WO1999047165A1 - Production d'enterotoxine mutante purifiee prevue pour etre utilisee comme adjuvant - Google Patents

Production d'enterotoxine mutante purifiee prevue pour etre utilisee comme adjuvant Download PDF

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Publication number
WO1999047165A1
WO1999047165A1 PCT/US1999/005625 US9905625W WO9947165A1 WO 1999047165 A1 WO1999047165 A1 WO 1999047165A1 US 9905625 W US9905625 W US 9905625W WO 9947165 A1 WO9947165 A1 WO 9947165A1
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subunit
vaccine
free
holotoxin
excess
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PCT/US1999/005625
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English (en)
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John D. Clements
Martin Friede
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The Administrators Of The Tulane Educational Fund
Smithkline Beecham
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Priority to AU30895/99A priority Critical patent/AU3089599A/en
Publication of WO1999047165A1 publication Critical patent/WO1999047165A1/fr

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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

Definitions

  • the present invention is directed towards 5 compositions and methods which provide a genetically distinct mutant of E. coli heat-labile enterotoxin (LT) .
  • the invention relates to formulations and methods for use of a mutant LT designated LT(R192G), modified by a single amino acid substitution that substantially
  • mutant holotoxin reduces its inherent toxicity but leaves intact the adjuvant properties of the molecule, provided as a single mutant A- subunit with five B-subunits, i.e., mutant holotoxin, which is shown to have adjuvanticity without the toxicity associated with wild type LT, said mutant holotoxin being
  • HIV for example, once the virus crosses the mucosal surface and enters the host cell, be that a dendritic cell, an epithelial cell, or a T-cell, the host-parasite relationship is moved decidedly in favor of the parasite (HIV) . In that case, as in many others, a vaccine strategy that does not prevent the initial infection of the host is unlikely to succeed.
  • slgA secretory IgA
  • mucosal immunization as a means of inducing secretory IgA (slgA) antibodies directed against specific pathogens of mucosal surfaces.
  • the rationale for this is the recognition that slgA constitutes greater than 80% of all antibodies produced in mucosal -associated lymphoid tissues in humans and that slgA may block attachment of bacteria and viruses, neutralize bacterial toxins, and even inactivate invading viruses inside of epithelial cells.
  • the existence of a Common Mucosal Immune System permits immunization on or at one mucosal surface to induce secretion of antigen-specific slgA at distant mucosal sites.
  • mucosal immunization may be an effective means of inducing not only slgA but also systemic antibody and cell-mediated immunity.
  • Tne mucosal immune response can be divided into two phases (McGhee and Kiyono, 1993, Infect Agents Dis 1 . 2:55-73) .
  • the inductive phase involves antigen presentation and the initiation events which dictate the subsequent immune response.
  • antigen-specific lymphocytes are primed and migrate from the inductive sites
  • mucosal immunization e . g. , Peyer's patches in the enteric mucosa
  • mucosal effector sites e . g. , lamina basement
  • mucosal immunization A significant difference between mucosal immunization and parenteral immunization is that both mucosal and systemic immunity can be induced by mucosal immunization while parenteral immunization generally results only in systemic responses .
  • Th helper T lymphocytes
  • Thl lymphocytes secrete substantial amounts of IL-2 and INF-gamma and execute cell-mediated immune responses (e . g. , delayed type hypersensitivity and macrophage
  • Th2 lymphocytes secrete IL-4, IL-5, IL-6 and IL-10 and assist in antibody production for humoral immunity.
  • antigenic stimulation of one T helper cell subset and not the other would result in production of a particular set of cytokines which would define the resulting immune response.
  • Thl type responses The presence of IL-2 and INF-gamma coupled with an antigenic stimulus presented by macrophages in the context of Class II MHC molecules can initiate Thl type responses.
  • the ability of Thl cells to secrete IL-2 and INF-gamma further amplifies the response by activating Thl cells in an
  • cytokines e . g. , IL-6
  • proliferation can release additional cytokines (e . g. , IL-6) which may induce the proliferation and
  • antigens such as allergens or parasites can effectively stimulate a Th2 lymphocyte response (the inductive phase) . Presentation of such antigens to Th2 cells can result in the production of the lymphokines IL-4 and IL-5 which can induce antigen specific B lymphocytes to secrete
  • IgE and IgGl induce eosinophillia, respectively (the 0 effector phase) .
  • stimulated Th2 cells can secrete IL-10 which has the ability to specifically inhibit secretion of IL-2 and INF-gamma by Thl lymphocytes and also to inhibit macrophage function.
  • T helper cell stimulated affects the resultant cellular immune response as well as the predominant immunoglobulin isotype secreted.
  • IL-4 stimulates switching to the IgE and IgGl isotypes
  • INF-gamma stimulates IgG2a secretion.
  • Mucosally administered antigens are frequently not immunogenic.
  • a number of strategies have been developed to facilitate mucosal immunization, including the use of attenuated mutants of bacteria ⁇ e . g. , Salmonella spp. ) as carriers of heterologous antigens, encapsulation of antigens into microspheres, gelatin capsules, different formulations of liposomes, adsorption onto nanoparticles, use of 0 lipophilic immune stimulating complexes, and addition of bacterial products with known adjuvant properties.
  • the two bacterial products with the greatest potential to function as
  • CT cholera toxin
  • LT heat-labile enterotoxin
  • LT and CT have many features in common, these are clearly distinct molecules with biochemical and immunologic differences which make them unique (see below) .
  • Both LT and CT are synthesized as multisubunit toxins with A and B components. On thiol reduction, the A component dissociates into two smaller polypeptide chains. One of 5 these, the Al piece, catalyzes the ADP-ribosylation of the stimulatory GTP-binding protein (GSa) in the adenylate cyclase enzyme complex on the basolateral surface of the epithelial cell resulting in increasing intracellular levels of cAMP.
  • GSa stimulatory GTP-binding protein
  • the resulting increase in cAMP causes secretion of 0 water and electrolytes into the small intestine through interaction with two cAMP-sensitive ion transport mechanisms involving 1) NaCl cotransport across the brush border of villous epithelial cells, and 2) electrogenic Na dependent Cl secretion by crypt cells (Field, 1980, Secretory Diarrhea _ pp21-30) .
  • the B-subunit binds to the host cell membrane receptor (ganglioside GM1) and facilitates the translocation of the A-subunit through the cell membrane.
  • CT and LT as a mucosal adjuvant against a variety of bacterial and viral pathogens using whole killed organisms or purified 0 subunits of relevant virulence determinants from these organisms.
  • Representative examples include tetanus toxoid (Xu-Amano et al . , 1993, Journal of Experimental Medicine ____78: 1309-1320; Yamamoto et al . , 1996, Annals of the New York
  • influenza virus Hashigucci et al . ,
  • the mutant of Clements et al . has been described more fully in PCT Publication WO96/06627, incorporated herein by reference.
  • the mutant LT holotoxin designated LT(R192G) was constructed using site-directed mutagenesis to create a single amino acid substitution within the disulfide subtended region of the A-subunit separating Al from A2. This single amino acid change altered the proteolytically sensitive site within this region, rendering the mutant insensitive to trypsin activation.
  • this mutant was examined by SDS-PAGE, its biological activity was examined on mouse Y-l adrenal tumor cells and Caco-2 cells, its enzymatic properties determined in an in vitro NAD:agmatine ADP-ribosyltransferase assay, and its immunogenicity and immunomodulating capabilities determined by testing for the retention of immunogenicity and adjuvanticity.
  • WO 96/06627 describes plasmid pBD95 which can be used to obtain the mutant LT(R192G) . Although not described in WO 96/06627, it has recently been discovered that when plasmid pBD95 is used to produce the mutant holotoxin,
  • LT(R192G) by expressing pBD95 in E. coli , varying amounts of free B-subunit can als be recovered as well as the holotoxin.
  • Microbiology 14:51-61) report mutant and wild-type AB5/AB5+B5 ratios that vary from 40% to 98% depending upon the type of mutation.
  • Such excess B-subunit can be separated from holotoxin by gel filtration chromatography due to the difference in molecular weight between the holotoxin and the free B-subunit pentamer (84 kd vs. 56 kd) .
  • LT(R192G) has been shown to possess the capability of enhancing an immune response (e . g. , IgG, IgA) to antigens unrelated to LT or LT(R192G) .
  • IgG immune response
  • IgA immune response
  • LT(R192G) has utility as an adjuvant for mucosally or parenterally administered antigens; such administration results in the production of serum IgG and/or mucosal slgA as well as cell-mediated immune responses against the antigen with which LT(R192G) is delivered and, more importantly, to protect against subsequent challenge with infectious organisms.
  • LT(R192G) has been shown to be an effective mucosal adjuvant and has recently been evaluated in humans in several Phase I safety studies . More recently, Tsuj i et al . (Tsuji et al . , 1997,
  • LT( ⁇ 192-194) was shown to have increased adjuvant activity for induction of serum IgG and mucosal IgA against measles virus when compared to native LT, LT-B, or LT(E112K).
  • LT( ⁇ 192-194) was effective when administered intranasally, subcutaneously, intraperitoneally, or orally although mucosal IgA responses were only demonstrated following mucosal administration.
  • the present invention provides an advantageously easily reproducible means of obtaining pure LT(R192G) free I 5 from excess B-subunit of LT, by providing a novel plasmid which expresses LT(R192G) free of excess B-subunit.
  • the present invention also provides for an expressable DNA encoding both a mutant A-subunit, which has arginine at position 192 changed to glycine, and a wild-type 20 B-subunit, which plasmid allows for the expression of LT(R192G) holotoxin with less than 10% excess B-subunit.
  • LT-B is able to enhance the adjuvanticity of LT(R192G) .
  • free B-subunit in combination with LT(R192G) was 25 found to qualitatively enhance the immunological outcome when LT(R192G) was used as an oral adjuvant. See U.S. Patent Application Serial No. 09/040,990, filed concurrently.
  • the present invention provides a method for producing pure LT(R192G), substantially free from excess B-
  • holotoxin refers to a complex of five B-subunits and one A-subunit of heat-labile enterotoxin.
  • free B-subunit refers to the B-subunit of heat-labile enterotoxin substantially free from the A-subunit of heat -labile enterotoxin.
  • Excess B-subunit refers to an amount of B-subunit which results in greater than a 5:1 ratio of B-subunits to A-subunit, 5:1 being the ratio of B:A subunits present in native heat-labile enterotoxin holotoxin.
  • the term "qualitatively enhanced” refers to an immune response which differs from the type of response elicited by adjuvant and immunogen without excess B- subunit .
  • immunogen with LT(R192G) with excess B-subunit elicits an enhanced T- cell response as compared to immunogen and LT(R192G) without excess B-subunit, which elicits a mostly humoral response.
  • the term “quantitatively enhanced” refers to an immune response which is greater than normal, but does not differ in the type of immune response elicited.
  • adjuvanticity of LT(R192G) is enhanced four fold, such that only one fourth the amount of LT(R192G) with excess B-subunit is required, as compared to LT(R192G) without excess B-subunit, to elicit a comparable immune response .
  • Figure 1 is a schematic diagram of plasmid pCS95, which encodes both subunits LT A and B under the control of the lac promoter.
  • Figure 1A illustrates the construction of plasmid pCS95 which contains the nucleotide sequence encoding mutant LT(R192G) .
  • Plasmid pCS95 was constructed by replacing the BamHI-Xbal of pBD95 with the BamHI-Xbal fragment of pDF82.
  • Figure IB shows the single amino acid change in
  • Plasmid pCS95 provides LT(R192G) which contains the single base substitution at amino acid residue 192 of subunit A, coding for Gly rather than Arg, which preserves the reading frame but eliminates the proteolytic site.
  • FIG. 2 is a graphic illustration of the effect of various ratios of free B-subunit to LT(R192G) in the patent mouse intestinal assay.
  • LT(R192G) with no excess B-subunit was admixed with different ratios of B-subunit and examined for enterotoxicity in the patent mouse assay.
  • Groups of mice were orally inoculated with native LT at 5, 25, 50 or 100 ⁇ g, or with 25 ⁇ g of LT(R192G) admixed with a different amount of free B-subunit.
  • the gut: carcass ratio of each animal was determined.
  • the gut-carcass ratio is defined as the intestinal weight divided by the remaining carcass weight.
  • Figure 3 is an additional graphic illustration of the effect of excess B-subunit in the patent mouse intestinal assay.
  • groups of mice were orally inoculated with native LT at 5 , 25, or 125 ⁇ , or with 25 ⁇ g of LT(R192G) .
  • Other groups received either 25 ⁇ g of native T or 25 ⁇ g of LT(R192G) admixed with a 3:1 or 10:1 excess of free B-subunit.
  • the gut: carcass ratio of each animal was determined. There were
  • Figure 4 is a graphic illustration of the effect of excess B-subunit on the ability of LT(R192G) to function as c an immunologic adjuvant for induction of serum IgG when administered intranasallv.
  • Mice were immunized intranasally with Ovalbumin (Ova) alone or in conjunction with 5 ⁇ g of Ovalbumin (Ova)
  • Serum anti -Ova IgG was determined by ELISA. There were seven animals per group and 0 the means for each data point are shown.
  • Figure 5 is a graphical illustration of the effect of excess B-subunit on the ability of LT(R192G) to function as an immunologic adjuvant for production of antigen-specific
  • Thl-type cytokines specifically, IFN-gamma
  • Mice were immunized intranasally with Ovalbumin (Ova) alone or in conjunction with 5 ⁇ g of LT(R192G) or 1.25 ⁇ g of LT(R192G) plus 3.75 ⁇ g of excess free B-subunit, designated 1AB5:3B5.
  • Cytokines were determined by ELISA following a o T-cell restimulation assay.
  • Figure 6 is a graphic illustration of the effect of excesss B-subunit on the ability of LT(R192G) to function as an immunologic adjuvant for production of antigen-specific
  • Th2-type cytokines specifically, IL-10
  • Mice 5 were immunized intranasally with Ovalbumin (Ova) alone or in conjunction with 5 ⁇ g of LT(R192G) or 1.25 ⁇ g of LT(R192G) plus 3.75 ⁇ g of excess free B-subunit, designated 1AB5:3B5.
  • Ovalbumin Ova
  • B-subunit enhances the ability of LT(R192G) to function as an immunologic adjuvant for induction of serum IgG when
  • mice 14 - administered orally.
  • Serum anti -CFAI IgG was determined by ELISA. There were seven animals per group and the means for each data point are shown.
  • Figure 8 is an additional graphic illustration that excess B-subunit enhances the ability of LT(R192G) to function as an immunologic adjuvant for induction of serum
  • B-subunit designated 1AB5:3B5.
  • Serum anti-Ova IgG was determined by ELISA. There were ten animals per group and the means for each data point are shown.
  • Figure 9 is a graphic demonstration that excess B-subunit enhances the ability of LT(R192G) to function as an immunologic adjuvant for production of antigen-specific Thl-type cytokines, specifically, IFN-gamma, by mononuclear cells from the spleens of animals immunized orally.
  • Mice were immunized orally with Ovalbumin (Ova) alone or in conjunction with 25 ⁇ g of LT(R192G) or 6.25 ⁇ g of LT(R192G) plus 18.75 ⁇ g of free B-subunit, designated 1AB5:3B5.
  • Ovalbumin Ova
  • Cytokines were determined by ELISA following a T-cell restimulation assay.
  • Figure 10 is a graphic demonstration that excess
  • B-subunit enhances the ability of LT(R192G) to function as an immunologic adjuvant for production of antigen-specific
  • Th2-type cytokines specifically, IL-10
  • Mice were immunized orally with Ovalbumin (Ova) alone or in conjunction with 25 ⁇ g of LT(R192G) or 6.25 ⁇ g of LT(R192G) plus 18.75 ⁇ g
  • Cytokines were determined by ELISA following a T-cell restimulation assay.
  • the present invention provides pure LT(R192G) by providing novel compositions of LT(R192G) and methods of using said compositions as adjuvants.
  • the present invention also provides for an expressable DNA encoding both a mutant A-subunit, which has arginine at position 192 changed to glycine, and a wild-type
  • LT is a member of the ADP-ribosylating family of bacterial toxins and, like other bacterial toxins that are members of the A-B toxin family, requires proteolysis of a trypsin sensitive bond to become fully active. That trypsin sensitive peptide is subtended by a disulfide interchange that joins the Al and A2 pieces of the A-subunit. In theory, if the Al and A2 pieces cannot separate, Al will not be able to find its target (adenylate cyclase) on the basolateral surface or assume the conformation necessary to bind or hydrolyze NAD. An unexpected finding was that LT(R192G) has greatly reduced toxicity, but retains the adjuvant properties of wild type LT.
  • LT(R192G) can be produced by a number of means apparent to those of skill in the art.
  • LT(R192G) can be isolated from E. coli expressing pBD95, a plasmid fully described in PCT Publication WO96/06627.
  • Example 6.1 can also be utilized to produce isolated or substantially pure LT(R192G) in E. coli .
  • LT(R192G) can be isolated by agarose affinity chromatography from bacteria expressing an LT(R192G) encoding plasmid. Alternate methods of purification will be apparent to those skilled in the art.
  • LT(R192G) produced by any means can be further purified by gel filtration chromatography, which allows for the separation of holotoxin from any free A or B subunits.
  • the present invention provides mutant LT(R192G) useful for compositions and methods to promote the production of serum and/or mucosal antibodies as well as cell -mediated immune responses against an antigen that is simultaneously administered with a genetically modified bacterial toxin, i.e. , LT(R192G) .
  • compositions of the present invention are useful for the formulation of adjuvant compositions through the mixing of a substantially pure preparation of LT(R192G), free LT-B subunit, and an antigen.
  • the product of this invention is useful to prepare formulations which can be administered as described below.
  • LT(R192G) in combination with B-subunit free of holotoxin at any B-subunit to LT(R192G) ratio of 1:1 or greater is administered in conjunction with any biologically relevant antigen and/or vaccine, such that an increased immune response to said antigen and/or vaccine is achieved.
  • LT(R192G) plus free B-subunit and antigen are administered simultaneously in a pharmaceutical composition
  • - 17 comprising an effective amount of LT(R192G) plus free B-subunit and an effective amount of antigen.
  • the antigen, the LT(R192G), and the free B-subunit free of holotoxin are administered separately within a short 5 time of each other.
  • the antigen is administered separately within a short time of the simultaneous administration of the LT(R192G) and the B-subunit free of holotoxin.
  • B-subunit is at a ratio of between 1:1 and 100:1 of B-subunit
  • LT(R192G) administered in combination with free B-subunit can be at a weight ratio of 2:1 to 10:1 of B-subunit to LT(R192G).
  • the LT(R192G) administered in combination with free B-subunit can be at a weight ratio of about 3:1 of B-subunit to LT(R192G).
  • the mode of administration is mucosal (i.e., intranasal, oral, rectal) or parenteral (i . e . , subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal) .
  • parenteral i . e . , subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal
  • LT(R192G) plus free B-subunit and antigen will vary depending upon the identity of the route of
  • LT(R192G) plus free B-subunit and antigen can be followed by a boost of the relevant antigen. Alternatively, no boost is given.
  • the timing of boosting may vary, depending on the route, antigen and the species being treated. The modifications in route,
  • ______ O dosage range and timing of boosting for any given species and antigen are readily determinable by routine experimentation.
  • the boost may be of antigen alone or in combination with LT(R192G) plus free B-subunit.
  • compositions of the present invention are intended for use both in immature and mature
  • antigens from pathogenic strains of bacteria ( Streptococcus pyogenes , Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningi tidis , Corynebacterium diphtheriae,
  • Klebsiella ozaenae Klebsiella rhinoscleromotis
  • Staphylococcus aureus Vibrio cholerae, Escherichia coli ,
  • Shigella dysenteriae Shigella flexneri , Shigella sonnei ,
  • Salmonella typhimurium, Treponema pallidum, Treponema per pneumonia, Treponema carateneum, Borrelia vincentii , Borrelia burgdorferi , Leptospira icterohemorrhagiae, Mycobacterium tuberculosis , Toxoplasma gondii , Pneumocystis carinii ,
  • Francisella tularensis Francisella tularensis , Brucella abortus, Brucella suis,
  • Rickettsia tsutsugumushi Chlamydia spp . , Helicobacter pylori
  • pathogenic fungi Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blasto yces dermati tidis , Cryptococcus neoformans , Histoplasma capsulatum
  • protozoa Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis , Trichomonas vaginal is , Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi , Leishmania donovani , Leishmania tropica, Leishmania braziliensis , Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria) ; or Helmin
  • Herpesviridae Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae,
  • vaccines include, but are not limited to, vaccines.
  • vaccines include, but are not limited to, influenza vaccine, pertussis vaccine, diphtheria and tetanus toxoid combined with pertussis vaccine, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, hepatitis E vaccine, Japanese encephalitis vaccine, herpes vaccine, measles vaccine, rubella vaccine, mumps vaccine, mixed vaccine of measles, mumps and rubella, papillomavirus vaccine, parvovirus vaccine, respiratory syncytial virus vaccine, Lyme disease vaccine, polio vaccine, varicella vaccine, gonorrhea vaccine, schistosomiasis vaccine, rotavirus vaccine, mycoplasma vaccine pneumococcal vaccine, meningococcal vaccine, campylobacter vaccine, helicobacter vaccine, cholera vaccine, enterotoxigenic E. coli vaccine, enterohemmorg
  • Such vaccines can be produced by known common processes.
  • such vaccines comprise either the entire organism or virus grown and isolated by techniques well known to the skilled c artisan or comprise relevant antigens of these organisms or viruses which are produced by genetic engineering techniques or chemical synthesis. Their production is illustrated by, but not limited to, the following:
  • Influenza vaccine a vaccine comprising the whole or part of hemagglutinin, neuraminidase, nucleoprotein and 0 matrix protein which are obtainable by purifying a virus, which is grown in embryonated eggs, with ether and detergent, or by genetic engineering techniques or chemical synthesis.
  • Pertussis vaccine a vaccine comprising the whole or a part of pertussis toxin, hemagglutinin and K-agglutinin 5 which are obtained from avirulent toxin with formalin which is extracted by salting-out or ultracentrifugation from the culture broth or bacterial cells of Bordetella pertussis, or by genetic engineering techniques or chemical synthesis.
  • Diphtheria and tetanus toxoid combined with 0 pertussis vaccine a vaccine mixed with pertussis vaccine, diphtheria and tetanus toxoid.
  • Japanese encephalitis vaccine a vaccine comprising the whole or part of an antigenic protein which is obtained by culturing a virus intracerebrally in mice and purifying the virus particles by centrifugation or ethyl 5 alcohol and inactivating the same, or by genetic engineering techniques or chemical synthesis.
  • Hepatitis B vaccine a vaccine comprising the whole or part of an antigen protein which is obtained by isolating and purifying the HBs antigen by salting-out or 0 ultracentrifugation, obtained from hepatitis carrying blood, or by genetic engineering techniques or by chemical synthesis.
  • Measles vaccine a vaccine comprising the whole or part of a virus grown in a cultured chick embryo cells or embryonated egg, or a protective antigen obtained by genetic engineering or chemical synthesis.
  • Rubella vaccine a vaccine comprising the whole or part of a virus grown in cultured chick embryo cells or embryonated egg, or a protective antigen obtained by genetic engineering techniques or chemical synthesis.
  • Mumps vaccine a vaccine comprising the whole or part of a virus grown in cultured rabbit cells or embryonated 0 egg, or a protective antigen obtained by genetic engineering techniques or chemical synthesis.
  • Rotavirus vaccine a vaccine comprising the whole or part of a virus grown in cultured MA 104 cells or isolated from the patient's feces, or a protective antigen obtained by genetic engineering techniques or chemical synthesis.
  • Mycoplasma vaccine a vaccine comprising the whole o or part of mycoplasma cells grown in a liquid culture medium for mycoplasma or a protective antigen obtained by genetic engineering techniques or chemical synthesis .
  • the vaccine preparation compositions of the present invention can be prepared by mixing the above illustrated antigens and/or vaccines with LT(R192G) at a desired ratio.
  • the antigen preparation of the 0 present invention can be used by preparing the antigen per se and the LT(R192G) separately or together.
  • the present invention encompasses a kit comprising an effective amount of antigen and an adjuvant effective amount of LT(R192G).
  • the components of the kit can either first be mixed together and then administered c or the components can be administered separately within a short time of each other.
  • compositions of the present invention can be combined with either a liquid or solid pharmaceutical carrier, and the compositions can be in the form of tablets, capsules, powders, granules, suspensions or 0 solutions.
  • the compositions can also contain suitable preservatives, coloring and flavoring agents, or agents that produce slow release.
  • Potential carriers that can be used in the preparation of the pharmaceutical compositions of this invention include, but are not limited to, gelatin capsules, 5 sugars, cellulose derivations such as sodium carboxymethyl cellulose, gelatin, talc, magnesium stearate, vegetable oil such as peanut oil, etc., glycerin, sorbitol, agar and water. Carriers may also serve as a binder to facilitate tabletting of the compositions for convenient administration. 0
  • the wild-type LT toxin is encoded on a naturally occurring plasmid found in strains of enterotoxigenic E. coli capable of producing this toxin. Clements et al . had 5 previously cloned the LT gene from a human isolate of E. coli designated H10407. This subclone consists of a 5.2 kb DNA fragment from the enterotoxin plasmid of H10407 inserted into the PstI site of plasmid pBR322 (Clements et al , 1983, Infect . Immun . 4 . 0:653).
  • pDF82 This recombinant plasmid, designated pDF82, has been extensively characterized and expresses LT under control of the native LT promoter. From pDF82, Clements et al . derived plasmid pBD95, which is fully described in PCT Publication WO96/06627.
  • Figure 1A shows the construction of plasmid pCS95, which was constructed by inserting the native LT-A subunit
  • Figure IB shows the Arg to Gly mutation at position
  • LT(R192G) was then purified by agarose affinity chromatography from bacteria expressing pCS95. This mutant
  • LT(R192G) with no free B-subunit was admixed with different ratios of B-subunit and examined for toxicity in 5 the patent mouse assay.
  • the addition of excess free B-subunit to LT(R192G) results in a reduction of the gut/carcass ratio as measured in the patent mouse assay.
  • free B-subunit was admixed with LT(R192G) and also with native LT at a ratio of 0 either 3:1 or 10:1.
  • free B-subunit suppresses the toxicity of native LT.
  • Ovalbumin (Ova) was selected as a representative antigen for these studies.
  • a number of investigations, including our own (Clements et al . , 1988, Vaccine 6 . : 269-277;
  • CFAI Colonizing Factor Antigen I
  • mice were immunized intranasally with Ova alone or in conjunction with
  • Serum anti -Ova IgG was determined by ELISA. There were seven animals per group and the means for each data point are shown. As shown in Figure
  • LT(R192G) containing excess B-subunit had serum anti-Ova IgG responses indistinguishable from animals immunized with Ova in conjunction with LT(R192G) without excess of B-subunit, even though a significantly lower total amount of LT(R192G) was administered (1.25 ⁇ g vs. 5 ⁇ g) .
  • mice were immunized orally with purified Colonizing Factor I (CFAI) from enterotoxigenic E. coli in conjunction with 6.25 ⁇ g of LT(R192G) or 6.25 ⁇ g of LT(R192G) plus 18.75 ⁇ g of free B-subunit, designated 1AB5:3B5.
  • Serum anti-CFAI IgG was determined by ELISA. There were seven animals per group and the means for each data point are shown. As shown in Figure
  • mice immunized orally with CFAI in conjunction with LT(R192G) containing excess B-subunit had serum anti-CFAI IgG responses significantly higher that did animals immunized with CFAI in conjunction with LT(R192G) without excess of c B-subunit.
  • excess B-subunit is able to alter the type of immune response elicited by oral administration of LT(R192G) with an antigen.
  • the CFAI response was unique because of the inherent ability of colonizing factors to bind to epithelial cells.
  • mice were immunized orally with Ova alone or in conjunction with 25 ⁇ g of LT(R192G) or 6.25 ⁇ g of LT(R192G) plus 18.75 ⁇ g of free B-subunit, designated 1AB5:3B5.
  • Serum anti-Ova IgG was 5 determined by ELISA. There were ten animals per group and the means for each data point are shown. As shown in Figure
  • mice immunized orally with Ova in conjunction with LT(R192G) containing excess B-subunit had significantly higher serum anti-Ova IgG responses than did animals 0 immunized with Ova in conjunction with LT(R192G) without excess of B-subunit, even though a significantly lower total amount of LT(R192G) was administered (6.25 ⁇ g vs. 25 ⁇ g) .
  • B-subunit was included in the adjuvant formulation compared 0 to LT(R192G) without free B-subunit.
  • ATCC American Type Culture Collection

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Abstract

La présente invention concerne des compositions et des procédés permettant d'obtenir un mutant génétiquement distinct d'entérotoxine labile à la chaleur d'E.Coli. De manière spécifique, l'invention concerne des formulations et des procédés permettant d'utiliser un mutant LT désigné LT (R192G) comme adjuvant sans la toxicité associée au type LT sauvage. Cette holotoxine mutante est obtenue en forme pure, sensiblement exempte d'excès de sous-unité B. Le LT (R192G) présente une grande utilité en association avec une sous-unité B libre pour induire une réponse immunitaire.
PCT/US1999/005625 1998-03-18 1999-03-17 Production d'enterotoxine mutante purifiee prevue pour etre utilisee comme adjuvant WO1999047165A1 (fr)

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Publication number Priority date Publication date Assignee Title
US8911742B2 (en) 1996-11-14 2014-12-16 The United States Of America As Represented By The Secretary Of The Army Transcutaneous immunization without heterologous adjuvant

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WO1992019265A1 (fr) * 1991-05-02 1992-11-12 Amgen Inc. Analogues de sous-unites de toxine du cholera derives d'adn recombine____________________________________________________________
WO1993013202A1 (fr) * 1991-12-31 1993-07-08 Biocine Sclavo Spa Mutants immunogenes detoxiques de la toxine du cholera et des toxines thermolabiles (lt), preparation et utilisation de ces mutant pour la preparation de vaccins
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8911742B2 (en) 1996-11-14 2014-12-16 The United States Of America As Represented By The Secretary Of The Army Transcutaneous immunization without heterologous adjuvant

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