U.S. patent application number 13/038021 was filed with the patent office on 2011-09-15 for vaccine.
Invention is credited to Lucy Amber Bird, Timothy Raymond HIRST, Andrew Morgan, Neil Andrew Williams, Andrew Douglas Wilson.
Application Number | 20110223194 13/038021 |
Document ID | / |
Family ID | 43316022 |
Filed Date | 2011-09-15 |
United States Patent
Application |
20110223194 |
Kind Code |
A1 |
HIRST; Timothy Raymond ; et
al. |
September 15, 2011 |
VACCINE
Abstract
A method for stimulating the immune response to a vaccine
applied to a mammalian subject includes the step of administering
to the subject an effective amount of EtxB or a molecule having
substantially equivalent activity, free from whole toxin and not
linked to an antigen.
Inventors: |
HIRST; Timothy Raymond;
(Taunton, GB) ; Williams; Neil Andrew; (Cross,
GB) ; Morgan; Andrew; (Henleaze, GB) ; Wilson;
Andrew Douglas; (Winscombe, GB) ; Bird; Lucy
Amber; (Chichester, GB) |
Family ID: |
43316022 |
Appl. No.: |
13/038021 |
Filed: |
March 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09674935 |
Dec 21, 2000 |
7914791 |
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PCT/GB99/01461 |
May 10, 1999 |
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13038021 |
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Current U.S.
Class: |
424/201.1 |
Current CPC
Class: |
A61K 2039/55544
20130101; A61P 31/00 20180101; A61K 2039/543 20130101; A61K 39/39
20130101; A61P 31/16 20180101; A61P 37/04 20180101; C12N 2710/16634
20130101; Y02A 50/30 20180101; A61P 31/22 20180101; Y02A 50/484
20180101; A61P 37/02 20180101; Y02A 50/412 20180101; A61P 31/12
20180101; A61P 31/20 20180101; Y02A 50/41 20180101; A61P 31/14
20180101 |
Class at
Publication: |
424/201.1 |
International
Class: |
A61K 39/295 20060101
A61K039/295; A61P 37/04 20060101 A61P037/04; A61P 31/22 20060101
A61P031/22 |
Foreign Application Data
Date |
Code |
Application Number |
May 8, 1998 |
GB |
9809958.3 |
Jun 3, 1998 |
GB |
9811954.8 |
Jun 8, 1998 |
GB |
9812316.9 |
Claims
1. A vaccine composition comprising a therapeutically effective
amount of Escherichia coli heat labile enterotoxin B subunit
(EtxB), and an antigen, wherein the EtxB is free from whole toxin
and is not linked to the antigen, wherein the antigen is a virus
antigen from the herpes virus family.
2. The composition according to claim 1, wherein the virus antigen
is an antigen of a virus selected from the group consisting of
Herpes Simplex Virus-1 (HSV-1), Herpes Simplex Virus-2 (HSV-2),
Epstein-Barr Virus (EBV), Varicella-zoster Virus (VZV),
Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human Herpes
Virus-7 (HHV-7) and Human Herpes Virus-8 (HHV-8).
3. The composition according to claim 1, wherein the virus antigen
is an antigen of a virus selected from the group consisting of
HSV-1, HSV-2, CMV or EBV.
4. A vaccine composition comprising 50 and 100 .mu.g of Escherichia
coli heat labile enterotoxin B subunit (EtxB), wherein the EtxB is
free from whole toxin, and an antigen, wherein the EtxB and antigen
are not linked to form a single active agent, wherein the antigen
is a virus antigen from the herpes virus family.
5. The composition according to claim 4, wherein the virus antigen
is an antigen of a virus selected from the group consisting of
Herpes Simplex Virus-1 (HSV-1), Herpes Simplex Virus-2 (HSV-2),
Epstein-Barr Virus (EBV), Varicella-zoster Virus (VZV),
Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human Herpes
Virus-7 (HHV-7) and Human Herpes Virus-8 (HHV-8).
6. The composition according to claim 4, wherein the virus antigen
is an antigen of a virus selected from the group consisting of
HSV-1, HSV-2, CMV or EBV.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/674,935 filed Dec. 21, 2000, which is a national stage entry
of International Application No. PCT/GB1999/001461 filed May 10,
1999, which claims priority to British Application Nos. GB9809958.3
filed May 8, 1999, GB9811954.8 filed Jun. 3, 1998, and GB9812316.9
filed Jun. 8, 1998, which are incorporated herein by reference in
their entireties.
[0002] This invention relates to an immunomodulator 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.
[0003] 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.
[0004] 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 GM1.
[0005] 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 Russell (1993) Infection and Immunity 61:
314-322, U.S. Pat. No. 5,182,109). 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 12:
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 al (1992) Eur. J.
Immunol. 22: 2277-2281, Douce et al (1997) Infection and Immunity
65: 2821-2828).
[0006] 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).
[0007] 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 al (1997) Proc. Natl. Acad. Sci.
94: 4610-4614, Williams et al (1997) Proc. Natl. Acad. Sci. 94:
5290-5295), suggesting that these molecules would be unsuitable for
use as adjuvants.
THE BASIS OF THE PRESENT INVENTION
[0008] 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 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:
[0009] 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);
[0010] ii) the distribution of antibodies produced was skewed
towards non-complement fixing antibodies, especially S-IgA and
IgG1;
[0011] iii) agents such as EtxB and CtxB also stimulated local and
systemic T-cell proliferative responses;
[0012] iv) agents such as CtxB and EtxB tend to shift the immune
response from a Th1-associated response to a Th2-associated
response;
[0013] 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;
[0014] vi) rEtxB is a more efficient immunomodulator than
rCtxB;
[0015] 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;
[0016] 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
[0017] ix) VtxB exerts similar immunomodulatory effects on
leukocyte populations in vitro to those exerted by EtxB and
CtxB.
[0018] 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
[0019] 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.
[0020] According to a first aspect of the present invention, there
is provided the use of:
[0021] (i) EtxB, CtxB or VtxB free from whole toxin;
[0022] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0023] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding;
[0024] as an immunomodulator for a vaccine against infectious
diseases.
[0025] 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:
[0026] (i) EtxB, CtxB or VtxB free from whole toxin;
[0027] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0028] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding;
[0029] wherein said antigenic determinant is an antigenic
determinant of said infectious agent.
[0030] 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.
[0031] 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:
[0032] a) one of the following agents:
[0033] (i) EtxB, CtxB or VtxB free from whole toxin;
[0034] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0035] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding; and
[0036] b) an antigenic determinant which is an antigenic
determinant of the infectious disease, for coadministration with
the said vaccine immunomodulator.
[0037] The vaccine composition of the second aspect of the
invention and the kit of the third aspect of the 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.
[0038] 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.
[0039] 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:
[0040] (i) EtxB, CtxB or VtxB free from whole toxin;
[0041] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0042] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding.
[0043] 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 in liquid
form.
[0044] The antigenic determinant and immunomodulator may be
administered to the subject as a single dose or in multiple
doses.
[0045] 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.
[0046] 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.
[0047] 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).
[0048] 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).
[0049] 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.
[0050] In this second embodiment, the antigenic determinant is
preferably an antigenic determinant of a viral coat protein (for
example haemagglutinin and neuraminidase) or of an internal protein
(for example, nucleoprotein).
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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,
Haemophilus influenzae type B and Streptococcus pneumoniae.
[0055] In a seventh 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
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.
[0056] In an eighth 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 trachomatis.
[0057] 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 enteritidis, Salmonella typhi, Helicobacter
pylori, Bacillus cereus, Campylobacter jejuni and Vibrio
cholerae.
[0058] 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
bacterial toxin or adhesion factor.
[0059] 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.
[0060] 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 Immune Responses
[0061] EtxB, CtxB, VtxB and other agents capable of binding to or
mimicking the effects of binding to GM1 or Gb3, are capable of
specifically upregulating mucosal antibody production.
[0062] 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, 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.
[0063] 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:
[0064] (i) EtxB, CtxB or VtxB free from whole toxin;
[0065] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0066] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or G3b binding.
[0067] Preferably the immunomodulator is EtxB.
[0068] 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:
[0069] a) a vaccine immunomodulator which is:
[0070] (i) EtxB, CtxB or VtxB free from whole toxin;
[0071] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0072] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or G3b binding; and
[0073] b) at least one HSV-1 antigenic determinant, for
coadministration with the said vaccine immunomodulator.
[0074] According to a fifth aspect of the invention there is
provided the use of:
[0075] (i) EtxB, CtxB or VtxB free from whole toxin;
[0076] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0077] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding
[0078] 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.
[0079] In this fifth aspect of the present invention, the agent may
be used in conjunction with one or more antigenic
determinant(s).
Downregulating the Pathological Components of Immune Responses
[0080] 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 Th1- 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.
[0081] According to a sixth aspect of the present invention, there
is provided the use of:
[0082] (i) EtxB, CtxB or VtxB free from whole toxin;
[0083] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0084] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding;
[0085] to downregulate the pathological components of
Th2-associated immune responses. The pathological components of
Th1-associated immune responses may also be down-regulated.
[0086] 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 Th1
associated reactions are down-regulated while Th2 associated
responses are upregulated. Th1 responses include the secretion of
.gamma.IFN 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.
[0087] 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.
[0088] 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.
[0089] 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.
Immunomodulation of the Antigen Processing Pathway
a) Prolonging Presentation
[0090] 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.
[0091] According to a seventh aspect of the present invention,
there is provided the use of:
[0092] (i) EtxB, CtxB or VtxB free from whole toxin;
[0093] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0094] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding;
[0095] as an immunomodulator in a vaccine, to prolong antigen
presentation and give sustained immunological memory in a mammalian
subject.
[0096] 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 from:
[0097] (i) EtxB, CtxB or VtxB free from whole toxin;
[0098] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0099] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding;
[0100] 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.
b) Intracellular Targeting of the Antigen to a MHC-I or MHC-II
Associated Pathway
[0101] 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 linkage of
the antigen to the immunomodulator.
[0102] 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.
[0103] The linkage of the antigen-immunomodulator conjugate can
also be chosen so that the antigen is delivered into the
nucleus.
[0104] 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:
[0105] (i) EtxB, CtxB or VtxB free from whole toxin;
[0106] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0107] (iii) an agent which has an effect on vesicular
internalisation mediated by GM1-binding or Gb3 binding.
[0108] 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:
[0109] (i) EtxB, CtxB or VtxB free from whole toxin;
[0110] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0111] (iii) an agent which has an effect on vesicular
internalisation mediated by GM1-binding or G3b binding;
[0112] wherein said antigen or antigenic determinant is an antigen
or antigenic determinant of said infectious agent.
[0113] 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-.gamma.(-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.
[0114] According to an eleventh aspect of the present invention
there is provided the use of:
[0115] (i) EtxB, CtxB or VtxB free from whole toxin;
[0116] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0117] (iii) an agent which has an effect on vesicular
internalisation mediated by GM1-binding or Gb3 binding;
[0118] in a conjugate with antigen or antigenic determinant to
target the delivery or said antigen or antigenic determinant to the
cytosol or nucleus of an antigen presenting cell.
[0119] According to a twelfth aspect of the present invention there
is provided the use of:
[0120] (i) EtxB, CtxB or VtxB free from whole toxin;
[0121] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0122] (iii) an agent which has an effect on vesicular
internalisation mediated by GM1-binding or Gb3 binding;
[0123] 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 1
molecules.
[0124] 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
[0125] 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.
EBV
[0126] 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 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.
[0127] Latently EBV-infected cells express a small number of
so-called "latent" proteins. These include six nuclear proteins
(EBNAs 1, 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.
[0128] 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.
[0129] 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 responses are
not detectable to the EBV latent protein EBNA1 and could explain
the apparent absence of such responses against LMP1.
[0130] According to an thirteenth aspect of the invention there is
provided a vaccine composition which comprises:
[0131] a) one of the following agents:
[0132] (i) EtxB, CtxB or VtxB free from whole toxin;
[0133] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0134] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding; and
[0135] b) an EBV antigen
[0136] for use in the treatment and/or prevention of EBV-associated
diseases.
[0137] 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.
[0138] According to a fourteenth aspect of the invention there is
provided a therapeutic composition which comprises:
[0139] (i) EtxB, CtxB or VtxB free from whole toxin;
[0140] (ii) an agent other than EtxB or CtxB, having GM1-binding
activity, or an agent other than VtxB having Gb3-binding activity;
or
[0141] (iii) an agent having an effect on intracellular signalling
events mediated by GM1-binding or Gb3 binding;
[0142] for use in the treatment of EBV-associated diseases.
[0143] 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 GM1-binding activity.
[0144] Based on the knowledge that EtxB cocaps with LMP1, and that
EtxB promotes fragmentation 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] The preferred agent for use in the thirteenth and fourteenth
aspects of the invention is EtxB.
[0149] 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-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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] GM-1 and Gb3-Associated Signalling
[0155] 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 GM1 associated 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 al (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
[0156] 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.
[0157] 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 second step by
administration of antigen alone.
[0158] 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.
[0159] There are a number of known methods by which it is possible
to identify antigenic determinants for a given infectious
agent.
[0160] 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,
[0161] 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;
[0162] 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 immuno blotting to detect those
antigen(s) recognised by the immune sera;
[0163] iii) serum samples from infected or convalescent patients or
infected or convalescent animals may be screened against whole cell
lysates derived from recombinant expression systems encoding one or
more antigens of interest, and using the standard techniques of
ELISA or Western blotting to detect those antigen(s) recognised by
the immune serum;
[0164] 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
[0165] 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.
[0166] 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.
[0167] There are a number of known methods by which it is possible
to isolate antigens for a given infectious agent.
[0168] 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,
ultrafiltration 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.
[0169] 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.
[0170] 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.
[0171] 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 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.
[0172] 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 mimic the effects of binding to GM1 or Gb3.
[0173] 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.
[0174] 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.
[0175] Humanised monoclonal antibodies may be preferred in the
present invention. Monoclonal antibodies may be prepared using any
technique which provides for 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 al
(1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma
technique (Cole et al (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 al (1984) Proc Natl Acad Sci
81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et
al (1985) Nature 314:452-454). Alternatively, techniques described
for the production of single chain antibodies (U.S. Pat. No.
4,946,779) can be adapted to produce target interaction component
specific single chain antibodies.
[0176] 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 al (1989, Proc Natl Acad Sci
86: 3833-3837), and Winter G and Milstein C (1991; Nature
349:293-299).
[0177] 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').sub.2 fragments which
can be produced by pepsin digestion of the antibody molecule and
the Fab fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity (Huse W D et al (1989) Science 256:1275-128 1).
[0178] 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.
[0179] 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.
[0180] 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).
[0181] 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 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.
[0182] 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].
[0183] Alternatively, phage display can be employed in the
identification of candidate agents which bind GM1 or Gb3.
[0184] 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
dimensional configuration, more closely resembling its naturally
occurring conformation. This allows for more specific and higher
affinity binding for screening purposes.
[0185] 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.
[0186] 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.
[0187] 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 mimicking the effect of GM1/Gb3 binding.
[0188] The invention will now be illustrated by reference to the
accompanying drawings and the following examples.
[0189] The examples refer to the figures in which:
[0190] FIG. 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.
[0191] FIG. 2: shows T cell proliferation of (mesenteric lymph
node) MLN or (cervical lymph node) CLN lymphocytes in mice
immunised with HSV-1/rEtxB.
[0192] FIG. 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 .mu.g EtxB.
[0193] FIG. 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.
[0194] FIG. 5A shows the reduction in the incidence of virus
shedding in mice immunized with HSV-1/rEtxB.
[0195] FIG. 5B shows the reduction in the incidence of clinical
disease and latency in mice immunized with HSV-1/rEtxB.
[0196] FIG. 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.
[0197] FIG. 7: shows the distribution of Ig subclasses following
intranasal administration of HSV-1 Gp with either rEtxB or rCtxB as
immunomodulator.
[0198] FIG. 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.
[0199] FIG. 9: shows serum immunoglobulin response following
immunisation of mice with HSV-1 or mock glycoproteins (gp) alone or
in the presence of adjuvant.
[0200] FIG. 10: shows mucosal IgA in eye washings following
intranasal immunisation of mice with HSV-1 or mock glycoproteins
alone or in the presence of adjuvant.
[0201] FIG. 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.
[0202] FIG. 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.
[0203] FIG. 13: shows the level of IgA in eye washings of mice
immunised with HSV-1 glycoproteins in the presence of varying
concentrations of rEtxB.
[0204] FIG. 14: shows the level of IgA in vaginal washings of mice
immunised with HSV-1 glycoproteins in the presence of varying
concentrations of rEtxB
[0205] FIG. 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.
[0206] FIG. 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.
[0207] FIG. 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
[0208] Mice were immunized intranasally three times with 10 .mu.g
HSV-1 glycoproteins (Gp) with either 10 or 20 .mu.g rEtxB. Controls
were either unmanipulated or given a mock preparation of viral
glycoprotein (mock) derived from HIV-uninfected tissue culture
cells. Antibody levels are expressed as 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 (FIG. 1). The present inventors have also shown
that doses of rEtxB as low as 0.1 .mu.g are also effective at
stimulating such responses.
[0209] 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
(FIG. 2).
[0210] The proliferation in response to HSV-1 Gp of T lymphocytes
from mesenteric lymph node (MLN) and cervical lymph node (CLN)
cells of mice immunized with HSV-1 Gp and varying amounts of EtxB
is shown in FIG. 3.
[0211] 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 FIG. 4.
[0212] Finally, mice immunised with HSV-1 and rEtxB were shown to
have a decrease in virus shedding following corneal scarification
with HSV-1 (FIG. 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
[0213] When EtxB is used as an immunomodulator, the Ig isotype
distribution is skewed (FIG. 6). The distribution of Ig subclasses
is different depending on whether rCtxB or rEtxB is used as an
immunomodulator (FIG. 7).
EXAMPLE 3
rEtxB is a More Efficient Immunomodulator than rCtxB
[0214] The levels of HSV-specific IgA (FIG. 8) and is greater
following stimulation with rEtxB/HSV-1 Gp that rCtxB/HSV-1 Gp.
EXAMPLE 4
FIG. 9
[0215] 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
.mu.g per immunisation, and these were combined with 10 .mu.g of
recombinant EtxB, or CtxB as adjuvant, or a mixture of 0.5 .mu.g of
Ctx and 10 .mu.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 10.sup.5 pfu HSV-1 strain SC16. The
data (shown in FIG. 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
FIG. 10
[0216] 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. The quantities of antibodies are expressed as a
percentage of the levels stimulated following ocular infection
induced by scarification with 10.sup.5 pfu HSV-1 strain SC16. The
data clearly demonstrates (FIG. 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
FIG. 11
[0217] 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
FIG. 12
[0218] Mice were immunised three times intranasally with HSV-1
glycoproteins (10 .mu.g) either alone or in the presence of
escalating doses of rEtxB as adjuvant. Three weeks after the final
immunisation blood was 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 10.sup.5 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 .mu.g of rEtxB. Further, it is clear that at
doses of 20 .mu.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
FIG. 13
[0219] 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 10.sup.5 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 .mu.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
FIG. 14
[0220] 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 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 .mu.g or above of rEtxB is used as
an adjuvant.
EXAMPLE 10
FIG. 15
[0221] Mice were either infected with 10.sup.5 pfu HSV-1 strain
SC16 by scarification into the cornea or immunised three times
intranasally with 10 .mu.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 IgG1 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 Th1 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, IgG1. This profile of the immune
response is clearly visible when the data is expressed as a ratio
of IgG1: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 IgG1. Significant levels of IgG2a are also produced
suggesting that Ctx/CtxB causes activation of Th1 and Th2 cells.
The activation of both responses and the relative dominance of Th2
is reflected in the IgG1:IgG2a ratio which is approximately 3.
Interestingly the nature of the response to HSV-1 stimulated by
rEtxB as adjuvant is almost exclusively Th2 dominated. High levels
of IgG1 are produced with only very low amounts of IgG2a. This
strong bias toward Th2 responsiveness is reflected in an IgG1:IgG2a
ratio of approximately 9.
EXAMPLE 11
FIG. 16
[0222] Mice were either infected with 10.sup.5 pfu HSV-1 strain
SC16 by scarification into the cornea or immunised three times
intranasally with 10 .mu.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 Th1
associated cytokine .gamma.-interferon (.gamma.-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 .gamma.IFN 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 Th1 activation
also occurs. These findings are consistent with those from the
analysis of antibody responses.
* * * * *