NZ507911A

NZ507911A - Immunomodulators for vaccines

Info

Publication number: NZ507911A
Application number: NZ507911A
Authority: NZ
Inventors: Neil Andrew Williams; Timothy Raymond Hirst; Andrew Morgan; Andrew Morgan Wilson; Lucy Amber Bird
Original assignee: Univ Bristol
Priority date: 1998-05-08
Filing date: 1999-05-10
Publication date: 2004-04-30
Also published as: CN1308546A; IL139467A; GB2353472A; EA200001134A1; JP2002514607A; JP4666761B2; IS5694A; CZ302333B6; EP1075274A2; BR9910305A; AU3939499A; PL344519A1; CZ20004147A3; WO1999058145A2; CN100335130C; GB0027072D0; EA004794B1; NO20005599L; KR20070067719A; MXPA00010934A

Abstract

The use of an immunomodulator selected from the group consisting of (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GM1-binding activity or an agent other than VtxB having Gb3-binding activity and (iii) an agent having effect on intracellular signaling events mediated by GM1-binding or Gb3 binding, in the preparation of a vaccine for treating one or more infectious diseases wherein if the vaccine also comprises an antigen or antigenic determinant the immunomodulator and antigen or antigenic determinant are separate moieties. Preferably the infectious disease is one for which the infectious agent is HSV-1, HSV-2, EBV, VZV, CMZ, HHV-6, HHV-7, HHV-8, influenza virus, parainfluenza virus, respiratory syncytial virus, hepatitis virus, meningitis, Streptococcus pneumonia, Legonella pneumophilia, mycobacterium tuberculosis, Neisseria meningitides, Haemophilus influenzae type B, Neisseria gonnorheae, HIV-1, HIV-2, chlamydia trachomatis, E. coli, rotavirus, Salmonella enteritidis, Salmonella typhi, Helicobacter pylori, Bacillus cereus, Campylobacter jejuni, Vibrio cholerae, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans, malaria, Trypanasom spp. Toxoplasma gondii, Leishmania donovani or Oncocerca spp. Also described is the use of at least one antigenic determinant and an immunomodulator, as described above, in the manufacture of a medicament for the prevention or treatment of a disease, to upregulate the production of antibodies at mucosal surfaces, to prolong antigen presentation and give sustained immunological memory, for use against an infectious diseases and for prolonging presentation of the antigenic determinant and giving sustained immunological memory. The immunomodulator in conjugate with the antigen or antigenic determinant can be used in the preparation of a medicament for targeting the delivery of the antigen to the cytosol or nucleus of an antigen presenting cell or upregulating the presentation of the antigenic determinant derived form the antigen by MHC class I molecules. A vaccine composition comprises EtxB, CtxB or agent other than EtxB or CtB which has GM1-binding activity and an EBV antigen.

Description

<div class="application article clearfix" id="description"> 507911 IMMDNOMODULATORS FOR VACCINES This invention relates to an iitimunomodulator for use in a vaccine which is intended for use against a range of infectious agents. Further this invention 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 heat-labile enterotoxin (Etx) are potent immunogens and 10 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 15 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. 20 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 (Oil), a glycosphingolipid found ubiquitously on the surface of mammalian cells. Vtx 25 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 30 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 35 attributable to the B subunit is indistinguishable from the adjuvant activity of the whole toxin (Wu and INTELLECTUAL PROPERTY OFFICE OF N.Z. -9 DEC 2003 WO 99/58145 PCT/GB99/01461 -2- Russell (1993) Infection and Immunity 61: 314-322, US-5182109). Subsequent studies using recombinant CtxB (rCtxB) have suggested that CtxB is a poor mucosal adjuvant and only the addition of native holotoxin cam 5 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 10 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). In another study, intranasal administration of 15 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 20 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 25 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. 30 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 Printed from Mimosa 11/02/2000 14:50:13 page -4- WO 99/58145 PCT/GB99/01461 -3- 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 5 responses to viral challenge. Specifically, the present inventors found that: i) agents such as EtxB and CtxB stimulate high levels of local (mucosal) antibody production (although immunization using rEtxB stimulated lower levels of 10 overall serum antibody production than Ctx/CtxB combined); ii) the distribution of antibodies produced was skewed towards non-complement fixing antibodies, especially S-IgA and IgGl; 15 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; 20 v) when agents such as CtxB and EtxB are used as immunomodulators some of the harmful effects of Th2-associated responses, such as the generation of IgE, are avoided; vi) rEtxB is a more efficient immunomodulator 25 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; 30 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 35 on leukocyte populations in vitro to those exerted by EtxB and CtxB. Printed from Mimosa 11/02/2000 14:50:13 page -5- -4- 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. iff ro^?aoa 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 an immunomodulator selected from the group consisting of: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having Oil-binding activity, or an agent other than VtxB having Gb3-binding activity; and (iii) an agent having -an effect on intracellular signalling events mediated by GM1-binding or Gb3 binding; in the preparation of a vaccine for treating one or more infectious diseases wherein, if the vaccine also comprises an antigen or an antigenic determinant, the immunomodulator and antigen or antigenic determinant are separate moieties. According to a second aspect of the present invention, there is provided a vaccine composition for ufce against an infectious disease, which infectious disease is caused by an infectious agent, wherein the vaccine composition comprises an antigenic determinant and an immunomodulator selected from: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having Oil-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 determinant of said infectious agent; and wherein the immunomodulator and the antigenic determinant are separate moieties. The antigent and immumodulator may be linked, for example covalently or genetically linked, to form a single effective agent. 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. Also described is a kit for vaccination of a mammalian subject, such as a human or veterinary subject, against an infectious disease, comprising: a) one of the following agents: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GM1 -binding activity, or an agent other than VtxB having Gb3-binding activity; or (iii) an agent having an effect on intracellular signalling events mediated by GMl-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 described INTELLECTUAL PROPERTY OFFICE OF N.Z - 2 FEB 2004 RECEIVED -6- may be used in a prophylactic or therapeutic vaccination method, where a "prophylactic vaccine" is administered to naive individuals to prevent disease development, and a "therapeutic vaccine" is administered to individuals with an existing infection ! to reduce or minimise the infection or to abrogate the immunopathological consequences of the disease. Agents such as EtxB have the capacity to alter the , nature of the immune response once infection has . occurred. A therapeutic vaccine (i.e. one which need not contain antigen) comprising such an agent may find particular use in circumstances in which the immune response has failed to get rid of an infection. This . application may be of particular use to treat a chronic , disease, for example a disease for which the causative agent is selected from the group consisting of herpes viruses, hepatitis viruses, HIV, TB and parasites. According to a third aspect of the present at least one antigenic determinant and an immunomodulator, where the immunomodulator is: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having 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 in the preparation of a vaccine for the prevention or treatment of a disease. 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 intellectual property office of im.z - 2 FEB 200*1 RECEIVED ;WO 99/58145 PCT/GB99/01461 ;-7- ;in liquid form. ;The antigenic determinant and immunomodulator may be. administered to the subject as a single dose or in multiple doses. ;5 In a first embodiment the immianomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, and the use of the third aspect of the invention is used against a disease for 10 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. 15 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 20 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 25 deteinminant 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 ;30 ;second aspect of the invention, and the use of the third aspect of the invention is used against a disease for which the infectious agent is an influenza virus. In this second embodiment, the antigenic 35 determinant is preferably an antigenic determinant of a viral coat protein (for example haemagglutinin and intellectual property office of n.z ;- 2 FEB 2094 RECEIVED ;WO 99/58145 PCT/GB99/01461 ;-8- ;neuraminidase) or of an internal protein (for example, nucleoprotein) . ;In a third embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the ;5 ;second aspect of the invention, and the use of the third aspect of the invention is used against a disease for which the infectious agent is a parainfluenza virus. ;In a fourth embodiment/ the immunomodulator of the 10 first aspect of the invention, the vaccine of the second aspect of the invention, and the use of the fourth aspect of the invention is used against a disease for which the infectious agent is respiratory syncytial 15 virus. ;In a fifth embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, and the use of the third ;20 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 25 C. ;In a sixth embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, and the use of the third ;30 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. ;35 In a seventh embodiment, the immunomodulator of i ntellectuaI "prop erty ' ;0fr0f OF N.z ;- 2 FEB 2004 RECEIVED ;WO 99/58145 ;PCT/GB99/01461 ;-9- ;the first aspect of the invention, the vaccine of the second aspect of the invention, and the use of the third aspect of the invention is used against pneumonia or a 5 respiratory tract infection. In this seventh embodiment, the infectious agent may be selected from the group consisting of Streptococcus pneumoniae, ;Legonella pneumophila and Mycobacterium tuberculosis. ;In an eighth embodiment, the immunomodulator of the 10 first aspect of the invention, the vaccine of the second aspect of the invention, and the use of the third aspect of the invention is used against a sexually-transmitted disease. In this eighth embodiment, the 15 infectious agent may be selected from the group consisting of Neisseria gonnorheae, HIV-1, HIV-2 and ;Chi amydi a tra choma tis. ;In an ninth embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the ;20 ;second aspect of the invention, and the use 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 25 consisting of enteropathogenic, enterotoxigenic and enteroinvasive E.coli, rotavirus, Salmonella enteritidis, Salmonella typhi, Helicobacter pylori, ;Bacillus cereus, Campylobacter jejuni and Vibrio cholerae. ;30 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 ;*" intelIectuaT^rope^ty" OFFICE OF M.Z - 2 FEB 2004 RECEIVED WO 99/58145 PCT/GB99/01461 -10- bacterial toxin or adhesion factor. In a tenth embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, and the use of the third 5 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 .0 Streptococcus mutans. In an eleventh embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, and the use of the third 15 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. 20 gtHrmilation of mucosal imwinne responses EtxB, CtxB, VtxB and other agents capable of binding to or mimicking the effects of binding to GM1 or Gb3, arfe capable of specifically upregulating 25 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 30 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 35 include, diseases caused by viruses (HIV, HSV, EBV, 1 intellectual property i office of n.z " 2 FEB 20M RECEIVED WO 99/58145 PCT/GB99/01461 -11- 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 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 against HSV-1 infection comprising at least one HSV-1 antigenic determinant and an immunomodulator, where the 10 immunomodulator is: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or 15 (iii) an agent having an effect on intracellular signalling events mediated by GMl-binding or G3b binding. Preferably the immunomodulator is EtxB. Also described is a kit for 20 vaccination of a mammalian subject against an HSV-1, comprising: a) a vaccine immunomodulator which is: (i) EtxB, CtxB or VtxB free from whole toxin; 25 (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 G3b 3 0 binding; and b) at least one HSV-1 antigenic determinant, for coadministration with the said vaccine immunomodulator. According to a fourth aspect of the invention there 35 is provided the use of: (i) EtxB, CtxB or VtxB free from whole toxin? OFFICE OF N.Z - 2 FEB 2004 RECEIVED -12- (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB haying Gb3-binding activity; or (iii) an agent having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding in the preparation of a medicament to upregulate the production of antibodies at mucosal surfaces. The production of non-complement-fixing serum antibodies may also be upregulated. Preferably, S-IgA is produced in accordance with the fifth aspect of the invention. In this fourth aspect of the present invention, the agent may be used in conjunction with one or more antigenic determinant(s). Downrecmlatinq the pathological components of •iTttmnne responses The inventors also found that when pure EtxB was used as an immunomodulator in the described way, the harmful effects of Th2 associated responses, such as the generation of high levels of potentially pathological IgE, were avoided. Despite this, the immune response triggered by the use of EtxB (or CtxB or VtxB) as an immunomodulator appears to favour the induction of Th2-associated cytokines. In other words EtxB (or CtxB) induces a shift from a Thl- to a Th2-type response. This has enabled the inventors to predict that pure EtxB, CtxB or VtxB,. as well as other agents capable of binding to or mimicking the effect of binding to GM1 or Gb3, will be capable of down regulating pathological components of the immune response associated with both Thl and Th2 activation. Described herein is,the use of: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an ^gent other than EtxB or CtxB, having INTELLECTUAL PROPERTY! OFFICE OF N.Z - 2 FEB 2004 RECEIVED WO 99/58145 PCT/GB99/01461 -13- 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 5 binding; to downregulate the pathological components of Th2-associated immune responses. The pathological components of Thl-associated immune responses may also be downregulated. 10 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 15 immune response such that inflammatory Thl associated reactions are down-regulated while Th2 associated responses are upregulated. Thl responses include the secretion of ylFN by activated T-cells leading to macrophage activation and delayed type hypersensitivity 20 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 25 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 30 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 35 non-complement fixing serum antibodies and secretory IgA production at the mucosal surfaces. Printed from Mimosa 11/02/2000 14:50:13 page -15- -14- The use of an agent can be particularly useful for therapeutic vaccination in diseases in which imraunopathological mechanisms are involved. Examples of such diseases are HSV-1, HSV-2, TB and HIV. The first aspect of the invention and the use described above 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. Tmrminnmodulafcion of the antigen processing pathway a) prolonging 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 B-subunit associated antigen presentation means that vaccines incorporating an agent in accordance with the present invention will have increased antigen persistence and lead to sustained immunological memory. According to a fifth aspect of the present invention, there is provided the use of: Ln%zlUrAL PROPERTY 1 OFFICE- OF fsj2 " 2 FE3 RECEIVED -15- (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or (iii) an agent having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding; in the preparation of a vaccine to prolong antigen presentation and give sustained immunological memory. According to a sixth aspect of the present invention, there is provided a use of an antigenic determinant and a immunomodulator selected from: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or (iii) an agent having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding; in the preparation of a vaccine for use against infectious disease. 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. bV intracellular targeting of the antigen to a MHC-I or MHC-II associated pathway As aforementioned, the antigen and immunomodulator in a therapeutic or prophylactic vaccine may be linked, for example covalently or genetically linked, to form a single effective agent. The present inventors have found that is possible to direct the antigen to different compartments of the cell and hence to different antigen presentation pathways IN lELLECTOAiPROPERTY OFFICE OF M.Z - 2 FEB 2004 RECEIVED -16- linkage of the antigen to the immunomodulator. By linking the antigen or antigenic determinant to the immunomodulator in a certain way, it is possible to facilitate translocation of the antigen across the 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 use of an antigen-immunomodulator conjugate can therefore be used to specifically enhance the 10 activation of cytotoxic T cells (CTL) . Induction of CTL is beneficial for the prevention and treatment of many diseases especially those caused by viruses, intracellular bacteria and parasites. The linkage of the antigen-immunomodulator 15 conjugate can also be chosen so that the antigen is delivered into the nucleus. Described herein is a conjugate comprising an antigen or antigenic determinant and an immunomodulator 20 selected from: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or 25 (iii) an agent which has an effect on vesicular internalisation mediated by GMl-binding or Gb3 binding. Also described herein is a vaccine composition for use against an infectious disease, which infectious 30 disease is caused by an infectious agent, which vaccine composition comprises a conjugate of an antigen or antigenic determinant and an immunomodulator selected from: (i) EtxB, CtxB or VtxB free from whole toxin; 35 (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB INTElj;KTtMl PROPER^ ofrcf op 2 1 AUG 2003 RECEIVED -17- 10 30 having Gb3-binding activity; or (iii) an agent which has an effect on vesicular internalisation mediated by GMl-binding or G3b binding; wherein said antigen or antigenic determinant is an antigen or antigenic determinant of said infectious agent. The antigen or antigenic determinant may be linked to the immunomodulator by a variety of methods including genetic linkage or chemical coniugation. The conjugate can be 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. The antigen or antigenic determinant may be chemically conjugated to the immunomodulator. Preferably the antigen or antigenic determinant is conjugated to the immunomodulator using a bifunctional cross-linking reagent, such as a heterobifunctional cross-linking reagent. More preferably the cross-linking agent is N-Y(-maleimido-butyroxyl)-succinimide ester (GMBS) or N-succinimidyl-(3-pyridyl-dithio)-propionate (SPDP). The vaccine composition may be administered by a number of different routes such as intranasal, oral, intra-vaginal, urethral or ocular administration. Intranasal immunisation is preferred. According to a seventh aspect of the present invention there is provided the use of: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-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 | iMiELLECTL OFPICl - 2 FI rec! -18- determinant in the preparation of a medicament for targeting the delivery or said antigen or antigenic determinant to the cytosol or nucleus of an antigen presenting cell. According to an eighth aspect of the present invention there is provided the use of: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-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 in the preparation of a medicament for upregulating the presentation of said antigenic determinant, or an antigenic determinant derived from said antigen, by MHC class I molecules. Preferably the use of the conjugate of the twelfth aspect of the invention is used is combination with the use of the agent in accordance with the fifth aspect of the invention to stimulate strong CTL responses and to upregulate mucosal antibody production. This activity would be particularly useful in the prevention and treatment of viral infections, for example influenza. EtxB is the preferred immunomodulator It has previously been thought that EtxB and CtxB have similar properties. However, the present inventors have found that rEtxB is a more potent and efficient immunomodulator than rCtxB. Hence the preferred immunomodulator is EtxB, or agents which mimic the effects of EtxB. EBV 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 WO 99/58145 PCT/GB99/01461 -19- 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 5 EBV and endemic Burkitt's lymphoma (BL) and undifferentiated nasopharyngeal carcinoma (NPC). Also, a large proportion of lymphomas that occur in immunocompromised patients are caused by EBV, and an association has been shown to exist between certain 10 Hodgkin's lymphomas and EBV. 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 15 two non-polyadenylated virus derived RNAs (EBERs) with a role in RNA splicing. EBV latent membrane protein 1 (LMP-1) is present in the plasma membrane of infected cells. It is also expressed in nasopharyngeal carcinomas (NPCs) and EBV-20 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-25 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. 3 0 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 35 immune system detection, thus allowing its persistence in the host. This explains why certain cellular immune Printed from Mimosa 11/02/2000 14:50:13 page -21- -20- (followed by -20a) responses are not detectable to the EBV latent protein EBNA1 and could explain the apparent absence of such responses against LMP1.. According to a ninth aspect of the invention there is provided a use-of: a) EtxB, CtxB, or an agent other than EtxB or Ctxb which has GMl-binding activity; and b) an EBV antigen in the manufacture of a vaccine composition for use in the treatment and/or prevention of EBV-associated diseases. Also described herein is a vaccine composition which comprises: a) one of the following agents: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having 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; and b) an EBV antigen for use in the treatment and/or prevention of EBV-associated diseases. In particular a preferred vaccine composition of the invention comprises EtxB, CtxB, or an agent other than EtxB or CtxB which has GMl-binding activity. According to a tenth aspect of the invention there is provided a therapeutic composition which comprises: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having 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_J3b3 binding; -20a- for use in the treatment of EBV-associated diseases. A preferred therapeutic composition of the present invention comprises EtxB, CtxB, or an agent other than EtxB or CtxB which has OPP'0F QP7uz • 2 FEB 2004 RECF=!\/cr\ -21- GM1-binding activity. 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 5 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. 10 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 15 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. 20 The vaccine of the ninth 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 25 as an adjuvant in its own right. The agents specified in the tenth 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. 30 The preferred agent for use in the ninth and tenth 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. 35 Preferably the antigen is an EBV latent membrane protein. Particularly preferred are the antigens LMP- 'NTEU^ECfu^pRop^-i office of n z ~ 2 FEB 2004 -22- 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. 5 The ninth and tenth 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 10 that these aspects of the invention will be particularly suited to the treatment and/or prevention of nasopharyngeal carcinomas and Hodgkin's lymphomas. The vaccine or the therapeutic composition according to the ninth and tenth aspects of 15 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 20 healthy EBV-infected or uninfected individual, an immunodeficient individual, or an individual with an EBV-associated disease. The vaccine may be administered by any suitable route. The agent and the antigen may be co-25 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. 3 0 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 35 suggests that EtxB interacts with at least one other receptor which is involved in the GM1 associated ' INIELLECTU^PR0PFW;V 1 off/cp of i\!.z - 2 FES 2004 Received WO 99/58145 PCT/GB99/01461 -23- 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 5 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 10 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 15 intracellular signalling events. Definitions An adjuvant is a substance which non-specifically enhances the immune response to an antigen, as distinct 20 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 25 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 3 0 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 3 5 immunomodulator may be administered together in a first step and then the immune response may be boosted in a Printed from Mimosa 11/02/2000 14:50:13 page -25- WO 99/58145 PCT/GB99/01461 -24- second step by administration of antigen alone. The term "antigenic determinant" as used herein refers to a site on an antigen which is recognised by an antibody or T-cell receptor. Preferably it is a 5 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 10 recognise the whole organism. There are a number of known methods by which it is possible to identify antigenic determinants for a given infectious agent. For example, potential protective antigens may be 15 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 2 0 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; 25 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 30 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 35 patients or infected or convalescent animals may be screened against whole cell lysates derived from Printed from Mimosa 11/02/2000 14:50:13 page -26- WO 99/58145 PCT/GB99/01461 -25- 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; 5 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 10 clones expressing antigens, or fragments thereof, that are recognised by the immune serum; or v) PBLs from the blood of infected or convalescent patients or PBL's, lymph node cells, spleen cells, or lamina propria cells from infected or convalescent 15 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-20 specific T-cell proliferative responses.. Alternatively it is possible to detect gene products which are essential for the in vivo survival of pathogens, as exemplified by ,the technique of signature tagged mutagenesis developed by Holden or the 25 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 30 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 35 activity. Printed from Mimosa 11/02/2000 14:50:13 page -27- WO 99/58145 PCT/GB99/01461 -26- There are a number of known methods by which it is possible to isolate antigens for a given infectious agent. For example, surface components of an infectious 5 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 10 sonication and/or detergent extraction. Centrifugation, ultrafiltation or precipitation may be used on collected antigen preparations. The antigen preparation containing HSV-1 glycoproteins described in Richards et al. , (1998) J. Infect. Dis. 177,-1451-7, 15 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 20 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 25 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, 3 0 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. 35 The recombinant form is particularly preferred. The recombinant form of the molecule may be produced by a Printed from Mimosa 11/02/2000 14:50:13 page -28- WO 99/58145 PCT/GB99/01461 -27- 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 5 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 10 genes expressing active mutant CtxB, EtxB or VtxB protein may be produced by known methods from the wild type gene. The terms "CtxB", "EtxB" and "VtxB" also include mutant molecules and other synthetic molecules 15 (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 20 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 25 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 3 0 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. 35 Humanised monoclonal antibodies may be preferred in the present invention. Monoclonal antibodies may be Printed from Mimosa 11/02/2000 14:50:13 page -29- WO 99/58145 PCT/GB99/01461 -28- 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 (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 GM1 or Gb3 may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the Printed from Mimosa 11/02/2000 14:50:13 page -30- WO 99/58145 PCT/GB99/01461 -29- disulfide bridges of the F(ab'>2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity 5 (Huse WD et al (1989) Science 256:1275-128 1). Peptide libraries or organic libraries may be made by combinatorial chemistry and then screened for their ability to bind GM1/Gb3. Synthetic compounds, natural products, and other sources of potentially biologically 10 active materials can be screened in a number of ways deemed to be routine to those of skill in the art. GM1 or Gb3 or fragments thereof can be used for screening peptides or molecules in any of a variety of screening techniques. The molecule may be free in 15 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 20 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 25 antibodies either directly or indirectly to an enzyme or radiolabel allows subsequent quantification of binding either using colorimetric or radioactivity based methods (ELISA or RIA respectively). Another way of determining binding to GM1/Gb3 30 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 35 alternatively, where mixtures of compounds are applied to the column, elution and subsequent analysis would Printed from Mimosa 11/02/2000 14:50:13 page -31- WO 99/58145 PCT/GB99/01461 -30- 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 5 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 GMl-affinity columns would be 10 analysed by HPLC and mass spectrometry of single homogenous peaks. Another way of determining the ability to bind to GM1/Gb3 and the precise affinity of the interaction would be by using plasmon surface resonance as 15 previously reported [Kuziemko et al (1996) Biochem 35:6375-6384] . Alternatively, phage display can be employed in the identification of candidate agents which bind GMl or Gb3. 20 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 25 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) 30 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 35 standard affinity ligand screening technologies. The phage surface displays the candidate agent in a three Printed from Mimosa 11/02/2000 14:50:13 page -32- WO 99/58145 PCT/GB99/01461 -31- dimensional configuration, more closely resembling its naturally occuring conformation. This allows for more specific and higher affinity binding for screening purposes. 5 Another technique for screening provides for high throughput screening of agents having suitable binding affinity to GMl 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 10 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 15 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 20 a solid support. In all aspects of the invention, the agent having GMl-binding activity or Gb3 binding activity may also be capable of cross-linking GMl or Gb3 receptors. EtxB is one such agent which is capable of cross-linking GMl 25 receptors by virtue of its pentameric form. There are various methods for identifying agents which have an effect on intracellular signalling events mediated by GM1/Gb3 binding but which do not themselves bind GMl or Gb3. For example, if an agent is shown to 3 0 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 GMl or Gb3 (by, for example, one of the binding assays described above), then it can 35 be concluded that the agent is capable of mimicing the effect of GM1/Gb3 binding. Printed from Mimosa 11/02/2000 14:50:13 page -33- WO 99/58145 PCT/GB99/01461 -32- The invention will now be illustrated by reference to the accompanying drawings and the following examples. The examples refer to the figures in which: 5 Figure 1: shows the stimulation of total Ig and IgA in the serum (MS) and IgA in the eye washings (EW) in mice immunised with HSV-1 glycoproteins/rEtxB. Figure 2: shows T cell proliferation of (mesenteric lymph node) MLN or (cervical lymph node) 10 CLN lymphocytes in mice immunised with HSV-l/rEtxB. Figure 3: shows T cell proliferation of cells from MLN and CLN of mice immunised intranasally with HSV-l Gp in the presence of 1-2 0/ug EtxB. Figure 4: shows the level of anti-HSV-1 serum Ig 15 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 2 0 HSV-1/rEtxB. Figure 6: shows the Ig isotype distribution in MS following infection with HSV-1 or immunisation with HSV-1 Gp in the presence of EtxB or CtxB as immunomodulator. 25 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 30 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 3 5 adj uvant. Figure 10: shows mucosal IgA in eye washings Printed from Mimosa 11/02/2000 14:50:13 page -34- WO 99/58145 PCT/GB99/01461 -33- following intranasal immunisation of mice with HSV-l or mock glycoproteins alone or in the presence of adjuvant. Figure 11: shows mucosal IgA in vaginal washings 5 following intranasal immunisation of mice with HSV-1 or mock glycoproteins (gp) alone or in the presence of adj uvant. Figure 12: shows the level of HSV-l-specific immunoglobulin in sera from mice immunised with HSV-1 10 glycoproteins in the presence of different doses of rEtxB as adjuvant. Figure 13: shows the level of IgA in eye washings of mice immunised with HSV-1 glycoproteins in the presence of varying concentrations of rEtxB. 15 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 20 immunisation with Ctx/CtxB or rEtxB or ocular infection with HSV-1. Figure 16: shows cytokine production from cultures of lymph node cells taken from mice which were either infected with HSV-1 by ocular scarification, or were 25 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 3 0 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 35 lOpig HSV-1 plycoproteins (Gp) with either 10 or 20/zg rEtxB. Controls were either unmanipulated or given a Printed from Mimosa 11/02/2000 14:50:13 page -35- 99/58145 PCT/GB99/01461 -34- 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 (Figure 1). The present inventors have also shown that doses of rEtxB as low as 0.1/xg are also effective at stimulating such responses. Also, T-lymphocytes from immunised mice from the cervical lymph node (which is local to the vaccination site) and from the mesenteric lymph node (which is distant to the vaccination site) were shown to proliferate when cultured in vitro with HSV-1, but not when cultured in vitro with mock HSV-1 Gp or without antigen (Figure 2). The proliferation in response to HSV-1 Gp of T lymphocytes from MLN and CLN of mice immunised with HSV-1 Gp and varying amounts of EtxB is shown in Fig 3. The production of Anti-HSV-1 serum Ig in mice following administration of HSV-1 glycoproteins at three day intervals with varying amounts of EtxB (or CtxB) is shown in Figure 4. Finally, mice immunised with HSV-1 and rEtxB were shown to have a decrease in virus shedding following corneal scarification with HSV-1 (Figure 5a), and a decrease in local spreading (oedema and lid disease), spreading to the trigeminal ganglion (zosteriform infection), spreading to the central nervous system (encephalitis) and latency compared to controls (5b). Example 2: rCtxB and rEtxB act as immunomodulators. When EtxB is used as an immunomodulator, the Ig isotype distribution is skewed (Figure 6). The distribution of Ig subclasses is different depending on whether rCtxB or rEtxB is used as an immunomodulator (Figure 7). Printed from Mimosa 11/02/2000 14:50:13 page -36- WO 99/58145 PCT/GB99/01461 -35- Example 3: rEtxB is a more efficient immunomodulator than rCtxB. The levels of HSV-specific IgA (Figure 8) and is greater following stimulation with rEtxB/HSV-1 Gp that 5 rCtxB/HSV-1 Gp. Example 4: (Figure 9) Mice were immunised three times intranasally with HSV-1 glycoproteins alone, a mock preparation of HSV-1 10 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 15 mucosal adjuvants. In each case the dose of HSV-1 glycoproteins was 10fig per immunisation, and these were combined with 10^g of recombinant EtxB, or CtxB as adjuvant, or a mixture of 0.5/xg of Ctx and 10/Ltg CtxB. Three weeks after the final immunisation, blood samples 20 were collected and total anti-HSV-1 antibodies were measured by ELISA. The quantities of antibodies are expressed as a percentage of the levels stimulated following ocular infection induced by scarification with 105 pfu HSV-1 strain SC16. The data (shown in 25 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. 30 Example 5: (Figure 10) Mice were immunised as described in example 4. Secretory IgA production in the eye was assessed by taking washings of the tears over consecutive days and 35 these samples were then pooled and subjected to ELISA analysis using a specific anti-IgA detecting antibody. Printed from Mimosa 11/02/2000 14:50:13 page -37- WO 99/58145 PCT/GB99/01461 -36- The quantities of antibodies are expressed as a percentage of the levels stimulated following ocular infection induced by scarification with 10^ pfu HSV-1 strain SC16. The data clearly demonstrates (Figure 10) 5 that high levels of secreted anti-HSV-l 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 10 between those animals immunised with Ctx/CtxB or EtxB as adjuvants. As with serum antibody, there was clear evidence that rCtxB is a very poor adjuvant. Example 6: (Figure 11) 15 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 2 0 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-l antibodies are . produced in distant mucosal sites following 25 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 3 0 was triggered by the use of rCtxB as adjuvant. Example 7: (Figure 12) Mice were immunised three times intranasally with HSV-1 glycoproteins (10/ig) either alone or in the 35 presence of escalating doses of rEtxB as adjuvant. Three weeks after the final immunisation blood was Printed from Mimosa 11/02/2000 14:50:13 page -38- WO 99/58145 PCT/GB99/01461 -37- taken, and the levels of anti-HSV-l antibodies were assessed by ELISA. The quantities of antibodies are expressed as a percentage of the levels stimulated following ocular infection induced by scarification 5 with 105 pfu HSV-1 strain-SC16. The data clearly demonstrates that the capacity of rEtxB to trigger antibody responses to heterologous added antigens is a dose dependent phenomenon with maximal responsiveness occurring at approximately 20-50ftg of rEtxB. Further, 10 it is clear that at doses of 20/xg rEtxB and above, the level of anti-HSV-l antibodies stimulated by intranasal infection is comparable or greater than that stimulated by a live virulent virus infection. 15 Example 8: (figure" 13) Mice were immunised as described in example 7. Secretory IgA production in the eye was assessed by taking washings of the tears over consecutive days and these samples were then pooled and subjected to ELISA 20 analysis using a specific anti-IgA detecting antibody. The quantities of antibodies are expressed as a percentage of the levels stimulated following ocular infection induced by scarification with 105 pfu HSV-1 strain SC1S. The data demonstrates that maximal IgA 25 responses in the eye are stimulated when HSV-1 glycoproteins are given in combination with 20/xg of rEtxB or above. At this dose the levels of IgA production are nevertheless lower than those triggered during virus infection of the eye. 30 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 35 days and these samples were then pooled and subjected to ELISA analysis using a specific anti-IgA detecting Printed from Mimosa 11/02/2000 14:50:13 page -39- WO 99/58145 PCT/GB99/01461 -38- antibody. The quantities of antibodies are expressed as endpoint titres which were calculated by linear regression analysis. The data shows that optimal anti-HSV-l responses are stimulated in the vagina when 20/ig 5 or above of rEtxB is used as an adjuvant. Example 10: (Figure 15) Mice were either infected with 105 pfu HSV-1 strain SC16 by scarification into the cornea or 10 immunised three times intranasally with 10/zg HSV-1 glycoproteins in combination with Ctx/CtxB or rEtxB. Three weeks after the final inoculation, serum was taken and was analysed by ELISA for the presence of IgGl and IgG2a against HSV-1. The quantities of 15 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 20 with HSV-1 predominantly activates Thl associated antibody production, as characterised by the high levels of the complement fixing antibody isotype, IgG2a. Infection stimulates relatively low levels of the Th2 associated IgG isotype, IgGl. This profile of 25 the immune response is clearly visible when the data is expressed as a ratio of IgGl: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, 30 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 IgGl:IgG2a ratio which is 35 approximately 3. Interestingly the nature of the response to HSV-1 stimulated by rEtxB as adjuvant is Printed from Mimosa 11/02/2000 14:50:13 page -40- WO 99/58145 PCT/GB99/01461 -39- almost exclusively Th2 dominated. High levels of IgGl are produced with only very low amounts of IgG2a. This strong bias toward Th2 responsiveness is reflected in an IgGl:IgG2a ratio of approximately 9. 5 Example 11: (Figure 16) Mice were either infected with 105 pfu HSV-1 strain SC16 by scarification into the cornea or immunised three times intranasally with 10/zg HSV-1 10 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 15 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 2 0 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 25 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-1. This indicates that although the response to these 30 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. Printed from Mimosa 11/02/2000 14:50:13 page -41- </div>

Claims

<div class="application article clearfix printTableText" id="claims"> • wnb i nvi < OFFICE OF N.Z. - 9 DEC 2003 RECEIVED -40- CLMltg 1 The us6 of an immunomodulator selected from the gr°upiconsisting Qf. 1 (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; and (iii) an agent having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding; in the preparation of a vaccine for treating one or more infectious diseases wherein, if the vaccine also comprises an antigen or an antigenic determinant, the immunomodulator and antigen or antigenic determinant are separate moieties.
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 HHV-8.
5. 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 l 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 WO 99/58145 PCT/GB99/01461 -41- the infectious disease is caused by an infectious agent, and the infectious agent is a hepatitis virus.
10. The use according to claim 9, wherein the infectious agent is selected from the group consisting 5 of hepatitis A, B, C and D viruses.
11. The use according to claim 10, wherein the infectious agent is a hepatitis A virus or a hepatitis C virus.
12. The use according to claim 1 or 2, wherein 10 the infectious disease is meningitis.
13. The use according to claim 12, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected form the group consisting of Neisseria, meningitidis, Haemophilus 15 influenzae type B and Streptococcus pneumoniae.
14. The use according to claim 1 or 2, wherein the infectious disease is pneumonia or a respiratory-tract infection.
15. The use according to claim 14, wherein the 20 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 25 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 30 consisting of Neisseria gonnorheae, HIV-1, HIV-2 and Chiamydia 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 35 infectious disease is caused by an infectious agent, Printed from Mimosa 11/02/2000 14:50:13 page -43- WO 99/58145 PCT/GB99/01461 -42- and the infectious agent is selected from the group consisting of enteropathogenic, enterotoxigenic, enteroinvasive, enterohaemorrhagic and enteroaggregative E.coli, rotavirus. Salmonella 5 enteritidis, Salmonella typhi, Helicobacter pylori, Bacillus cereus, Campylobacter jejuni and Vibrio cholerae.
20. The use according to claim 1 or 2, wherein the infectious disease is a superficial infection. 10
21. The use according to claim 20, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes and Streptococcus mutans. 15
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 20 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 25 comprises an antigenic determinant and an immunomodulator selected from: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having 30 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 Printed from Mimosa 11/02/2000 14:50:13 page -44- - 43 - determinant of said infectious agent, and wherein the immunomodulator and the antigenic determinant are separate moieties.
25. A vaccine composition according to claim 24 in which the infectious disease is HSV-1 infection and wherein the antigenic determinant is an antigenic determinant of HSV-1.
26. A vaccine composition according to claim 24 or 25 in which the immunomodulator is EtxB free from whole toxin.
27. A vaccine composition according to claim 24 which comprises an antigen, the antigenic determinant being a site on the antigen.
28. A use of at least one antigenic determinant and an immunomodulator, where the immunomodulator is: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having 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 in the preparation of a vaccine for the prevention or treatment of a disease.
29. The use of: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or 81150 l.DOC iiV'tLLECTUAT PROPEH-IV" fj o^-rlc qp «\;2 - 2 FED 2004 .received - 44 - (iii)an agent having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding in the preparation of a medicament to upregulate the production of antibodies at mucosal surfaces.
30. The use of: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or (iii)an agent having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding in the preparation of a vaccine to prolong antigen presentation and give sustained immunological memory.
31. A use of an antigenic determinant and a immunomodulator selected from: (i) EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or (iii)an agent having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding in the preparation of a vaccine for use against an infectious disease wherein said antigenic determinant is an antigenic determinant of said infectious agent and wherein the immunomodulator prolongs presentation of the antigenic determinant and gives sustained immunological memory. k ii\l7tLLcC+ual' prgpfrty* h OWDF OP N1" ';- 2 FES 2004 received;81150_1.DOC;- 45 -;32. The use of:;(i) EtxB, CtxB or VtxB free from whole toxin;;(ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or;(iii)an agent which has an effect on vesicular internalisation mediated by GMl-binding or Gb3 binding;;in a conjugate with antigen or antigenic determinant in the preparation of a medicament for targeting the delivery or said antigen or antigenic determinant to the cytosol or nucleus of an antigen presenting cell.;33. The use of:;(i) EtxB, CtxB or VtxB free from whole toxin;;(ii) an agent other than EtxB or CtxB, having GMl-binding activity, or an agent other than VtxB having Gb3-binding activity; or;(iii)an agent which has an effect on vesicular internalisation mediated by GMl-binding or Gb3 binding;;in a conjugate with antigen or antigenic determinant in the preparation of a medicament for upregulating the presentation of said antigenic determinant, or an antigenic determinant derived from said antigen, by MHC class I molecules.;34. A vaccine composition which comprises:;a) EtxB, CtxB, or an agent other than EtxB or CtxB which has GMl-binding activity; and;- 46 -b) an EBV antigen for use in the treatment and/or prevention of EBV-associated diseases.;35. The use of EtxB, CtxB or an agent other than EtxB or CtxB which has GMl-binding activity in the manufacture of a therapeutic composition for use in the treatment of EBV-associated diseases.;36. The use defined in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.;37. A vaccine composition as defined in claim 24 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.;38. A use as claimed in any one of claims 28 to 33 or 35 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.;39. A vaccine composition as claimed in claim 34 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings.;M f?.*1 ^ By the authorised agents ft w't'llectual PROPp'ryv'>• Qp-iOf nr~ N2~' I - 2 FED 2004 81150_1.DOC </div>