Note: Descriptions are shown in the official language in which they were submitted.
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VACCINE
This invention relates to an immunornodulator for
use in a vaccine which is intended for use against a
range of infectious agents. Further this invention
relates to a vaccine composition comprising the
immunomodulator, preferably in combination with antigen
and a vaccination method using the vaccine composition.
Cholera toxin (Ctx) and its close relative E. coli
heat-labile enterotoxin (Etx) are potent immunogens and
mucosal adjuvants. However, their inherent toxicity
makes them unsuitable for human use. For example,
although Ctx is the most commonly used mucosal adjuvant
in experimental animals, it is unsuitable for use in
humans because of its potent diarrhoea-inducing
properties. Attempts have been made to separate
toxicity from adjuvant activity, for example by using
components of Ctx and Etx as replacements for the
holotoxins themselves. E. coli verotoxin (Vtx) is
another known bacterial toxin.
Ctx and Etx are heterohexameric proteins composed
of a an enzymatically active A subunit and a pentameric
B subunit. CtxB and EtxB are known to bind GM1-
ganglioside (GM1), a glycosphingolipid found
ubiquitously on the surface of mammalian cells. Vtx
binds to Gb3 which is a similar type of receptor to
GMl.
In an attempt to circumvent the problem of
toxicity for vaccine development, the adjuvant activity
of the non-toxic B subunits has previously been
investigated. However, many of the reports describe
experiments in which a commercial preparation of CtxB
or EtxB was used. These preparations are inevitably
contaminated with a small but biologically significant
amount of active toxin, so the adjuvant activity
attributable to the B subunit is indistinguishable from
the adjuvant activity of the whole toxin (Wu and
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Russell (1993) Infection and Immunity 61: 314-322, US-
5182109). Subsequent studies using recombinant CtxB
(rCtxB) have suggested-that CtxB is a poor mucosal
adjuvant and only the addition of native holotoxin can
provoke strong bystander responses (Tamura et al (1994)
Vaccine I2: 419-426). Other studies have suggested
that rCtxB lacks the ADP-ribosylating and the cAMP-
stimulating activities of the holotoxin and that, as
adjuvant mechanism is linked to these abilities, .CtxB
would be unsuitable for use as an adjuvant (Vajdy and
Lycke (1992) Immunology 75: 488-492, Lycke et a1 (1992)
Eur. J. Immunol. 22: 2277-2281, Douce et al
(1997)Infection and Immunity 65: 2821-2828).
In another study, intranasal administration of
ovalbumin using rCtxB as an adjuvant resulted in poor
antibody responses. A non-toxic derivative of Ctx with
a mutation in the A subunit also generated weak
responses to bystander antigens, whereas the presence
of an active A subunit dramatically enhanced adjuvant
activity, suggesting that an active A subunit is
essential (Douce et al (1997) as above).
It has also been shown that rCtxB and rEtxB can be
used to promote tolerance to heterologous antigens (Sun
et al (1994) Proc. Natl. Acad. Sci. 91: 4610-4614, Sun
et al (1996) Proc. Natl. Acad. Sci. 93: 7196-7201,
Bergerot et a1 (1997) Proc. Natl. Acad. Sci. 94: 4610-
4614, Williams et a1 (1997) Proc. Natl. Acad. Sci. 94:
5290-5295), suggesting that these molecules would be
unsuitable for use as adjuvants.
The basis o~ the present invention
In spite of the teaching in the art that CtxB and
EtxB have poor adjuvanticity and can, in fact, act as
tolerogens, the present inventors nevertheless
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investigated the use of rEtxB (thus containing no
residual holotoxin or A subunit) in an intranasal
vaccine for HSV in a murine model and surprisingly
found that it is able to stimulate protective immune
responses to viral challenge. Specifically, the
present inventors found that:
i) agents such as EtxB and CtxB stimulate high
levels of local (mucosal) antibody production (although
immunization using rEtxB stimulated lower levels of
overall serum antibody production than Ctx/CtxB
combined);
ii) the distribution of antibodies produced was
skewed towards non-complement fixing antibodies,
especially S-IgA and IgGl;
iii) agents such as EtxB and CtxB also stimulated
local and systemic T-cell proliferative responses;
iv) agents such as CtxB and EtxB tend to shift
the immune response from a Thl-associated response to a
Th2-associated response;
v) when agents such as CtxB and EtxB are used
as immunomodulators some of the harmful effects of Th2-
associated responses, such as the generation of IgE,
are avoided;
vi) rEtxB is a more efficient immunomodulator
than rCtxB;
vii) agents such as EtxB and CtxB are capable of
altering the way in which an antigen presenting cell
internalises and processes antigen, increasing antigen
persistence;
viii) if an agent such as EtxB and CtxB is linked
to an antigen, it is possible to alter the processing
route of the antigen by altering the linkage to the
immunomodulator; and
ix) VtxB exerts similar immunomodulatory effects
on leukocyte populations in vitro to those exerted by
EtxB and CtxB.
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These important discoveries are the basis of the
various aspects of the present invention and enabled
the inventors to predict that pure EtxB, CtxB and VtxB,
as well as other agents capable of binding to or
mimicking the effect of binding to GM1 or Gb3, will be
useful as immunomodulators for use in vaccines in the
prophylactic and therapeutic vaccination against HSV-1
infection, as well as other infections, the prevention
or treatment of which would benefit from
immunomodulation of the types listed above.
Stimulation of immune responses
EtxB, CtxB, VtxB and other agents capable of
binding to or mimicking the effects of binding to GM1
or Gb3, are capable of acting as immunomodulators and
stimulate specific immune responses to antigenic
challenge.
According to a first aspect of the present
invention, there is provided the use of:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having .an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding;
as an immunomodulator for a vaccine against
infectious diseases.
According to a second aspect of the present
invention, there is provided a vaccine composition for
use against an infectious disease, which infectious
disease is caused by an infectious agent, wherein the
vaccine composition comprises an antigenic determinant
and an immunomodulator selected from:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
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GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding;
wherein said antigenic determinant is an antigenic
determinant of said infectious agent.
The antigen and immunomodulator may be linked, for
example covalently or genetically linked, to form a
single effective agent. In a specific embodiment of
this invention the antigen and immunomodulator may be
chemically conjugated. For example, the antigen and
immunomodulator may be chemically conjugated using
heterobifunctional cross-linking reagents. In most
applications of this aspect of the invention, separate
administration (in which the antigen and
immunomodulator are not so linked) is preferred because
it enables separate administration of the different
moieties.
According to a third aspect of the present
invention, there is provided a kit for vaccination of a
mammalian subject, such as a human or veterinary
subject, against an infectious disease, comprising:
a) one of the following agents:
(i)' EtxB,.CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding; and
b) an antigenic determinant which is an antigenic
determinant of the infectious disease, for
coadministration with the said vaccine immunomodulator.
The vaccine composition of the second aspect of
the invention and the kit of the third aspect of the
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invention may be used in a prophylactic or therapeutic
vaccination method, where a "prophylactic vaccine" is
administered to naive-individuals to prevent disease
development, and a "therapeutic vaccine" is
administered to individuals with an existing infection
to reduce or minimise the infection or to abrogate the
immunopathological consequences of the disease.
Agents such as EtxB have the capacity to alter the
nature of the immune response once infection has
occurred. A therapeutic vaccine (i.e. one which need
not contain antigen) comprising such an agent may find
particular use in circumstances in which the immune
response has failed to get rid of an infection. This
application may be of particular use to treat a chronic
disease, for example a disease for which the causative
agent is selected from the group consisting of herpes
viruses, hepatitis viruses, HIV, TB and parasites.
According to a fourth aspect of the present
invention there is provided a method of preventing or
treating a disease in a host, which method comprises
the step of inoculating said host with a vaccine
comprising at least one antigenic determinant and an
immunomodulator, where the immunomodulator is:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding.
The vaccine may be packaged for coadministration
and may be administered by a number of different routes
such as intranasal, oral, intra-vaginal, urethral or
ocular administration. Intranasal immunisation is
presently preferred. When a vaccine is administered
intranasally, it may be administered as an aerosol or
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in liquid form.
The antigenic determinant and immunomodulator may
be.administered to the subject as a single dose or in
multiple doses.
In a first embodiment the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a disease for
which the infectious agent is a member of the herpes
virus family. For example, the infectious agent may be
selected from the group consisting of HSV-1, HSV-2,
EBV, VZV, CMV, HHV-6, HHV-7 and HHV-8. In particular,
the infectious agent may be HSV-1, HSV-2, CMV or EBV.
In this first embodiment, the antigenic
determinant is preferably an antigenic determinant of
an immediate early, early or late gene product (for
example a surface glycoprotein) of the herpes virus.
If the infectious agent is HSV-1 or HSV-2, the
antigenic determinant may be an antigenic determinant
of a gene product selected from the following group:
gD, gB, gH, gC or a latency associated transcript
( LAT ) .
If the infectious agent is EBV, the antigenic
determinant may be an antigenic determinant of gp340 or
gp350 or of a latent protein (for example EBNAs 1,2 ,
3A, 3B, 3C and -LP, LMP-1, -2A and 2B or an EBER).
In a second embodiment, the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a disease for
which the infectious agent is an influenza virus.
In this second embodiment, the antigenic
determinant is preferably an antigenic determinant of a
viral coat protein (for example haemagglutinin and
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neuraminidase) or of an internal protein (for example,
nucleoprotein).
In a third embodiment, the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a disease for
which the infectious agent is a parainfluenza virus.
In a fourth embodiment, the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a disease for
which the infectious agent is respiratory syncytial
virus.
In a fifth embodiment, the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a disease for
which the infectious agent is a hepatitis virus. For
example, the infectious agent may be selected from the
group consisting of hepatitis A, B, C and D. In
particular the infectious agent may be hepatitis A or
C.
In a sixth embodiment, the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against meningitis. In
this sixth embodiment, the infectious agent may be
selected from the group consisting of Neisseria
meningitidis, Haernophilus influenzae type B and
Streptococcus pneumoniae.
In a seventh embodiment, the immunomodulator of
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the first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against pneumonia or a
respiratory tract infection. In this seventh
embodiment, the infectious agent may be selected from
the group consisting of Streptococcus pneumoniae,
Legonella pneumophila and Mycobacterium tuberculosis.
In an eigth embodiment, the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a sexually-
transmitted disease. In this eighth embodiment, the
infectious agent may be selected from the group
consisting of Neisseria gonnorheae, HIV-1, HIV-2 and
Chlamydia trachomat.is.
In an ninth embodiment, the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a
gastrointestinal disease. In this ninth embodiment,
the infectious agent may be selected from the group
consisting of enteropathogenic, enterotoxigenic and
enteroinvasive E.coli, rotavirus, Salmonella
enteri tidis, Salmonella typhi, Helicobacter pylori,
Eacillus cereus, Campylobacter jejuni and Vibrio
cholerae.
If the infectious agent is selected from the group
consisting of enteropathogenic, enterotoxigenic,
enteroinvasive, enterohaemorrhagic and
enteroaggregative E.coli, then the antigenic
determinant may be an antigenic determinant of a
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bacterial toxin or adhesion factor.
In a tenth embodiment, the immunomodulator of the
first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a superficial
infection. In this tenth embodiment, the infectious
agent may be selected from the group consisting of
Staphylococcus aureus, Streptococcus pyogenes and
Streptococcus mutans.
In an eleventh embodiment, the immunomodulator of
the first aspect of the invention, the vaccine of the
second aspect of the invention, the kit of the third
aspect of the invention and the method of the fourth
aspect of the invention is used against a parasitic
disease. In this eleventh embodiment, the infectious
agent may be selected from the group consisting of
malaria, Trypanasoma spp., Toxoplasma gondii,
Leishmania donovani and Oncocerca spp.
Stimulation of mucosal ia~une responses
EtxB, CtxB, VtxB and other agents capable of
binding to or mimicking the effects of binding to GM1
or Gb3, ara capable of specifically upregulating
mucosal antibody production.
The vaccine immunomodulator of the first aspect of
the invention, the vaccine composition of the second
aspect of the invention and the kit of the third aspect
of the invention are particularly effective against
diseases where protection from infection or treatment
is effected in vivo by a mucosal immune response. For
example, against diseases in which, during infection,
the infectious agent binds to, colonises or gains
access across the mucosa. Examples of such diseases
include, diseases caused by viruses (HIV, HSV, EBV,
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CMV, influenza, measles, mumps, rotavirus etc),
diseases caused by bacteria (E. coli, Salmonella,
Shigella, Chlamydia, N. gonnorhoea, T. pallidium,
Streptococcus species including those which cause
dental caries), and diseases caused by parasites.
In a preferred embodiment of the second aspect
of the present invention there is provided a vaccine
against HSV-1 infection comprising at least one HSV-1
antigenic determinant and an immunomodulator, where the
immunomodulator is:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or G3b
binding.
Preferably the immunomodulator is EtxB.
In a preferred embodiment of the third aspect of
the present invention there is provided a kit for
vaccination of a mammalian subject against an HSV-1,
comprising:
a) a vaccine immunomodulator which is:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or G3b
binding; and
b) at least one HSV-1 antigenic determinant,
for coadministration with the said vaccine
immunomodulator.
According to a fifth aspect of the invention there
is provided the use of:
(i) EtxB, CtxB or VtxB free from whole toxin;
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(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding
to upregulate the production of antibodies at
mucosal surfaces. The production of non-complement-
fixing serum antibodies may also be upregulated.
Preferably, S-IgA is produced in accordance with the
fifth aspect of the invention.
In this fifth aspect of the present invention, the
agent may be used in conjunction with one or more
antigenic determinant(s).
Dov~rnreaulating~ the pathological components of immune
responses
The inventors also found that when pure EtxB was
used as an immunomodulator in the described way, the
harmful effects of Th2 associated responses, such as
the generation of high levels of potentially
pathological IgE, were avoided. Despite this, the
immune response triggered by the use of EtxB (or CtxB
or VtxB) as an immunomodulator appears to favour the
induction of Th2-associated cytokines. In other words
EtxB (or CtxB) induces a shift from a Thl- to a Th2-
type response. This has enabled the inventors to
predict that pure EtxB, CtxB or VtxB, as well as other
agents capable of binding to or mimicking the effect of
binding to GM1 or Gb3, will be capable of down
regulating pathological components of the immune
response associated with both Th1 and Th2 activation.
According to a sixth aspect of the present
invention, there is provided the use of:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
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GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding;
to downregulate the pathological components of
Th2-associated immune responses. The pathological
components of Thl-associated immune responses may also
be downregulated.
It is known that EtxB and CtxB bind to GM1 and
induce differential effects on lymphocyte populations,
including a specific depletion of CD8+ T cells and an
associated activation of B cells (WO 97/02045). Hence,
EtxB and CtxB are thought to alter the balance of the
immune response such that inflammatory Thl associated
reactions are down-regulated while Th2 associated
responses are upregulated. Thl responses include the
secretion of yIFN by activated T-cells leading to
macrophage activation and delayed type hypersensitivity
reactions. Such responses may be an important cause of
pathology during infections with a number of pathogens.
Th2 responses include the activation of T-cells to
produce cytokines such as IL-4, IL-5, IL-10, and are
known to promote the secretion of high levels of
antibody, especially IgA.
It has now surprisingly been found that when EtxB
is used as an immunomodulator in the described way, the
harmful effects of Th2 associated responses, such as
the generation of high levels of potentially
pathological IgE, are avoided. Therefore, EtxB and
CtxB are capable of down regulating pathological
components of the immune response associated both with
Th1 and Th2 activation. Such responses are modulated
in favour of the production of high levels of
non-complement fixing serum antibodies and secretory
IgA production at the mucosal surfaces.
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The use of an agent in accordance with the sixth
aspect of the invention is particularly useful for
therapeutic vaccination in diseases in which
immunopathological mechanisms are involved. Examples
of such diseases are HSV-1, HSV-2, TB and HIV.
The first and sixth aspects of the invention can
be combined. In other words, agents such as EtxB can
be used simultaneously as an immunomodulator and a
therapeutic agent. For example in diseases where
immunopathological mechanisms are involved, the use of
a vaccine incorporating agents such as EtxB or CtxB may
act not only to limit infection, but also to abrogate
the pathological disease processes. The
immunomodulating agent is thus acting both
prophylactically and therapeutically. Examples of
infections where vaccination in this way is therefore
likely to be of particular value include those caused
by the herpes virus family, gastrointestinal and
respiratory tract pathogens.
Iuununomodulation of the antigen processing pathway
aZ prolonqing~resentation
The present inventors have also found that when
EtxB (or CtxB or VtxB? is used as an immunomodulator,
the antigen internalisation and processing pathway is
altered. The presence of the B subunit causes
prolonged presentation, possibly by altering antigen
trafficking inside the antigen presenting cell such
that antigen degradation is delayed and therefore
maintained over longer periods. This feature of B-
subunit associated antigen presentation means that
vaccines incorporating an agent in accordance with the
present invention will have increased antigen
persistence and lead to sustained immunological memory.
According to a seventh aspect of the present
invention, there is provided the use of:
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(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GMl-binding activity, -or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GMl-binding or Gb3
binding;
as an immunomodulator in a vaccine, tv prolong
antigen presentation and give sustained immunological
memory in a mammalian subject.
According to an eighth aspect of the present
invention, there is provided a vaccine composition for
use against an infectious disease, comprising an
antigenic determinant and a immunomodulator selected
f rom
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding;
wherein said antigenic determinant is an antigenic
determinant of said~infectious disease and wherein the
immunomodulator prolongs presentation of the antigenic
determinant and gives sustained immunological memory.
intracellular targeting of the antigren to a I~iC-I
or l~iC-II associated pathway
As aforementioned, the antigen and immunomodulator
in a therapeutic or prophylactic vaccine may be linked,
for example covalently or genetically linked, to form a
single effective agent. The present inventors have
found that is possible to direct the antigen to
different compartments of the cell and hence to
different antigen presentation pathways by altering the
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linkage of the antigen to the immunomodulator.
By linking the antigen or antigenic determinant to
the immunomodulator in a certain way, it is possible to
facilitate translocation of the antigen across the
endosomal membrane into the cytosol. The present
inventors predict that this would enhance loading of
antigenic peptides on to MHC class I molecules. The
use of an antigen-immunomodulator conjugate can
therefore be used to specifically enhance the
activation of cytotoxic T cells (CTL). Induction of
CTL is beneficial for the prevention and treatment of
many diseases especially those caused by viruses,
intracellular bacteria and parasites.
The linkage of the antigen-immunomodulator
conjugate can also be chosen so that the antigen is
delivered into the nucleus.
According to a ninth aspect of the present
invention there is provided a conjugate comprising an
antigen or antigenic determinant and an immunomodulator
selected from:
(i) EtxB, CtxB or VtxB tree from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent which has an effect on vesicular
internalisation mediated by GM1-binding or Gb3 binding.
According to a tenth aspect of the present
invention there is provided a vaccine composition for
use against an infectious disease, which infectious
disease is caused by an infectious agent, which vaccine
composition comprises a conjugate of an antigen or
antigenic determinant and an immunomodulator selected
from:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
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having Gb3-binding activity; or
(iii) an agent which has an effect on vesicular
internalisation mediated by GM1-binding or G3b binding;
wherein said antigen or antigenic determinant is
an antigen or antigenic determinant of said infectious
agent.
The antigen or antigenic determinant may be linked
to the immunomodulator by a variety of methods
including genetic linkage or chemical conjugation. In
a first preferred embodiment the conjugate is a fusion
protein made by genetic linkage of the antigen or
antigenic determinant to the immunomodulator.
Preferably the antigen or antigenic determinant is
genetically linked to the C-terminus of the
immunomodulator. In a second preferred embodiment the
antigen or antigenic determinant is chemically
conjugated to the immunomodulator. Preferably the
antigen or antigenic determinant is conjugated to the
immunomodulator using a bifunctional cross-linking
reagent, such as a heterobifunctional cross-linking
reagent. More preferably the cross-linking agent is N-
Y(-maleimido-butyroxyl)-succinimide ester (GMBS) or N-
succinimidyl-(3-pyridyl-dithio)-propionate (SPDP).
The vaccine composition may be administered by a number
of different routes~such as intranasal, oral, intra-
vaginal, urethral or ocular administration. Intranasal
immunisation is preferred.
According to an eleventh aspect of the present
invention there is provided the use of:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent which has an effect on vesicular
internalisation mediated by GM1-binding or Gb3 binding;
in a conjugate with antigen or antigenic
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determinant.to target the delivery or said antigen or
antigenic determinant to the cytosol or nucleus of an
antigen presenting cell.
According to a twelfth aspect of the present
invention there is provided the use of:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent which has an effect on vesicular
internalisation mediated by GM1-binding or Gb3 binding;
in a conjugate with antigen or antigenic
determinant to upregulate the presentation of said
antigenic determinant, or an antigenic determinant
derived from said antigen, by MHC class I molecules.
Preferably the use of the conjugate of the twelfth
aspect of the invention is used is combination with the
use of the agent in accordance with the fifth aspect of
the invention to stimulate strong CTL responses and to
upregulate mucosal antibody production. This activity
would be particularly useful in the prevention and
treatment of viral infections, for example influenza.
EtxB is the preferred immunomodulator
It has previously been thought that EtxB and CtxB
have similar properties. However, the present
inventors have found that rEtxB is a more potent and
efficient immunomodulator than rCtxB. Hence the
preferred immunomodulator is EtxB, or agents which
mimic the effects of EtxB.
EHV
EBV is one of the eight known human herpes
viruses. Infection usually occurs in early childhood;
however, clinical symptoms are usually weak or
undetectable at this stage. Primary infection with EBV
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later in life is associated with infectious
mononucleosis (IM), which is the second most frequent
disease in adolescence in the US. EBV also has
oncogenic potential. There is a strong link between
EBV and endemic Burkitt's lymphoma (BL) and
undifferentiated nasopharyngeal carcinoma (NPC). Also,
a large proportion of lymphomas that occur in immuno-
compromised patients are caused by EBV, and an
association has been shown to exist between certain
Hodgkin's lymphomas and EBV.
Latently EBV-infected cells express a small number
of so-called "latent" proteins. These include six
nuclear proteins (EBNAs l, 2, 3A, 3B, 3C and -LP),
three integral membrane proteins (LMP-1, 2A and 2B) and
two non-polyadenylated virus derived RNAs (EBERs) with
a role in RNA splicing.
EBV latent membrane protein 1 (LMP-1) is present
in the plasma membrane of infected cells. It is also
expressed in nasopharyngeal carcinomas (NPCs) and EBV-
positive Hodgkin's lymphomas (HD) which indicates a
role for LMP-1 in the development of these tumours.
The LMP-1 gene can alter the phenotype of uninfected
cells causing the upregulation of cell surface
activation markers, promoting cell proliferation. LMP-
1 can also alter signalling pathways and has anti-
apoptotic effects. An cellular immune response
directed against this viral antigen has not been
demonstrated with any degree of certainty in either
healthy carriers or tumour patients.
Many animal viruses have evolved mechanisms to
avoid detection by the host immune system. Commonly,
these mechanisms involve interference with the TAP-
associated peptide translocation system. It is thought
that EBV has also evolved similar mechanisms to avoid
immune system detection, thus allowing its persistence
in the host. This explains why certain cellular immune
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responses are not detectable to the EBV latent protein
EBNA1 and could explain the apparent absence of such
responses against LMP1.
According to an thirteenth aspect of the invention
there is provided a vaccine composition which
comprises:
a) one of the following agents:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding; and
b) an EBV antigen
for use in the treatment and/or prevention of EBV-
associated diseases.
In particular the vaccine composition of the
thirteenth aspect of the invention comprises EtxB,
CtxB, or an agent other than EtxB or CtxB which has
GM1-binding activity.
According to a fourteenth aspect of the invention
there is provided a therapeutic composition which
comprises:
(i) EtxB, CtxB or VtxB free from whole toxin;
(ii) an agent other than EtxB or CtxB, having
GM1-binding activity, or an agent other than VtxB
having Gb3-binding activity; or
(iii) an agent having an effect on intracellular
signalling events mediated by GM1-binding or Gb3
binding;
for use in the treatment of EBV-associated
diseases.
In particular the therapeutic composition of the
fourteenth aspect of the invention comprises EtxB,
CtxB, or an agent other than EtxB or CtxB which has
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GM1-binding activity.
Based on the knowledge that EtxB cocaps with LMP1,
and that EtxB promotesfragmentation of LMP-1, it is
theorised that EtxB (and other agents like CtxB having
GM1 binding activity) will be useful to stimulate anti-
EBV immune responses. This activity has applications
in vaccines to prevent EBV associated diseases, and in
therapeutic treatments to treat such diseases once they
have developed.
Without wishing to be bound by theory, it is
believed that when EtxB cocaps with LMP-1 the antigen
is processed by a different intracellular route, which
enables the antigen to by-pass the normal processing
route which is blocked by the virus. The antigen is
thus presented efficiently on the cell surface. The
action of EtxB may also cause different epitopes of the
antigen to be presented at the cell surface, from those
which are presented if the antigen were processed by
the conventional route.
The vaccine of the thirteenth aspect of the
invention may be used to prevent infection by EBV, or
development of EBV-associated diseases in EBV-infected
individuals. The vaccine may~also comprise a separate
adjuvant, or the agent (such as EtxB or CtxB) can act
as an adjuvant in its own right.
The agents specified in the fourteenth aspect of
the present invention may be used alone (i.e. without
antigen) in the treatment of a EBV-associated disease
which has already developed in a subject.
The preferred agent for use in the thirteenth and
fourteenth aspects of the invention is EtxB.
The EBV antigen is an antigen derivable from EBV
itself or an antigen which is caused to be expressed by
an EBV-infected host cell by the action of EBV.
Preferably the antigen is an EBV latent membrane
protein. Particularly preferred are the antigens LMP-
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1, LMP-2A, LMP-2B, and EBNA-1 as well as antigenic
fragments thereof. The antigen may be isolated
directly from EBV infected cells, or be made by
synthetic or recombinant means.
The thirteenth and fourteenth aspects of present
invention are particularly suited for the treatment
and/or prevention of the following diseases: infectious
mononucleosis, Burkitt's lymphoma, nasopharyngeal
carcinomas, and Hodgkin's lymphomas. It is believed
that these aspects of the invention will be
particularly suited to the treatment and/or prevention
of nasopharyngeal carcinomas and Hodgkin's lymphomas.
The vaccine or the therapeutic composition
according to the thirteenth and fourteenth aspects of
the invention may be used to prevent development of, or
treat, an EBV-associated disease in a mammalian
subject, by administration of an immunologically
effective amount to the subject.
The mammalian subject may be, for example, a
healthy EBV-infected or uninfected individual, an
immunodeficient individual, or an individual with an
EBV-associated disease.
The vaccine may be administered by any suitable
route. The agent and the antigen may be co-
administered to the mammalian subject or administered
separately. The agent and the antigen may be separate
or linked, for example covalently or genetically
linked, to form a single effective agent.
GM-1 and Gb3-associated signalling
Without wishing to be bound by theory, it is
believed that GM1 or Gb3 binding may trigger
intracellular signalling directly or indirectly. The
present inventors have also found evidence which
suggests that EtxB interacts with at least one other
receptor which is involved in the GMl associated
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intracellular signalling event. It may be that binding
of EtxB (or CtxB) to GM1 facilitates binding to a
protein, which protein triggers intracellular
signalling. It is not known what specifically triggers
the signalling event, it may be phosphorylation of GM1
or the protein. When EtxB/CtxB binds GM1 on the cell
surface, bound GM1 is internalised in vesicles
(Williams et a1 (1999) Immunology Today 20;95-101).
GM1 and other glycolipids (such as Gb3) are known to be
preferentially located in "membrane rafts" in which key
protein receptors are also found. It is therefore
possible that internalisation of GM1 as a result of B-
subunit binding causes cocapping of such proteins
leading to their being triggered to mediate
intracellular signalling events.
Definitions
An adjuvant is a substance which non-specifically
enhances the immune response to an antigen, as distinct
from a vaccine carrier, the purpose of which is to
target the antigen to a desired site. The term
"immunomodulator" is used herein to indicate an agent
which acts, like an adjuvant, to stimulate certain
immune responses, but which also directs the immune
response in a particular direction.
The term "coadministration" is used to mean that
the site and time of administration of the antigen and
immunomodulator are such that the necessary immune
response is stimulated. Thus, while the antigen and
the immunomodulator may be administered at the same
moment in time and at the same site, there may be
advantages in administering the antigen at a different
time and/or at a different site from the
immunomodulator. For example, antigen and
immunomodulator may be administered together in a first
step and then the immune response may be boosted in a
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second step by administration of antigen alone.
The term "antigenic determinant" as used herein
refers to a site on an antigen which is recognised by
an antibody or T-cell receptor. Preferably it is a
short peptide derived from or as part of a protein
antigen, however the term is also intended to include
glycopeptides and carbohydrate antigenic determinants.
The term also includes modified sequences of amino
acids or carbohydrates which stimulate responses which
recognise the whole organism.
There are a number of known methods by which it is
possible to identify antigenic determinants for a given
infectious agent.
For example, potential protective antigens may be
identified by elevating immune responses in infected or
convalescent patients, in infected or convalescent
animals , or by monitoring in vitro immune responses to
antigen containing preparations. For example,
i) serum samples from infected or convalescent
patients or infected or convalescent animals may be
screened against whole cell lysates of an infectious
agent, or lysates of cells infected by the said agent,
by the standard technique of Western blotting to detect
those.antigen(s) recognised by the immune serum;
ii) serum samples from infected or convalescent
patients or infected or convalescent animals may be
screened against partial or highly purified antigens
from an infectious agent; or lysates of cells infected
by the said agent, by the standard technique s of
ELISA, in which partial or highly purified antigens
are used to coat microtitre wells, or by iimuno
blotting to detect those antigens) recognised by the
immune sera;
iii) serum samples from infected or convalescent
patients or infected or convalescent animals may be
screened against whole cell lysates derived from
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recombinant expression systems encoding one or more
antigens of interest, and using the standard
techniques of ELISA or Western blotting to detect
those antigens) recognised by the immune serum;
iv) serum samples from infected or convalescent
patients or infected or convalescent animals may be
screened against an expression library containing
cloned genes from the infectious agent of interest,
using colony blot immunodectection to identify that
clones expressing antigens, or fragments thereof, that
are recognised by the immune serum; or
v) PBLs from the blood of infected or convalescent
patients or PBL~s, lymph node cells, spleen cells, or
lamina propria cells from infected or convalescent
animals may be cultured in vitro in the presence of
partial or highly purified antigens derived from either
an infectious agent, or lysates of cells infected by
the said agent, or a recombinant expression system
encoding one or more antigens, so as detect antigen-
specific T-cell proliferative responses.
Alternatively it is possible to detect gene
products which are essential for the in vivo survival
of pathogens, as exemplified by,the technique of
signature tagged mutagenesis developed by Holden or the
detection of gene products specifically induced in
vivo, such as IVET (In Vivo Expression Technology)
developed by Mekalanos or differential fluorescence
induction developed by Falkow, identify a subset of
genes amongst which are likely to potential protective
antigens. Using these methods the gene products may be
screened as outlined above. The genes may be cloned
into expression vectors and the antigens recovered for
inclusion into vaccine formulations together with
agents that modulate a glycosphingolipid-associated
activity.
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There are a number of known methods by which it is
possible to isolate antigens for a given infectious
agent.
For example, surface components of an infectious
agent comprising one or more potential protective
antigens may be extracted from the agent, or from cells
infected by the agent, by use of procedures that allow
the recovery of the antigens. This may include the use
of cell disruption techniques to lyse cells such as
sonication and/or detergent extraction. Centrifugation,
ultrafiltation or precipitation may be used on
collected antigen preparations. The antigen
preparation containing HSV-1 glycoproteins described in
Richards et al., (1998) J. Infect. Dis. 177;1451-7,
exemplifies such a method.
Also, antigens of an infectious agent, or from
cells infected by a said agent may be extracted by a
variety of procedures, including but not limited to,
urea extraction, alkali or acid extraction, or
detergent extraction and then subjected to
chromatographic separation. Material recovered in
void or elution peaks comprising one or more potential
protective antigens may used in vaccine formulations.
Alternatively, genes encoding one or more
potential protective antigens may be cloned into a
variety of expression vectors suitable for antigen
production. These may include bacterial or eukaryotic
expression systems, for example Escherichia coli,
Bacillus spp., Vibrio spp. Sacarromyces cerevisiae,
mammalian and insect cell lines. Antigens may be
recovered by conventional extraction, separation and/or
chromatographic procedures.
The terms "CtxB", "EtxB" and "VtxB" as used herein
include natural and recombinant forms of the molecule.
The recombinant form is particularly preferred. The
recombinant form of the molecule may be produced by a
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method in which the gene or genes coding for the
specific polypeptide chain (or chains) from which the
protein is formed, is inserted into a suitable vector
and then used to transfect a suitable host. For
example, the gene coding for the polypeptide chain from
which the EtxB assemble may be inserted into, for
example, plasmid pMM68, which is then used to transfect
host cells, such as Vibrio sp.60. The protein is
purified and isolated in a manner known per se. Mutant
genes expressing active mutant CtxB, EtxB or VtxB
protein may be produced by known methods from the wild
type gene.
The terms "CtxB", "EtxB" and "VtxB" also include
mutant molecules and other synthetic molecules
(containing parts of CtxB, EtxB or VtxB) which retain
the capacity to bind GM1 or Gb3 or the capacity to
mimick the effects of binding to GM1 or Gb3.
Agents other than EtxB and CtxB which retain GM1
binding activity, and agents other than VtxB which
retain Gb3 binding activity include antibodies which
bind GM1 or Gb3.
For the production of antibodies, various hosts
including goats, rabbits, rats, mice, etc. may be
immunized by injection with GM1 or Gb3 or any
derivative or homologue thereof. Depending on the host
species, various adjuvants may be used to increase
immunological response. Such adjuvants include, but
are not limited to, Freund's, mineral gels such as
aluminium hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol. BCG (Bacilli Calmette-Guerin) and
Corynebacterium parvum are potentially useful human
adjuvants.
Humanised monoclonal antibodies may be preferred
in the present invention. Monoclonal antibodies may be
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prepared using any technique which provides fox the
production of antibody molecules by continuous cell
lines in culture. These include, but are not limited
to, the hybridoma technique originally described by
Koehler and Milstein (1975 Nature 256:495-497), the
human B-cell hybridoma technique (Kosbor et al (1983)
Immunol Today 4:72; Cote et a1 (1983) Proc Natl Acad
Sci 80:2026-2030) and the EBV-hybridoma technique (Cole
et aI (1985) Monoclonal Antibodies and Cancer Therapy,
Alan R Liss Inc, pp 77-96). In addition, techniques
developed for the production of "chimeric antibodies",
the splicing of mouse antibody genes to human antibody
genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used
(Morrison et a1 (1984) Proc Natl Acad Sci 81:6851-6855;
Neuberger et al (1984) Nature 312:604-608; Takeda et a1
(1985) Nature 314:452-454). Alternatively, techniques
described for the production of single chain antibodies
(US Patent No. 4,946,779) can be adapted to produce
target interaction component specific single chain
antibodies.
Antibodies may also be produced by inducing in
vivo production in the lymphocyte population or by
screening recombinant immunoglobulin libraries or
panels of highly specific binding reagents as disclosed
in Orlandi et a1 (1989, Proc Natl Acad Sci 86: 3833-
3837), and Winter G and Milstein C (1991; Nature
349:293-299).
Antibody fragments which contain specific binding
sites for GM1 or Gb3 may also be generated. For
example, such fragments include, but are not limited
to, the F(ab')2 fragments which can be produced by
pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the
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disulfide bridges of the F(ab~)2 fragments.
Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity
(Huse WD et a1 (1989) Science 256:1275-128 1).
Peptide libraries or organic libraries may be made
by combinatorial chemistry and then screened for their
ability to bind GM1/Gb3. Synthetic compounds, natural
products, and other sources of potentially biologically
active materials can be screened in a number of ways
deemed to be routine to those of skill in the art.
GM1 or Gb3 or fragments thereof can be used for
screening peptides or molecules in any of a variety of
screening techniques. The molecule may be free in
solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The abolition of
activity or the formation of binding complexes between
GM1 or Gb3 and the agent being tested may be measured.
Another way of determining binding to GM1/Gb3
would be by using purified GM1/Gb3 to coat microtiter
plates. Following blocking, the agent under
investigation is applied to the plate and allowed to
interact prior to washing and detection with specific
antibodies to said agent. Conjugation of the
antibodies either directly or indirectly to an enzyme
or radiolabel allows subsequent quantification of
binding either using colorimetric or radioactivity
based methods (ELISA or RIA respectively).
Another way of determining binding to GM1/Gb3
would be by binding the saccharide moiety of GM1/Gb3 to
a suitable column matrix in order to allow standard
affinity chromatography to be performed. Removal of
known compounds applied to the column from the diluent
would be used as evidence for binding activity, or
alternatively, where mixtures of compounds are applied
to the column, elution and subsequent analysis would
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determine the properties of the ganglioside binding
agent. In the case of proteins, analysis would involve
peptide sequencing and tryptic digest mapping followed
by comparisons with available databases. In the event
that eluted proteins cannot be identified in this way
then standard biochemical analysis, for example mass
determination by laser desorption mass spectrometry
would be used to further characterise the compound.
Non-proteins eluted from GM1-affinity columns would be
analysed by HPLC and mass spectrometry of single
homogenous peaks.
Another way of determining the ability to bind to
GM1/Gb3 and the precise affinity of the interaction
would be by using plasmon surface resonance as
previously reported [Kuziemko et al (1996) Biochem
35:6375-6384].
Alternatively, phage display can be employed in
the identification of candidate agents which bind GM1
or Gb3.
Phage display is a protocol of molecular screening
which utilises recombinant bacteriophage. The
technology involves transforming bacteriophage with a
gene that encodes an appropriate ligand (in this case a
candidate agent) capable of reacting with GM1/Gb3 (or a
derivative or homologue thereof) or the nucleotide
sequence (or a derivative or homologue thereof)
encoding same. The transformed bacteriophage (which
preferably is tethered to a solid support) expresses
the appropriate ligand (such as the candidate agent)
and displays it on their phage coat. The entity or
entities (such as cells) bearing the target molecules
which recognises the candidate agent are isolated and
amplified. The successful candidate agents are then
characterised. Phage display has advantages over
standard affinity ligand screening technologies. The
phage surface displays the candidate agent in a three
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dimensional configuration, more closely resembling its
naturally occuring conformation. This allows for more
specific and higher affinity binding for screening
purposes.
Another technique for screening provides for high
throughput screening of agents having suitable binding
affinity to GM1 or Gb3 and is based upon the method
described in detail in WO 84/03564. In summary, large
numbers of different small peptide test compounds are
synthesized on a solid substrate, such as plastic pins
or some other surface. The peptide test agents are
reacted with the target interaction component fragments
and washed. A bound target interaction component is
then detected - such as by appropriately adapting
methods well known in the art. A purified target
interaction component can also be coated directly onto
plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies
can be used to capture the peptide and immobilize it on
a solid support.
In all aspects of the invention, the agent having
GM1-binding activity or Gb3 binding activity may also
be capable of cross-linking GM1 or Gb3 receptors. EtxB
is one such agent which is capable of cross-linking GM1
receptors by virtue of its pentameric form.
There are various methods for identifying agents
which have an effect on intracellular signalling events
mediated by GM1/Gb3 binding but which do not themselves
bind GM1 or Gb3. For example, if an agent is shown to
upregulate CD25 or MHC class II on B cells, or to
upregulate CD25 or promote apoptosis of CD8+ T cells,
or to upregulate IL-10 secretion by monocytes, but the
agent is shown not to bind GM1 or Gb3 (by, for example,
one of the binding assays described above), then it can
be concluded that the agent is capable of mimicing the
effect of GM1/Gb3 binding.
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The invention will now be illustrated by reference
to the accompanying drawings and the following
examples.
The examples refer to the figures in which:
Figure 1: shows the stimulation of total Ig and
IgA in the serum (MS) and IgA in the eye washings (EW)
in mice immunised with HSV-1 glycoproteins/rEtxB.
Figure 2: shows T cell proliferation of
(mesenteric lymph node) MLN or (cervical lymph node)
CLN lymphocytes in mice immunised with HSV-1/rEtxB.
Figure 3: shows T cell proliferation of cells from
MLN and CLN of mice immunised intranasally with HSV-1
Gp in the presence of 1-20~.g EtxB.
Figure 4: shows the level of anti-HSV-1 serum Ig
in mice following administration of HSV-1 glycoproteins
three times at 10 day intervals with variable amounts
of rEtxB or rCtxB as adjuvant.
Figure 5: shows the reduction in virus shedding,
clinical disease and latency in mice immunised with
HSV-1/rEtxB.
Figure 6: shows the Ig isotype distribution in MS
following infection with HSV-1 or immunisation with
HSV-1 Gp in the presence of EtxB or CtxB as
immunomodulator.
Figure 7: shows the distribution of Ig subclasses
following intranasal administration of HSV-1 Gp with
either rEtxB or rCtxB as immunomodulator.
Figure 8: shows the immunogenic effect of
different amounts of rEtxB or rCtxB on the level of
HSV-1 specific IgA in eye washings following
administration with HSV-1 glycoproteins.
Figure 9: shows serum immunoglobulin response
following immunisation of mice with HSV-1 or mock
glycoproteins (gp) alone or in the presence of
adjuvant.
Figure 10: shows mucosal IgA in eye washings
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following intranasal immunisation of mice with HSV-1 or
mock glycoproteins alone or in the presence of
adjuvant.
Figure 11: shows mucosal IgA in vaginal washings
following intranasal immunisation of mice with HSV-1 or
mock glycoproteins (gp) alone or in the presence of
adjuvant.
Figure 12: shows the level of HSV-1-specific
immunoglobulin in sera from mice immunised with HSV-1
glycoproteins in the presence of different doses of
rEtxB as adjuvant.
Figure 13: shows the level of IgA in eye washings
of mice immunised with HSV-1 glycoproteins in the
presence of varying concentrations of rEtxB.
Figure 14: shows the level of IgA in vaginal
washings of mice immunised with HSV-1 glycoproteins in
the presence of varying concentrations of rEtxB
Figure 15: shows IgG subclass distribution of the
serum antibody response to HSV-1 following intranasal
immunisation with Ctx/CtxB or rEtxB or ocular infection
with HSV-1.
Figure 16: shows cytokine production from cultures
of lymph node cells taken from mice which were either
infected with HSV-1 by ocular scarification, or were
immunised by intranasal administration of HSV-1
glycoproteins with Ctx/CtxB or rEtxB as adjuvant.
Figure 17: shows the level of protection against
ocular HSV-1 infection in mice immunised intranasally
with a mixture of HSV-1 or mock glycoproteins in the
presence of rEtxB as immunomodulator.
Example 1: rEtxB can be used in conjunction with
HSV-1 Gp for immunisation.
Mice were immunised intranasally three times with
IO~.g HSV-1 plycoproteins (Gp) with either 10 or 20~g
rEtxB. Controls were either unmanipulated or given a
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mock preparation of viral glycoprotein (mock) derived
from HIV-uninfected tissue culture cells. Antibody
levels are expressed a-s a percentage of post-infection
levels. The production of total Ig and IgA in the
serum and IgA in eye washings was stimulated by HSV-1
glycoproteins/rEtxB (Figure 1). The present inventors
have also shown that doses of rEtxB as low as O.lug are
also effective at stimulating such responses.
Also, T-lymphocytes from immunised mice from the
cervical lymph node (which is local to the vaccination
site) and from the mesenteric lymph node (which is
distant to the vaccination site) were shown to
proliferate when cultured in vitro with HSV-1, but not
when cultured in vitro with mock HSV-1 Gp or without
antigen (Figure 2).
The proliferation in response to HSV-1 Gp of T
lymphocytes from MLN and CLN of mice immunised with
HSV-1 Gp and varying amounts of EtxB is shown in Fig 3.
The production of Anti-HSV-1 serum Ig in mice
following administration of HSV-1 glycoproteins at
three day intervals with varying amounts of EtxB (or
CtxB) is shown in Figure 4.
Finally, mice immunised with HSV-1 and rEtxB were
shown to have a decrease in virus shedding following
corneal scarification with HSV-1 (Figure 5a), and a
decrease in local spreading (oedema and lid disease),
spreading to the trigeminal ganglion (zosteriform
infection), spreading to the central nervous system
(encephalitis) and latency compared to controls (5b).
Example 2: rCtxB and rEtxB act as
immunomodulators.
When EtxB is used as an immunomodulator, the Ig isotype
distribution is skewed (Figure 6). The distribution of
Ig subclasses is different depending on whether rCtxB
or rEtxB is used as an immunomodulator (Figure 7).
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Example 3: rEtxB is a more efficient
immunomodulator than rCtxB.
The levels of HSV-specific IgA (Figure 8) and is
greater following stimulation with rEtxB/HSV-1 Gp that
rCtxB/HSV-1 Gp.
Example 4: (Figure 9)
Mice were immunised three times intranasally with
HSV-1 glycoproteins alone, a mock preparation of HSV-1
glycoproteins (prepared by taking uninfected tissue
culture cells and subjecting them to identical
treatment regimes as those employed for the isolation
and purification of HSV-1 proteins), or HSV-1
glycoproteins in combination with a variety of putative
mucosal adjuvants. In each case the dose of HSV-1
glycoproteins was 10~,g per immunisation, and these were
combined with 10~.g of recombinant EtxB, or CtxB as
adjuvant, or a mixture of 0.5ug of Ctx and 10~.g CtxB.
Three weeks after the final immunisation, blood samples
were collected and total anti-HSV-1 antibodies were
measured by ELISA. The quantities of antibodies are
expressed as a percentage of the levels stimulated
following ocular infection induced by scarification
with 105 pfu HSV-1 strain SC16. The data (shown in
Figure 9) shows that the strongest serum antibody
response is stimulated when antigen is combined with a
mixture of whole Ctx and CtxB. However, a high level
response is also stimulated when rEtxB is used as an
adjuvant. In contrast, rCtxB is a very weak adjuvant.
Example 5: (Figure 10)
Mice were immunised as described in example 4.
Secretory IgA production in the eye was assessed by
taking washings of the tears over consecutive days and
these samples were then pooled and subjected to ELISA
analysis using a specific anti-IgA detecting antibody.
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The quantities of antibodies are expressed as a
percentage of the levels stimulated following ocular
infection induced by scarification with 105 pfu HSV-1
strain SC16. The data clearly demonstrates (Figure 10)
that high levels of secreted anti-HSV-1 antibodies are
produced following immunisation in the presence of
either Ctx/CtxB or EtxB. In contrast to the results
from analysis of serum antibody responses, there was no
difference in the level of antibodies in the eye
between those animals immunised with Ctx/CtxB or EtxB
as adjuvants. As with serum antibody, there was clear
evidence that rCtxB is a very poor adjuvant.
Example 6: (Figure 11)
Mice were immunised as described in example 4.
Secretory IgA production in the vagina was assessed by
taking washings from the genital tract over consecutive
days and these samples were then pooled and subjected
to ELISA analysis using a specific anti-IgA detecting
antibody. The quantities of antibodies are expressed as
endpoint titres which were calculated by linear
regression analysis. The data clearly demonstrates
that high levels of secreted anti-HSV-1 antibodies are
produced in distant mucosal'sites following
~ immunisation in the presence of either Ctx/CtxB or
EtxB. In the vagina, the highest levels of antibodies
were released following immunisation in the presence of
rEtxB. Lower levels were released following
immunisation with Ctx/CtxB and very little secretion
was triggered by the use of rCtxB as adjuvant.
Example 7: (Figure 12)
Mice were immunised three times intranasally with
HSV-1 glycoproteins (10~.g) either alone or in the
presence of escalating doses of rEtxB as adjuvant.
Three weeks after the final immunisation blood was
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taken, and the levels of anti-HSV-1 antibodies were
assessed by ELISA. The quantities of antibodies are
expressed as a percentage of the levels stimulated
following ocular infection induced by scarification
with 105 pfu HSV-1 strain-SC16. The data clearly
demonstrates that the capacity of rEtxB to trigger
antibody responses to heterologous added antigens is a
dose dependent phenomenon with maximal responsiveness
occurring at approximately 20-50~,g of rEtxB. Further,
it is clear that at doses of 20~.g rEtxB and above, the
level of anti-HSV-1 antibodies stimulated by intranasal
infection is comparable or greater than that stimulated
by a live virulent virus infection.
Example 8 : ( f figure 13 )
Mice were immunised as described in example 7.
Secretory IgA production in the eye was assessed by
taking washings of the tears over consecutive days and
these samples were then pooled and subjected to ELISA
analysis using a specific anti-IgA detecting antibody.
The quantities of antibodies are expressed as a
percentage of the levels stimulated following ocular
infection induced by scarification with 105 pfu HSV-1
strain SC16. The data demonstrates that maximal IgA
responses in the eye are stimulated when HSV-1
glycoproteins are given in combination with 20~,g of
rEtxB or above. At this dose the levels of IgA
production are nevertheless lower than those triggered
during virus infection of the eye.
Example 9: (Figure 14)
Mice were immunised as described in example 7.
Secretory IgA production in the vagina was assessed by
taking washings from the genital tract over consecutive
days and these samples were then pooled and subjected
to ELISA analysis using a specific anti-IgA detecting
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antibody. The quantities of antibodies are expressed as
endpoint titres which were calculated by linear
regression analysis. 'The data shows that optimal anti-
HSV-1 responses are stimulated in the vagina when 20~g
or above of rEtxB is used as an adjuvant.
Example 10: (Figure 15?
Mice were either infected with 105 pfu HSV-1
strain SC16 by scarification into the cornea or
immunised three times intranasally with 10~,g HSV-1
glycoproteins in combination with Ctx/CtxB or rEtxB.
Three weeks after the final inoculation, serum was
taken and was analysed by ELISA for the presence of
IgGl and IgG2a against HSV-1. The quantities of
antibodies are expressed as endpoint titres which were
calculated by linear regression analysis (fig. 7a).
The data clearly shows that the nature of the antibody
response to HSV-1 is influenced by the way in which the
antigens are presented to the immune system. Infection
with HSV-1 predominantly activates Thl associated
antibody production, as characterised by the high
levels of the complement fixing antibody isotype,
IgG2a. Infection stimulates relatively low levels of
the Th2 associated IgG isotype, IgGl. This profile of
the immune response is clearly visible when the data is
expressed as a ratio of IgGI:IgG2a as shown in fig. 7b.
The ratio is substantially less than 1 following
infection. Intranasal immunisation in the presence of
Ctx/CtxB as adjuvant triggers the release,
predominantly, of Th2 associated IgGl. Significant
levels of IgG2a are also produced suggesting that
Ctx/CtxB causes activation of Thl and Th2 cells. The
activation of both responses and the relative dominance
of Th2 is reflected in the IgGI:IgG2a ratio which is
approximately 3. Interestingly the nature of the
response to HSV-1 stimulated by rEtxB as adjuvant is
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almost exclusively Th2 dominated. High levels of IgGl
are produced with only very low amounts of IgG2a. This
strong bias toward Th2- responsiveness is reflected in
an IgGl:IgG2a ratio of approximately 9.
Example 11: (Figure 16)
Mice were either infected with 105 pfu HSV-1
strain SC16 by scarification into the cornea or
immunised three times intranasally with 10~,g HSV-1
glycoproteins in combination with Ctx/CtxB or rEtxB.
Three weeks after the final inoculation lymph nodes
were removed from animals and were used to generate
single cell suspensions that were cultured either in
the presence of killed HSV-1 or a mock preparation of
virus from non-infected tissue culture cells. On days
4 to 7 of the cultures, samples of cells were removed
and subjected to cELISA analysis to reveal the
secretion of cytokines. The data clearly shows that T-
cells in the cultures were capable of responding to
HSV-1, but not significantly to mock virus
preparations. Lymph node cells taken from mice which
had been infected with HSV-1 produced predominantly the
Thl associated cytokine Y-interferon (Y-IFN). Lymph
node cells taken from animals that were immunised
intranasally produced high levels of the Th2 associated
cytokines,.IL-4 and IL-10. In addition, both Ctx/CtxB
and rEtxB had led to the activation of T-cells which
secreted yIFN upon in vitro stimulation with HSV-1.
This indicates that although the response to these
adjuvants is dominated by the production of Th2
cytokines some Thl activation also occurs. These
findings are consistent with those from the analysis of
antibody responses.
