaccines. Generating new vaccines is an expensive business that often involves a complex evaluation of vaccine safety, immunogenicity and efficacy in the field. This barrier has delayed the appearance of new vaccines on the market and has discouraged some investigators from following-up vaccine research into vaccine development. 558 Bntnh Medical Bulletin 1998,54 (No 3) Vaccine strategies for emerging diseases Modern vaccine approaches There is a clear move in the field of vaccinology towards better characterised and more defined vaccines8. The rapid development of molecular biology and immunology in the 1980s generated great optimism that we would develop quickly a new generation of vaccines based on genetic engineering. This has been slow to bear fruit due to unexpected problems associated with the poor immunogenicity of some defined antigens. A good example of this were the attempts to generate a subunit foot and mouth disease vaccine9. However, some recombinant vaccines did reach the market in a relatively short period of time. Perhaps the most dramatic example of this type of success are the subunit hepatitis B vaccines based on recombinant surface antigen10. There is even a school of thought that increased vaccination with more defined vaccines may impair the natural evolution of the immune system as we encounter less antigens in a disease situation. Lack of, or inappropriate, immune insult may be a cause of auto-immune responses or diseases involving immune disfunction, such as asthma or inflammatory bowel disease. In spite of these problems, new vaccines are reaching the market with a regular frequency. When considering new vaccine design, there are several factors to take into account, some of which will differ according to whether a live or dead vaccine is to be used. For both types of vaccine, the potential route of immunisation is important. Many live vaccines can .be given by natural routes, including mucosal (intranasal, oral, etc.) where it may be possible to induce local and secretory immunity. The mucosal route has proved more difficult to exploit for non-living vaccines, where the problem of overcoming tolerance exists. We have already mentioned the potential problem of reversion for live vaccines, although this can often be eliminated using molecular approaches. However, there is the important issue of the increasing number of immunocompromised individuals to consider with live immunisation. For inactivated vaccines, the issue of antigenic composition and the requirement for immunopotentiators (adjuvants) is a priority. For all types of vaccines the issue of vaccine competition is of increasing importance. Let us consider some of these issues in more detail in relation to emerging problems. Route of immunisation Most commercial vaccines are delivered parenterally by injection. The few existing examples of widely used mucosal vaccines (e.g. oral polio) British Medical Bulletin 1998,54 (No 3) 559 Resurgent/emergent infectious diseases are based on live attenuated organisms. Mucosal vaccines are attractive because of the potential to stimulate natural immunity at the mucosal sites normally colonised by the virulent pathogen. The induction of local immunity might be particularly important for designing vaccines against emerging mucosal pathogens such as H. pylori and this has been recognised in early attempts to design vaccines11. For reasons that are not well understood, non-living vaccines are frequently poorly immunogenic when delivered mucosally. The reason may be linked to the way that antigens are processed by gut associated lymphoid tissues and the need to avoid eliciting damaging immune responses to environmental or dietary antigens. The mucosal route is attractive because of the potential to prevent colonisation at mucosal surfaces which provides an additional barrier that contributes to protective immunity. There are also advantages associated with the avoidance of the use of needles. We are learning how to induce active immunity to mucosally delivered antigens but progress is slow12. However, the discovery of mucosal adjuvants, such as cholera toxin, is providing some potential for advance (see later). Live vaccines Live vaccines have been in use since the early days of vaccinology. Indeed vaccinia, the smallpox vaccine, was used successfully to eradicate the disease13. Live vaccines offer the advantage that they do not require adjuvants and can stimulate potent cellular and humoral immune responses. Cellular immunity is likely to be important for protection against many resurgent and emergent diseases, including AIDS and malaria. Thus, live vaccines are potentially useful against pathogens that incorporate an intracellular stage into their lifestyle in vivo which includes many emerging/resurging pathogens. However, live vaccines have been associated with some of the major disasters in vaccine practice. The need to design safe, non-reverting, and fully characterised live vaccines is of the utmost importance. BCG, the current tuberculosis vaccine, was originally used as an oral vaccine but a switch was made to parenteral vaccination in many countries after a major accident14. M. tuberculosis is perceived as a resurgent problem because of the increase in drug resistance and its increasing occurrence in deprived areas in developed countries (it has always been a problem in developing countries). BCG is an undefined vaccine with a somewhat chequered history in terms of its performance in vaccine efficacy trials14. However, BCG vaccine is still in use in many countries including the UK. Intensive efforts are underway to construct a defined new live tuberculosis vaccine 560 British Medical Bulletin 1998,54 (No 3) Vaccine strategies for emerging diseases to replace BCG, based on a rationally attenuated strain of M. tuberculosis harbouring defined gene deletions. This work is being facilitated by the availability of the complete sequence of the M. tuberculosis genome15. Genomic information can be used to identify candidate genes for attenuating mutations or as a source of possible protective antigens. In addition, recent progress has been made in gene replacement technology in M. tuberculosis that was previously a barrier to vaccine development16. Several modern defined live vaccines are currently at different stages of development and evaluation in clinical trials. These include vaccines targeted against Shigella, Salmonella, Vibrio cholerae and even HTV17"20. These vaccines normally harbour deletion mutations in more than one virulence-associated gene. For example, in the case of new live cholera vaccines, the cholera enterotoxin gene is deleted19. These new live vaccines are often designed to be deliverable mucosally as a single protective dose and, as they are so potently immunogenic, live vaccines are now attracting interest as vectors to deliver heterologous protective antigens from other pathogens to the immune system. There are many examples in the literature of both Salmonella and BCG strains being engineered to express antigens from pathogens, including other bacteria (tetanus, Borrelta, Streptococci, etc.), viruses (HIV, influenza, etc.) and parasites (malaria, schistosomes, etc.). These vector bacteria can also be engineered to deliver simultaneously antigens from more than one additional pathogen21"23 which could allow the development of multicomponent, single dose mucosal vaccines. Recombinant derivatives of viruses including polio24 and vaccinia25 have also been adapted for use as vectors for heterologous antigen delivery. Nevertheless, there are still some worries associated with the use of live organisms as vaccines. The problem of potential reversion to virulence can be virtually eliminated from bacterial vaccines but the plastic nature of many viral genomes and their ability to integrate into the genome has generated some concern. For example, there are plans to evaluate attenuated mutants of HTV in man and these concerns have even been expressed in the popular press by experienced scientists. An additional concern is the use of live vaccines in lmmunocompromised individuals. As the AIDS pandemic spreads, more people will display increased susceptibility to infection and some attenuated pathogens may be fully virulent in immunocompromised hosts. A good example is the increased virulence of vaccinia in HlV-infected individuals. Hence, in future it will be important to design live vaccine strains that are fully attenuated in the absence of a functional immune system. One way this may be accomplished is to test new vaccine candidates in animals harbouring knock out mutations in genes involved in immunity26'27. British Medical Bulletin 1998,54 (No 3) 561 Resurgent/emergent infectious diseases Acellular vaccines The move towards safer vaccines has focused attention on acellular vaccines of defined antigenic composition. Many early vaccines were based on whole inactivated bacteria, e.g. pertussis (whooping cough), or viruses. These whole cell vaccines are often reactogenic and contain antigens that do not contribute to a protective immune response. Tetanus and diphtheria vaccines were early examples of acellular vaccines, being based on fractions harbouring inactivated toxins. Recently there has been a re-emergence of diphtheria in former states of the Soviet Union, although both tetanus and diphtheria can still be seen as fairly frequent infections in some developing countries (see Eskola et al., this issue). An unusual feature of the Soviet diphtheria outbreak was the high number of older individuals infected, suggesting that immunity was waning in the population. This outbreak was almost certainly due, in part, to poor vaccine uptake but may also have been a consequence of poor vaccine quality. Early studies estimated diphtheria vaccine efficacy at about 80%,28 and it may be possible to improve on this by incorporating more efficient immunopotentiators into diphtheria toxoid vaccines (alum is currently the adjuvant of choice). Since some individuals may simply not respond to diphtheria toxin as an antigen, due to immunological restriction, it may be possible to incorporate additional Corynebactertum diphtheriae antigens into the vaccine. For example, antigens might be selected from the surface layer of C. diphtheriae in addition to the extracellular toxm. This may help reduce the potential for carnage of the pathogen, or even improve long-term protection. The problem of poor immunogenicity is a common trait associated with many purified antigens and is compounded by the poor range of immunopotentiators suitable for use in humans. Even the existing hepatitis B vaccine is poorly immunogenic in older individuals. Poor immunogenicity, and the inability to induce protective humoral and cellular responses, has impaired acellular vaccine development for several emerging diseases. This is particularly true for some of the viral diseases, e.g. HIV. There is also the problem of identifying potentially protective immunogens. In the case of bacterial pathogens, there are often literally hundreds of potential antigens to choose from, and many of the most promising candidates may be integral membrane proteins, compounding the problem of purification in an appropriate immunogenic form. The inability to identify or handle protein antigens has, in the past, encouraged exploration of the potential of polysaccharides, as protective immunogens. This approach has been successful for several diseases caused by encapsulated bacterial pathogens including Haemophilus influenzae type B,29i3° but antigenic diversity or poor immunogenicity of capsular polysaccharides has 562 British Medical Bulletin 1998,54 (No 3) Vaccine strategies for emerging diseases compromised progress with other pathogens. Many companies have developed pneumococcal vaccines based on capsular polysaccharides, but antigenic diversity means that vaccines must incorporate polysaccharides from multiple capsular types31. Conjugate vaccines (see later) offer a route towards improving the immunogenicity of polysaccharide vaccines. Since many antigens are poorly immunogenic in humans, even though they may show promise as protective antigens in animal models, the need to identify adjuvants suitable for use in man has intensified. It is inappropriate to review this complex area here, but many of the large vaccine companies have experimental adjuvants in clinical trials and these will undoubtedly have a major impact on this area. Many of these adjuvants offer promise for assisting the development of improved vaccines for use against emergent and resurgent infectious agents, including malaria. Resurgent and emergent infectious agents are often in a phase of genome evolution that has enabled them to adapt to a new environmental niche. A good example of this may be the enterohaemorrhagic E. coli that have combined new virulence determinants into the existing E. colt genome backbone32. Pathogens that exhibit the ability to adapt or evolve rapidly present a particularly difficult challenge to vaccine developers. Many emergent pathogens can display complex patterns of antigenic variation. This may be in individual genes, such as the HTV envelope protein, or may be in multiple genes as in the case of Borrelia (several genes) or Plasmodium (scores of genes)33"35. Designing acellular vaccines based on antigenically variable proteins is an enormously difficult task. For many of these diseases, we have so far been unsuccessful. One approach to overcome this problem is to incorporate several different antigens into a vaccine in an attempt to raise efficacy levels and compensate for antigenic variation. This approach has been used for Plasmodium vaccines36. A second approach is to incorporate antigenic variants of a single protein into a vaccine. Both approaches may be applicable. Other attempts have involved the use of non-variable antigens from highly antigenically variable pathogens. So far, this approach has generated little practical success, but may be productive in the future. Again, even if appropriate antigens are identified they must be delivered in a manner that elicits an appropriate immune response. Novel vaccine formulations The immunogenicity of vaccine preparations can be enhanced using adjuvants. This has been recognised for many years but still there are sparingly few adjuvants available for use in humans. Many traditional vaccines incorporate aluminium salts (alhydrogel) as adjuvant but this British Medical Bulletin 1998,54 (No 3) 563 Resurgent/emergent infectious diseases formulation is not suitable for many vaccines8. Alhydrogel apparently tends to push immune responses towards a Th2 type immune response, which favours antibody production over cellular responses. Vaccines against many emerging diseases will require cellular immunity involving Thl or cytotoxic T-cell responses (CTL). Although we are able to generate such responses in experimental systems, or with live vaccines, it will be a challenge to induce Thl or CTL responses routinely in humans for some disease agents using defined acellular vaccine preparations. This area is high on the priorities of many companies. Some of the new adjuvants involve the use of non-toxic oil preparations or even detoxified forms of lipopolysaccharide37. Some early clinical data look promising, but many clinical studies are still incomplete. Other approaches involve the use of powder formulations or immunomodulators based on bacterial cell wall components. A major recent development has been the unexpected recognition that vaccines can be based upon DNA rather than protein, so called DNA or nucleic acid immunisation38'39. The nucleic acid immunisation approach involves the injection of DNA or RNA encoding a protective antigen from a pathogen, expressed from a functional eukaryotic promoter. The DNA can be injected into muscle tissue or can be forced under the skin using a so-called gene gun. Apparently, the injected DNA is assimilated by host cells and the protective antigen is expressed by these cells. This type of immunisation can evoke strong cellular responses as well as the production of antibodies. DNA immunisation has been used to elicit immune responses to a wide variety of antigens, including those from bacteria (tetanus, Mycobacterta), viruses (hepatitis B, HIV) and parasites (malaria)38. The beauty of DNA immunisation is the simplicity of the approach, only requiring DNA purification which is not technically challenging. However, the technology remains unproven in the clinic and there are many safety and ethical considerations associated with the use of DNA as an immunogen. For example, there may be long-term consequences for health associated with potential integration of foreign DNA into the genome. Nevertheless, it is likely that there will be an explosion of clinical data on DNA immunisation in the next few years. Conjugate vaccines A more established method for overcoming poor immunogenicity has been used in relation to polysaccharide or peptide-based vaccines. It relies on the hapten-carrier concept. Many polysaccharides are poorly immunogenic because they are so-called T-cell independent antigens. It is often possible to raise short-term antibody, mostly IgM, against many polysaccharides but immunological memory is poor, and it is difficult to 564 British Medical Bulletin 1998,54 (No 3) Vaccine strategies for emerging diseases immunise young children whose immune systems are apparently not fully matured. If polysaccharides are chemically cross-linked to immunogenic carrier proteins, such as tetanus toxoid, their immunogenicity can be improved dramatically and even young children can raise an IgG response that has a longer duration. Conjugate vaccines have been of great value for designing vaccines against encapsulated bacteria. Perhaps the most spectacular example is H. influenzae, where licensed conjugate vaccines are now well established. However, this type of approach is also likely to play a significant role in new vaccine design against emerging or resurging pathogens. A good example is the current intensive clinical evaluation of Streptococcus pneumoniae conjugate vaccines29"31. Conjugate vaccines may in the future find use against parasitic infections. Here, this may involve protein-protein conjugates rather than polysaccharide-protein conjugates. For example, fusion proteins have been made using proteins from the different stages of the Plasmodium life-cycle. Non-living mucosal vaccines It would be attractive to design vaccines based on pure antigens that could be delivered as a spray, or in an encapsulated form, and taken by mouth. There are both practical and immunological reasons for this, but it has proved extremely difficult to generate real practical vaccines of this type, as most pure proteins are poorly immunogenic when delivered via mucosal surfaces. This may be due to a combination of factors. Mucosally delivered antigens will be subjected to extreme conditions (acid environment) or proteolytic degradation at the body surfaces. If they reach immune inductive tissues, such as GAIT, they may be processed as environmental or dietary antigens and fail to stimulate appropriate immunity. Several experimental approaches have been taken to try to overcome some of these barriers. Antigens can be incorporated into biodegradable or lipid-based particles that offer some protection against degradation and improve mucosal targeting40. Encapsulated vaccines have been evaluated in the clinic, but, so far, have not been fully commercialised, in part due to production problems and in part due to poor immunogenicity. However, results with some antigens have been encouraging and recent data suggest that DNA vaccines may be deliverable in this way41. A different approach to tackle this problem is to use specific mucosal adjuvants that can actively modulate immunity at mucosal surfaces. Very few mucosal adjuvants have been identified. Perhaps the best characterised are cholera enterotoxin (CT) and the heat-labile (LT) enterotoxin of E. coli that have been shown to activate immune British Medical Bulletin 1998,54 (No 3) 565 Resurgent/emergent infectious diseases responses to co-administered antigens following mucosal (intranasal, oral) delivery42. These toxins are potent immunomodulators and mucosal adjuvants, activating local and systemic immune responses. However, they are too toxic for consideration as practical vaccine components. Recently, some non-toxic derivatives of both CT and LT have been shown to retain mucosal adjuvant activity opening up a possible route towards clinical evaluation43"46. Non-toxic derivatives of LT have been incorporated into an experimental H. pylori vaccine that can be administered orally. Experiments in a murine model have shown that several H. pylori antigens can be combined with mutant LTs to make effective oral vaccines17. These types of vaccine are likely to be a prototype for other emerging diseases. Conclusions It is clear that there is a great deal of activity in the field of vaccine development and research and much of the progress has potential application to emergent and resurgent diseases. We have been able to discuss some of the most exciting developments here and have eluded to how these might be applied. References Scheld WM, Armstrong D, Hughes JM (eds) Emerging Infections 1. Washington, DC American Society for Microbiology, 1998 Anderson BE, Dawson JE, Jones DC, Wilson KH. 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