AU2006249261A1 - Vaccine - Google Patents

Description

08/12 2006 16:10 FAX 61 2 82311099 F.B. RICE CO 1a004/072 1

AUSTRALIA

Patents Act 1990 UNIVERSITY OF BRISTOL COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Vaccine The following statement is a full description of this invention including the best method of performing it known to us:- COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:10 FAX 61 2 82311099 F.B. RICE 8 CO R005/072 l a

VACCINE

This invention relates to an immunomodulator for Suse in a vaccine which is intended for use against a Srange of infectious agents. Further this invention 00 5 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 C( heat-labile enterotoxin (Etx) are potent immunogens and mucosal adjuvants. However, their inherent toxicity C makes them unsuitable for human use. For example, o although Ctx is the most commonly used mucosal adjuvant C 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 GM1ganglioside (GM1), a glycosphingolipid found ubiquitously on the surface of mammalian cells. Vtx binds to Gb3 which is a similar type of receptor to GM1.

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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:11 FAX 61 2 82311099 F.B. RICE CO 0006/072 \0 0 -2- 0 Ci Russell (1993) Infection and Immunity 61: 314-322, US- 5182109). Subsequent studies using recombinant CtxB 0 (rCtxB) have suggested that CtxB is a poor mucosal 00 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 \D that rCtxB lacks the ADP-ribosylating and the cAMPstimulating activities of the holotoxin and that, as Sadjuvant mechanism is linked to these abilities, CtxB V\ 10 would be unsuitable for use as an adjuvant (Vajdy and 0 Lycke (1992) Immunology 75; 488-492, Lycke et al (1992) C 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 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 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:11 FAX 61 2 82311099 F.B. RICE CO R007/072 o -3- CA investigated the use of rEtxB (thus containing no Sresidual holotoxin or A subunit) in an intranasal 0 vaccine for HSV in a murine model and surprisingly 00 'found that it is able to stimulate protective immune o 5 responses to viral challenge. Specifically, the present inventors found that: i) agents such as EtxB and CtxB stimulate high c levels of local (mucosal) antibody production (although immunization using rEtxB stimulated lower levels of C( 10 overall serum antibody production than Ctx/CtxB

VO

Scombined); Sii) 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 Th2associated 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.

COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:12 FAX 61 2 82311099 F.B. RICE CO R 008/072 Va o -4- CA These important discoveries are the basis of the Svarious aspects of the present invention and enabled 0 the inventors to predict that pure EtxB, CtxB and VtxB, 00 as well as other agents capable of binding to or O S 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

NO

C infection, as well as other infections, the prevention O or treatment of which would benefit from Cl 10 immunomodulation of the types listed above.

VO

o Stimulation of ijnne resnonses ci 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: 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: EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtXB or CtxB, having COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:12 FAX 61 2 82311099 F.B. RICE CO R009/072 o (CN GM1-binding activity, or an agent other than VtxB Shaving Gb3-binding activity; or S(iii) an agent having an effect on intracellular 00 signalling events mediated by GMl-binding or Gb3 o 5 binding; wherein said antigenic determinant is an antigenic determinant of said infectious agent.

i The antigen and immunomodulator may be linked, for example covalently or genetically linked, to form a 10 single effective agent. In a specific embodiment of IO this invention the antigen and immunomodulator may be Schemically 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: 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:12 FAX 61 2 82311099 F.B. RICE CO 010/072

NO

o -6invention may be used in a prophylactic or therapeutic 0 vaccination method, where a "prophylactic vaccine" is Sadministered to naive -individuals to prevent disease 0 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.

NO Agents such as EtxB have the capacity to alter the Snature of the immune response once infection has occurred. A therapeutic vaccine one which need .0 not contain antigen) comprising such an agent may find Sparticular 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: 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2008 16:13 FAX 61 2 82311099 F.B. RICE CO R011/072 -7o in liquid form.

C The antigenic determinant and immunomodulator may be O administered to the subject as a single dose or in multiple doses.

0 5 In accordance with each of the previously described 00 o aspects of the invention, it is preferred that the immunomodulator EtxB, CtxB and/or VtxB) and the antigenic determinant are separate moieties they are

N

O separate distinct components). Preferably, the C 10 immunomodulator and the antigenic determinant are not Cg fusion proteins, or proteins that have been cross-linked.

NO In a first embodiment the immunomodulator of the o first aspect of the invention, the vaccine of the second C 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:13 FAX 61 2 82311099 F.B. RICE CO 11012/072 -7a- 0 invention is used against a disease for which the 0- infectious agent is an influenza virus.

o In this second embodiment, the antigenic determinant Sis preferably an antigenic determinant of a viral coat protein (for example haemagglutinin and ci cO 0O COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:13 FAX 61 2 82311099 F.B. RICE CO R013/072 -8- 0 neuraminidase) or of an internal protein (for example, o nucleoprotein).

SIn a third embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the o 5 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

N

D 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 ND second aspect of the invention, the kit of the third Saspect of the invention and the method of the fourth C 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, Haemophilus influenzae type B and Streptococcus pneumoniae.

In a seventh embodiment, the immunomodulator of COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:14 FAX 61 2 82311099 F.B. RICE CO 0014/072 o the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third Saspect of the invention and the method of the fourth 0 0 aspect of the invention is used against pneumonia or a respiratory tract infection. In this seventh embodiment, the infectious agent may be selected from \O the group consisting of Streptococcus pneumoniae, e Legonella pneumophila and Mycobacteriurm tuberculosis.

SIn an eigth embodiment, the immunomodulator of the \0 10 first aspect of the invention, the vaccine of the o second aspect of the invention, the kit of the third C aspect of the invention and the method of the fourth aspect of the invention is used against a sexuallytransmitted 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.

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.

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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:14 FAX 61 2 82311099 F.B. RICE CO 015/072

O

0 O Sbacterial toxin or adhesion factor.

SIn a tenth embodiment, the immunomodulator of the first aspect of the 00 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 \O from the group consisting of Staphylococcus aureus, Streptococcus pyogenes and C Streptococcus mutans.

In an eleventh embodiment, the immunomodulator of the first aspect of the C invention, the vaccine of the second aspect of the invention, the kit of the third aspect 0 10 of the invention and the method of the fourth aspect of the invention is used against a C 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.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Stimulation of mncosal 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 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, COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:14 FAX 61 2 82311099 F.B. RICE CO i016/072 Va 0 -11- 0 (C CMV, influenza, measles, mumps, rotavirus etc), O diseases caused by bacteria coli, Salmonella, SShigella, Chlamydia, N. gonnorhoea, T. pallidium, 00 Streptococcus species including those which cause o 5 dental caries), and diseases caused by parasites.

In a preferred embodiment of the second aspect of the present invention there is provided a vaccine CN against HSV-i infection comprising at least one HSV-1 Ch antigenic determinant and an immunomodulator, where the pC 10 immunomodulator is: O EtxB, CtxB or VtxB free from whole toxin; o (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: 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 GMl-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: EtxB, CtxB or VtxB free from whole toxin; COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 S08/12 2006 16:15 FAX 61 2 82311099 F.B. RICE CO @017/072 -12- (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-complementfixing 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).

Downrequlating the pathological components of immune reasonses 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 Th2type 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 Thl and Th2 activation.

According to a sixth aspect of the present invention, there is provided the use of: EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, ,having COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:15 FAX 61 2 82311099 F.B. RICE CO l018/072 0 -13- ^C GM1-binding activity, or an agent other than VtxB having Gb3-binding activity; or 0 (iii) an agent having an effect on intracellular 00 signalling events mediated by GMl-binding or Gb3 O 5 binding; to downregulate the pathological components of Th2-associated immune responses. The pathological

VO

Ci components of Thl-associated immune responses may also be downregulated.

Ci 10 It is known that EtxB and CtxB bind to GM1 and o induce differential effects on lymphocyte populations, Sincluding 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 Thl 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.

COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:15 FAX 61 2 82311099 F.B. RICE CO l019/072

NO

o -14- (C The use of an agent in accordance with the sixth aspect of the invention is particularly useful for 0 therapeutic vaccination in diseases in which 00 immunopathological mechanisms are involved. Examples 0 5 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

VO

c- be used simultaneously as an immunomodulator and a ntherapeutic agent. For example in diseases where 10 immunopathological mechanisms are involved, the use of

VO

o a vaccine incorporating agents such as EtxB or CtxB may O 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.

l-mmun mdulation of the antigen processing pathway a pnrolonging presentation 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 Bsubunit 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: COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:16 FAX 61 2 82311099 F.B. RICE CO I020/072 Va o EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having SGM1-binding activity, .or an agent other than VtxB 00 having Gb3-binding activity; or (iii) an agent having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding; CN as an immunomodulator in a vaccine, to prolong Santigen presentation and give sustained immunological C 10 memory in a mammalian subject.

O

o According to an eighth aspect of the present 0 invention, there is provided a vaccine composition for use against an infectious disease, comprising an antigenic determinant and a immunomodulator selected from: 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 inmunomodulator prolongs presentation of the antigenic determinant and gives sustained immunological memory.

hl intracellular tar etina.f the antigen to a MC-I or MHC-II associated pathay 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:16 FAX 61 2 82311099 F.B. RICE CO I021/072 o -16- C- linkage of the antigen to the immunomodulator.

SBy linking the antigen or antigenic determinant to Sthe immunomodulator in a certain way, it is possible to 00 facilitate translocation of the antigen across the O 5 endosomal membrane into the cytosol. The present inventors predict that this would enhance loading of antigenic peptides on to MHC class I molecules. The

VO

pg use of an antigen-immunomodulator conjugate can Oh therefore be used to specifically enhance the C- 10 activation of cytotoxic T cells (CTL). Induction of \O CTL is beneficial for the prevention and treatment of o 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: 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.

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: 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:16 FAX 61 2 82311099 F.B. RICE CO a022/072 Va O -17- C- having Gb3-binding activity; or o (iii) an agent which has an effect on vesicular

C)

Sinternalisation mediated by GM1-binding or G3b binding; 00 wherein said antigen or antigenic determinant is an antigen or antigenic determinant of said infectious agent.

The antigen or antigenic determinant may be linked -C to the immunomodulator by a variety of methods Ch including genetic linkage or chemical conjugation. In e< 10 a first preferred embodiment the conjugate is a fusion SN protein made by genetic linkage of the antigen or Santigenic 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 Ny(-maleimido-butyroxyl)-succinimide ester (GMBS) or Nsuccinimidyl-(3-pyridyl-dithio)-propionate (SPDP).

The vaccine composition may be administered by a number of different routes such as intranasal, oral, intravaginal, urethral or ocular administration- Intranasal immunisation is preferred.

According to an eleventh aspect of the present invention there is provided the use of: 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:17 FAX 61 2 82311099 F.B. RICE CO a023/072

NO

0 -z L 0 determinant to target the delivery or said antigen or o antigenic determinant to the cytosol or nucleus of an Santigen presenting cell.

According to a twelfth aspect of the present invention there is provided the use of: EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having SGM1-binding activity, or an agent other than VtxB having Gb3-binding activity; or (iii) an agent which has an effect on vesicular NO internalisation mediated by GM1-binding or Gb3 binding; Sin a conjugate with antigen or antigenic C 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.

Etf is the preferred immnnomodulator 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.

EBy 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:17 FAX 61 2 82311099 F.B. RICE CO a024/072 o -19later in life is associated with infectious mononucleosis which is the second most frequent 0 disease in adolescence in the US. EBV also has 00 oncogenic potential. There is a strong link between EBV and endemic Burkitt's lymphoma (BL) and undifferentiated nasopharyngeal carcinoma (NPC). Also, \O a large proportion of lymphomas that occur in immuno- C( compromised patients are caused by EBV, and an Sassociation has been shown to exist between certain C 10 Hodgkin's lymphomas and EBV.

NO

o Latently EBV-infected cells express a small number C 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.

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 EBVpositive 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 antiapoptotic 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 TAPassociated 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:18 FAX 61 2 82311099 F.B. RICE CO f025/072 Va 0 C( responses are not detectable to the EBV latent protein o EBNA1 and could explain the apparent absence of such 0 responses against LMP1..

00 According to an thirteenth aspect of the invention O 5 there is provided a vaccine composition which comprises: a) one of the following agents: C EtxB, CtxB or VtxB free from whole toxin; n (ii) an agent other than EtxB or CtxB, having Ci 10 GM1-binding activity, or an agent other than VtxB

VO

O having Gb3-binding activity; or O (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 EBVassociated 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: 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; 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:18 FAX 61 2 82311099 F.B. RICE CO I026/072

NO

0 -21- GM1-binding activity.

SBased on the knowledge that EtxB cocaps with LMP1, 0 and that EtxB promotes. fragmentation of LMP-1, it is 00 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 Va C therapeutic treatments to treat such diseases once they Shave developed.

Cq 10 Without wishing to be bound by theory, it is

NO

Sbelieved that when EtxB cocaps with LMP-1 the antigen o 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 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- COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:18 FAX 61 2 82311099 F.B. RICE CO a027/072 V -22- S1, LMP-2A, LMP-2B, and EBNA-1 as well as antigenic O fragments thereof. The antigen may be isolated Sdirectly from EBV infected cells, or be made by synthetic or recombinant means.

0 5 The thirteenth and fourteenth aspects of present invention are particularly suited for the treatment and/or prevention of the following diseases: infectious IO mononucleosis, Burkitt's lymphoma, nasopharyngeal 0 carcinomas, and Hodgkin's lymphoras. It is believed that these aspects of the invention will be \D particularly suited to the treatment and/or prevention o of nasopharyngeal carcinomas and Hodgkin's lymphomas.

Ci 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 EV-associated disease.

The vaccine may be administered by any suitable route. The agent and the antigen may be coadministered 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 GM1 associated COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 18:19 FAX 61 2 82311099 F.B. RICE CO 9028/072 Va 0 -23- C intracellular signalling event. It may be that binding Sof EtxB (or CtxB) to GMI facilitates binding to a Sprotein, which protein triggers intracellular 00 signalling. It is not known what specifically triggers the signalling event, it may be phosphorylation of GMl or the protein. When EtxB/CtxB binds GM1 on the cell surface, bound GMI is internalised in vesicles

NO

Ci (Williams et al (1999) Immunology Today 20;95-101).

SGM1 and other glycolipids (such as Gb3) are known to be N 10 preferentially located in "membrane rafts" in which key Sprotein receptors are also found. It is therefore pC possible that internalisation of GM1 as a result of Bsubunit binding causes cocapping of such proteins leading to their being triggered to mediate intracellular signalling events.

Definitiona 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:19 FAX 61 2 82311099 F.B. RICE CO I029/072 -24- 0 second step by administration of antigen alone.

o The term "antigenic determinant" as used herein Srefers to a site on an antigen which is recognised by an antibody or T-cell receptor. Preferably it is a 00 short peptide derived from or as part of a protein antigen, however the term is also intended to include glycopeptides and carbohydrate antigenic determinants.

NO The term also includes modified sequences of amino acids or carbohydrates which stimulate responses which recognise the whole organism.

IO There are a number of known methods by which it is Spossible to identify antigenic determinants for a given C 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 antigen(s) 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:19 FAX 61 2 82311099 F.B. RICE CO 030/072

NO

o recombinant expression systems encoding one or more Santigens of interest, and using the standard 0 techniques of ELISA or Western blotting to detect 00 those antigen(s) 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

VO

Ci cloned genes from the infectious agent of interest, n using colony blot immunodectection to identify that C 10 clones expressing antigens, or fragments thereof, that

VO

O are recognised by the immune serum; or Sv) 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 antigenspecific 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.

COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:20 FAX 61 2 82311099 F.B. RICE CO I031/072 Va o -26- C There are a number of known methods by which it is Spossible to isolate antigens for a given infectious agent.

00 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 C( the recovery of the antigens. This may include the use Sof cell disruption techniques to lyse cells such as 10 sonication and/or detergent extraction. Centrifugation,

NO

o ultrafiltation or precipitation may be used on o 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:20 FAX 61 2 82311099 F.B. RICE CO 0032/072

O

o -27- (C method in which the gene or genes coding for the O specific polypeptide chain (or chains) from which the 0 protein is formed, is inserted into a suitable vector 00 and then used to transfect a suitable host. For O 5 example, the gene coding for the polypeptide chain from which the EtxB assemble may be inserted into, for Sexample, plasmid pMM68, which is then used to transfect

NO

host cells, such as Vibrio sp.60. The protein is N purified and isolated in a manner known per se. Mutant ci 0 10 genes expressing active mutant CtxB, EtxB or VtxB 0 protein may be produced by known methods from the wild eC 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:20 FAX 61 2 82311099 F.B. RICE CO R033/072 0 .I NO S-28prepared using any technique which provides for the Sproduction of antibody molecules by continuous cell Slines in culture. These include, but are not limited 00 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) ND 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 o 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 Nati 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 (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 al (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 GMI 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:21 FAX 61 2 82311099 F.B. RICE CO a034/072 0 -29-

C

disulfide bridges of the F(ab') 2 fragments.

Alternatively, Fab expression libraries may be 0 constructed to allow rapid and easy identification of 00 monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1).

Peptide libraries or organic libraries may be made O by combinatorial chemistry and then screened for their CN ability to bind GMl/Gb3. Synthetic compounds, natural products, and other sources of potentially biologically C 10 active materials can be screened in a number of ways o deemed to be routine to those of skill in the art.

0 QGMI 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 GMl/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 GMl/Gb3 would be by binding the saccharide moiety of GMl/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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:21 FAX 61 2 82311099 F.B. RICE CO 035/072 N0 0- determine the properties of the ganglioside binding O agent. In the case of proteins, analysis would involve peptide sequencing and tryptic digest mapping followed O0 by comparisons with available databases. In the event o 5 that eluted proteins cannot be identified in this way then standard biochemical analysis, for example mass determination by laser desorption mass spectrometry NO would be used to further characterise the compound.

Non-proteins eluted from GM1-affinity columns would be 10 analysed by HPLC and mass spectrometry of single NO homogenous peaks.

SAnother way of determining the ability to bind to C GM1/Gb3 and the precise affinity of the interaction would be by using plasmon surface resonance as previously reported [Kuziemko at al (1996) Biochem 35:6375-G384].

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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:22 FAX 61 2 82311099 F.B. RICE CO 0036/072 Va o -31- Ci dimensional configuration, more closely resembling its 0 naturally occuring conformation. This allows for more

C)

0 specific and higher affinity binding for screening 00 purposes.

o 5 Another technique for screening provides for high throughput screening of agents having suitable binding affinity to GMI or Gb3 and is based upon the method CN described in detail in WO 84/03564. In summary, large C numbers of different small peptide test compounds are C 10 synthesized on a solid substrate, such as plastic pins S 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 GM-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 GMI/Gb3 binding but which do not themselves bind GMl 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 CDS+ 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.

COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:22 FAX 61 2 82311099 F.B. RICE CO 037/072 Va o -32ci The invention will now be illustrated by reference o to the accompanying drawings and the following Sexamples.

00 The examples refer to the figures in which: O 5 Figure 1: shows the stimulation of total Ig and IgA in the serum (MS) and IgA in the eye washings (EW) w- in mice immunised with HSV-1 glycoproteins/rEtxB.

Va Figure 2: shows T cell proliferation of h (mesenteric lymph node) MLN or (cervical lymph node) C 10 CLN lymphocytes in mice immunised with HSV-1/rEtxB.

NO Figure 3: shows T cell proliferation of cells from SMLN and CLN of mice immunised intranasally with HSV-1 Gp in the presence of 1-20kg 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-l/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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:22 FAX 61 2 82311099 F.B. RICE CO I 038/072 Va o -33- (C following intranasal immunisation of mice with HSV-1 or 8 mock glycoproteins alone or in the presence of 0 adjuvant.

00 Figure 11: shows mucosal IgA in vaginal washings 0 5 following intranasal immunisation of mice with HSV-1 or mock glycoproteins (gp) alone or in the presence of adjuvant.

(C Figure 12: shows the level of HSV-1-specific immunoglobulin in sera from mice immunised with HSV-1 C 10 glycoproteins in the presence of different doses of

\O

SrEtxB as adjuvant.

O Figure 13: shows the level of IgA in eye washings of mice immunised with HSV-I 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-i1 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 10pg HSV-I plycoproteine (Gp) with either 10 or rEtxB. Controls were either unmanipulated or given a COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:23 FAX 61 2 82311099 F.B. RICE CO a039/072 0 -34- C-i mock preparation of viral glycoprotein (mock) derived 0 from HIV-uninfected tissue culture cells. Antibody 0 levels are expressed as a percentage of post-infection 00 levels. The production of total Ig and IgA in the 0 5 serum and IgA in eye washings was stimulated by HSV-1 glycoproteins/rEtxB (Figure The present inventors have also shown that doses of rEtxB as low as 0.lpg are IND also effective at stimulating such responses.

Also, T-lymphocytes from immunised mice from the 10 cervical lymph node (which is local to the vaccination ID site) and from the mesenteric lymph node (which is o 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 Op and varying amounts of EtxB is shown in Fig 3.

The production of Anti-HSV-1 serum Ig in mice following administration of HSV-l 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 Sa), 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 Example 2: rCtxB and rEtxB act as immunomodulators.

When EtxB is used as an immunomodulator, the Ig isotype distribution is skewed (Figure The distribution of Ig subclasses is different depending on whether rCtxB or rEtxB is used as an immunomodulator (Figure 7).

COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:23 FAX 61 2 82311099 F.B. RICE CO a 040/072 0 C( Example 3: rEtxB is a more efficient Simmunomodulator than rCtxB.

0 The levels of HSV-specific IgA (Figure 8) and is OO greater following stimulation with rEtxB/HSV-1 Gp that o 5 rCtxB/HSV-1 Gp.

Example 4: (Figure 9)

NO

ri Mice were immunised three times intranasally with HSV-1 glycoproteins alone, a mock preparation of HSV-1 C 10 glycoproteins (prepared by taking uninfected tissue

VO

Sculture cells and subjecting them to identical o 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 10g per immunisation, and these were combined with 10fg of recombinant EtxB, or CtxB as adjuvant, or a mixture of 0 .5Mg of Ctx and 10pg CtxB.

Three weeks after the final immunisation, blood samples were collected and total anti-HSV-l 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 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.

COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:23 FAX 61 2 82311099 F.B. RICE CO 1041/072 0 o The quantities of antibodies are expressed as a o percentage of the levels stimulated following ocular Sinfection induced by scarification with 105 pfu HSV-1 0 strain SC16. The data clearly demonstrates (Figure 00 5 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 \O from analysis of serum antibody responses, there was no F difference in the level of antibodies in the eye between those animals immunised with Ctx/CtxB or EtxB 0 as adjuvants. As with serum antibody, there was clear Sevidence 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 (10og) either alone or in the presence of escalating doses of rEtxB as adjuvant.

Three weeks after the final immunisation blood was COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2008 16:24 FAX 81 2 82311099 F.B. RICE CO a 042/072

O

O0 a 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 infecion induced by scarification with 105 pfu HSV-1 strain-SCl6. The data clearly demonstrates that the capacity of r2txB to trigger antibody responses to heterologous added antigens is a dose dependent phenomenon with maximal responsivneness occurring at approximately 20-50pg of rEtxB- Further, )N 10 it is clear that at doses of 20pg rEtxB and above, the olevel of anti-Hay-1 antibodies stimulated by intranasal infection is comparable or greater than that stimulated by a live virulent virus infection.

Example' 5: (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 satmples were then pooled and subjected to ELISA Analysis usin 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 cata demonstrates that maximal IgA responses in the eye are stimulated when HSV-l glycoproteins are given in combination with 20Ag 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:24 FAX 81 2 82311099 F.B. RICE CO j043/072

\O

O -6 an tibody. The quantities of antibodies are expressed as endpoint titres which were calculated by linear 00 regression anhalysis. The data bshows that optimal anti- HSV-l responses are stimulated in the vagina when or above of rEtxB is used as an adjuvant.

Va Example 10: (Figure Mice were eicher infected with 105 pfu 9Sv-1 strain sc16 by scarification into the conea or O 10 immunised three times intranasally with IOpg HSV-1 o glycoproteins in combination with Ctx/CtxB or rEtB.- Three weeks after the final inoculation, serum was taken and was analysed by ELISA for the preSence of IgG1 and IgC2a against RSV-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 RESV-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 Ig isotype, IgOl. This profile of the immune response is clearly visible when the dta is expressed as a ratio of IgG:IgG2a as shown in fig. 7hb.

The ratio i substantially less than 1 following infection. Intranasal immunisation in the presence of Ct/CtB 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 Iglg11 9 G2a ratio whih is approximately 3- Interestingly the nature of the response to Rsv-1 stimulated by rEtB as adjuvant is COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:24 FAX 61 2 82311099 F.B. RICE CO a044/072 Va 0 -39almost exclusively Th2 dominated. High levels of IglG are produced with only very low amounts of IgG2a. This Sstrong bias toward Th2 responsiveness is reflected in 00 an IgG1:IgG2a ratio of approximately 9.

Example 11: (Figure 16) 9 Mice were either infected with 105 pfu HSV-1 C( strain SC16 by scarification into the cornea or Simmunised three times intranasally with 10g HSV-1 10 glycoproteins in combination with Ctx/CtxB or rEtxB,

NO

o Three weeks after the final inoculation lymph nodes C 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 Tcells 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 ylFN upon in vitro stimulation with HSV-I.

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.

COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08

Claims (26)

1. The use of: EtxB, CtxB or VtxB free from whole toxin; 00 S(ii) an agent other than EtxB or CtxB, having GM1- binding activity, or an agent other than VtxB having Gb3-binding activity; or N0 (iii) an agent having an effect on intracellular signalling events mediated by GMl-binding 'or Gb3 binding; ci 10 as an immunomodulator for a vaccine against infectious diseases. C- 2. The use according to claim 1, wherein the immunomodulator is EtxB free from whole toxin.

3. The use according to claim 1 or 2, wherein the infectious disease is one for which the infectious agent is a member of the herpes virus family.

4. The use according to claim 3, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of HSV-1, HSV-2, EBV, VZV, CMV, HHV-6, HHV-7 and RHV-8. The use according to claim 4, wherein the infectious agent is selected from the group consisting of HSV-1, HSV-2, CMV or EBV.

6. The use according to claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is an influenza virus.

7. The use according to claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is a parainfluenza virus.

8. The use according to claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is a respiratory syncytial virus.

9. The use according to claim 1 or 2, wherein COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:25 FAX 61 2 82311099 F.B. RICE CO 9046/072 0 0 -41- Sthe infectious disease is caused by an infectious agent, and the infectious agent is a hepatitis virus. The use according to claim 9, wherein the 00 o infectious agent is selected from the group consisting of hepatitis A, B, C and D viruses.

11. The use according to claim 10, wherein the kN infectious agent is a hepatitis A virus or a hepatitis N C virus. 01

12. The use according to claim 1 or 2, wherein the infectious disease is meningitis. S13. The use according to claim 12, wherein the Sinfectious disease is caused by an infectious agent, and the infectious agent is selected form the group consisting of Neisseria meningitidis, Haemophilus influenzae type B and Streptococcus pneumuniae,

14. The use according to claim 1 or 2, wherein the infectious disease is pneumonia or a respiratory tract infection. The use according to claim 14, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of Streptococcus pneumoniae, Legonella pneumophila and Mycobacterium tuberculosis.

16. The use according to claim 1 or 2, wherein the infectious disease is a sexually-transmitted disease.

17. The use according to claim 16, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of Neisseria gonnorheae, HIV-1, HIV-2 and Chlamydia trachomatis.

18. The use according to claim 1 or 2, wherein the infectious disease is a gastrointestinal disease.

19. The use according to claim 18, wherein the infectious disease is caused by an infectious agent, COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:25 FAX 61 2 82311099 F.B. RICE CO l047/072 Va 0 S-42- Sand the infectious agent is selected from the group consisting of enteropathogenic, enterotoxigenic, enteroinvasive, enterohaemorrhagic and 00 o enteroaggregative E.coli, rotavirus, Salmonella enteritidis, Salmonella typhi, Helicobacter pylori, Bacillus cereus, Campylobacter jejuni and Vibrio C cholerae. The use according to claim 1 or 2, wherein C the infectious disease is a superficial infection. VO

21. The use according to claim 20, wherein the oinfectious disease is caused by an infectious agent, ci and the infectious agent is selected from the group consisting of StaphylococCus aureue, Streptococcus pyogenes and Streptococcus mutans.

22. The use according to claim 1 or 2, wherein the infectious disease is a parasitic disease.

23. The use according to claim 22, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of malaria, Trypanasoma spp., Toxoplasma gondii, Leishmania donovani and Oncocerca spp.

24. 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: 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 GMl-binding or Gb3 binding; wherein said antigenic determinant is an antigenic COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:26 FAX 61 2 82311099 F.B. RICE CO @I048/072 NO 0 S-43- o determinant of said infectious agent. A vaccine composition according to claim 24 in which the infectious disease is HSV-1 infection and o wherein the antigenic determinant is an antigenic determinant of HSV-1.

26. A vaccine composition according to claim 24 \O or 25 in which the immunomodulator is EtxB free from whole toxin.

27. A vaccine composition according to claim 24, 10 25 or 26 in which the immunomodulator and the antigenic NO o determinant are separate moieties. 0 28. A vaccine composition according to claim 24, or 26 in which the immunomodulator and the antigenic determinant are linked by a bifunctional crosslinking reagent.

29. A kit for vaccination of a mammalian subject against an infectious disease, which kit comprises: a) one of the following agents: 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- 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: 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 COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:26 FAX 61 2 82311099 F.B. RICE CO 1049/072 \O 0 S-44- o Gb3-binding activity; or (iii) an agent having an effect on intracellular signalling events mediated by GMl-binding 00 Sor Gb3 binding.

31. The use of: EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GM1- C binding activity, or an agent other than VtxB having Gb3-binding activity; or (iii) an agent having an effect on Sintracellular signalling events mediated by GM1-binding Sor Gb3 binding to upregulate the production of antibodies at mucosal surfaces.

32. The use oft 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 in a vaccine, to prolong antigen presentation and give sustained immunological memory in a mammalian subject. 33, A vaccine composition for use against an infectious disease, which infectious disease is caused by an infectious agent, which vaccine comprises an antigenic determinant and a immunomodulator selected from: 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 GMl-binding COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:26 FAX 61 2 82311099 F.B. RICE CO I050/072 VO O o or Gb3 binding; Swherein said antigenic determinant is an antigenic determinant of said infectious agent and wherein the o immunomodulator prolongs presentation of the antigenic determinant and gives sustained immunological memory.

34. The use of: OD EtxB, CtxB or vtxB free from whole toxin; 0' (ii) an agent other than EtxB or CtxB, having GM1- binding activity, or an agent other than VtxB having I\N 10 Gb3-binding activity; or (iii) an agent which has an effect on c- vesicular internalisation mediated by GM1-binding or Gb3 binding; 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. The use of: 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 GMl-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.

36. A vaccine composition which comprises: a) EtxB, CtxB, or an agent other than EtxB or CtxB which has GM1-binding activity; and b) an EBV antigen for use in the treatment and/or prevention of EBV- associated diseases.

37. A therapeutic composition which comprises: COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08 08/12 2006 16:27 FAX 61 2 82311099 F.B. RICE CO 051/072 ON -46- o EtxB, CtxB or an agent other than EtxB or CtxB which has GMl-binding activity for use in the treatment of EBV-associated 00 e- diseases. Dated this eighth day of December 2006 University of Bristol Patent Attorneys for the Applicant: F B RICE CO COMS ID No: SBMI-05618420 Received by IP Australia: Time 16:34 Date 2006-12-08