WO1999058145A2 - Vaccin - Google Patents

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Publication number
WO1999058145A2
WO1999058145A2 PCT/GB1999/001461 GB9901461W WO9958145A2 WO 1999058145 A2 WO1999058145 A2 WO 1999058145A2 GB 9901461 W GB9901461 W GB 9901461W WO 9958145 A2 WO9958145 A2 WO 9958145A2
Authority
WO
WIPO (PCT)
Prior art keywords
agent
etxb
infectious
ctxb
binding
Prior art date
Application number
PCT/GB1999/001461
Other languages
English (en)
Other versions
WO1999058145A3 (fr
Inventor
Timothy Raymond Hirst
Neil Andrew Williams
Andrew Morgan
Andrew Douglas Wilson
Lucy Amber Bird
Original Assignee
University Of Bristol
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9809958.3A external-priority patent/GB9809958D0/en
Priority claimed from GBGB9812316.9A external-priority patent/GB9812316D0/en
Priority to PL34451999A priority Critical patent/PL344519A1/xx
Priority to HU0104842A priority patent/HUP0104842A3/hu
Priority to CA 2331832 priority patent/CA2331832A1/fr
Priority to US09/674,935 priority patent/US7914791B1/en
Priority to AU39394/99A priority patent/AU3939499A/en
Priority to NZ507911A priority patent/NZ507911A/en
Priority to JP2000547996A priority patent/JP4666761B2/ja
Priority to BR9910305A priority patent/BR9910305A/pt
Application filed by University Of Bristol filed Critical University Of Bristol
Priority to GB0027072A priority patent/GB2353472A/en
Priority to MXPA00010934A priority patent/MXPA00010934A/es
Priority to EP19990922284 priority patent/EP1075274A2/fr
Priority to EA200001134A priority patent/EA004794B1/ru
Priority to KR1020007012486A priority patent/KR20010043441A/ko
Publication of WO1999058145A2 publication Critical patent/WO1999058145A2/fr
Publication of WO1999058145A3 publication Critical patent/WO1999058145A3/fr
Priority to IS5694A priority patent/IS5694A/is
Priority to IL139467A priority patent/IL139467A/en
Priority to NO20005599A priority patent/NO20005599L/no
Priority to AU2003261492A priority patent/AU2003261492B2/en
Priority to US13/038,021 priority patent/US20110223194A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to an immunomodulator for use in a vaccine which is intended for use against a range of infectious agents. Further this invention relates to a vaccine composition comprising the immunomodulator, preferably in combination with antigen and a vaccination method using the vaccine composition.
  • Ctx Cholera toxin
  • Etx heat-labile enterotoxin
  • Vtx E. coli verotoxin
  • Ctx and Etx are heterohexameric proteins composed of a an enzymatically active A subunit and a pentameric B subunit.
  • CtxB and EtxB are known to bind GM1- ganglioside (GM1) , a glycosphingolipid found ubiquitously on the surface of mammalian cells.
  • GM1- ganglioside GM1
  • Vtx binds to Gb3 which is a similar type of receptor to GM1.
  • CtxB is a poor mucosal adjuvant and only the addition of native holotoxin can provoke strong bystander responses (Tamura et al (1994)
  • Vaccine 12: 419-426 Other studies have suggested that rCtxB lacks the ADP-ribosylating and the cAMP- stimulating activities of the holotoxin and that, as adjuvant mechanism is linked to these abilities, CtxB would be unsuitable for use as an adjuvant (Vajdy and
  • rCtxB and rEtxB can be used to promote tolerance to heterologous antigens (Sun et al (1994) Proc. Natl. Acad. Sci. 91: 4610-4614, Sun et al (1996) Proc. Natl. Acad. Sci. 93: 7196-7201,
  • agents such as EtxB and CtxB stimulate high levels of local (mucosal) antibody production (although immunization using rEtxB stimulated lower levels of overall serum antibody production than Ctx/CtxB combined) ; ii) the distribution of antibodies produced was skewed towards non-complement fixing antibodies, especially S-IgA and IgGl; iii) agents such as EtxB and CtxB also stimulated local and systemic T-cell proliferative responses; iv) agents such as CtxB and EtxB tend to shift the immune response from a Thl-associated response to a Th2-associated response; v) when agents such as CtxB and EtxB are used as immunomodulators some of the harmful effects of Th2- associated responses, such as the generation of IgE, are avoided; vi) rEtxB is a more efficient immunomodulator than rCtxB; vii) agents such as EtxB and C
  • EtxB, CtxB, VtxB and other agents capable of binding to or mimicking the effects of binding to GMl or Gb3 are capable of acting as immunomodulators and stimulate specific immune responses to antigenic challenge.
  • a vaccine composition for use against an infectious disease, which infectious disease is caused by an infectious agent, wherein the vaccine composition comprises an antigenic determinant and an immunomodulator selected from: (i) EtxB, CtxB or VtxB free from whole toxin;
  • the antigen and immunomodulator may be linked, for example covalently or genetically linked, to form a single effective agent.
  • the antigen and immunomodulator may be chemically conjugated.
  • the antigen and immunomodulator may be chemically conjugated using heterobifunctional cross-linking reagents.
  • separate administration in which the antigen and immunomodulator are not so linked is preferred because it enables separate administration of the different moieties .
  • kits 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;
  • the vaccine composition of the second aspect of the invention and the kit of the third aspect of the invention may be used in a prophylactic or therapeutic vaccination method, where a "prophylactic vaccine” is administered to naive individuals to prevent disease development, and a "therapeutic vaccine” is administered to individuals with an existing infection to reduce or minimise the infection or to abrogate the immunopathological consequences of the disease.
  • a therapeutic vaccine i.e. one which need not contain antigen
  • a therapeutic vaccine 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.
  • a method of preventing or treating a disease in a host comprises the step of inoculating said host with a vaccine comprising at least one antigenic determinant and an immunomodulator, where the immunomodulator is:
  • EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having
  • the vaccine may be packaged for coadministration and may be administered by a number of different routes such as intranasal, oral, intra-vaginal , urethral or ocular administration. Intranasal immunisation is presently preferred. When a vaccine is administered intranasally, it may be administered as an aerosol or in liquid form.
  • the antigenic determinant and immunomodulator may be administered to the subject as a single dose or in multiple doses .
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a disease for which the infectious agent is a member of the herpes virus family.
  • 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.
  • the infectious agent may be HSV-1, HSV-2, CMV or EBV.
  • 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 .
  • the infectious agent is HSV-1 or HSV-2
  • the antigenic determinant may be an antigenic determinant of a gene product selected from the following group: gD, gB, gH, gC or a latency associated transcript (LAT) .
  • the antigenic determinant may be an antigenic determinant of gp340 or gp350 or of a latent protein (for example EBNAs 1,2 , 3A, 3B, 3C and -LP, LMP-1, -2A and 2B or an EBER) .
  • a latent protein for example EBNAs 1,2 , 3A, 3B, 3C and -LP, LMP-1, -2A and 2B or an EBER
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a disease for which the infectious agent is an influenza virus .
  • the antigenic determinant is preferably an antigenic determinant of a viral coat protein (for example haemagglutinin and neuraminidase) or of an internal protein (for example, nucleoprotein) .
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a disease for which the infectious agent is a parainfluenza virus.
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a disease for which the infectious agent is respiratory syncytial virus.
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a disease for which the infectious agent is a hepatitis virus.
  • the infectious agent may be selected from the group consisting of hepatitis A, B, C and D.
  • the infectious agent may be hepatitis A or C.
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against meningitis.
  • the infectious agent may be selected from the group consisting of Neisseria meningitidis, Haemophilus influenzae type B and
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against pneumonia or a respiratory tract infection.
  • the infectious agent may be selected from the group consisting of Streptococcus pneumoniae,
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a sexually- transmitted disease.
  • the infectious agent may be selected from the group consisting of Neisseria gonnorheae, HIV-1, HIV-2 and
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a gastrointestinal disease.
  • the infectious agent may be selected from the group consisting of enteropathogenic, enterotoxigenic and enteroinvasive E. coli , rotavirus, Salmonella enteri tidis, Salmonella typhi , Helicobacter pylori ,
  • the infectious agent is selected from the group consisting of enteropathogenic, enterotoxigenic, enteroinvasive, enterohaemorrhagic and enteroaggregative E.coli
  • the antigenic determinant may be an antigenic determinant of a bacterial toxin or adhesion factor.
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a superficial infection.
  • the infectious agent may be selected from the group consisting of Staphylococcus aureus , Streptococcus pyogenes and Streptococcus mutans .
  • the immunomodulator of the first aspect of the invention, the vaccine of the second aspect of the invention, the kit of the third aspect of the invention and the method of the fourth aspect of the invention is used against a parasitic disease.
  • the infectious agent may be selected from the group consisting of malaria, Trypanasoma spp., Toxoplasma gondii ,
  • EtxB, CtxB, VtxB and other agents capable of binding to or mimicking the effects of binding to GMl or Gb3 are capable of specifically upregulating mucosal antibody production.
  • the vaccine immunomodulator of the first aspect of the invention, the vaccine composition of the second aspect of the invention and the kit of the third aspect of the invention are particularly effective against diseases where protection from infection or treatment is effected in vivo by a mucosal immune response.
  • diseases in which, during infection, the infectious agent binds to, colonises or gains access across the mucosa examples include, diseases caused by viruses (HIV, HSV, EBV, CMV, influenza, measles, mumps, rotavirus etc), diseases caused by bacteria (E. coli, Salmonella, Shigella, Chlamydia, N. gonnorhoea, T. pallidium, Streptococcus species including those which cause dental caries), and diseases caused by parasites.
  • a vaccine against HSV-1 infection comprising at least one HSV-1 antigenic determinant and an immunomodulator, where the immunomodulator is:
  • EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having 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 binding.
  • the immunomodulator is EtxB.
  • kits for vaccination of a mammalian subject against an HSV-1 comprising: a) a vaccine immunomodulator which is:
  • EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having
  • 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
  • S-IgA is produced in accordance with the fifth aspect of the invention.
  • the agent may be used in conjunction with one or more antigenic determinant (s) .
  • 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 .
  • the pathological components of Thl-associated immune responses may also be downregulated. It is known that EtxB and CtxB bind to GMl and induce differential effects on lymphocyte populations, including a specific depletion of CD8+ T cells and an associated activation of B cells (WO 97/02045) . Hence, EtxB and CtxB are thought to alter the balance of the immune response such that inflammatory Thl associated reactions are down-regulated while Th2 associated responses are upregulated.
  • Thl responses include the secretion of ⁇ IFN by activated T-cells leading to macrophage activation and delayed type hypersensitivity reactions. Such responses may be an important cause of pathology during infections with a number of pathogens.
  • Th2 responses include the activation of T-cells to produce cytokines such as IL-4, IL-5, IL-10, and are known to promote the secretion of high levels of antibody, especially IgA.
  • EtxB and CtxB are capable of down regulating pathological components of the immune response associated both with Thl and Th2 activation. Such responses are modulated in favour of the production of high levels of non-complement fixing serum antibodies and secretory IgA production at the mucosal surfaces.
  • the use of an agent in accordance with the sixth aspect of the invention is particularly useful for therapeutic vaccination in diseases in which immunopathological mechanisms are involved. Examples of such diseases are HSV-1, HSV-2, TB and HIV.
  • agents such as EtxB can be used simultaneously as an immunomodulator and a therapeutic agent.
  • 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 .
  • 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.
  • 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.
  • 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 having an effect on intracellular signalling events mediated by GMl-binding or Gb3 binding; as an immunomodulator in a vaccine, to prolong antigen presentation and give sustained immunological memory in a mammalian subject.
  • a vaccine composition for use against an infectious disease comprising an antigenic determinant and a immunomodulator selected from:
  • EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having 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; 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.
  • the antigen and immunomodulator in a therapeutic or prophylactic vaccine may be linked, for example covalently or genetically linked, to form a single effective agent.
  • the present inventors have found that is possible to direct the antigen to different compartments of the cell and hence to different antigen presentation pathways by altering the linkage of the antigen to the immunomodulator.
  • CTL cytotoxic T cells
  • the linkage of the antigen-immunomodulator conjugate can also be chosen so that the antigen is delivered into the nucleus.
  • a conjugate comprising an antigen or antigenic determinant and an immunomodulator selected from:
  • EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having 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.
  • a vaccine composition for use against an infectious disease which infectious disease is caused by an infectious agent
  • which vaccine composition comprises a conjugate of an antigen or antigenic determinant and an immunomodulator selected from:
  • EtxB, CtxB or VtxB free from whole toxin; (ii) an agent other than EtxB or CtxB, having
  • the antigen or antigenic determinant may be linked to the immunomodulator by a variety of methods including genetic linkage or chemical conjugation.
  • the conjugate is a fusion protein made by genetic linkage of the antigen or antigenic determinant to the immunomodulator.
  • the antigen or antigenic determinant is genetically linked to the C-terminus of the immunomodulator.
  • the antigen or antigenic determinant is chemically conjugated to the immunomodulator.
  • the antigen or antigenic determinant is conjugated to the immunomodulator using a bifunctional cross-linking reagent, such as a heterobifunctional cross-linking reagent.
  • 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.
  • 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 to upregulate the presentation of said antigenic determinant, or an antigenic determinant derived from said antigen, by MHC class I molecules.
  • 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 is one of the eight known human herpes viruses. Infection usually occurs in early childhood; however, clinical symptoms are usually weak or undetectable at this stage. Primary infection with EBV later in life is associated with infectious mononucleosis (IM) , which is the second most frequent disease in adolescence in the US. EBV also has oncogenic potential. There is a strong link between EBV and endemic Burkitt ' s lymphoma (BL) and undifferentiated nasopharyngeal carcinoma (NPC) . Also, a large proportion of lymphomas that occur in immuno- compromised patients are caused by EBV, and an association has been shown to exist between certain Hodgkin's lymphomas and EBV.
  • BL Burkitt ' s lymphoma
  • NPC undifferentiated nasopharyngeal carcinoma
  • Latently EBV- infected cells express a small number of so-called "latent” proteins. These include six nuclear proteins (EBNAs 1, 2, 3A, 3B, 3C and -LP) , three integral membrane proteins (LMP-1, 2A and 2B) and two non-polyadenylated virus derived RNAs (EBERs) with a role in RNA splicing.
  • EBNAs 1, 2, 3A, 3B, 3C and -LP three integral membrane proteins
  • LMP-1, 2A and 2B three integral membrane proteins
  • EBERs non-polyadenylated virus derived RNAs
  • LMP-1 EBV latent membrane protein 1
  • NPCs nasopharyngeal carcinomas
  • HD EBV- positive Hodgkin's lymphomas
  • the LMP-1 gene can alter the phenotype of uninfected cells causing the upregulation of cell surface activation markers, promoting cell proliferation.
  • LMP- 1 can also alter signalling pathways and has anti- apoptotic effects. An cellular immune response directed against this viral antigen has not been demonstrated with any degree of certainty in either healthy carriers or tumour patients. Many animal viruses have evolved mechanisms to avoid detection by the host immune system.
  • a vaccine composition which comprises : a) one of the following agents:
  • the vaccine composition of the thirteenth aspect of the invention comprises EtxB, CtxB, or an agent other than EtxB or CtxB which has
  • a fourteenth aspect of the invention there is provided a therapeutic composition which comprises: (i) EtxB, CtxB or VtxB free from whole toxin;
  • the therapeutic composition of the fourteenth aspect of the invention comprises EtxB, CtxB, or an agent other than EtxB or CtxB which has GM1-binding activity.
  • EtxB cocaps with LMP1, and that EtxB promotes fragmentation of LMP-1
  • EtxB and other agents like CtxB having GMl binding activity
  • This activity has applications in vaccines to prevent EBV associated diseases, and in therapeutic treatments to treat such diseases once they have developed.
  • EtxB cocaps with LMP-1 the antigen is processed by a different intracellular route, which enables the antigen to by-pass the normal processing route which is blocked by the virus. The antigen is thus presented efficiently on the cell surface.
  • the action of EtxB may also cause different epitopes of the antigen to be presented at the cell surface, from those which are presented if the antigen were processed by the conventional route.
  • the vaccine of the thirteenth aspect of the invention may be used to prevent infection by EBV, or development of EBV-associated diseases in EBV- infected individuals.
  • the vaccine may also comprise a separate adjuvant, or the agent (such as EtxB or CtxB) can act as an adjuvant in its own right.
  • the agents specified in the fourteenth aspect of the present invention may be used alone (i.e. without antigen) in the treatment of a EBV-associated disease which has already developed in a subject.
  • the preferred agent for use in the thirteenth and fourteenth aspects of the invention is EtxB.
  • the EBV antigen is an antigen derivable from EBV itself or an antigen which is caused to be expressed by an EBV-infected host cell by the action of EBV.
  • the antigen is an EBV latent membrane protein.
  • Particularly preferred are the antigens LMP- 1, LMP-2A, LMP-2B, and EBNA-1 as well as antigenic fragments thereof.
  • the antigen may be isolated directly from EBV infected cells, or be made by synthetic or recombinant means.
  • the thirteenth and fourteenth aspects of present invention are particularly suited for the treatment and/or prevention of the following diseases : infectious mononucleosis, Burkitt ' s lymphoma, nasopharyngeal carcinomas, and Hodgkin's lymphomas. It is believed that these aspects of the invention will be particularly suited to the treatment and/or prevention of nasopharyngeal carcinomas and Hodgkin's lymphomas.
  • the vaccine or the therapeutic composition according to the thirteenth and fourteenth aspects of the invention may be used to prevent development of, or treat, an EBV-associated disease in a mammalian subject, by administration of an immunologically effective amount to the subject.
  • the mammalian subject may be, for example, a healthy EBV-infected or uninfected individual, an immunodeficient individual, or an individual with an EBV-associated disease.
  • the vaccine may be administered by any suitable route.
  • the agent and the antigen may be co- administered to the mammalian subject or administered separately.
  • the agent and the antigen may be separate or linked, for example covalently or genetically linked, to form a single effective agent.
  • GMl or Gb3 binding may trigger intracellular signalling directly or indirectly.
  • the present inventors have also found evidence which suggests that EtxB interacts with at least one other receptor which is involved in the GMl associated intracellular signalling event. It may be that binding of EtxB (or CtxB) to GMl facilitates binding to a protein, which protein triggers intracellular signalling. It is not known what specifically triggers the signalling event, it may be phosphorylation of GMl or the protein.
  • EtxB/CtxB binds GMl on the cell surface, bound GMl is internalised in vesicles (Williams et al (1999) Immunology Today 20; 95-101) .
  • GMl and other glycolipids are known to be preferentially located in "membrane rafts" in which key protein receptors are also found. It is therefore possible that internalisation of GMl as a result of B- subunit binding causes cocapping of such proteins leading to their being triggered to mediate intracellular signalling events.
  • An adjuvant is a substance which non-specifically enhances the immune response to an antigen, as distinct from a vaccine carrier, the purpose of which is to target the antigen to a desired site.
  • the term "immunomodulator” is used herein to indicate an agent which acts, like an adjuvant, to stimulate certain immune responses, but which also directs the immune response in a particular direction.
  • 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.
  • antigen and the immunomodulator may be administered at the same moment in time and at the same site, there may be advantages in administering the antigen at a different time and/or at a different site from the immunomodulator.
  • antigen and immunomodulator may be administered together in a first step and then the immune response may be boosted in a second step by administration of antigen alone.
  • antigenic determinant refers to a site on an antigen which is recognised by an antibody or T-cell receptor. Preferably it is a short peptide derived from or as part of a protein antigen, however the term is also intended to include glycopeptides and carbohydrate antigenic determinants. The term also includes modified sequences of amino acids or carbohydrates which stimulate responses which recognise the whole organism.
  • potential protective antigens may be identified by elevating immune responses in infected or convalescent patients, in infected or convalescent animals , or by monitoring in vi tro immune responses to antigen containing preparations.
  • serum samples from infected or convalescent patients or infected or convalescent animals may be screened against whole cell lysates of an infectious agent, or lysates of cells infected by the said agent, by the standard technique of Western blotting to detect those antigen (s) recognised by the immune serum
  • serum samples from infected or convalescent patients or infected or convalescent animals may be screened against partial or highly purified antigens from an infectious agent, or lysates of cells infected by the said agent, by the standard technique s of ELISA, in which partial or highly purified antigens are used to coat microtitre wells, or by iimuno blotting to detect those antigen (s) recognised by the immune sera
  • surface components of an infectious agent comprising one or more potential protective antigens may be extracted from the agent, or from cells infected by the agent, by use of procedures that allow the recovery of the antigens .
  • This may include the use of cell disruption techniques to lyse cells such as sonication and/or detergent extraction. Centrifugation, ultrafiltation or precipitation may be used on collected antigen preparations.
  • antigens of an infectious agent may be extracted by a variety of procedures, including but not limited to, urea extraction, alkali or acid extraction, or detergent extraction and then subjected to chromatographic separation. Material recovered in void or elution peaks comprising one or more potential protective antigens may used in vaccine formulations.
  • genes encoding one or more potential protective antigens may be cloned into a variety of expression vectors suitable for antigen production. These may include bacterial or eukaryotic expression systems, for example Escherichia coli ,
  • Antigens may be recovered by conventional extraction, separation and/or chromatographic procedures .
  • CtxB “EtxB” and “VtxB” as used herein include natural and recombinant forms of the molecule.
  • the recombinant form is particularly preferred.
  • the recombinant form of the molecule may be produced by a method in which the gene or genes coding for the specific polypeptide chain (or chains) from which the protein is formed, is inserted into a suitable vector and then used to transfect a suitable host.
  • the gene coding for the polypeptide chain from which the EtxB assemble may be inserted into, for example, plasmid pMM68, which is then used to transfect host cells, such as Vibrio sp . 60.
  • the protein is purified and isolated in a manner known per se .
  • Mutant genes expressing active mutant CtxB, EtxB or VtxB protein may be produced by known methods from the wild type gene .
  • CtxB also include mutant molecules and other synthetic molecules (containing parts of CtxB, EtxB or VtxB) which retain the capacity to bind GMl or Gb3 or the capacity to mimick the effects of binding to GMl or Gb3.
  • Agents other than EtxB and CtxB which retain GMl binding activity, and agents other than VtxB which retain Gb3 binding activity include antibodies which bind GMl or Gb3.
  • various hosts including goats, rabbits, rats, mice, etc. may be immunized by injection with GMl or Gb3 or any derivative or homologue thereof.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol .
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants .
  • Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983)
  • 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-
  • Antibody fragments which contain specific binding sites for GMl or Gb3 may also be generated.
  • 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 disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1).
  • Peptide libraries or organic libraries may be made by combinatorial chemistry and then screened for their ability to bind GMl/Gb3. Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways deemed to be routine to those of skill in the art .
  • GMl or Gb3 or fragments thereof can be used for screening peptides or molecules in any of a variety of screening techniques .
  • the molecule may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of activity or the formation of binding complexes between GMl or Gb3 and the agent being tested may be measured.
  • Another way of determining binding to GM1/Gb3 would be by using purified GMl/Gb3 to coat microtiter plates. Following blocking, the agent under investigation is applied to the plate and allowed to interact prior to washing and detection with specific antibodies to said agent. Conjugation of the antibodies either directly or indirectly to an enzyme or radiolabel allows subsequent quantification of binding either using colorimetric or radioactivity based methods (ELISA or RIA respectively) .
  • Another way of determining binding to GM1/Gb3 would be by binding the saccharide moiety of GMl/Gb3 to a suitable column matrix in order to allow standard affinity chromatography to be performed. Removal of known compounds applied to the column from the diluent would be used as evidence for binding activity, or alternatively, where mixtures of compounds are applied to the column, elution and subsequent analysis would determine the properties of the ganglioside binding agent. In the case of proteins, analysis would involve peptide sequencing and tryptic digest mapping followed by comparisons with available databases. In the event that eluted proteins cannot be identified in this way then standard biochemical analysis, for example mass determination by laser desorption mass spectrometry would be used to further characterise the compound. Non-proteins eluted from GMl-affinity columns would be analysed by HPLC and mass spectrometry of single homogenous peaks .
  • Phage display can be employed in the identification of candidate agents which bind GMl or Gb3.
  • Phage display is a protocol of molecular screening which utilises recombinant bacteriophage .
  • the technology involves transforming bacteriophage with a gene that encodes an appropriate ligand (in this case a candidate agent) capable of reacting with GMl/Gb3 (or a derivative or homologue thereof) or the nucleotide sequence (or a derivative or homologue thereof) encoding same.
  • the transformed bacteriophage (which preferably is tethered to a solid support) expresses the appropriate ligand (such as the candidate agent) and displays it on their phage coat.
  • Phage display has advantages over standard affinity ligand screening technologies.
  • the phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occuring conformation. This allows for more specific and higher affinity binding for screening purposes .
  • Another technique for screening provides for high throughput screening of agents having suitable binding affinity to GMl or Gb3 and is based upon the method described in detail in WO 84/03564.
  • large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test agents are reacted with the target interaction component fragments and washed.
  • a bound target interaction component is then detected - such as by appropriately adapting methods well known in the art.
  • a purified target interaction component can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • 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 receptors by virtue of its pentameric form.
  • Figure 1 shows the stimulation of total Ig
  • Figure 2 shows T cell proliferation of (mesenteric lymph node) MLN or (cervical lymph node) CLN lymphocytes in mice immunised with HSV-1/rEtxB.
  • Figure 3 shows T cell proliferation of cells from MLN and CLN of mice immunised intranasally with HSV-1 Gp in the presence of l-20 ⁇ g EtxB.
  • Figure 4 shows the level of anti-HSV-1 serum Ig in mice following administration of HSV-1 glycoproteins three times at 10 day intervals with variable amounts of rEtxB or rCtxB as adjuvant.
  • Figure 5 shows the reduction in virus shedding, clinical disease and latency in mice immunised with HSV-1/rEtxB.
  • Figure 6 shows the Ig isotype distribution in MS following infection with HSV-1 or immunisation with HSV-1 Gp in the presence of EtxB or CtxB as immunomodulator .
  • Figure 7 shows the distribution of Ig subclasses following intranasal administration of HSV-1 Gp with either rEtxB or rCtxB as immunomodulator.
  • Figure 8 shows the immunogenic effect of different amounts of rEtxB or rCtxB on the level of HSV-1 specific IgA in eye washings following administration with HSV-1 glycoproteins.
  • Figure 9 shows serum immunoglobulin response following immunisation of mice with HSV-1 or mock glycoproteins (gp) alone or in the presence of adjuvant .
  • Figure 10 shows mucosal IgA in eye washings following intranasal immunisation of mice with HSV-1 or mock glycoproteins alone or in the presence of adjuvant .
  • Figure 11 shows mucosal IgA in vaginal washings following intranasal immunisation of mice with HSV-1 or mock glycoproteins (gp) alone or in the presence of adjuvant .
  • Figure 12 shows the level of HSV-1-specific immunoglobulin in sera from mice immunised with HSV-1 glycoproteins in the presence of different doses of rEtxB as adjuvant.
  • Figure 13 shows the level of IgA in eye washings of mice immunised with HSV-1 glycoproteins in the presence of varying concentrations of rEtxB.
  • Figure 14 shows the level of IgA in vaginal washings of mice immunised with HSV-1 glycoproteins in the presence of varying concentrations of rEtxB
  • Figure 15 shows IgG subclass distribution of the serum antibody response to HSV-1 following intranasal immunisation with Ctx/CtxB or rEtxB or ocular infection with HSV-1.
  • Figure 16 shows cytokine production from cultures of lymph node cells taken from mice which were either infected with HSV-1 by ocular scarification, or were immunised by intranasal administration of HSV-1 glycoproteins with Ctx/CtxB or rEtxB as adjuvant.
  • Figure 17 shows the level of protection against ocular HSV-1 infection in mice immunised intranasally with a mixture of HSV-1 or mock glycoproteins in the presence of rEtxB as immunomodulator.
  • Example 1 rEtxB can be used in conjunction with HSV-1 Gp for immunisation.
  • mice were immunised intranasally three times with lO ⁇ g HSV-1 plycoproteins (Gp) with either 10 or 20 ⁇ g rEtxB .
  • Controls were either unmanipulated or given a mock preparation of viral glycoprotein (mock) derived from HIV-uninfected tissue culture cells.
  • Antibody levels are expressed as a percentage of post-infection levels .
  • the production of total Ig and IgA in the serum and IgA in eye washings was stimulated by HSV-1 glycoproteins/rEtxB ( Figure 1) .
  • the present inventors have also shown that doses of rEtxB as low as 0. l ⁇ g are also effective at stimulating such responses.
  • 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) .
  • 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 .
  • HSV-1 glycoproteins mice were immunised three times intranasally with HSV-1 glycoproteins alone, a mock preparation of HSV-1 glycoproteins (prepared by taking uninfected tissue culture cells and subjecting them to identical treatment regimes as those employed for the isolation and purification of HSV-1 proteins) , or HSV-1 glycoproteins in combination with a variety of putative mucosal adjuvants.
  • the dose of HSV-1 glycoproteins was lO ⁇ g per immunisation, and these were combined with lO ⁇ g of recombinant EtxB, or CtxB as adjuvant, or a mixture of 0.5 ⁇ g of Ctx and lO ⁇ g CtxB.
  • mice were immunised as described in example 4. Secretory IgA production in the eye was assessed by taking washings of the tears over consecutive days and these samples were then pooled and subjected to ELISA analysis using a specific anti-IgA detecting antibody. The quantities of antibodies are expressed as a percentage of the levels stimulated following ocular infection induced by scarification with 10 5 pfu HSV-1 strain SC16. The data clearly demonstrates ( Figure 10) that high levels of secreted anti-HSV-1 antibodies are produced following immunisation in the presence of either Ctx/CtxB or EtxB.
  • Example 6 ( Figure 11) Mice were immunised as described in example 4.
  • EtxB In the vagina, the highest levels of antibodies were released following immunisation in the presence of rEtxB. Lower levels were released following immunisation with Ctx/CtxB and very little secretion was triggered by the use of rCtxB as adjuvant.
  • mice were immunised three times intranasally with HSV-1 glycoproteins (lO ⁇ g) either alone or in the presence of escalating doses of rEtxB as adjuvant. Three weeks after the final immunisation blood was taken, and the levels of anti-HSV-1 antibodies were assessed by ELISA. The quantities of antibodies are expressed as a percentage of the levels stimulated following ocular infection induced by scarification with 10 5 pfu HSV-1 strain SC16. The data clearly demonstrates that the capacity of rEtxB to trigger antibody responses to heterologous added antigens is a dose dependent phenomenon with maximal responsiveness occurring at approximately 20-50 ⁇ g of rEtxB . Further, it is clear that at doses of 20 ⁇ g rEtxB and above, the level of anti-HSV-1 antibodies stimulated by intranasal infection is comparable or greater than that stimulated by a live virulent virus infection.
  • mice were immunised as described in example 7.
  • Secretory IgA production in the eye was assessed by taking washings of the tears over consecutive days and these samples were then pooled and subjected to ELISA analysis using a specific anti- IgA detecting antibody.
  • the quantities of antibodies are expressed as a percentage of the levels stimulated following ocular infection induced by scarification with 10 5 pfu HSV-1 strain SC16.
  • the data demonstrates that maximal IgA responses in the eye are stimulated when HSV-1 glycoproteins are given in combination with 20 ⁇ g of rEtxB or above. At this dose the levels of IgA production are nevertheless lower than those triggered during virus infection of the eye.
  • mice were immunised as described in example 7.
  • Secretory IgA production in the vagina was assessed by taking washings from the genital tract over consecutive days and these samples were then pooled and subjected to ELISA analysis using a specific anti- IgA detecting antibody.
  • the quantities of antibodies are expressed as endpoint titres which were calculated by linear regression analysis. The data shows that optimal anti- HSV-1 responses are stimulated in the vagina when 20 ⁇ g or above of rEtxB is used as an adjuvant.
  • mice were either infected with 10 ⁇ pfu HSV-1 strain SC16 by scarification into the cornea or immunised three times intranasally with lO ⁇ g HSV-1 glycoproteins in combination with Ctx/CtxB or rEtxB .
  • serum was taken and was analysed by ELISA for the presence of IgGl and IgG2a against HSV-1.
  • the quantities of antibodies are expressed as endpoint titres which were calculated by linear regression analysis (fig. 7a) .
  • the data clearly shows that the nature of the antibody response to HSV-1 is influenced by the way in which the antigens are presented to the immune system.
  • mice were either infected with 10 5 pfu HSV-1 strain SC16 by scarification into the cornea or immunised three times intranasally with lO ⁇ g HSV-1 glycoproteins in combination with Ctx/CtxB or rEtxB .
  • lymph nodes were removed from animals and were used to generate single cell suspensions that were cultured either in the presence of killed HSV-1 or a mock preparation of virus from non-infected tissue culture cells.
  • samples of cells were removed and subjected to cELISA analysis to reveal the secretion of cytokines .
  • the data clearly shows that T- cells in the cultures were capable of responding to HSV-1, but not significantly to mock virus preparations.
  • Lymph node cells taken from mice which had been infected with HSV-1 produced predominantly the Thl associated cytokine ⁇ -interferon ( ⁇ -IFN) .
  • Lymph node cells taken from animals that were immunised intranasally produced high levels of the Th2 associated cytokines, IL-4 and IL-10.
  • both Ctx/CtxB and rEtxB had led to the activation of T-cells which secreted ⁇ IFN upon in vitro stimulation with HSV-1. This indicates that although the response to these adjuvants is dominated by the production of Th2 cytokines some Thl activation also occurs.

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Abstract

L'invention concerne l'utilisation (i) de EtxB, CtxB ou de VtxB exempt de toute toxine, (ii) d'un agent autre que EtxB ou CtxB ayant une activité de liaison GM1, ou d'un agent autre que VtxB ayant une activité de liaison Gb3, ou (iii) d'un agent ayant un effet sur des événements de transmission de signaux intracellulaires dont la médiation est assurée par liaison GM1 ou Gb3, comme immunomodulateur pour un vaccin contre des maladies infectieuses.
PCT/GB1999/001461 1998-05-08 1999-05-10 Vaccin WO1999058145A2 (fr)

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EA200001134A EA004794B1 (ru) 1998-05-08 1999-05-10 Вакцина
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EP19990922284 EP1075274A2 (fr) 1998-05-08 1999-05-10 Vaccin
CA 2331832 CA2331832A1 (fr) 1998-05-08 1999-05-10 Sous-unite b d'enterotoxine thermolabile d'e. coli (etxb) en tant qu'immunomodulateur
US09/674,935 US7914791B1 (en) 1998-05-08 1999-05-10 Vaccine
AU39394/99A AU3939499A (en) 1998-05-08 1999-05-10 Vaccine
NZ507911A NZ507911A (en) 1998-05-08 1999-05-10 Immunomodulators for vaccines
JP2000547996A JP4666761B2 (ja) 1998-05-08 1999-05-10 ワクチンのための免疫調節因子
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IL139467A IL139467A (en) 1998-05-08 2000-11-05 USE OF EtxB, CtxB OR VtxB IN THE MANUFACTURE OF A VACCINE AGAINST INFECTIOUS DISEASES
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AU2003261492A AU2003261492B2 (en) 1998-05-08 2003-11-07 Vaccine
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Cited By (9)

* Cited by examiner, † Cited by third party
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WO2002047727A1 (fr) * 2000-12-11 2002-06-20 University Of Bristol Agent therapeutique comprenant une sous-unite 'b' d'une toxine proteique
WO2003000899A1 (fr) * 2001-06-22 2003-01-03 University Of Bristol FORMES MUTANTES DE EtxB ET CtxB ET LEUR UTILISATION EN TANT QUE PORTEURS
US7056521B2 (en) 1997-03-21 2006-06-06 Chiron Corporation Detoxified mutants of bacterial ADP-ribosylating toxins as parenteral adjuvants
US7070781B2 (en) * 1993-12-22 2006-07-04 Chiron Srl Nontoxic mucosal adjuvant
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US7588763B2 (en) 1995-07-05 2009-09-15 Trident Pharmaceuticals, Inc. Methods for the prevention against or treatment of diabetes with ETXB and/or insulin
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US7872114B2 (en) 1991-12-31 2011-01-18 Novartis Vaccines And Diagnostics Srl Immunogenic detoxified mutants of cholera toxin
US7485304B2 (en) 1993-12-22 2009-02-03 Novartis Vaccines And Diagnostics Srl Non-toxic mucosal adjuvant
US7070781B2 (en) * 1993-12-22 2006-07-04 Chiron Srl Nontoxic mucosal adjuvant
US7632513B2 (en) 1995-06-30 2009-12-15 Novartis Vaccines And Diagnostics Srl Immunogenic detoxified mutants of cholera toxin
US7588763B2 (en) 1995-07-05 2009-09-15 Trident Pharmaceuticals, Inc. Methods for the prevention against or treatment of diabetes with ETXB and/or insulin
US7291588B2 (en) 1996-10-31 2007-11-06 Chiron Srl Immunogenic detoxified mutant E. coli LT-A toxin
US7056521B2 (en) 1997-03-21 2006-06-06 Chiron Corporation Detoxified mutants of bacterial ADP-ribosylating toxins as parenteral adjuvants
US7914791B1 (en) 1998-05-08 2011-03-29 Trident Pharmaceuticals, Inc. Vaccine
US7115730B1 (en) 1999-04-27 2006-10-03 Chiron Srl Immunogenic detoxified mutant E. coli LT-A-toxin
AU2002222153B2 (en) * 2000-12-11 2006-10-05 University Of Bristol Therapeutic agent comprising a B-subunit of a protein toxin
WO2002047727A1 (fr) * 2000-12-11 2002-06-20 University Of Bristol Agent therapeutique comprenant une sous-unite 'b' d'une toxine proteique
US7422752B2 (en) 2001-06-22 2008-09-09 Hunter Immunology Ltd. Mutant forms of EtxB and CtxB and their use as carriers
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US8425917B2 (en) 2001-06-22 2013-04-23 Hunter Immunology Ltd Mutant forms of EtxB and CtxB and their use as carriers

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PL344519A1 (en) 2001-11-05
AU3939499A (en) 1999-11-29

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