MXPA00010934A

MXPA00010934A - Vaccine.

Info

Publication number: MXPA00010934A
Application number: MXPA00010934A
Authority: MX
Inventors: Timothy Raymond Hirst
Original assignee: Univ Bristol
Priority date: 1998-05-08
Filing date: 1999-05-10
Publication date: 2003-07-14
Also published as: EP1075274A2; IL139467A; CN100335130C; JP2002514607A; PL344519A1; WO1999058145A3; IS5694A; IL139467A0; KR20070067719A; CN1308546A; CA2331832A1; NO20005599L; JP4666761B2; WO1999058145A2; EA004794B1; BR9910305A; CZ20004147A3; GB2353472A; HUP0104842A2; EA200001134A1

Abstract

The use of: (i) EtxB, CtxB or VtxB free of whole toxin is described, (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB that has binding activity to Gb3; or (iii) an agent that has an effect on the events of intracellular signaling mediated by binding to GMI or binding to Gb3, as an immuno-modulator for a vaccine against infectious diseases

Description

VACCINE DESCRIPTIVE MEMORY This invention relates to an immunomodulator for use in a vaccine which is intended for use against a range of infectious agents. Furthermore, this invention relates to a vaccine composition comprising the immunomodulator, preferably in combination with antigen and a method of vaccination using the vaccine composition. Cholera toxin (Ctx) and the molecule closely related to it, the heat-labile enterotoxin (Etx) of E. coli, are impotent immunogens and adjuvants in mucous membranes. However, their inherent toxicity makes them unsuitable for use in humans. For example, although Ctx is the mucosal adjuvant that is most commonly used in experimental animals, it is unsuitable for use in humans because of its potent properties to induce diarrhea. Attempts have been made to separate the toxicity from the adjuvant activity, for example, using components of Ctx and Etx as replacements of the holotoxins themselves. Verotoxin (Vtx) from E. coli is another known bacterial toxin. Ctx and Etx are heterohexamer proteins composed of an enzymatically active A subunit and a pentamer B subunit. It is known that CtxB and EtxB bind to ganglioside GM1 (GM1), a glycosphingolipid ubiquitously present on the surface of mammalian cells. Vtx binds to Gb3 which is a similar type of receptor for GM1. In an attempt to avoid the problem of toxicity for the development of vaccines, the adjuvant activity of non-toxic subunits B has been previously 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 that the adjuvant activity attributable to the B subunit is indistinguishable from the adjuvant activity of the intact toxin (Wu and Russell (1993) Infection and Immunity 61: 314 -322, US-5182109). Subsequent studies in which recombinant CtxB (rCtxB) has been used, have suggested that CtxB is a poor mucosal adjuvant, and only the addition of native holotoxin can elicit strong zinc responses (Tamura et al (1994) Vaccine 12: 419-426 ). Other studies have suggested that rCtxB lacks the activities of ADP ribosylation and cAMP stimulation of holotoxin and that, as an adjuvant mechanism linked to these capabilities, CtxB would be inadequate for its 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 ovalbumin using rCtxB as an adjuvant resulted in poor antibody responses. A non-toxic derivative of Ctx with a mutation in subunit A also generated weak responses to zinc antigens, while the presence of an active A subunit dramatically increased adjuvant activity, suggesting that an active A subunit is essential (Douce et al (1997 ), previously mentioned). It has also been shown that rCtxB and rEtxB can be used to promote tolerance to heterologous antigens (Sun et al (1994) Proc. Nati Acad Sci 91: 4610-4614, Sun et al (1996) Proc. Nati. Acad. Sci 93: 7196-720, Bergerot et al (1997) Proc Nati Acad Sci 94: 4610-4614, Williams et al (1997) Proc Nati Acad Sci 94: 5290-5295), suggesting that these molecules would be inadequate for their use as adjuvants.

BACKGROUND OF THE INVENTION In spite of the teaching in the art that CtxB and EtxB have poor adjuvant capacity and can, in fact, function as tolerogens, the present inventors investigated the use of rEtxB (thus not containing subunit A). or residual holotoxin) in an intranasal vaccine for HSV in a murine model, and they surprisingly found that it is able to stimulate protective immune responses to viral challenge. Specifically, the present inventors found that: i) agents such as EtxB and CtxB stimulate high levels of local antibody production (in mucous membranes) (although immunization using rEtxB stimulated lower levels of overall antibody production in serum than combined Ctx / CtxB); ii) the distribution of produced antibodies was diverted to binding antibodies in the absence of complement, especially S-IgA and IgG1; iii) agents such as EtxB and CtxB stimulated also local and systemic T cell proliferative responses, iv) agents such as CtxB and EtxB tended to change the immune response of a Th1-associated response to a Th2-associated response; v) when agents such as CtxB and EtxB are used as immunomodulators, some of the deleterious effects of the responses associated with Th2, 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 a cell presenting antigen incorporates and processes antigen, increasing the persistence of the antigen; viii) if an agent such as EtxB and CtxB is linked to an antigen, it is possible to alter the antigen processing path by altering the binding to the immunomodulator; and ix) VtxB exerts similar immunomodulatory effects on leukocyte populations in vitro to those exerted by EtxB and CtxB. These important findings are the basis of the various aspects of the present invention, and allowed the inventors to predict that pure EtxB, CtxB and VtxB, as well as other agents capable of binding to or simulating the binding effect to GM1 or Gb3, They will be useful as immunomodulators for use in vaccines in prophylactic and therapeutic vaccination against HSV-1 infection, as well as other infections, whose prevention or treatment would benefit from immunomodulation of the types mentioned above.

Stimulation of immune responses EtxB, CtxB, VtxB and other agents capable of binding to or simulating the effects of binding to GM1 or Gb3, are able to function as immunomodulators and stimulate specific immune responses to antigenic challenge. In accordance with a first aspect of the present invention, the use is provided of: (i) EtxB, CtxB or VtxB free of intact 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 that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; as an immunomodulator for a vaccine against infectious diseases.

In accordance with a second aspect of the present invention, there is provided a vaccine composition for use against an infectious disease, wherein the infectious disease is caused by an infectious agent and wherein the vaccine composition comprises an antigenic determinant and a selected immunomoducer. from: (i) EtxB, CtxB or VtxB free of intact 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 that has an effect on signaling events Intracellular mediated by binding to GM1 or binding to Gb3; wherein said antigenic determinant is an antigenic determinant of said infectious agent. The antigen and the immunomoducer can be linked, for example, covalently or genetically linked, to form an individual effective agent. In a specific embodiment of this invention, the antigen and immunomoducer can be chemically conjugated. For example, the antigen and the immunomoducer can be chemically conjugated using heterobifunctional crosslinking reagents. In most applications of this aspect of the invention, separate administration (in which the antigen and the immunomoducer is not so linked) is preferred, because it allows separate administration of different portions.

In accordance with a third aspect of the present invention, a kit for vaccination of a mammalian subject, such as a human or veterinary subject, against an infectious disease is provided, which comprises: (a) one of the following agents: (i) ) EtxB, CtxB or VtxB free of intact 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 that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; and (b) an antigenic determinant which is an antigenic determinant of the infectious disease, for co-administration with said vaccine immunomodulator. The vaccine composition of the second aspect of the invention, and the equipment of the third aspect of the invention, can be used in a prophylactic or therapeutic vaccination method, wherein a "prophylactic vaccine" is administered to unaffected individuals to prevent development. of disease, and a "therapeutic vaccine" is administered to individuals with an existing infection to reduce or minimize infection, or to nullify the immunopathological consequences of the disease. Agents such as EtxB have the ability to alter the nature of the immune response once the infection has occurred. A therapeutic vaccine (ie, one that does not need to contain antigen) comprising said agent, may find particular use in circumstances in which the immune response has not been able to get rid of an infection. This application may be of particular use for treating a chronic disease, for example, a disease for which the causative agent is selected from the group consisting of herpes virus, hepatitis virus, HIV, TB and parasites. According to a fourth aspect of the present invention, there is provided a method for preventing or treating a disease in a host, which comprises the step of inoculating said host with a vaccine comprising at least one antigenic determinant and one immunomodulator, wherein the immunomodulator is: (i) EtxB, CtxB or VtxB free of intact 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 that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3. The vaccine may be packaged for co-administration, and may be administered by a number of different routes such as intranasal, oral, intra-vaginal, urethral or ocular administration. Currently, intranasal immunization is preferred. When a vaccine is administered intranasally, it can be administered as an aerosol or in liquid form.

The antigenic determinant and the immunomodulator can be administered to the subject as a single dose or in multiple doses. In a first embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a disease for which the infectious agent is a member of the herpes virus family. For example, the infectious agent can 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 can be HSV-1, HSDV-2, CMV or EBV. In this first embodiment, the antigenic determinant is preferably an antigenic determinant of an immediate, early or late early gene product (eg, a surface glycoprotein) of the herpes virus. If the infectious agent is HSV-1 or HSV-2, the antigenic determinant can 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 can be an antigenic determinant of gp340 or gp350 or a latent protein (for example, EBNAs 1, 2, 3A, 3B, 3C and -LP, LMP-1, -2A and 2B or EBER). In a second embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a disease for which the infectious agent is an influenza virus. In this second embodiment, the antigenic determinant is preferably an antigenic determinant of a viral coat protein (e.g., hemagglutinin and neuraminidase), or of an internal protein (e.g., nucleoprotein). In a third embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a disease for which the infectious agent is a parainfluenza virus. In a fourth embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a disease for which the Infectious agent is a 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 equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a disease for which the infectious agent is a hepatitis virus. For example, the infectious agent can be selected from the group consisting of hepatitis A, B, C and D. In particular, the infectious agent can 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 equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against meningitis. In this sixth embodiment, the infectious agent can be selected from the group consisting of Neisseria meningitidis, Haemophilus influenzae type B and Streptococcus pneumoniae. In a seventh embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against pneumonia or a tract infection respiratory. In this seventh embodiment, the infectious agent can be selected from the group consisting of Streptococcus pneumoniae, Legonella pneumophila and Mycobacterium tuberculosis. In an eighth embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a sexually transmitted disease. In this eighth embodiment, the infectious agent can be selected from the group consisting of Neisseria gonorrhoeae, HIV-1, HIV-2 and Chiamydia trachomatis. In a ninth embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a gastrointestinal disease. In this ninth embodiment, the infectious agent can 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, enterohemorrhagic and enteroaggregative E. coli, then the antigenic determinant may be an antigenic determinant of an adhesion factor or bacterial toxin. In a tenth embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a surface infection. In this tenth embodiment, the infectious agent can be selected from the group consisting of Staphylococcus aureus, Streptococcus pyogenes and Streptococcus mutans. In an eleventh embodiment, the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the equipment of the third aspect of the invention and the method of the fourth aspect of the invention, are used against a parasitic disease. In this eleventh embodiment, the infectious agent can be selected from the group consisting of malaria, Trypanosoma spp., Toxoplasma gondii, Leishmania donovani and Oncocerca spp.

Stimulation of immune responses in mucous membranes EtxB, CtxB, VtxB and other agents capable of binding to or simulating the effects of binding to GM1 or Gb3, are capable of specifically upregulating the production of antibodies in mucous membranes. The vaccine immunomodulator of the first aspect of the invention, the vaccine composition of the second aspect of the invention and the equipment of the third aspect of the invention, are particularly effective against diseases where protection against infection or treatment is carried out in vivo. by an immune response in mucous membranes, for example, against diseases in which during infection, the infectious agent binds, colonizes or has access to the mucosa. Examples of such diseases include diseases caused by viruses (HIV, HSV, EBV, CMV, influenza, measles, mumps, rotavirus, etc.), diseases caused by bacteria (species of E. coli, Salmonella, Shigella, Chlamydia, N. gonorrhoeae , T. pallidum, Streptococcus, including those that cause dental caries), and diseases caused by parasites. In a preferred embodiment of the second aspect of the present invention, a vaccine against HSV-1 infection is provided, comprising at least one antigenic determinant of HSV-1 and an immunomodulator, wherein the immunomodulator is: (i) EtxB, CtxB or VtxB free of intact toxin; (i) 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 that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3. Preferably, the immunomodulator is EtxB. In a preferred embodiment of the third aspect of the present invention, a kit for vaccination of a mammalian subject against an HSV-1 is provided, which comprises: a) a vaccine immunomodulator which is: (i) EtxB, CtxB or VtxB free of intact 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 that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; and b) at least one antigenic determinant of HSV-1, for co-administration with said vaccine immunomodulator. According to a fifth aspect of the invention, the use is provided of: (i) EtxB, CtxB or VtxB free of intact toxin; (i) 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 that has an effect on intracellular signaling events mediated by binding to G 1 or binding to Gb 3, to upregulate the production of antibodies on mucosal surfaces. One can also upregulate the production of binding antibodies in the absence of complement. Preferably, S-IgA is produced in accordance with the fifth aspect of the invention. In this fifth aspect of the present invention, the agent can be used in conjunction with one or more antigenic determinants.

Subrequlation of the pathological components of immune responses The inventors also found that when pure EtxB was used as an immunomodulator in the manner described, the deleterious effects of Th2-associated responses, such as the generation of potentially high levels of IgE, were avoided. Despite this, the immune response induced by the use of EtxB (or CtxB or VtxB) as an immunomodulator seems to favor the induction of Th2-associated cytokines. In other words, EtxB (or CtxB) induces a change of a Th1-type response to Th2. This has allowed the inventors to predict that pure EtxB, CtxB or VtxB, as well as other agents capable of binding to or simulating the binding effect to GM1 or Gb3, will be able to downregulate pathological components of the immune response associated with the activation of Th1 and Th2. According to a sixth aspect of the present invention, the use of: (i) EtxB, CtxB or VtxB free of intact toxin is provided.; (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 that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; to downregulate the pathological components of immune responses associated with Th2. The pathological components of immune responses associated with Th1 can also be down-regulated. It is known that EtxB and CtxB bind to GM1 and induce differential effects on lymphocyte populations, including specific depletion of CD8 + T cells and activation of associated B cells (see WO 97/02045). Therefore, it is thought that EtxB and CtxB alter the balance of the immune response, so that the inflammatory reactions associated with Th1 are down-regulated, while the responses associated with Th2 are up-regulated. Th1 responses include the secretion of yIFN by activated T cells, which leads to activation of macrophages and delayed-type hypersensitivity reactions. These responses can 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 antibodies, especially IgA. It has now surprisingly been found that when EtxB is used as an immunomodulator in the manner described, the deleterious effects of Th2-associated responses are avoided, such as the generation of potentially high levels of IgE pathologies. Therefore, EtxB and CtxB are able to down-regulate the pathological components of the immune response associated with the activation of Th1 and Th2. These responses are modulated in favor of the production of high levels of binding antibodies in the absence of complement, and production of secretory IgA on mucosal surfaces. The use of an agent according to the sixth aspect of the invention is particularly useful for therapeutic vaccination in diseases in which immunopathological mechanisms are involved. Examples of such diseases are HSV-1, HSV-2, TB and HIV. The first and sixth aspects of the invention can be combined. In other words, agents such as EtxB can be used simultaneously as an immunomodulator and therapeutic agent. For example, in diseases where immunopathological mechanisms intervene, the use of a vaccine that incorporates agents such as EtxB or CtxB can not only act to limit the infection, but also to suppress the pathological processes of the disease. The immunomodulation agent acts in this manner both prophylactically and therapeutically. Examples of infections where vaccination is thus likely to be of this particular value form, include those caused by the herpes virus family, as well as pathogens from the gastrointestinal and respiratory tracts.

Immunomodulation of antigenic processing pathway a) Prolongation of the presentation The present inventors have also found that when EtxB (or CtxB or VtxB) is used as an immunomodulator, the route of processing and incorporation of the antigen is altered. The presence of subunit B causes prolonged presentation, possibly altering antigen trafficking within antigen-presenting cells, so that antigen degradation is delayed and therefore maintained for prolonged periods. This characteristic of presentation of the antigen associated with subunit B means that vaccines that incorporate an agent according to the present invention will have increased persistence of the antigen and will lead to sustained immunological memory. According to a seventh aspect of the present invention, the use is provided of: (i) EtxB, CtxB or VtxB free of intact 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 that has an effect on intracellular signaling effects mediated by binding to GM1 or binding to Gb3; as an immunomodulator in a vaccine, to prolong the presentation of the antigen and provide a sustained immunological memory in a mammalian subject. According to an eighth aspect of the present invention, there is provided a vaccine composition for use against an infectious disease, which comprises an antigenic determinant and an immunomodulator selected from: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB having GM1 binding activity, or an agent other than VtxB having binding activity to Gb3; or (iii) an agent that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; wherein said antigenic determinant is an antigenic determinant of said infectious disease, and wherein the immunomodulator prolongs the presentation of the antigenic determinant and provides a sustained immunological memory. b) Intracellular localization of the antigen to a pathway associated with MHC-I or MHC-II As mentioned above, the antigen and the immunomodulator in a therapeutic or prophylactic vaccine can be linked, for example, covalently or genetically linked, to form an agent infectious individual. The present inventors have found that it is possible to direct the antigen to different compartments of the cell, and therefore different ways of presenting antigen by altering the binding of the antigen to the immunomodulator. By binding the antigen or the antigenic determinant to the immunomodulator it is possible to facilitate, in some way, the translocation of the antigen through the endosomal membrane into the cytosol. The present inventors predict that this would increase the loading of antigenic peptides on the MHC class I molecules. The use of an antigen-immunomodulatory conjugate can therefore be used to specifically increase the activation of cytotoxic T cells (CTL). The induction of CTL is beneficial for the prevention and treatment of many diseases, especially those caused by viruses, intracellular bacteria, and parasites. The binding of the antigen-immunomodulatory conjugate can also be chosen, so that the antigen is delivered in the nucleus.

According to a ninth aspect of the present invention, there is provided a conjugate comprising an antigen or antigenic determinant and an immunomodulator selected from: (i) EtxB, CtxB or VtxB free of intact 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 that has an effect on vesicular incorporation mediated by binding to GM1 or binding to Gb3. According to a tenth aspect of the present invention, there is provided a vaccine composition for use against an infectious disease, wherein the infectious disease is caused by an infectious agent, whose vaccine composition comprises a conjugate of an antigen or antigenic determinant and an immunomodulator selected from: (i) EtxB, CtxB or VtxB free of intact toxin; (I) 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 that has an effect on vesicular incorporation mediated by binding to GM1 or binding to Gb3; wherein said antigen or antigenic determinant is an antigen or antigenic determinant of said infectious agent.

The antigen or antigenic determinant can be linked to the immunomodulator by various methods including genetic ligament or chemical conjugation. In a first preferred embodiment, the conjugate is a fusion protein obtained by genetic ligation of the antigen or binding of the antigenic determinant to the immunomodulator. Preferably, the antigen or antigenic determinant is genetically linked to the C terminus of the immunomodulator. In a second preferred embodiment, the antigen or antigenic determinant is chemically conjugated to the immunomodulator. Preferably, the antigen or antigenic determinant is conjugated to the immunomodulator using a bifunctional crosslinking reagent, such as a heterobifunctional crosslinking reagent. More preferably, the entanglement agent is N-y (-maleimido-butyroxyl) -succinimide (GMBS) ester or N-succinimidyl (3-pyridyl-dithio) -propionate ester (SPDP). The vaccine composition can be administered by a number of different routes such as intranasal, oral, intra-vaginal, urethral or ocular administration. Intranasal immunization is preferred. In accordance with an eleventh aspect of the present invention, the use is provided of: (i) EtxB, CtxB or VtxB free of intact toxin; (I) an agent other than EtxB or CtxB having GM1 binding activity, or an agent other than VtxB having Gb3 binding activity; or (ii) an agent that has an effect on vesicular incorporation mediated by binding to GM1 or binding to Gb3; in a conjugate with antigen or antigenic determinant to direct the delivery of said antigen or antigenic determinant to the cytosol or nucleus of a cell presenting antigen. According to a twelfth aspect of the present invention, the use is provided of: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB having GM1 binding activity, or an agent other than VtxB having binding activity to Gb3; or (iii) an agent that has an effect on vesicular incorporation mediated by binding to GM1 or binding to Gb3; in a conjugate with antigen or antigenic determinant to upregulate the presentation of said antigenic determinant, or an antigenic determinant derived from said antigen, by MHC class I molecules. Preferably, the use of the conjugate of the twelfth aspect of the invention is used in combination with the use of the agent according to the fifth aspect of the invention to stimulate strong CTL responses, and to up-regulate mucosal antibody production. This activity would be particularly useful in the prevention and treatment of viral infections, for example, influenza.

EtxB as the preferred immunomodulator It was mentioned above that EtxB and CtxB have similar properties. However, the present inventors have found that rEtxB is a more potent and efficient immunomodulator than rCtxB. Accordingly, the preferred immunomodulator is EtxB, or agents that simulate the effects of EtxB.

EBV EBV is one of the eight known herpes viruses in humans. The infection usually occurs in early childhood; however, clinical symptoms are usually weak or undetectable at this stage. The primary infection with EBV in later stages of life is associated with infectious mononucleosis (IM), which is the second most frequent disease in adolescence in the United States. EBV also has oncogenic potential. There is a strong link between EBV and Burkitt's endemic lymphoma (BL) and undifferentiated nasopharyngeal carcinoma (NPC). Also, a large proportion of the lymphomas that occur in immunocompromised patients is caused by EBV, and it has been shown that there is an association between certain Hodgkin lymphomas and EBV. Cells infected in a latent form by EBV express a small number of so-called "latent" proteins. These include six nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C and -LP), three integral membrane proteins (LMP-1, 2A and 2B) and two molecules of non-polyadenylated RNA derived from viruses (EBERs) with a function in RNA splicing. The latent EBV membrane protein 1 (LMP-1) is present in the plasma membrane of infected cells. It is also expressed in nasopharyngeal carcinomas (NPCs) and EBV-positive Hodgkin lymphomas (HD), which indicates a role for LMP-1 in the development of these tumors. The LMP-1 gene can alter the phenotype of uninfected cells causing upregulation of cell surface activation markers, promoting cell proliferation. LMP-1 can also alter the signaling pathways, and has antiapoptotic effects. A cellular immune response directed against this viral antigen has not been demonstrated with a certain degree of certainty in healthy carriers or patients with tumors. Many animal viruses have evolved mechanisms to prevent their detection by the host's immune system. Commonly, these mechanisms involve interference with the translocation system of peptides associated with TAP. It is thought that EBV has also evolved similar mechanisms to avoid its detection by the immune system, thus allowing its persistence in the host. This explains why certain cellular immune responses are not detectable for the latent EBV protein, EBNA1, and could explain the apparent absence of such responses against LMP1. According to a thirteenth aspect of the invention, a vaccine composition is provided which comprises: a) one of the following agents: (!) EtxB, CtxB or VtxB free of intact 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 that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; and b) an EBV antigen, for use in the treatment and / or prevention of diseases associated with EBV. In particular, the vaccine composition of the thirteenth aspect of the present invention comprises EtxB, CtxB, or an agent other than EtxB or CtxB having GM1 binding activity. According to a fourteenth aspect of the present invention, there is provided a therapeutic composition comprising: (i) EtxB, CtxB or VtxB free of intact 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 that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; for its use in the treatment of diseases associated with EBV.

In particular, the therapeutic composition of the fourteenth aspect of the present invention comprises EtxB, CtxB, or an agent other than EtxB or CtxB, which has GM1 binding activity. Based on the knowledge that EtxB performs joint blockade with LMP1, and that EtxB promotes fragmentation of LMP-1, the theory has been formulated that EtxB (and other agents such as CtxB having GM1 binding activity) will be useful for stimulating anti-EBV immune responses. This activity has applications in vaccines to prevent diseases associated with EBV, and in therapeutic treatments to treat these diseases once they have been developed. Without wishing to be limited by theory, it is thought that when EtxB performs joint blockade with LMP-1, the antigen is processed by a different intracellular pathway, which allows the antigen to avoid the normal processing pathway that is blocked by the virus . The antigen is presented in this way efficiently on the cell surface. The action of EtxB can also cause epitopes different from the antigen to be presented on the cell surface, from those that are presented if the antigen was processed through the conventional route. The vaccine of the thirteenth aspect of the present invention can be used to prevent infection with EBV, or the development of diseases associated with EBV in individuals infected with EBV. The vaccine may also comprise a separate adjuvant, or the agent (such as EtxB or CtxB) may act as an adjuvant by itself.

The agents specified in the fourteenth aspect of the present invention can be used alone (ie, without antigen) in the treatment of an EBV-associated disease, which has already developed in a subject. The preferred agent for use in the thirteenth and fourteenth aspect of the invention is EtxB. The EBV antigen is an antigen derived from EBV itself, or an antigen that is made to be expressed by a host cell infected with EBV, by the action of EBV. Preferably, the antigen is a latent EBV membrane protein. Particularly preferred are the antigens LMP-1, LMP-2A, LMP-2B and EBNA-1, as well as the antigenic fragments thereof. The antigen can be isolated directly from cells infected with EBV, or it can be obtained by synthetic or recombinant means. The thirteenth and fourteenth aspects of the present invention are particularly suitable for the treatment and / or prevention of the following diseases: infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinomas and Hodgkin lymphomas. It is thought that these aspects of the invention will be particularly suitable for the treatment and / or prevention of nasopharyngeal carcinomas and Hodgkin lymphomas. The vaccine or therapeutic composition according to the thirteenth and fourteenth aspects of the invention, can be used to prevent the development of, or to treat, an EBV-associated disease in a mammalian subject, by administering an immunologically effective amount to the subject. The mammalian subject may be, for example, a healthy uninfected individual or one infected with EBV, an immunodeficient individual, or an individual with an EBV-associated disease. The vaccine can be administered by any suitable route. The agent and the antigen can be co-administered to the mammalian subject, or administered separately. The agent and the antigen can be separated or linked, for example, covalently bound or genetically linked, to form an individual effective agent.

Signaling associated with GM-1 and Gb3 Without wishing it to be limited by theory, it is thought that binding to GM1 or Gb3 can directly or indirectly trigger intracellular signaling. The present inventors have also found evidence to suggest that EtxB interacts with at least some other receptor involved in the intracellular signaling event associated with GM1. It may be that the binding of EtxB (or CtxB) to GM1 facilitates binding to a protein, the same protein that triggers intracellular signaling. It is unknown what specifically triggers the signaling event, it can be phosphorylation of GM1 or the protein. When EtxB / CtxB binds to GM1 on the cell surface, bound G1 is incorporated into vesicles (Williams ef al (1999) Immunoloy Today 20; 95-101). GM1 and other glycolipids (such as Gb3) are known to be preferably located in "floating membrane masses" in which key protein receptors are also found. Therefore, it is possible that the incorporation of GM1 as a result of binding to subunit B causes a joint blocking of such proteins which leads to their being triggered to mediate intracellular signaling events.

Definitions An adjuvant is a substance which does not specifically increase the immune response to an antigen, unlike a vehicle of a vaccine, whose purpose is to direct the antigen to a desired site. The term "immunomodulatory" is used herein to mean an agent which acts, as an adjuvant, to stimulcertain immune responses, but which also directs the immune response in a particular direction. The term "co-administration" is used to mean that the site and time of administration of the antigen and immunomodulator are such that the necessary immune response is stimul. Thus, although the antigen and the immunomodulator may be administered at the same time and at the same site, there may be advantages in administering the antigen at a different time and / or at a different site of the immunomodulator. For example, the antigen and immunomodulator can be administered together in a first step and then the immune response can be taken to a second step by the administration of the antigen alone.

The term "antigenic determinant" as used herein, refers to a site on an antigen which is recognized by an antibody or T cell receptor. Preferably, it is a small peptide derived from or as part of an antigen of protein, however, the term also aims to include glycopeptides and carbohydr as antigenic determinants. The term also includes modified sequences of amino acids or carbohydr which stimulresponses recognized by the intact organism. There are a number of known methods by which it is possible to identify antigenic determinants for a particular infectious agent. For example, potential protective antigens can be identified by elevating immune responses in infected or convalescent patients, in infected or convalescent animals, or by monitoring in vitro immune responses to preparations containing antigen. For example, i) serum samples from infected or convalescent patients or infected or convalescent animals can be selected against lys of intact cells of an infectious agent, or lys of cells infected by said agent, using the standard Western blotting technique to detect those antigens recognized by immune serum; ii) serum samples from infected or convalescent patients or infected or convalescent animals can be selected against partially or highly purified antigens of an infectious agent, or lys of cells infected by said agent, using the standard ELISA technique, in which they are used partially or highly purified antigens to cover cavities for microtitre, by immunoblotting to detect those antigens recognized by immune sera; iii) serum samples from infected or convalescent patients or infected or convalescent animals can be selected against intact cell lys derived from recombinant expression systems that code for one or more antigens of interest, and using standard ELISA or Western blotting techniques to detect those antigens recognized by the immune serum; iv) serum samples from infected or convalescent patients or infected or convalescent animals can be screened against an expression library containing cloned genes of the infectious agent of interest, using colony blot immunodetection to identify clones expressing antigens, or fragments of the same, which are recognized by the immune serum; ov) PBL from the blood of infected or convalescent patients or PBL, lymph node cells, spleen cells or laminae cells of infected or convalescent animals can be cultured in vitro in the presence of partially or highly purified antigens derived from either a infectious agent, or lys of cells infected by said agent, or a recombinant expression system that codes for one or more antigens, to detect proliferative responses of antigen-specific T cells. Alternatively, it is possible to detect gene products which are essential for the in vivo survival of pathogens, as exemplified by the technique of mutagenesis labeled with identification developed by Holden or the detection of gene products specifically induced in vivo such as IVET ( of In Vivo Expression) developed by Mekalanos or differential fluorescence induction developed by Falkow, identify a subset of genes among which potential protective antigens are likely. Using these methods, the gene products can be selected as noted above. The genes can be cloned into expression vectors and the antigens recovered for inclusion in vaccine formulations together with agents that modulate an activity associated with glycosphingolipids. There are a number of known methods by which it is possible to isolate antigens for a particular infectious agent. For example, surface components of an infectious agent comprising one or more potential protective antigens can be extracted from the agent, or from cells infected by the agent, by the use of methods that allow the recovery of the antigens. This may include the use of cell disruption techniques to lyse cells such as sound treatment and / or detergent extraction. Centrifugation, ultrafiltration or precipitation can be used in collected antigen preparations. The antigen preparation containing HSV-1 glycoproteins described in Richards et al., (1998) J. Infect. Dis. 177; 1451-7, exemplifies said method. In addition, the antigens of an infectious agent or of cells infected by said agent can be extracted through a variety of procedures, including but not limited to, extraction with urea, extraction with alkali or acid, or extraction with detergent and then undergo chromatographic separation. The material recovered in null or elution peaks comprising one or more potential protective antigens in vaccine formulations can be used. Alternatively, genes encoding one or more potential protective antigens can 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. Saccharomyces cerevisiae, insect and mammalian cell lines. The antigens can be recovered by conventional extraction, chromatographic and / or separation methods. 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 can be produced through a method in which the gene or genes encoding the chain (or chains) of specific polypeptides from which the protein is formed, is inserted into a suitable vector and then it is used to transfect a suitable host. For example, the gene encoding the polypeptide chain from which the EtxB assembly can be inserted, for example plasmid pMM68, which is then used to transfect host cells, such as Vibrio sp. 60. The protein is purified and isolated in a manner known per se. Mutant genes expressing active mutant CtxB, EtxB or VtxB proteins can be produced by methods known from the wild-type gene. The terms "CtxB", "EtxB" and "VtxB" also include mutant molecules and other synthetic molecules (containing parts of CtxB, EtxB or VtxB) which retain the ability to bind to G 1 or Gb3 or the ability to simulate effects of binding to GM1 or Gb3. Agents other than EtxB and CtxB which retain the GM1 binding activity, and agents other than VtxB which retain the Gb3 binding activity include antibodies that bind to GM1 or Gb3. For the production of antibodies, several hosts, including goats, rabbits, rats, mice, etc., can be immunized by injection. with GM1 or Gb3 or any derivative or homologue thereof. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to Freund's adjuvants, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, limpet hemocyanin, and dinitrophenol. BCG (Bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants. Humanized monoclonal antibodies may be preferred in this invention. Monoclonal antibodies can be prepared using any technique that 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 (1993) Proc Nati Acad Sci 80: 2026-2030) and the EBV-hybridoma technique (Colé 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 can be used to obtain a molecule with specific antigen-specific character and biological activity (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 (U.S. Patent No. 4,946,779) can be adapted to produce single chain antibodies specific for target interaction component. The antibodies can also be produced by inducing in vivo production in the lymphocyte population or by selecting recombinant immunoglobulin libraries or panels from highly specific binding reagents as described in Orlandi et al (1989, Proc Nati Acad Sci 86: 3833-3837 ), and Winter G and Milstein C (1991; Nature 349: 293-299). Antibody fragments containing specific binding sites for GM1 or Gb3 can also be generated. For example, such fragments include, but are not limited to F (ab ') 2 fragments which can be produced by digestion of pepsin from the antibody molecule and Fab fragments which can be generated by reducing the bisulfide bridges of the F (ab ') 2 fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specific character (Huse WD et al (1989) Science 256: 1275-128 1). Peptide libraries or organic libraries can be made by combinatorial chemistry and then analyzed for their binding capacity to GM1 / Gb3. Synthetic compounds, natural products and other sources of potentially active materials at the biological level can be selected in a number of ways considered routine for those skilled in the art. GM1 or Gb3 or fragments thereof can be selected to analyze peptides or molecules in any of a variety of selection techniques. The molecule can be free in solution, fixed to a solid support, carried on a cell surface or located intracellularly. The abolition of activity or the formation of binding complexes between GM1 or Gb3 and the agent under test can be measured.

Another way to determine binding to GM1 / Gb3 would be to use purified GM1 / Gb3 to cover microtiter plates. Following blocking, the agent under investigation is applied to the plate and allowed to interact before washing and detection with antibodies specific to said agent. The conjugation of the antibodies either directly or indirectly to an enzyme or radiolabel allows subsequent quantification of binding either using collimetic methods or based on radioactivity (ELISA or RIA, respectively). Another way to determine binding to GM1 / Gb3 would be to bind the saccharide portion of GM1 / Gb3 to a suitable column matrix in order to allow standard affinity matography to be performed. Removal of known compounds applied to the diluent column can be used as evidence of binding activity or alternatively, where mixtures of compounds are applied to the column, elution and subsequent analysis would determine the properties of the ganglioside binding agent. . In the case of proteins, the analysis would involve peptide sequencing and tryptic mapping by digestion followed by comparisons with available databases. In the case that the eluted proteins can not be identified in this way, then standard biochemical analysis can be used, for example, mass determination by laser desorption mass spectrometry to subsequently characterize the compound. Molecules that are not proteins eluted from affinity columns for G 1 would be analyzed by HPLC and mass spectrometry of individual homogeneous peaks. Another way to determine the binding capacity of GM1 / Gb3 and the precise affinity of the interaction would be to use plasmon surface resonance as reported previously [Kuziemko et al (1996) Biochem 35: 6375-6384). Alternatively, phage display can be employed in the identification of candidate agents which bind to GM1 or Gb3. The phage display is a molecular selection protocol, which uses recombinant bacteriophages. The technology involves the transformation of bacteriophages with a gene encoding 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 thereof). or homologous thereof) that codes for it. The transformed bacteriophage (which is preferably adhered to a solid support) expresses the appropriate ligand (such as the candidate agent) and deploys it in its phage coat. The entity or entities (such as cells) that possess the target molecules which recognize the candidate agent are isolated and amplified. Successful candidate agents are then characterized. Phage display has advantages over standard affinity ligand analysis technologies. The phage surface deploys the candidate agent in a three-dimensional configuration, closely resembling its natural conformation. This allows a more specific and higher affinity binding for analysis purposes. Another analysis technique provides high throughput analysis of agents that have adequate binding affinity to GM1 or Gb3 and is based on the method described in detail in WO 84/03564. In summary, large amounts of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test agents react with the target interaction component fragments and are washed. A linked objective interaction component is then detected - for example by suitably adapting methods well known in the art. A purified objective interaction component can also be directly coated on plates for use in the aforementioned drug analysis techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. In all aspects of the invention, the agent having GM1 binding activity or Gb3 binding activity may be able to bind to GM1 or Gb3 receptors. EtxB is said agent which is capable of intertwining with GM1 receptors by virtue of its pentameric form. There are several methods to identify agents which have an effect on intracellular signaling events mediated by binding to GM1 / Gb3 but which do not bind themselves to G1 or Gb3. For example, if an agent upregulates CD25 or MHC class II in B cells, or upregulates CD25 or promotes CD8 + T cell apoptosis, or upregulates secretion of IL-10 by monocytes, but the agent shows no binding to GM1 or Gb3 (e.g. , by means of one of the binding tests described above), then it can be concluded that the agent is capable of simulating the binding effect to GM1 / Gb3. The invention will now be illustrated with reference to the accompanying drawings, tables and the following examples. The examples refer to the figures in which: Figure 1 shows the stimulation of total Ig and IgA in serum (MS) and IgA in eye washings (EW) in mice immunized with HSV-1 / rEtxB glycoproteins. Figure 2 shows proliferation of MLN T cells (mesenteric lymph node) or CLN lymphocytes (cervical lymph node) in mice immunized with HSV-1 / rEtxB. Figure 3 shows proliferation of T cells from MLN and CLN cells from mice immunized intranasally with HSV-1 GP in the presence of 1-20 g of EtxB. Figure 4 shows the level of anti-HSV-1 Ig in serum in mice followed by administration of HSV-1 glycoproteins three times at 10 day intervals with varying amounts of rEtxB or rCtxB as adjuvant. Figure 5 shows the isotype distribution of Ig in MS followed by infection with HSV-1 or immunization with Gp of HSV-1 in the presence of EtxB or CtxB as immunomodulator.

Figure 6 shows the distribution of subclasses of Ig followed by intranasal administration of Gp of HSV-1 with either rEtxB or rCtxB as immunomodulator. Figure 7 shows the immunogenic effect of different amounts of rEtxB or rCtxB on the level of HSV-1 specific IgA in ocular washings followed by administration with HSV-1 glycoproteins. Figure 8 shows immunoglobulin response in serum followed by immunization of mice with HSV-1 or imitation glycoproteins (gp) alone or in the presence of adjuvant. Figure 9 shows mucosal IgA in ocular washes followed by intranasal immunization of mice with HSV-1 or imitation glycoproteins alone or in the presence of adjuvant. Figure 10 shows mucosal IgA in vaginal washings followed by intranasal immunization of mice with HSV-1 or imitation glycoproteins (gp) alone or in the presence of adjuvant. Figure 11 shows the level of HSV-1 specific immunoglobulin in sera from mice immunized with HSV-1 glycoproteins in the presence of different doses of rEtxB as an adjuvant. Figure 12 shows the level of IgA in ocular washes of mice immunized with HSV-1 glycoproteins in the presence of varying concentrations of rEtxB.

Figure 13 shows the level of IgA in vaginal washings of mice immunized with HSV-1 glycoproteins in the presence of varying concentrations of rEtxB. Figure 14 shows IgG subclass distribution of the antibody response in serum to HSV-1 followed by intranasal immunization with Ctx / CtxB or rEtxB or ocular infection with HSV-1. Figure 15 shows cytokine production from cultures of lymph node cells taken from mice which were either infected with HSV-1 by ocular scarification, or immunized by intranasal administration of HSV-1 glycoproteins with Ctx / CtxB or rEtxB as adjuvant .

EXAMPLE 1 rEtxB can be used together with HSV-1 Gp for immunization Mice were immunized intranasally three times with 10 pg of HSV-1 (Gp) glycoproteins with either 10 or 20 pg of rEtxB. The controls were unmanipulated or were given an imitation preparation of viral glycoprotein (imitation) derived from cells not infected with HIV tissue culture. Antibody levels are expressed as a percentage of post-infection levels. The production of total Ig and IgA in the serum and IgA in ocular washes was stimulated by HSV-1 / rEtxB glycoproteins (figure 1). The present inventors have also shown that doses of rEtxB as low as 0.1 g are also effective in stimulating such responses. In addition, T lymphocytes from mice immunized from the cervical lymph node (which is local to the vaccination site) and from the mesenteric lymph node (which is distant from the vaccination site) were shown to proliferate when cultured in vitro with HSV-1, but not when they were cultured in vitro with imitation HSV-1 Gp or without antigen (figure 2). The proliferation in response to HSV-1 Gp of MLN and CLN T lymphocytes from mice immunized with HSV-1 Gp and variable amounts of EtxB is shown in Figure 3. The production of anti-HSV-1 Ig in serum in mice followed by administration of HSV-1 glycoproteins at three-day intervals with varying amounts of EtxB (or CtxB) is shown in Figure 4. Finally, mice immunized with HSV-1 and rEtxB showed to have a decrease in virus diffusion followed by corneal scarification with HSV-1 (Table 1) and a decrease in local spread (edema and eyelid disease), spreading to the trigeminal ganglion (zosteriform infection), spread to the central nervous system (encephalitis) and latency compared to controls (table 2). Tables 1 and 2 show the reduction in virus diffusion, clinical disease and latency in mice immunized with HSV-1 rEtxB.

TABLE 1 Incidence of virus diffusion of the eye followed by corneal scarification of mice with HSV-1 (SC16) Day after 10μ? of rEtxB + gp 20μ? of rEtxB + gp 20μ? of rEtxB + gp2 of HSV-1 (%) 'infection of HSV-1 (%) imitation (%) 1 0 30 60 2 60 80 95 3 60 80 95 6 10 0 70 7 10 0 70 8 0 0 10 9 0 0 0 Percentage of animals from which the fluid washing the ocular secretions revealed the presence of live viral particles in a plaque test. 2A Imitation infected animals were given a glycoprotein inoculum prepared from uninfected tissue culture cells.

TABLE 2 Clinical disease followed by corneal scarification of mice with HSV-1 (SC16) Ulcers2 Edema Disease Infection Encephalitis Latency corneas1 of Zosteriforme TG1 TG2 TG3 eyelids K ^ g of 80% 0% 0% 0% 0% 22% 11% 0 % rEtxB + gp of HSV-1 2 (Vg of 70% 0% 0% 0% 0% 80% 10% 0% rEtxB + gp of HSV-1 20Mg of 80% 45% 55% 40% 40% 83% 30 % 16% rEtxB + imitation gp 1Latency was determined by extraction of the trigeminal ganglion (TG) from surviving mice 2 months after infection and co-culture with Vero cells. The figures given are for each of the TG lobes (TG1, TG2 and TG3). 2The figures are the percentage of animals that show signs of the symptoms described at any point during acute infection. Each mouse was examined on a daily basis during the first 11 days of infection. N = 15 per group EXAMPLE 2 rCtxB and rEtxB act as immunomodulators When EtxB is used as an immunomodulator, the Ig isotype distribution is tilted (Figure 5). The distribution of Ig subclasses differs depending on whether rCtxB or rEtxB is used as an immunomodulator (Figure 6).

EXAMPLE 3 rEtxB is an immunomodular more efficient than rCtxB The HSV specific IgA levels (Figure 7) are higher followed by stimulation with rEtxB / Gp of HSV-1 than with rCtxB / Gp of HSV-1.

EXAMPLE 4; (FIGURE 8) The mice were immunized three times intranasally with HSV-1 glycoproteins alone, an imitation preparation of HSV-1 glycoproteins (prepared by taking uninfected tissue culture cells and subjecting them to treatment regimens identical to those employed for isolation and protein purification of HSV-1), or HSV-1 glycoproteins in combination with a variety of putative mucosal adjuvants. In each case, the HSV-1 glycoprotein dose was 10μ9 per immunization, and these were combined with 10μg of recombinant EtxB, or CtxB as adjuvant, or a mixture of? .dμ? of Ctx and 10 g of CtxB. Three weeks after the final immunization, blood samples were collected and anti-HSV-1 total antibodies were measured by ELISA. Amounts of antibodies are expressed as a percentage of the stimulated levels followed by ocular infection induced by scarification with 105 pfu of strain SC16 of HSV-1. The data (shown in Figure 9) show that the strongest antibody response in serum is stimulated when antigen is combined with a mixture of intact 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 9) The mice were immunized as described in the example 4. The production of secretory IgA in the eye was evaluated by taking tear washes for consecutive days and then these samples were pooled and subjected to ELISA analysis using a specific anti-IgA detector antibody. Amounts of antibodies are expressed as a percentage of stimulated levels followed by ocular infection induced by scarification with 105 pfu of strain SC16 of HSV-1. The data clearly show (Figure 10) that high levels of secreted anti-HSV-1 antibodies are produced followed by immunization in the presence of either Ctx / CtxB or EtxB. Unlike the results of the analysis of antibody responses in serum, there was no difference in the level of antibodies in the eye between those animals immunized with Ctx / CtxB or EtxB as adjuvants. As with the serum antibody, there was clear evidence that rCtxB is a very poor adjuvant.

EXAMPLE 6: (FIGURE 10) The mice were immunized as described in Example 4. The production of secretory IgA in the vagina was evaluated by taking washes from the genital tract for consecutive days and then these samples were pooled and subjected to ELISA analysis using an anti-cell specific antibody. -lgA. The amounts of antibodies are expressed as endpoint titers which were calculated by linear regression analysis. The data clearly demonstrate that high levels of secreted anti-HSV-1 antibodies occur at distant sites in mucosal tracts followed by immunization in the presence of either Ctx / CtxB or EtxB. In the vagina, the highest levels of antibodies were released followed by immunization in the presence of rEtxB. The lowest levels were released followed by immunization with Ctx / CtxB and very little secretion was triggered by the use of rCtxB as an adjuvant.

EXAMPLE 7: (FIGURE 11) Mice were immunized three times intranasally with HSV-1 C ^ g glycoproteins either alone or in the presence of intensified doses of rEtxB as an adjuvant. Three weeks after the final immunization, blood was taken and anti-HSV-1 antibody levels were evaluated by ELISA. Amounts of antibodies are expressed as a percentage of stimulated levels followed by ocular infection induced by scarification with 105 pfu of strain SC16 of HSV-1. The data clearly demonstrate that the ability of rEtxB to elicit antibody responses to added heterologous antigens is a dose-dependent phenomenon with maximal response occurring at approximately 20-50μg of rEtxB. Furthermore, it is clear that at doses of 20μg rEtxB and more, the level of anti-HSV-1 antibodies stimulated by intranasal infection is comparable or superior to that stimulated by a live virulent virus infection.

EXAMPLE 8: (FIGURE 12) The mice were immunized as described in example 7. The production of secretory IgA in the eye was evaluated by taking washings of tears for consecutive days and then these mixtures were pooled and subjected to ELISA analysis using an anti-cell specific antibody. -lgA. Amounts of antibodies are expressed as a percentage of stimulated levels followed by ocular infection induced by scarification with 105 pfu of strain SC16 of HSV-1. The data demonstrate that the maximal IgA responses in the eye are stimulated when HSV-1 glycoproteins are given in combination with 20μ9 rEtxB or more. However, in this dose the levels of IgA production are lower than those triggered during eye virus infection.

EXAMPLE 9. (FIGURE 13) The mice were immunized as described in the example 7. The production of secretory IgA in the vagina was evaluated by taking washings of the genital tract for consecutive days and then these samples were pooled and subjected to ELISA analysis using a specific anti-IgA detector antibody. The amounts of antibodies are expressed as endpoint titers which were calculated by linear regression analysis. The data show that optimal anti-HSV-1 responses are stimulated in the vagina when 20 μg or more of rEtxB is used as an adjuvant.

EXAMPLE 10: (FIGURE 14) Mice were infected with either 105 pfu of SCV strain of HSV-1 by scarification in the cornea or immunized three times intranasally with 10 of HSV-1 glycoproteins in combination with Ctx / CtxB or rEtxB. Three weeks after the final inoculation, serum was taken and analyzed by ELISA for the presence of IgG1 and IgG2 = against HSV-1. The amounts of antibodies are expressed as endpoint titers which were calculated by linear regression analysis (Figure 7A). The data clearly show that the nature of the antibody response to HSV-1 is influenced by the way in which antigens present in the immune system. Infection with HSV-1 predominantly activates the production of the antibody associated with Th1, as characterized by the high levels of the isotype of the complement binding antibody, IgG2a. The infection stimulates relatively low levels of the IgG isotype associated with Th2, IgG1. This profile of the immune response is clearly visible when the data is expressed as a ratio of IgG1: IgG2a as shown in Figure 7b. The ratio is substantially less than 1 followed by infection. Intranasal immunization in the presence of Ctx / CtxB as an adjuvant triggers the release, predominantly, of lgG1 associated with Th2. Important levels of IgG2a also occur suggesting that Ctx / CtxB causes the activation of Th1 and Th2 cells. The activation of both responses and the relative dominance of Th2 is reflected in the ratio lgG1: IgG2a which is approximately 3. Interestingly, the nature of the response to HSV-1 stimulated by rEtxB as adjuvant is almost exclusively dominated by Th2 . Only high levels of lgG2a produce high levels of IgG1. This strong deviation towards the Th2 response is reflected in an IgG1: IgG2a ratio of approximately 9.

EXAMPLE 11: (FIGURE 15) Mice were either infected with 105 pfu of SCV strain HSV-1 by scarification in the cornea or immunized 3 times intranasally with 10 μ? of HSV-1 glycoproteins in combination with Ctx / CtxB or rEtxB. Three weeks after the final inoculation, lymph nodes were removed from animals and used to generate individual cell suspensions that were cultured either in the presence of destroyed HSV-1 or an imitation preparation of virus from uninfected cells of culture. tissues. On days 4 to 7 of the cultures, cell samples were removed and subjected to ELISA analysis to reveal the secretion of cytokines. The data clearly show that the T cells in the cultures were able to respond to HSV-1, but not in an important way to preparations of virus mimicry. Lymph node cells taken from mice that had been infected with HSV-1 produced predominantly? -interferon (y-IFN) -cycline associated with Th1. The lymph node cells taken from animals that were immunized intranasally produced high levels of the cytosines associated with Th2, IL-4 and IL-10. In addition, both Ctx / CtxB and rEtxB have led to the activation of T cells which secreted ylFN in vitro stimulation with HSV-1. This indicates that although the response to these adjuvants is dominated by the production of Th2 cytokines, Th1 activation also occurs. These findings are consistent with those of the antibody response analyzes. Table 3 shows the level of protection against infection of Ocular HSV-1 in mice immunized intranasally with a mixture of HSV-1 or imitation glycoproteins in the presence of rEtxB as an immunomodulator.

I heard OR I heard PICTURE 3 1n = 29 2n = 30

Claims

NOVELTY OF THE INVENTION CLAIMS

1- The use of: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB having Gb3 binding activity; or (iii) an agent that has an effect on intracellular signaling events mediated by binding to G 1 or binding to Gb 3; as an immunomodulator for a vaccine against infectious diseases.

2 - The use as claimed in claim 1, wherein the immunomodulator is EtxB free of intact toxin. 3. - The use as claimed in 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 as claimed in 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 as claimed in claim 4, wherein the infectious agent is selected from the group consisting of HSV-1, HSV-2, CMV or EBV. 6. - The use as claimed in 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 as claimed in 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 as claimed in 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 as claimed in claim 1 or 2, wherein the infectious disease is caused by an infectious agent, and the infectious agent is a hepatitis virus. 10. - The use as claimed in claim 9, wherein the infectious agent is selected from the group consisting of hepatitis A, B, C, and D viruses. 11. - Use as claimed in Claim 10, wherein the infectious agent is a hepatitis A virus or a hepatitis C virus. 12. The use as claimed in claim 1 or 2, wherein the infectious disease is meningitis. 13. - The use as claimed in claim 12, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of Neisseria meningitidis, Haemophilus influenzae type B and Streptococcus pneumoniae. 14. Use as claimed in claim 1 or 2, wherein the infectious disease is pneumonia or an infection of the respiratory tract. 15. The use as claimed in 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 as claimed in claim 1 or 2, wherein the infectious disease is a sexually transmitted disease. 17. - The use as claimed in 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 as claimed in claim 1 or 2, wherein the infectious disease is a gastrointestinal disease. 19. The use as claimed in claim 18, wherein the infectious disease is caused by an infectious agent, and the infectious agent is selected from the group consisting of enteropathogenic, enterotoxigenic, enteroinvasive, enterohemorrhagic and enteroaggregative E. coli , rotavirus, Salmoella enteritidis, Salmoella typhi, Helicobacter pylori, Bacillus cereus, Campylobacter jejuni and Vibrio cholerae. 20 - The use as claimed in claim 1 or 2, where the infectious disease is a superficial infection. 21. The use as claimed in 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. 22 - The use as claimed in claim 1 or 2, wherein the infectious disease is a parasitic disease. 23. The use as claimed in 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, infectious disease which 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 intact toxin; (ti) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB having Gb3 binding activity; or (i and /) an agent that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; wherein said antigenic determinant is an antigenic determinant of said infectious agent. 25. The vaccine composition according to claim 24, further characterized in that the infectious disease is HSV-1 infection and wherein the antigenic determinant is an antigenic determinant of HSV-1. 26. - The vaccine composition according to claim 24 or 25, further characterized in that the immunomodulator is EtxB free of intact toxin. 27. The vaccine composition according to claim 24, 25 or 26, further characterized in that the immunomodulator and the antigenic determinant are separate portions. 28. The vaccine composition according to claim 24, 25 or 26, further characterized in that 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 comprises: a) one of the following agents: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB having Gb3 binding activity; or (iii) an agent that has an effect on intracellular signaling events mediated by binding to G 1 or binding to Gb 3; and b) an antigenic determinant which is an antigenic determinant of the infectious disease, for co-administration with said vaccine immunomodulator. 30. - A method for 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, wherein the immunomodulator is: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB that has binding activity to Gb3; or (iii) an agent that has an effect on intracellular signaling events mediated by binding to G 1 or binding to Gb 3. 31. - The use of: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB having Gb3 binding activity; or (iii) an agent that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3 to upregulate the production of antibodies on mucosal surfaces. 32. - The use of: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB having Gb3 binding activity; or (iii) an agent that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; as an immunomodulator in a vaccine, to prolong the presentation of antigen and to give sustained immunological memory in a mammalian subject. 33. - A vaccine composition for use against an infectious disease, infectious disease which is caused by an infectious agent, whose vaccine comprises an antigenic determinant and an immunomodulator selected from: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB that has binding activity to Gb3; or (iii) an agent that has an effect on intracellular signaling events mediated by binding to GM1 or binding to Gb3; wherein said antigenic determinant is an antigenic determinant of said infectious agent and wherein the immunomodulator prolongs the presentation of the antigenic determinant and provides sustained immunological memory. 34.- The use of: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB having Gb3 binding activity; or (iii) an agent that has an effect on vesicular incorporation mediated by binding to GM1 or binding to Gb3; in a conjugate with antigen or antigenic determinant to direct the delivery or said antigen or antigenic determinant to the cytosol or nucleus of a cell presenting antigen. 35 - The use of: (i) EtxB, CtxB or VtxB free of intact toxin; (ii) an agent other than EtxB or CtxB, which has GM1 binding activity, or an agent other than VtxB having Gb3 binding activity; or (iii) an agent that has an effect on vesicular incorporation mediated by binding to GM1 or binding to Gb3; 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 comprising: a) EtxB, CtxB or an agent different from EtxB or CtxB which has GM1 binding activity; and b) an EBV antigen for use in the treatment and / or prevention of diseases associated with EBV. 37. A therapeutic composition which comprises: EtxB, CtxB or an agent other than EtxB or CtxB which has GM1 binding activity for use in the treatment of diseases associated with EBV.