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British Medical Bulletin 62:163-173 (2002)
© 2002 The British Council
Vaccines against dangerous pathogens
DSTL, Chemical and Biological Sciences, Porton Down, Salisbury, UK
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Abstract |
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Top
Abstract
Defining a dangerous pathogenDangerous pathogens are defined by the UK Health and Safety Executive's advisory committee as category 3 (those which cause severe human disease for which prophylaxis or therapy is usually available) or category 4 (as for category 3, but for which prophylaxis or therapy is not available). Research and development of vaccines for such pathogens is challenging, due to the safety constraints in the manipulation of these pathogens. This chapter discusses the various approaches which can be taken to develop candidate vaccines for these pathogens, including the potential impact of genome sequencing on shortening the time required for R&D. For these pathogens, a direct test of the efficacy of the candidate vaccines in man is not ethical and, therefore, particular emphasis is placed on the demonstration of efficacy in animal models. Emphasis is also placed on the derivation of surrogate markers of efficacy and a demonstration that these correlate with protection in the animal model.
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Defining a dangerous pathogen |
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A wide range of micro-organisms have the potential to cause serious and sometimes fatal diseases in humans. Indeed, with the growing population of immunocompromised individuals, many micro-organisms which were previously considered to be non-pathogenic might now be labelled as ‘emerging’ or ‘opportunistic’ pathogens. This changing relationship between micro-organisms and humans challenges our definition of the term ‘pathogen’. The label of ‘dangerous pathogen’ further challenges our use of terminology, since it might be argued that any micro-organism capable of causing disease poses some danger to the unfortunate host. How then do we define dangerous pathogens? One source of guidance in the UK is the classification of micro-organisms according to the hazard posed to human health which is provided by the Advisory Committee on Dangerous Pathogens (ACDP) for the Health and Safety Executive (HSE)1. This classification system broadly allocates pathogens into one of four groups (Table 1) depending on the likely nature and severity of the resultant disease in immunocompetent hosts, the likelihood that disease could be transmitted from infected to naive individuals, and the availability of some form of disease therapy. Similar classification systems are now used by all countries in the Western World and some standardisation of classification is being encouraged. Therefore, such guidelines might provide one way of defining a ‘dangerous pathogen’. For the purposes of this review, pathogens in ACDP hazard groups 3 and 4 are considered to constitute ‘dangerous pathogens’. Disease which is caused by these pathogens is likely to have some or all of the following attributes: (i) it is life-threatening even in immunocompetent hosts; (ii) it is easily contracted; (iii) it may be transmitted in an epidemic manner; and (iv) some form of prophylaxis or therapy may not be available. The balance of these various considerations determines whether candidate pathogens are assigned to ACDP hazard group 3 or 4.
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The origins of dangerous pathogens |
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What of the origins of such pathogens? Conventional dogma states that a pathogen which causes the death of the host, and possibly the decimation of an entire host population, is not well adapted to this life-style. The elimination of a host population prevents the long-term establishment of a micro-organism in this environmental niche, surely the goal of any successful pathogen? In contrast, pathogens which cause mild infections in the host, possibly without eliciting a host response and are highly transmissible, are well adapted to the human host–pathogen relationship. In the context of human disease, we might consider that dangerous pathogens of humans have arisen in two ways which may not be mutually exclusive.
First, they might have evolved from existing micro-organisms which cause a relatively mild disease in humans, as a consequence of genetic changes. Significant insights into this evolutionary pathway have been provided by the genome sequences of certain pathogens and their ancestors. In the case of Yersinia pestis, the causative agent of plague, there is good evidence that the bacterium evolved between 1500 and 20,000 years ago from the closely related bacterium Yersinia pseudotuberculosis2. Whereas the former causes a serious and often fatal disease of humans which is generally transmitted by a flea bite, the latter generally causes a relatively mild enteric infection as a consequence of the ingestion of contaminated foodstuffs. The evolution of Y. pestis might have occurred relatively rapidly, with the acquisition of novel DNA sequences in the form of plasmids which encode a range of virulence factors and the loss of other gene functions which are required for enteric disease. The genetic signatures of these large scale genetic changes are still obvious in the Y. pestis genome3. Escherichia coli O157:H7 provides another example of the evolution of a dangerous pathogen by the acquisition of a range of virulence determinants including the shiga-like toxin4. In this case, the putative ancestral strain is E. coli K12, and divergence is thought to have occurred 4.5 million years ago5.
Second, dangerous pathogens might appear as a consequence of the exposure of a human population to a pathogen which causes a relatively mild disease in a different host species. Many viral pathogens fall into this group including HIV, Lassa fever virus and Ebola virus. In the case of HIV1 and HIV2, the common chimpanzee and sooty mangabey monkey, respectively, appear to have provided the progenitor virus6. However, this virus has undergone additional genetic changes, and continues to adapt to evade the immune responses of its host, making it a highly successful pathogen.
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The need for vaccines against dangerous pathogens |
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By their very nature, dangerous pathogens cause serious and often fatal diseases in humans. Control of such diseases might be achieved by reducing the possibility of contact between the pathogen and susceptible hosts. Such control measures – which might involve improved water or food supplies, changes in sewage disposal systems, reduced contact with animal reservoirs or insect vectors, or some form of physical barrier between infected and naive humans – have been used to great effect in the past. However, these measures alone cannot provide complete control of these diseases. Therapeutic intervention is one strategy which might be used to deal with known cases of infection. However, because of the potential for disease to spread in an epidemic manner, such control measures may be logistically difficult to use on a large scale and are in any case not available for some dangerous pathogens (especially those in ACDP hazard group 4). In addition, for many diseases such as pneumonic plague or Lassa fever, symptomatic individuals may be beyond any form of therapy. Vaccines offer the potential not only to protect individuals from the disease but also to provide herd immunity preventing the epidemic spread of disease.
Leaving aside HIV, the number of cases of disease caused by other dangerous pathogens world-wide is relatively low, and epidemics rarely occur, unless in the wake of some major physical disaster such as an earthquake. The occurrence of the latter which upsets the ecosystem and which could allow the re-emergence of latent diseases, should trigger at least increased disease surveillance and, in vulnerable areas for certain diseases, the instigation of anti-microbial prophylaxis. However, generally it is inappropriate to immunise large numbers of individuals against diseases with which they will never come into contact. Conversely, vaccination might be indicated for individuals living or travelling to an area where a disease is endemic or because of their occupational involvement with certain pathogens or infected individuals, e.g. research and clinical scientists and medical personnel. Increasingly, there is concern globally about the deliberate spread of disease by bioterrorists and a need to have vaccines and therapies available to neutralise this threat. Therefore, for many diseases caused by dangerous pathogens, vaccines fulfil the following needs: first, on a small scale, for the immunisation of at-risk groups; and second as a key element of disease control, either to eliminate endemic disease or to prevent the active spread of disease either in situations where the ecosystem has been upset by natural disaster or where there is the threat of deliberate spread by bioterrorist activity.
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Vaccine development strategies |
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The development of vaccines against diseases caused by dangerous pathogens poses a number of problems which are not encountered by those working with diseases caused by less hazardous micro-organisms. The requirements for biocontainment mean that only some laboratories are able to undertake work with dangerous pathogens, and work cannot proceed at the same pace as with other pathogens. A key element of the vaccine research and development programme is to demonstrate the efficacy of the candidate vaccine in an appropriate animal model of the disease caused by the dangerous pathogen, which under the constraints of biocontainment, is a particularly taxing requirement. These constraints inevitably mean that the biology of most dangerous pathogens is poorly understood and vaccine development can, therefore, be a protracted and expensive process.
In the case of bacterial pathogens, recent initiatives to sequence their genomes might go some way towards providing a body of background information which can be exploited for vaccine development. This information might be directly exploited to identify surface located proteins which might form the basis of effective sub-unit vaccines7–9. The feasibility of this approach has already been proven for pathogens such as Neisseria menigitidis10, Streptococcus pneumoniae11 and Porphyromonas gingivalis12. Alternatively, the in silico re-construction of biochemical pathways might indicate genes which can be inactivated for the generation of rationally attenuated live vaccines13,14. For the reasons outlined above, these approaches have a particular utility for the development of vaccines against diseases caused by dangerous pathogens.
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Vaccine R&D: difficulties particular to dangerous pathogens |
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In the research phase, it may well be useful to pursue several approaches towards the identification of a candidate vaccine(s), depending on what is known of the pathogen. The starting point is the whole organism which may be heat-killed or irradiated prior to screening for immunogenicity and protective efficacy. The approaches discussed above may yield some surface antigens to trial as a potential sub-unit vaccine(s) or to express from a live vaccine vector. Alternatively, it may be possible to construct a rationally attenuated live vaccine by the selective mutation of key genes, e.g. by transposon mutagenesis. If genome sequencing information is not available, a technique such as signature-tagged mutagenesis may be employed to select avirulent mutants of the organism at random, which can then be screened for immunogenicity and protective efficacy.
In the case of viral pathogens, the isolation of structural and non-structural proteins from the parent virus may be possible. Such proteins could be expressed from a live viral vaccine vector such as adenovirus or vaccinia.
If a positive lead emerges from any of these approaches, the next requirement is to demonstrate the safety and efficacy of the approach being pursued. For the dangerous pathogens, the direct demonstration of the efficacy in man of a candidate vaccine, as yet unlicensed, will not be possible for ethical and legal reasons. Thus the use of animal models in which the disease process is as close as possible to that seen in man, will be required. Further, the demonstration of efficacy in more than one authentic animal model will be required15. If one of these is a rodent model, then ideally data should be supplied also from a non-rodent model. It may be very difficult to replicate the human pattern of disease with certainty, particularly where the pathogen is well-adapted to its human host. Since a direct test of the efficacy of the candidate vaccine in man is not possible, it will be necessary to identify some correlates of protective immunity in the selected animal models and to propose these as surrogate markers of efficacy of the candidate vaccine in man. It will be required to test the appearance of these surrogate markers of efficacy in the individuals immunised during clinical trialling.
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The identification of correlates of protective immunity in animal models |
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The identification of correlates will be informed by knowledge of the life-style of the pathogen and also of the mechanism of protective immunity induced by the candidate vaccine. For example, Y. pestis has a mixed extra- and intracellular life-style in its mammalian host16. On first introduction via the flea bite, the Y. pestis bacteria are extracellular, but will be phagocytosed preferentially by macrophages. Within macrophages, the switch to mammalian body temperature and low calcium conditions, allow the caf and lcr genes of Y. pestis to be activated, respectively, to export and assemble the F1 protein as a part of the capsule forming on the bacterial cell's surface and to secrete Yersinia outer proteins (Yops)17. Amongst the latter is the V protein which is thought to have a pivotal role in co-ordinating the translocation of certain Yops with cytotoxic and antiphagocytic activity into the host cell17,18 and which has itself been visualised on the bacterial cell surface19. The F1 and V proteins have been pursued as recombinant protein components of a new subunit vaccine for plague either in free association20–23 or as a genetic fusion24 or indeed as a genetic fusion expressed from a live vaccine vector25. The F1 + V vaccine is now in advanced development26 and correlates of protective immunity have been identified. There is no doubt that the mechanism of protection following immunisation with the F1 + V proteins also involves T-cell memory and this has been demonstrated in the mouse model22,27. In the mouse, the T-cell response to alhydrogel adsorbed F1 + V is biased towards Th2 and this response is highly protective. However, recent work has illustrated that although delivery of the F1 + V proteins formulated in the Ribi adjuvant system to IL-4T mice (genetic knock-outs for the IL-4 receptor) induced predominantly a Th1 response, the passive transfer of their antiserum into B-cell deficient knock-out mice, with no intrinsic antibody, protected the latter fully against live organism challenge28. Such experimental data suggest that protection against plague in the mouse can be afforded by either the Th1 or Th2 pathways and that optimum protection is expected from a balanced Th1/Th2 response.
Murine B-cell epitopes on the V antigen have been identified29,30 and a region containing a neutralising epitope identified with the derivation of a monoclonal antibody which is protective in the mouse against challenge with plague bacteria29.
Such correlates of protective immunity can be exploited to yield surrogate markers of efficacy. For example, the passive transfer of immune serum from F1 + V immunised Balb/c mice into severe combined immunodeficient mice with the beige mutation (SCID-bge) mice was demonstrated to protect the latter from challenge with Y. pestis31. Having no inherent functional immune system, and no natural killer cells (due to the beige mutation), the observed protection in the SCID-bge mice could be attributed only to the donated antibody and indicated the importance of neutralising the two key virulence factors F1 and V, in order to protect against plague infection. Further, the passive transfer of a combination of B plus CD4+ cells from the immune Balb/c mouse into the naïve SCID-Bge mouse was significantly more protective than transfer of B plus CD8+ cells31.
Further, competitive ELISA assays can be established based on the identification of the neutralising B-cell epitope on the V antigen29 and similarly epitopes on the F1 antigen32,34. In these assays, serum from immunised animals is competed against the respective neutralising polyclonal or monoclonal antibodies for binding to the respective protein, F1 or V. If serum from man immunised with F1 + V is demonstrated to compete with murine monoclonal antibody for binding to the V antigen, this will indicate that the neutralising B-cell epitope on the V antigen first identified in the mouse29 is conserved in man. This fact will augur well for the exploitation of this assay as a surrogate marker of efficacy in man.
Finally, the ability of the V antigen to translocate Yops into host cells could be exploited as a surrogate marker of efficacy in an in vitro assay to measure the inhibitory potential of serum from individuals immunised with F1 + V to prevent the cytotoxic effect of the V protein for cells in tissue culture33.
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The testing of surrogate markers of efficacy during clinical trialling |
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Under UK and European law, the clinical trialling of vaccines proceeds in phases in which various aspects of the eventual vaccine use in man are addressed. In phase 1, the safety of the vaccine in a small cohort of vaccinees is determined. This phase can also be used to achieve some dose-ranging in man.
Permission to carry out the trial must be sought, and a justification for the collection of samples made. Under these constraints, it will be possible to test the proposed surrogate markers of efficacy. Assuming no adverse events are reported during phase 1 trialling, and permission is granted to proceed to phase 2 trialling, this will allow larger scale testing of the surrogate markers of efficacy using samples drawn from a larger number of individuals in possibly a multicentre trial. Clinical trialling would normally progress into phase 3 (demonstration of efficacy in man) after completion of phase 2. For vaccines against dangerous pathogens, phase 3 trialling will not be possible. Therefore, more emphasis will be placed on proving the surrogate markers of efficacy in the phase 2 trial. Assuming that this is achieved, and the marketing authorisation application (MAA) is granted for the vaccine, some post-MAA safety surveillance studies will be required. These studies also provide an opportunity to apply the surrogate markers of efficacy identified for each vaccine pre-licensure, to determine the duration and degree of protection the vaccine provides in an expanded and diverse population of vaccinees.
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Vaccine formulation options |
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The fact that some pathogens can infect man by more than one route, adds to the danger they pose. For example, although in nature Y. pestis is adapted to be transmitted by flea bite, it can also be transmitted by the inhalational route in aerosol form from an infected animal or another individual with pneumonic plague. Similarly, the sporulating bacteria causative of anthrax (Bacillus anthracis) or botulism (Clostridium botulinum) can be acquired by the inhalational route, and many viruses can be transmitted by this route (e.g. Ebola, Lassa fever, and the encephalitic viruses). Since the lung and respiratory tract are vulnerable to first exposure to an inhaled pathogen, it is important that adequate protective immunity is present here. In immunised individuals, this may be achieved either by transudation into the respiratory tract and the lung of high levels of IgG circulating in the blood, or by the induction of local immunity (IgA) or by a combination of both. Parenteral immunisation will induce IgG systemically and it has been demonstrated in the mouse that a high titre response to the F1 + V vaccine given intramuscularly will protect against an inhalational challenge with Y. pestis21. Similar findings have been made for parenterally administered recombinant subunit vaccines for anthrax. However, nasal or inhalational delivery of subunit vaccines may be even more effective than parenteral delivery to protect against mucosal exposure to dangerous pathogens and considerable effort is being placed in a next generation of vaccines which can effectively induce both mucosal and systemic immunity. For a vaccine against the encephalitic virus VEEV, such an approach may be essential to achieve local immunity rapidly to prevent the neurotropism of the virus34. Similarly, for an effective vaccine against HIV, the induction of protective local immunity in the vaginal35 or rectal mucosa, as well as a high level of systemic immunity is essential to counter the virus.
For the parenteral delivery of sub-unit vaccines, the usual approach is to co-formulate the protein subunits with alum adjuvants, either by adsorption to aluminium hydroxide or by precipitation with aluminium phosphate. Options for mucosal delivery include the encapsulation of sub-units in polymeric nano- or microspheres which in research has achieved particularly effective mucosal vaccine formulations for Y. pestis27,36,37 and B. anthracis38, liposomal encapsulation39 or live vaccine vector delivery, e.g. from salmonella40.
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Key points for clinical practice |
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The R&D of vaccines against dangerous pathogens is enormously challenging. Although only those laboratories around the world which have the infrastructure to handle safely the causative organisms are active in R&D of vaccine approaches for the dangerous pathogens, this is an extremely important, if minority, activity. The output from this R&D could have a large impact on mankind. Some of the poorest peoples of the world are affected by diseases caused by such dangerous pathogens, e.g. HIV has decimated the adult populations of countries in Africa41, where Ebola virus also resurges regularly and natural plague foci exist in the southern regions of the continents of Asia, Africa and America with seven countries reporting cases of plague every year for the last 44 years41. These are Brazil, the Democratic Republic of the Congo, Madagascar, Myanmar, Peru, USA and Vietnam. World-wide over the past 44 years, there have been 80,613 cases of plague reported with 6587 deaths42. The development of effective vaccines against such dangerous pathogens could lead to the eradication of the natural disease in endemic areas and relieve human anxiety and suffering as well as significantly impacting on the economy of these regions. The development of effective vaccines would also counter the threat of deliberate use of such dangerous pathogens by intending bioterrorists, which threatens to undo some of the major medical advances of the last century.
The challenge of the 21st century is to bring some of the current vaccine candidates against dangerous pathogens, which are in development, into clinical use so that the benefits of the R&D effort expended on them, may be reaped in terms of the alleviation of human suffering.
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Footnotes |
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Correspondence to: Dr E D Williamson, DSTL, Chemical and Biological Sciences, Porton Down, Salisbury SP4 0JQ, UK
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References |
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