Download Prospects of new vaccines for resurgent and emergent diseases

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
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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.
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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)
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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
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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.
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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
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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
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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
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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
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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.
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