Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
  • C07K14/245—Escherichia (G)
• • 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
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • 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
  • A61P35/00—Antineoplastic agents
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • 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
• • 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
• • 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
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S424/00—Drug, bio-affecting and body treating compositions
  • Y10S424/832—Drug, bio-affecting and body treating compositions involving bacterial toxin that has modified amino acid sequence

Definitions

• the present invention relates to an adjuvant useful for the administration of vaccines to organisms.
• the adjuvant of the invention allows the delivery of vaccines to mucosal surfaces to raise a secretory and systemic immune response.
• an oral vaccine which may be fed to subjects is easier to administer on a large scale in the absence of specialised equipment, especially to subjects which may be difficult to handle or even locate, such as livestock and wild animals. The spread of infection by the re-use of needles in developing countries would thereby be avoided.
• an oral vaccine may be provided in the form of an edible solid, which is easier to handle under extreme conditions and is more stable than liquid suspensions as currently used.
• the secretory immune response mainly IgA-mediated, appears to be substantially separate from the systemic immune response.
• Systemic vaccination is ineffective for raising a secretory immune response. This is a considerable disadvantage when considering immunisation against pathogens, which often enter the subject across a mucosal surface such as the gut or lung.
• pathogens which often enter the subject across a mucosal surface such as the gut or lung.
• no adjuvant capable of eliciting a secretory immune response to a given antigen is currently available.
• the apparent difficulty is largely due to a phenomenon known as oral tolerance.
• the linings of the gut and the lungs are naturally tolerant to foreign antigens, which prevents an immune response being raised to ingested or inhaled substances, such as food and airborne particulate matter.
• the ADP-ribosylating bacterial toxins namely diphtheria toxin, pertussis toxin (PT) , cholera toxin (CT) , the E.coli heat-labile toxin (LT1 and LT2) , Pseudomonas endotoxin A, C. botulinum C2 and C3 toxins as well as toxins from C. perfringens, C. spiriforma and C. difficile are potent toxins in man.
• These toxins are composed of a monomeric, enzymatically active A subunit which is responsible for ADP-ribosylation of GTP-binding proteins, and a non-toxic B subunit which binds receptors on the surface of the target cell and delivers the A subunit across the cell membrane.
• a subunit is known to increase intracellular cAMP levels in target cells, while the B subunit is pentameric and binds to GM1 ganglioside receptors.
• CT cholera toxoid
• CT induces systemic and mucosal immunity to co-administered antigens, in other words functions as a mucosal adjuvant
• Elson Curr. Top. Microbiol. Immunal, 1989; 146: 29; Elson and Ealding, J. Immunol. 1984; 133: 2892-2897; Elson and Ealding, Ibid. 1984; 132: 2736-2741; Elson et al. , J. Immunol. Methods 1984; 6_7: 101-118; Lycke and Homgren, Immunology 1986; 59: 301-338
• CTB was covalently coupled to horseradish peroxidase (HRP) and administered to mice intraduodenally. This gave rise to a powerful mucosal immune response to HRP (McKenzie and Halsey, J. Immunol 1984; 133: 1818-1824).
• HRP horseradish peroxidase
• CTB is not useful as a mucosal adjuvant.
• a second approach to eliminating the toxicity of CT has been to mutate the CT holotoxin in order to reduce or eliminate its toxicity.
• the toxicity of CT resides in the A subunit and a number of mutants of CT and its homologue, LT, comprising point mutations in the A subunit are known in the art. See, for example, International Patent Application 092/19265 (Amgen) . It is accepted in the art that CT and LT are generally interchangeable, showing considerable homology.
• an active mucosal adjuvant which may be used to increase the immunogenicity of an antigen when administered to a mucosal surface, such as orally or intranasally.
• the present invention in a first aspect, provides a pharmaceutical composition comprising a non-toxic mucosal adjuvant in admixture with a second antigen.
• the non-toxic mucosal adjuvant is a detoxified mutant of a bacterial ADP-ribosylating toxin, optionally comprising one or more amino acid additions, deletions or substitutions.
• Particularly suitable are detoxified mutants of CT or LT.
• a mutant LT in accordance with the invention may possess an Arg7 to Lys7 substitution at position 7 of the A subunit, the so-called LTK7 mutant.
• Alternative mutants are known to those skilled in the art and are preferred molecules for use in the present invention. Examples include PT mutated at position 129, in particular PT having a Glu 129->Gly mutation. Further mutants include PT mutated at one or both of Trp 26 and Arg 9, optionally in combination with the Glu 129 mutation.
• the mutant used in the invention may moreover be a mutant wherein the mutation has been effected in a part of the molecule which results in the prevention of proteolytic cleavage of the A subunit of the toxin, such that enzymatic activity is not brought about.
• Such mutants are described in Grant et al . Inf. and Immunity (1994) 62(10 ⁇ 4270-4278.
• the mutant may comprise an Arg 192->Gly mutation in LT or a corresponding mutation in another ADP- ribosylating toxin.
• the mutant of the invention is preferably in the form of a holotoxin, comprising the mutated A subunit and the B subunit, which may be oligomeric, as in the wild-type holotoxin.
• the B subunit is preferably not mutated.
• a mutated A subunit may be used in isolation from the B subunit, either in an essentially pure form or complexed with other agents, which may replace the B subunit and/or its functional contribution.
• the adjuvant of the invention is preferably administered in admixture with a suitable antigen against which it is desired to raise an immune response. If the antigen and the adjuvant are not in admixture, it is preferred that they be administered within a relatively short time of each other, at the same site of administration. It has been observed that the adjuvant effect provided by wild-type CT is short lived (see Lycke and Homgren, Immunology 1986; j>9_; 301-308).
• the mucosal adjuvant of the invention may be administered, optionally in isolation from other antigens, as a boost following systemic or mucosal administration of a vaccine.
• the precise formulation of the vaccine may vary in accordance with the nature of the im unogen.
• the mucosal adjuvant may be similarly enclosed so that the antigen and the adjuvant may interact simultaneously with the mucosal immune system.
• separate mucosal administration of the adjuvant of the invention may be used to enhance mucosal response to parentally-administered vaccines.
• the present invention provides the use of a non-toxic mutant of CT or LT as a mucosal adjuvant in the preparation of a composition for mucosal administration.
• the composition is a vaccine and is useful for the immunisation of a subject against a disease or the treatment of a subject suffering from a disease.
• the mutant comprises one or more a ino acid additions, substitutions or deletions in the amino acid sequence of the A subunit of CT or LT which is or are effective to abolish the toxicity of the toxin.
• a method for the prevention or treatment of a disease in a subject comprising administering to the subject an immunologically effective dose of an antigen adjuvanted with a non-toxic CT or LT mutant by contacting a mucosal surface of the subject with said adjuvanted antigen.
• the mucosal surface may be any suitable mucosal surface of the subject.
• the administration may be carried out by inhalation, by means of a rectal or vaginal suppository, or a pessary, by feeding or other buccal administration, by means of an aerosol, by intranasal delivery or direct application to mucosal surfaces.
• oral and intranasal administration are especially preferred.
• the subject may be any organism susceptible to immunisation. Especially indicated are humans and other mammals such as livestock, pets and wildlife.
• Diseases against which the subject may be immunised include all diseases capable of being treated or prevented by immunisation, including viral diseases, allergic manifestations, diseases caused by bacterial or other pathogens which enter through or colonise mucosal surfaces, AIDS, autoimmune diseases such as systemic Lupus Erythe - atosus, Alzheimer's disease and cancers.
• viral infections which may be treated or prevented using the invention include infection by DNA viruses, such as EBV and VZV, and in particular herpesviridae, for example HSV and HCMV, adenoviridae, papovaviridae, such as HPV,hepadna -viridae, such as HBV, infection by RNA viruses, such as picorvaviridae, especially polivirus and HAV, rhinoviruses and FMDV, togaviridae, flaviviridae, coronaviridae, paramyxo -viridae, such as RSV, ortho yoxoviridae, such as influenza virus, and retroviridae, especially HIV.
• DNA viruses such as EBV and VZV
• herpesviridae for example HSV and HCMV
• adenoviridae such as HPV,hepadna -viridae, such as HBV
• RNA viruses such as picorvaviridae, especially polivirus and
• bacterial infections suitable for treatment or prophylaxis by the invention include infection with Helicobacter pylori, streptococci, meningococcus A, B, and C, bordetella pertussis and chlamydia and trachomatis.
• Vaccine formulation suitable for delivery at mucosal surfaces may be prepared as set out hereinbelow, while further formulations will be apparent to those of skill in the art. Suitable administration regimes are, likewise, set out below while modifications of the exemplified values will be apparent to those of skill in the art.
• the invention provides a mutant of CT or LT which is a non-toxic mucosal adjuvant and a second antigen for simultaneous separate or sequential administration. Simultaneous administration of the adjuvant and the second antigen when combined in a single vehicle, carrier or particle, as exemplified below, is particularly preferred.
• the second antigen may be any antigen to which it is desired to raise an immune response in the subject.
• Suitable antigens comprise bacterial, viral and protozoan antigens derived from pathogenic organisms, as well as allergens, allogens and antigens derived from tumours and self-antigens.
• the antigen will be a protein, polypeptide or peptide antigen, but alternative antigenic structures, such as nucleic acid antigens, carbohydrate antigens, and whole or attenuated or inactivated organisms such as bacteria, viruses or protozoa are not excluded.
• the invention further provides a method for the manufacture of an adjuvanted vaccine comprising the steps of: a) performing site-diected mutagenesis on the A-subunit of a bacterial ADP-ribosylating toxin in order to detoxify the toxin; and b) bringing the detoxified toxin into association with a second antigen, such that it functions as a mucosal adjuvant.
• antigens useful in the present invention include HSV gD, gB and other glycoproteins; HIV gpl20 and other proteins; CMV gB or gH; HCV antigens; HDV delta antigen; HAV antigens; EBV and VZV antigens; B. pertussis antigens and H.
• the second antigen may be the immunogenic component of the vaccine intended for injection.
• Such vaccines, and the immunogenic components thereof may be subunit vaccines, whole inactivated or attenuated organisms or polynucleotide vaccines.
• the vaccines according to the invention may either be prophylactic (to prevent infection) or therapeutic (to treat disease after infection) .
• These vaccines may either be prophylactic (to prevent infection) or therapeutic (to treat disease after infection) .
• Such vaccines comprise antigen or antigens, usually in combination with "pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
• Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplet emulsions or liposomes) , and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. In preferred aspects of the invention, these carriers may function as immunostimulating agents ("adjuvants") . Furthermore, the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
• a bacterial toxoid such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
• Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum) , such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components) , such as for example (a) MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below) , although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer
• RibiTM adjuvant system Ribi Immunochem, Hamilton, MT
• MPL monophosphorylipid A
• TDM trehalose dimycolate
• CWS cell wall skeleton
• saponin adjuvants such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes)
• CFA Complete Freunds Adjuvant
• INFRAS RibiTM adjuvant system
• muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-thre ⁇ nyl-D -isoglutamine (thr-MDP) ,N-acetyl-normuramyl-l-alanyl -d-isoglutamine (nor-MDP) ,N-acetylmuramyl-1-alanyl-d-isoglutaminyl-l-alani ne-2-(1'-2 '-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy )-ethylamine (MTP-PE), etc.
• the immunogenic compositions e.g., the antigen, pharmaceutically acceptable carrier, and adjuvant
• auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
• the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
• the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
• Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic polypeptide ⁇ , as well as any other of the above-mentioned components, as needed.
• "i munolog " cally effective amount” it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
• the immunogenic compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule.
• the vaccine may be administered in conjunction with other immunoregulatory agents.
• immunostimulatory agents include interleukins, such as interleukins 1,2, 4-7 and 12, and interferons, especially 7 -interferon.
• interleukins such as interleukins 1,2, 4-7 and 12
• interferons especially 7 -interferon.
• Figure la shows the titre of total ovalbumin specific antibody in BALB/c mice immunised i/n or s/c with either ovalbumin alone or ovalbumin together with toxin derivatives;
• Figure lb shows the titre of total toxin-specific antibody in the mice of Figure la;
• Figure 2 shows a measurement of ovalbumin-specific IgA in nasal and lung lavages of mice injected as in Figure l;
• Figure 3 shows the presence of tetanus toxoid-specific antibodies in the serum of BALB/c mice immunised i/n or s/c with tetanus toxin fragment C alone or together with toxin derivatives.
• LTK7 Site-directed mutagenesis was used to replace the arginine residue at position seven of the A subunit of LT with lysine in order to construct a non-toxic LT mutant that could still assemble as a holotoxin with cell binding activity.
• the mutant protein named LTK7
• LTK7 was purified and tested for ADP-ribosyltransferase and toxic activity in several assays. LTK7 was still able to bind GM1 ganglioside receptor but showed a complete loss of enzymatic activity, in agreement with published data (Lobet et al . , Infect. Immun. 1991; 5_9:2870-2879) . Further, LTK7 was inactive in the mouse ileal loop assay and in vitro on Yl cells, even when a dose equivalent to 10 7 toxic units of wild-type LT was tested (Table 1) .
• mice were separated into groups and immunised using ovalbumin as a reporter antigen. Animals were immunised intranasally (i/n) or subcutaneously (s/c) using lO ⁇ g of ovalbumin alone or ovalbumin mixed with either l ⁇ g CT, LT or LTK7. Mice were split into four groups of six mice. Four mice from each group were lightly anaesthetised and immunised with either lO ⁇ g of ovalbumin or lO ⁇ g of ovalbumin with l ⁇ g of toxins, delivered in a total volume of 30 ⁇ l. The remaining two mice were immunised with the same amount of proteins s/c in a total volume of lOO ⁇ l. Proteins given subcut were first adsorbed to 2% A1(0H) 3 .
• Quantitation of antibody was estimated by ELISA.
• 96-well EIA plates (costar) were coated overnight with 60 ⁇ g/ml of ovalbumin. Measurement of toxin-specific antibodies was performed using a GM1 capture ELISA. Toxin-specific antibodies were measured against the antigen used in the immunisations. No single toxin was used in the measurements of toxin-specific antibody from each group, and as such the titres between these groups can not be compared directly.
• Sera from each group were pooled from four and two mice respectively. Samples were prepared in duplicate from a dilution of 1:50. Absorbences were read at 450nm using the kineticalc version 2.13 programme (Biotek instruments). This programme calculates the rate of change of substrate over thirty time points ten seconds apart.
• mice including those immunised with the mutant toxin, developed high levels of antibodies to these toxin in their sera.
• the local secretory antibody levels to ovalbumin were measured using both lung and nose washings (Fig. 2) .
• animals were culled by cardiac puncture and dissected so that the trachea was exposed. An ultra-thin pipette was then inserted into a small nick in the trachea.
• Lung washes were collected by repeated flushing and aspiration of 1.5 ml of 0.1% bovine serum albumin (Sigma), in PBS, into the lungs. Nose washes were collected by flushing 1ml of 0.1% BSA in PBS through the nasal cavity.
• Ovalbumin-specific IgA antibodies were measured by ELISA using an anti-mouse alpha-chain-specific conjugate antibody (Serotec) .
• Samples were prepared from individual animals and columns in this figure represent the mean rate of change of substrate, using kineticalc, for four and two mice immunised i/n and s/c respectively.
• the figures are constructed using the raw absorbence data at a dilution of 1:3 with respect to lung washes. These correspond to titres of between 1:2 and 1:6 for nose washes and between 1:70 and 1:120 for lung washes. These titres were calculated using the method described above.
• mice immunised s/c or i/n with ovalbumin alone contained no detectable ovalbumin-specific IgA in the washings sampled. All individual mice immunised with ovalbumin in combination with CT, LT or LTK7, showed detectable levels of anti-ovalbumin IgA. Thus both a local and systemic anti-ovalbumin response are detectable in these animals.
• mice were immunised i/n with a) lO ⁇ g of fragment C alone; b) lO ⁇ g of fragment C + l ⁇ g of LT; c) lO ⁇ g of fragment C + l ⁇ g of LTK7 and d) PBS only, all in a final volume of 30 ⁇ l.
• Five mice were immunised i/n with a) l ⁇ g of LT and b) l ⁇ g of LTK7. The remaining two groups of mice were immunised s/c with either no protein or lO ⁇ g of fragment C in a dose volume of lOO ⁇ l.
• These vaccines were prepared as described in Figure 1. Animals were immunised on day 1 and 22.
• mice that sero-convert to Fragment C can be protected against toxin challenge the groups were challenged with active tetanus toxin. All mice immunised s/c with Fragment C alone were protected whereas all mice immunised i/n were highly susceptible. All mice i/n immunised with Fragment C combined with either LT or LTK7 survived the challenge (Table 2) .
• the titre of anti-Fragment C antibodies in the serum of mice was on average about 1/3,000 in mice vaccinated with the K7 mutant + Fragment C and 1/12,000 for LT + Fragment C.

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