Download Helicobacter pylori vaccines and mechanisms of effective

Immunology and Cell Biology (2001) 79, 67–73
Theoretical Article
Helicobacter pylori vaccines and mechanisms of effective
immunity: Is mucus the key?
P H I L I P S U T TO N
School of Microbiology and Immunology, University of New South Wales, Sydney, New South Wales, Australia
Summary In this theoretical article, the hypothesis is proposed that immunization against gastric helicobacter
infection is mediated by CD4+ T-cell induced changes in mucus production. Vaccine development for the gastric
pathogen Helicobacter pylori has encountered several problems. Resolving these problems is impeded by our
lack of understanding of the mechanisms by which the immune response influences bacterial colonization.
Protective immunity requires CD4+ T cells, but the majority of helicobacters are located in the mucus of the gastric
lumen, away from the epithelial surface. Evidence suggests that this mechanism functions independently of antibodies, so how this is achieved is unknown.
Clues to this mechanism may be provided by immune clearance of nematode infection. Similar to H. pylori,
expulsion of the intestinal nematode, Nippostrongylus brasiliensis, in rodents is mediated by CD4+ T-cell changes
in the numbers of goblet cells and the type of mucins secreted into the gut. Immune-mediated changes in secretion
of gastric mucins could similarly be responsible for the reductions in helicobacter colonization seen in immunized
animals. Helicobacter pylori are highly motile bacteria that have evolved to inhabit their specialized niche. Alterations in their mucus environment could influence their motility, such that the bacteria cannot remain efficiently
within the mucus and are flushed away.
Key words: effector mechanism, Helicobacter pylori, immunisation, mucus.
Introduction
Over the last decade, there has been significant effort focused
upon the development of a vaccine against the human gastric
pathogen, Helicobacter pylori. This bacterium was discovered
18 years ago in Perth, Australia, in the stomachs of individuals exhibiting chronic gastritis.1,2 Following initial scepticism,
it has now been conclusively demonstrated that H. pylori is a
key aetiological factor in the majority of peptic ulcers3 and is
listed as a class I carcinogen due to its association with the
development of gastric adenocarcinoma and mucosa associated lymphoid tissue (MALT) lymphoma.4–6 Although antimicrobial therapy is available, the cost and the emerging drug
resistance7 mean that an effective vaccine would be of great
benefit, particularly in the poorer developing countries, which
have the highest incidence of gastric cancer. Unfortunately,
some issues regarding the final vaccine remain to be resolved;
a situation made more complicated by our lack of understanding regarding the effector mechanisms induced by
immunization. This theoretical article discusses the evidence
that may explain the immune events that influence colonization by gastric helicobacters.
Helicobacters and vaccines
The first animal model developed to investigate helicobacters
in gastric pathology used a mouse infected with Helicobacter
Correspondence: P Sutton, School of Microbiology and
Immunology, University of New South Wales, Sydney, NSW 2052,
Australia. Email: [email protected]
Received 15 June 2000; accepted 21 August 2000.
felis, a very close relative of H. pylori, which was isolated
from a cat.8 Helicobacter felis was found to not only colonize
mouse gastric tissue in abundance, but also to induce severe
gastritis. Mouse-colonizing strains of H. pylori are now available,9,10 although they do not induce a severe inflammatory
response in mice, which make them rather poor models for
studying helicobacter-induced gastritis. They do, however,
provide an ideal model for investigating protective immunity
against H. pylori infection.
The use of these helicobacter–animal models for immunization studies has raised a number of important issues.
First, although it was initially believed that vaccination
induced sterilizing immunity, which completely prevented or
cleared infection, it has subsequently been realized following
the development of more sensitive assays that although bacterial colonization is greatly reduced, some bacteria always
remain.11,12
The second issue is the requirement for a mucosal adjuvant, such as cholera toxin (CT) or heat labile toxin from
Escherichia coli (LT), for effective immunity. Unfortunately
humans are more sensitive to these reagents than are rodents,
so they are considered too toxic for use in a final human
vaccine. Currently there is no mucosal adjuvant that is suitable for use in humans. Some progress has been made with
the use of detoxified forms of these adjuvants13 and with live
vectors, such as recombinant attenuated salmonella expressing helicobacter antigen.14–17
The third potential problem involves a phenomenon known
as postimmunization gastritis. Prophylactic immunization of
mice with subsequent challenge with helicobacters can induce
a more severe gastritis than caused by infection alone.11,18,19
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P Sutton
Given that it is the inflammation that leads to the associated
diseases, this would not be a desirable outcome. Postimmunization gastritis appears to be driven by the residual bacteria
that are not cleared by immunization, because treatment of the
immunized challenged mice with antimicrobials to remove the
remaining H. felis prevented the exacerbation of gastritis.11
Thus, in order to create a human vaccine it may be
necessary to improve immunization to produce sterilizing
immunity, circumvent the adjuvant problem and ensure that
the immunizations will not, in fact, increase the likelihood of
an individual developing helicobacter-induced pathologies.
All of these are difficult to achieve without an understanding
of the effective immune mechanisms, induced by vaccination,
that influence colonization by H. pylori.
Effective immunity against H. pylori infection:
What is known?
In humans, infection with H. pylori normally occurs during
childhood and, if left untreated, will generally remain in the
gastric lumen for the lifetime of the individual. A small proportion of these organisms adhere to the luminal surface of
the gastric epithelium, but the majority of the colonizing
bacteria are ‘free-swimming.’ The presence of these bacteria
induces a chronic gastritis, which lasts for decades. This
inflammation is a cell-mediated response driven by Th1-type
cytokines, as shown by numerous studies using both infected
humans and animal models.20,21 Despite this extremely longterm immune response, the infection persists.
Antibodies
Given the location of the infection in the gastric lumen, it was
initially expected that antibodies would mediate protective
immunity. Even before animal models became available,
Czinn and Nedrud found that immunization of mice and
ferrets with killed H. pylori and CT produced a significant
intestinal and serum antibody response.22 When the H. felis
mouse model was developed and protective immunity demonstrated,23 it was found that oral delivery of helicobacterspecific IgA monoclonal antibodies at the same time as challenge with H. felis protected mice against colonization.24 Many
of the following studies found an association between protective immunity and serum or secretory antibody responses.19,25,26
It was an early paper from 1986 by Wyatt et al., who
looked at tissue from individuals infected with Campylobacter
pyloridis (now H. pylori), which provided the first suggestion
that antibodies may not be effective.27 Bacteria colonizing
the gastric tissue of patients with gastritis were coated with
IgA.27 They also found that the bacteria infecting patients with
active gastritis (a form with neutrophil infiltration, which is
associated with greater progression to helicobacter-induced
diseases) were frequently coated with IgG and IgM.27 The
antibody response in these individuals was clearly having no
success in clearing the infection.
Doubts regarding the role of antibodies were further
raised by several immunization studies that found no association, or even an inverse correlation, between protective
immunity and antibody production.20,28 Most convincingly,
immunization of antibody-deficient mice produces the same
level of protection against both H. felis and H. pylori as is
present in wild-type mice.29–31 Thus, normal protection can
occur even in the complete absence of antibodies. This
demonstrates that antibody independent immune mechanisms
exist that are induced by immunization and can influence the
colonization of bacteria that inhabit the gastric lumen.
T cells
In contrast, there is clear evidence that T lymphocytes are
critically required for protective immunity against helicobacter infection. Experiments using major histocompatibility
complex (MHC)-deficient mice have demonstrated that CD4+,
but not CD8+ T cells, are essential for effective immunity
against H. pylori in mice. Immunization of mice deficient in
MHC class I molecules produces the same protective immunity as wild-type mice. However, if mice are deficient in MHC
class II molecules, used by the immune system to present
antigen to CD4+ Th cells, then no protection against bacterial
colonization results.29,32
It remains uncertain whether it is the Th1- or Th2-type
response that is important. Some evidence suggests a role for
Th2-type immunity. Adoptive transfer of helicobacter-specific
Th1 cell lines has no effect on bacterial colonization after
challenge, but transfer of helicobacter-specific Th2 cell lines
induces a significant reduction in infection levels.33 Additionally, protection following immunization is associated with
increased IL-4 and decreased IFN-γ secretion by splenic and
gastric lymphocytes.20,34
Gastric infection with helicobacters naturally produces an
ineffective Th1 inflammatory response,20,21 so it came as no
surprise that Th2 cells were found to be associated with protection. However, evidence is accumulating that questions
this role for Th2 immunity. Immunizations on IL-4-deficient
mice have found that although protective efficacy is reduced,
it is not lost, suggesting that some protection is possible in
the absence of Th2 immunity.35 In contrast, several studies
have suggested that protection is lost in mice deficient in
IFN-γ or its receptor.36,37 In addition, a reduction in H. felis
colonization, induced by adenovirus co-infection, is dependent on IFN-γ and IL-12.38
Whether it eventuates to be Th1, Th2 or both CD4+ cell
types that induce protective immunity, it is likely that they
only constitute the trigger of the immune response. With the
demonstration that immune mechanisms exist that can
function independently of antibodies, it is necessary then to
speculate on alternative effector mechanisms that can prevent
or inhibit helicobacter colonization of the gastric mucosa. To
do this we need to consider what is known regarding the
interaction of the bacterium and its habitat.
Helicobacter and the gastric mucosa
Helicobacter pylori inhabits the gastric mucosa and is mainly
found located free in the mucus layer, with some binding to
the epithelial surface both in infected humans39,40 and in
mice.10 Figure 1 shows a gastric mucus scraping taken from a
person infected with H. pylori and the spiral bacteria are
clearly present in large numbers in the mucus. Only a low proportion of colonizing H. pylori actually adhere to the epithelium.39,41 Schreiber et al., using a microinjection system, have
analysed the location of H. felis within the mucus of infected
Immunity and H. pylori: A role for mucus?
69
Figure 1 Light micrograph showing a gastric mucus scraping from
an individual infected with Helicobacter pylori.
mouse stomachs: three-quarters were found between 5 and
20 µm from the epithelial surface and no bacteria were located
closer than 5 µm.42 Thus, H. felis does not appear to adhere at
all to the epithelium. Despite this, effective immunity against
infection with H. felis induced by immunization appears to
parallel that obtained with the H. pylori mouse model.
Effects of H. pylori infection on gastric mucus production
Mucus is a viscous gel secreted by mucosal cells lining the
gastrointestinal tract, including goblet cells and epithelial
cells. The secreted mucus forms a protective barrier at the
mucosal surface and its main components are heavily glycosylated proteins called mucins. The type of mucin produced
and the way in which it is glycosylated varies between different sections of the gastrointestinal tract, reflecting the different demands required of the mucus at individual sites. The
luminal surface of the stomach is lined with gastric columnal
epithelial cells, which secrete neutral mucopolysaccharides.
There is much evidence to suggest that H. pylori changes
mucus production by the host. Before the discovery of
H. pylori, it was noted that individuals with gastritis or peptic
ulcer disease exhibited changes in mucin production at the
gastric surface.43 There then followed many studies that
proposed that these changes may be due to infection with
H. pylori, although most of these were performed in vitro.
Micots et al. found that culture of H. pylori with a human
mucus secreting cell line strongly inhibited mucin secretion
induced by acute stimulation with secretagogue agents.44
They concluded that H. pylori can directly impair the secretory functions of mucous cells. Supporting this, it has been
found, using cultured gastric epithelial cells in vitro, that
H. pylori lysate inhibits mucus production.45
Slomiany et al. have found that H. pylori produces active
enzymes, both proteases and lipases, that degrade gastric
mucins isolated from healthy individuals, in vitro.46,47
However, the significance of this has been contended by
Markesich et al., who have examined the mucus from patients
infected with H. pylori before and after bacterial eradication.
In this in vivo study, they found that the gastric mucus was
actually more viscous after the loss of H. pylori infection than
the mucus obtained from the same individual while still
infected.48 Slomiany et al., themselves later supported the
Markesich et al. study, in a paper where they demonstrated
that the physiological and mechanical protective properties of
mucus were restored after H. pylori eradication.49
Adhesion between H. pylori and gastric mucins
Numerous studies have investigated the binding properties of
H. pylori. In vitro experiments have provided evidence that
the adhesion of H. pylori to epithelial cell lines50,51 and to
mucins52–54 may involve sialic acid and sulfated oligosaccharides. This has been supported in vivo by Genta et al., who
have found that the adhesion of H. pylori to areas of intestinal
metaplasia is associated with the expression of sulfomucins
on the gastric tissue.55
Another bacterium that can infect the mucosal surface is
Pseudomonas aeroginosa, an important pathogen in cystic
fibrosis. This bacterium infects the lungs and expresses mucinbinding proteins with which it adheres to bronchial mucins.
This binding is inversely correlated with iron availability, so
in conditions of low iron, mucin-binding protein expression
and thus mucin binding increase.56 One unusual feature about
H. pylori, which was revealed upon genomic sequencing of
two strains, is the high number of iron-scavenging molecules
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P Sutton
it produces.57,58 This observation suggests that iron performs
an important role for H. pylori, and it is tempting to speculate
that this is perhaps related to mucin-interactions.
A lesson from a nematode?
Nippostrongylus brasiliensis is a nematode which inhabits the
mucus of the jejunum of rats. Like H. pylori, the organism
is topographically distant from immune cells. However, this
infection does induce a protective immune response. So can
we gain clues regarding immunity against H. pylori, by
looking at what is known about N. brasiliensis?
The theory on how the immune system of rats can induce
expulsion of N. brasiliensis is intriguing. The current hypothesis, first proposed by Ogilvie and Love, is that this
expulsion is a two-step process, whereby specific antibodies
cause damage to the worm and a subsequent lymphocytemediated non-specific response leads to worm expulsion.59
Evidence to support this hypothesis comes from the use of
‘damaged’ worms; that is, worms that are in the process
of being expelled. If damaged worms are used to infect naïve
rats, then the worms are expelled in an accelerated manner.60
Evidence suggests that this latter non-specific step is caused
by a T-cell mediated alteration in goblet cell numbers and
mucin production.
Infection of immunocompetent rats with larvae of N. brasiliensis produces an acute infection, which the host begins
to clear after 10 days, with most worms expelled by 14 days
postinfection.61 If purified immune T cells from rats infected
with N. brasiliensis for 10 days are adoptively transferred
into naïve rats, which are challenged at the same time with
N. brasiliensis, the response to the nematode is greatly accelerated. In rats receiving these specific T cells, clearance
begins after day six, with all worms completely expelled by
day eight.61,62
As early as 1963, it was noted that goblet cells in the
intestinal mucosa of rats increased in number just prior to and
during expulsion of N. brasiliensis.63 It was later found that a
similar increase in goblet cell number was induced on transfer of purified immune T cells from 10 day N. brasiliensis
infected rats into newly infected recipient rats, but not when
these T cells were transferred into uninfected controls.62
Histochemical staining revealed that during expulsion of the
nematode by a rat, there was a change in the composition of
goblet cell mucins.64 By the use of lectin immunostaining,
Oinuma et al. have demonstrated that at the time of expulsion, goblet cell mucins begin to strongly express a variety
of glycoconjugates, including sialic acid, N-acetyl-Dgalactosamine (GalNAc) and N-acetyl-D-glucosamine.65
When nude rats, which lack normal T cells, were infected
with N. brasiliensis, the worms were not cleared and there
was no increase in either goblet cell numbers or the type of
mucins produced.60 The same authors, using a mouse model,
have found that spontaneous cure of N. brasiliensis infection
is inhibited by treatment of mice with antibodies against the
CD4, but not CD8 T cell marker.66 Most significantly, they
have found that the in vivo depletion of CD4+ cells reduces
the secretion of intestinal mucus, which is associated with the
loss of worm expulsion.
There are at least two hypotheses explaining how the
two-step model may work. The first proposes that a fully
functional N. brasiliensis in some way inhibits the mucinaltering activity of the T cells. Thus, in the first step, an
antibody mediated attack occurs by which the worm is
damaged and prevents the production of the inhibitory
signal, leaving the T cells free to induce expulsion. The
second hypothesis states that the damage inflicted on the
worm causes release of a stimulatory signal that leads T cells
to induce worm expulsion.
Why changes in host mucin production lead to expulsion
of a reasonably complex organism, such as a nematode worm,
is also unknown, but has some degree of specificity. When
rats were co-infected with N. brasiliensis and the related
nematode Strongyloides ratti, the worms were expelled at
different times.67 Expulsion of N. brasiliensis correlated with
changes in mucus, whereas loss of S. ratti infection occurred
on development of a significant mast cell response. The
authors have proposed that there is an interaction between the
mucins and a specific function of N. brasiliensis.67
Bringing it all together: The conclusion
Helicobacter pylori is highly evolved to survive and indeed
thrive in a specialized ecological niche, which is uninhabitable to almost all other infectious organisms. This environment in which H. pylori exists is one of low pH, such that
even the gastric tissue protects itself by secretion of a thick
mucus layer. Part of the evolution of H. pylori into this niche
involves the utilization of this mucus, at least in part for
protection against the harsh environment of the stomach. The
majority of H. pylori are localized in the mucus away from
the epithelial surface and, in the case of H. felis colonization
of mice, no bacteria adhere to the gastric tissue.
These bacteria not only directly interact with mucus and
mucin proteins, but infection can also alter their synthesis.
One possible interpretation is that the host is responding to
limit infection by altering the mucus in an attempt to flush out
the bacteria. Alternatively, perhaps it is part of the strategy of
the bacteria, to favour its colonization and in fact reduce its
expulsion. Given the lifelong infection that is the normal
result, the latter is perhaps the most likely explanation.
Using animal models of helicobacter infection, it has been
shown that immunization can induce an effective immune
response to often quite dramatically reduce bacterial colonization. The mechanism of this process is unknown, but
involves CD4+ T cells and is antibody independent. Despite
the finding that there is a clear distance between the helicobacters and the epithelial surface, effective immunity is
still achievable. It is unlikely that a direct cell action can be
responsible for influencing colonization.
There are many similarities between the situation of
protective immunity against helicobacter infection and the
clearance of N. brasiliensis infection by rodents. Both organisms live in gastrointestinal mucus, predominantly distal
from the epithelial surface, and clearance is mediated by a
process involving CD4+ T cells. It is interesting that the main
association of worm expulsion and host immunity is the
CD4+ T-cell driven alteration in mucus production.
Considering these facts, the hypothesis is presented here
that immunization against gastric helicobacter infection
induces CD4+ T-cell mediated changes in mucus production.
If true, how could changes in mucus lead to expulsion of
Immunity and H. pylori: A role for mucus?
Figure 2
71
Electron micrographs of gastric mucus scrapings from mice showing (a) Helicobacter pylori and (b) Helicobacter felis.
the bacteria? There are several possibilities. Perhaps a change
in mucus consistency could produce greater exposure of
the helicobacters to the stomach’s acid secretions. This is
supported by our knowledge that H. pylori is sensitive to
environmental pH.68
It is more likely, however, that the environmental change
is such that the bacteria cannot remain efficiently within the
mucus and are flushed away. Gastric helicobacters are very
motile organisms, possessing flagella for propulsion and a
spiral morphology (shown in Fig. 2), which allows rapid
corkscrew movement through their viscous surroundings.
Hazell et al. have examined the movement of H. pylori in
various solutions of methyl cellulose and have found that in
a highly viscous environment the bacteria has amazing motility, moving up to 78 µm/s.40 If for a moment we imagined
H. pylori as 2 m long, this would equate to a speed of approximately 90 km/h, while travelling through a dense gel. When
the viscosity of the environment is increased or decreased, the
velocity of the spiral helicobacter rapidly declines.40 The
mucus layer of the stomach is a dynamic habitat, with a
constant flow of mucus and other gastric secretions down the
alimentary tract. It is not unreasonable to assume that the
motility of gastric helicobacters is essential for them to
remain in the gastric mucosa and that impediment of this
motility by changes in mucus viscosity could well lead to
rapid expulsion. Motility impediment could possibly result
from either an increase or a decrease in viscosity. It is also
possible, though less likely, that the altered mucins bind to
the flagella, producing the same result.
In a paper written over 20 years ago, and 5 years before
the discovery of H. pylori, Nawa et al.62 wrote:
‘Even though N. brasiliensis causes severe pathological
changes in the intestine, the parasites remain on the surface
of the mucosa and do not penetrate the epithelium. This
observation raises important questions as to how lymphoid
cells can exert a direct effect on the parasites. Most workers
agree that a specific antibody has an important role, although
it is generally thought that the final phase of expulsion is
brought about by nonspecific mediators.’
It appears that helicobacter researchers today are asking
the same questions that have been asked for over two decades
by immunologists studying nematode clearance. We are now
in a position to use this knowledge obtained from nematodes
to examine and unravel a mystery of immune function and
H. pylori infection. Solving this mystery, to explain this
poorly recognized and understood mucosal immune function,
will be invaluable in vaccine design for protection against
H. pylori. Current development is occurring very much in the
dark, with improvements difficult in the face of our ignorance regarding the effector mechanisms. Beyond that, understanding this mechanism will have great implications in the
development of immune strategies for combatting other
lumenal dwelling pathogens.
Acknowledgements
I would like to gratefully acknowledge the significant contribution of Professor Chris Parish, who brought to my attention
the concept that T cells can mediate effective immunity by
alterations in mucus production. My appreciation also goes to
Jani O’Rourke for the photo and electron micrographs of the
bacteria.
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