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WO1999047164A1 - Utilisation de l'enterotoxine mutante avec un exces de sous-unite b comme adjuvant - Google Patents

Utilisation de l'enterotoxine mutante avec un exces de sous-unite b comme adjuvant Download PDF

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Publication number
WO1999047164A1
WO1999047164A1 PCT/US1999/005622 US9905622W WO9947164A1 WO 1999047164 A1 WO1999047164 A1 WO 1999047164A1 US 9905622 W US9905622 W US 9905622W WO 9947164 A1 WO9947164 A1 WO 9947164A1
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Prior art keywords
subunit
holotoxin
antigen
mutant
virus
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PCT/US1999/005622
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English (en)
Inventor
John D. Clements
Martin Friede
Original Assignee
The Administrators Of The Tulane Educational Fund
Smithkline Beecham
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Application filed by The Administrators Of The Tulane Educational Fund, Smithkline Beecham filed Critical The Administrators Of The Tulane Educational Fund
Publication of WO1999047164A1 publication Critical patent/WO1999047164A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—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
    • A61K39/39—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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • 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

Definitions

  • the present invention is directed towards 5 compositions and methods which provide enhanced adjuvanticity of 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 0 substantially 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, together with an excess of B-subunits which is shown to have qualitatively enhanced adjuvanticity to induce 5 both antigen-specific antibody and T-cell responses when administered orally, and quantitatively enhanced adjuvanticity when administered intranasally, when compared to a formulation containing the mutant holotoxin without excess B-subunits.
  • HIV for example, once the virus crosses the mucosal surface and enters the host cell, be that a dendritic cell, c 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 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 5 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 0 of antigen-specific slgA at distant mucosal sites. It is only now being appreciated that mucosal immunization may be an effective means of inducing not only slgA but also systemic antibody and cell-mediated immunity.
  • the mucosal immune response can be divided into two phases (McGhee and Kiyono, 1993, Infect Agents Dis 12 . : 55-73) . 5
  • 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 and parenteral immunization A significant difference between mucosal immunization and parenteral immunization is that both mucosal and systemic immunity can be induced by mucosal immunization while c parenteral immunization generally results only in systemic responses .
  • helper T 0 lymphocytes secrete substantial amounts of IL-2 and INF-gamma and execute cell -mediated immune responses (e . g. , delayed type hypersensitivity and macrophage activation), whereas Th2 lymphocytes secrete IL-4, IL-5, IL-6 and IL-10 and assist in antibody production for humoral immunity.
  • Th helper T 0 lymphocytes
  • 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.
  • 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 autocrine fashion and macrophages in a paracrine fashion.
  • IL-6 which may induce the proliferation and differentiation of antigen specific B lymphocytes to secrete antibody (the effector phase) .
  • the predominant isotype secreted by murine B lymphocytes is often
  • 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 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 whereas
  • INF-gamma stimulates IgG2a secretion.
  • IL-6 and TGF-beta can cause isotype switching to IgA.
  • 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
  • CT cholera toxin
  • LT heat-labile enterotoxin
  • LT and CT have many features in common, these are clearly distinct molecules with biochemical and 5 immunologic differences which make them unique (see below) .
  • Both LT and CT are synthesized as multisubunit toxins with A and B components.
  • the A component dissociates into two smaller polypeptide chains.
  • the Al piece catalyzes the ADP-ribosylation of the 0 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.
  • the resulting increase in cAMP causes secretion of 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 0 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 subunits of relevant virulence determinants from these organisms.
  • Representative examples include tetanus toxoid
  • Infectious Diseases 175 : 839-846) Norwalk virus capsid 5 protein, synthetic peptides from measles virus (Hathaway et al., 1995, Vaccine 11:1495-1500), and the HIV-1 C4/V3 peptide T1SP10 MN(A) (Staats et al . , 1996, Journal of Immunology 157 :462-472) .
  • LT and CT have significant potential for use as 0 adjuvants for mucosally administered antigens (see Dickinson and Clements, 1996, Mucosal Vaccines pp73-87 for a recent review) .
  • toxins can stimulate a net lumenal secretory response may prevent their use. For instance, as little as 5 ⁇ g of purified CT was sufficient to induce significant diarrhea in volunteers while 25 ⁇ g was shown to elicit a full 20-liter 0 cholera purge (Levine et al . , 1983, Microbiological Reviews
  • 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.
  • 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.
  • the physical characteristics of this mutant were 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 adj uvanticity .
  • 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,
  • 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, IgA an 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.
  • ADP-ribosylagmatine activity has a ten-fold reduction in enterotoxicity in rabbit ligated ileal loops, and a 50% reduction and delayed onset of cAMP induction in cultured myeloma cells.
  • LT( ⁇ 192-194) was shown to have increased
  • LT(E112K) was effective when administered intranasally, subcutaneously, intraperitoneally, or orally c although mucosal IgA responses were only demonstrated following mucosal administration. These investigators also demonstrated increased adjuvant activity for mucosally administered LT( ⁇ 192-194) in conjunction with KLH, BCG, and
  • isolated B-subunit of LT exhibits adjuvanticity when administered intranasally, but not orally. In most studies, 5 however, the isolated B-subunits of LT does not exhbiit adjuvanticity.
  • isolated recombinant B-subunit does not have adjuvant activity. Where activity has been observed for isolated B-subunit, it has typically been with B-subunit prepared from LT holotoxin by 0 dissociation chromatography by gel filtration in the presence of a dissociating agent (i.e., guanidine HC1 or formic acid) .
  • a dissociating agent i.e., guanidine HC1 or formic acid
  • B-subunit prepared by this technique is invariably contaminated with trace amounts of A-subunit such that upon renaturation a small amount of holotoxin is 5 reconstituted.
  • LT-B (Clements et al . , 1988, Vaccine 6 : 269 -211 ) support that conclusion. It is not, however, a universally accepted conclusion.
  • LT-B with 0.5% LT holotoxin was shown to function as an immunologic adjuvant for influenza virus vaccine when administered intranasally. This represents a B-subunit to LT holotoxin ratio of 200:1 and no effect on toxicity was determined. Moreover, there was no indication that free B-subunit qualitatively changes the outcome when admixed with native LT.
  • the methods and compositions provide a qualitatively enhanced immunological outcome when administered orally.
  • the present invention is based on the surprising discovery that an amount of free B-subunit of the heat-labile
  • LT-B E. coli
  • LT(R192G) a protease-site mutant of LT
  • LT(R192G) a protease-site mutant of LT
  • the present invention also provides a method for further enhancing the immune response to a co-administered antigen when LT(R192G) is used as an oral adjuvant by including an excess amount of free B-subunit of LT.
  • the invention also provides a composition useful in these methods.
  • the composition comprises an effective amount of LT(R192G) in combination with free B-subunit of LT and an effective amount of antigen.
  • the present invention supersedes the prior art in that LT(R192G) in the presence of free B-subunit has enhanced adjuvanticity for both antigen-specific antibody and T-cell responses when administered orally and enhanced adjuvanticity when administered intranasally in comparison to LT(R192G) holotoxin without excess free B-subunit.
  • the utility of this surprising discovery is that an adjuvant effective amount of
  • LT(R192G) may be utilized in an effective immunization program against a variety of pathogens involving the administration of an effective amount of LT(R192G) adjuvant plus excess B-subunit in admixture with killed or attenuated pathogens or relevant virulence determinants of specific pathogens .
  • the present invention further supersedes the prior art in that the present invention may be used to specifically increase levels of antigen-specific Thl- and Th2-type cytokines and serum antibody responses when LT(R192G) in combination with excess free B-subunit of LT is used an oral adjuvant. This finding is totally unexpected, given current
  • holotoxin refers to a complex of five B-subunits and one A-subunit of heat-labile 0 enterotoxin.
  • free B-subunit refers to the B-subunit of heat-labile enterotoxin substantially free from the A-subunit of heat -labile enterotoxin.
  • the term "excess B-subunit” refers 5 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 holotoxin, i.e. natural heat- labile enterotoxin.
  • the term "qualitatively enhanced” o 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. 5
  • 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 LT(R192G).
  • 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 I 5 the reading frame but eliminates the proteolytic site.
  • Figure 2 is a graphic illustration of the effect of various ratios of free B-subunit to LT(R192G) in the patent mouse intestinal assay. For these studies, LT(R192G) with no excess B-subunit was admixed with different ratios of
  • mice 20 B-subunit and examined for toxicity 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 25 intestinal weight divided by the remaining carcass weight. There were three animals per group and the means for each data point are shown.
  • Figure 3 is an additional graphic illustration of the effect of excess B-subunit in the patent mouse intestinal
  • mice were orally inoculated with native LT at 5, 25, or 125 ⁇ g, or with 25 ⁇ g of LT(R192G) .
  • Other groups received either 25 ⁇ g of native LT(R192G) .
  • Figure 4 is a graphic illustration of the effect of excess B-subunit on the ability of LT(R192G) to function as an immunologic adjuvant for induction of serum IgG when administered intranasally. Mice were immunized intranasally with Ovalbumin (Ova) alone or in conjunction with 5 ⁇ g of 0
  • Serum anti -Ova IgG was determined by ELISA. There were seven animals per group and the means for each data point are shown.
  • Figure 5 is a graphical illustration of the effect 5 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, by mononuclear cells from the spleens of animals immunized intranasally.
  • Mice were immunized intranasally with Ovalbumin (Ova) alone o or in conjunction with 5 ⁇ g of LT(R192G) or 1.25 ⁇ g of
  • Cytokines were determined by ELISA following a
  • Figure 6 is a graphic illustration of the effect of excesss B-subunit on the ability of LT(R192G) to function as 5 an immunologic adjuvant for production of antigen-specific
  • Th2-type cytokines specifically, IL-10
  • Mice were immunized intranasally with Ovalbumin (Ova) alone or in conjunction with 5 ⁇ g of LT (R192G) -AB5 or 1.25 ⁇ g of 0 LT(R192G) plus 3.75 ⁇ g of excess free B-subunit, designated
  • Cytokines were determined by ELISA following a
  • B-subunit enhances the ability of LT(R192G) to function as an immunologic adjuvant for induction of serum IgG when 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. 0
  • 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
  • IgG when administered orally. Mice were immunized orally with Ovalbumin (Ova) alone or in conjunction with 25 ⁇ g of 5 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 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 0 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
  • Mice were immunized orally with Ovalbumin (Ova) alone or in conjunction with 25 ⁇ g of LT(R192G) or 6.25 ⁇ g of LT(R192G) 5 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 0 immunologic adjuvant for production of antigen-specific
  • Th2-type cytokines specifically, IL-10
  • the present invention provides novel compositions of LT(R192G) combined with free B-subunit and compositions thereof, and methods of using LT(R192G) and free B-subunit as an adjuvant having advantages for use to induce an immune 10 response to a co-administered antigen.
  • 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. Subsequent to the effective priority date of WO96/06627, others have had success in isolating LT(R192G) from E. coli expressing other plasmid constructs. (Grant et al . , (1994),
  • Plasmid pCS95 fully described in 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
  • 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 B-subunit of LT can be produced by a number of means apparent to those of skill in the art.
  • B-subunit of LT can be produced by a number of means apparent to those of skill in the art.
  • B-subunit of LT can be produced by a number of means apparent to those of skill in the art.
  • - 18 - subunit can be isolated from E. coli expressing pJC217, a plasmid fully described in U.S. Patent No. 5,308,835.
  • LT-B has also been isolated from bacteria expressing other plasmid constructs. For examples, see European Patent Application 5 Serial No. 0060129; Yamamoto et al . , 1981, J " . Bacteriol .
  • LT-B can be obtained from holotoxin obtained from E. coli or recombinantly expressed or from recombinantly expressed B subunit only.
  • LT-B can be purified by agarose affinity chromatography from bacteria expressing any plasmid encoding the B-subunit of LT. Alternate methods of purification will be apparent to those skilled in the art.
  • the present invention encompasses compositions and methods for use of the compositions 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,
  • compositions of the present invention is carried out through the mixing of a substantially pure preparation of LT(R192G) and LT-B subunit in amounts which yield the desired ratio of B-subunit to LT(R192G) .
  • the LT(R192G) in combination with free B-subunit is at a weight ratio of 1:1 to 100:1 of
  • LT(R192G) in combination with free B-subunit is at a weight ratio of 2:1 to 10:1 of B-subunit to LT(R192G).
  • the LT(R192G) in combination with free B-subunit is at a weight ratio of about 3:1 of B-subunit to LT(R192G) .
  • LT(R192G) has been shown to function as an effective adjuvant when administered on different mucosal c surfaces, the effect of free B-subunit on both intranasal and oral adjuvanticity was examined. The outcome of those studies revealed that LT(R192G) in the presence of free B-subunit had quantitatively enhanced adjuvanticity when administered intranasally and, surprisingly, both quantitatively and qualitatively enhanced adjuvanticity when 0 administered orally.
  • LT(R192G) 5 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.
  • the LT(R192G) plus free B-subunit and antigen are administered simultaneously in a pharmaceutical composition comprising an effective amount of LT(R192G) plus free B-subunit and an effective amount of antigen.
  • the antigen, the 5 LT(R192G), and the free B-subunit free of holotoxin are administered separately within a short 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 0 holotoxin.
  • the LT(R192G) administered in combination with free B-subunit is at a ratio of between 1:1
  • the LT(R192G) administered in combination with free B-subunit is at a weight ratio of 2:1 to 10:1 of
  • the LT(R192G) c administered in combination with free B-subunit is 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) .
  • the respective amounts of LT(R192G) plus free B-subunit and 0 antigen will vary depending upon the identity of the route of administration, antigen employed and the species of animal to be immunized.
  • the initial administration of LT(R192G) plus free B-subunit and antigen is followed by a boost of the relevant antigen.
  • no 5 boost is given.
  • the timing of boosting may vary, depending on the route, antigen and the species being treated. The modifications in route, 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 0 or i n combination with LT(R192G) plus free B-subunit.
  • compositions of the present invention are intended for use both in immature and mature vertebrates, in particular birds, mammals, and humans.
  • Useful 5 antigens include antigens from pathogenic strains of bacteria ( Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, 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 immi tis, Aspergillus fumigatus, Candida albicans, Blastomyces dermati tidis,
  • Trypanosoma rhodesiense Trypanosoma cruzi , Leishmania donovani , Leishmania tropica, Leishmania braziliensis,
  • Trichinella spiralis Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni , Schistosoma haematobium, and hookworms
  • Trichinella spiralis Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni , Schistosoma haematobium, and hookworms
  • pathogenic viruses as examples and not by limitation: Poxviridae,
  • 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, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B c Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus) either presented to the immune system in whole or in part isolated from media cultures designed to grow such viruses which are well known in the art or protective antigens therefrom obtained by genetic engineering techniques or by chemical synthesis.
  • Arboviridae Rhabdoviridae, Arenaviridae
  • Hepatitis A virus Hepatitis B virus
  • Hepatitis C virus Hepatitis E virus
  • Non-A/Non-B c Hepatitis virus Rhinoviridae, Coronaviridae, Rotoviridae,
  • 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, 5 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 0 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, entero
  • 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 artisan or comprise relevant antigens of these organisms or viruses which are produced by genetic engineering techniques 0 or chemical synthesis. Their production is illustrated by, but not limited to, as follows:
  • Influenza vaccine a vaccine comprising the whole or part of hemagglutinin, neuraminidase, nucleoprotein and matrix protein which are obtainable by purifying a virus, which is grown in embryonated eggs, with ether and detergent, c 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 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 0 by genetic engineering techniques or chemical synthesis.
  • Diphtheria and tetanus toxoid combined with pertussis vaccine a vaccine mixed with pertussis vaccine, diphtheria and tetanus toxoid.
  • Japanese encephalitis vaccine a vaccine 5 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 alcohol and inactivating the same, or by genetic engineering techniques or chemical synthesis.
  • 0 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 ultracentrifugation, obtained from hepatitis carrying blood, or by genetic engineering techniques or by chemical synthesis . 5
  • 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 0 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 egg, or a protective antigen obtained by genetic engineering techniques or chemical synthesis.
  • Mixed vaccine of measles, rubella and mumps a vaccine produced by mixing measles, rubella and mumps vaccines .
  • 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 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. Those conditions for which effective prevention may be achieved by the present method will be obvious to the skilled artisan.
  • the vaccine preparation compositions of the present invention can be prepared by mixing the above illustrated antigens and/or vaccines with LT(R192G) and excess free B- subunit at a desired ratio. Pyrogens or allergens should naturally be removed as completely as possible.
  • the antigen preparation of the present invention can be used by preparing the antigen per se and the LT(R192G) together with excess free B-subunit separately or together.
  • the present invention encompasses a kit comprising an effective amount of antigen and an adjuvant effective amount of LT(R192G) plus excess free B-subunit.
  • the components of the kit can either first be mixed together and then administered or the components can be administered separately within a short time of each other.
  • the vaccine preparation compositions of the present invention can be combined with either a liquid or solid
  • compositions can be in the form of tablets, capsules, powders, granules, suspensions or solutions.
  • the compositions can also contain suitable preservatives, coloring and flavoring agents, or agents that c 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, 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 tablettmg of the compositions for convenient administration.
  • the wild-type LT toxin is encoded on a naturally 20 occurring plasmid found in strains of enterotoxigenic E. coli capable of producing this toxin. Clements et al . had 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 Pstl site of plasmid pBR322 (Clements et al , 1983,
  • 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 regulatory region upstream from the LT-A coding region of
  • Figure IB shows the Arg to Gly mutation at position 192.
  • the BamHI and Xbal restriction sites referred to in the diagram as "new" were added by site directed mutagenesis, as described in PCT Publication WO96/06627.
  • the new Xbal site c was added through a silent mutation, resulting in no alteration of the amino acid sequence of the peptide encoded by the gene .
  • LT(R192G) was then purified by agarose affinity chromatography from bacteria expressing pCS95. This mutant
  • LT designated LT(R192G) was then examined by 0
  • the wild-type LT toxin is encoded on a naturally occurring plasmid found in strains of enterotoxigenic E. coli 0 capable of producing this toxin. Clements et al . had 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 Pstl site of plasmid pBR322 (Clements et al , 1983, Infect . Immun . 4 . 0:653).
  • This recombinant plasmid designated pDF82, has been extensively characterized and expresses LT under control of the native LT promoter.
  • the next step in this process was to place the LT-B gene under the control of a strong promoter, in this case the lac promoter on plasmid pUC18. This was accomplished by isolating the gene for LT-B 0 from pDF87 and recombining it in a cassette in the vector plasmid.
  • This plasmid, designated pJC217 is fully described in U.S. Patent No. 5,308,835.
  • LT(R192G) with no free B-subunit was admixed with different ratios of B-subunit and examined for toxicity in the patent mouse assay. The results are shown in Figure 2.
  • free B-subunit was admixed with LT(R192G) and also with native LT at a ratio of either 3:1 or 10 10:1. The results are shown in Figure 3.
  • Ovalbumin (Ova) was selected as a representative antigen for these studies. A number of investigations,
  • CFAI enterotoxigenic E. coli
  • mice were immunized intranasally with Ova alone or in conjunction with
  • 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, 5 even though a significantly lower total amount of LT(R192G) was administered (1.25 ⁇ g vs. 5 ⁇ g) .
  • 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 enhance the 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 (LT) pouvant être plus facilement utilisé comme adjuvant. De manière spécifique, l'invention concerne des formulations et des procédés permettant d'utiliser un mutant LT désigné LT (R192G) sans excès de sous-unité LT-B. Les compositions selon l'invention peuvent être utilisées beaucoup plus facilement, du point de vue qualitatif, comme adjuvants pour induire des réponses des lymphocytes T et des anticorps spécifiques à des antigènes lors de leur administration orale. Par voie intranasale, elles présentent une meilleure capacité d'administration comme adjuvant d'un point de vue quantitatif, par comparaison à une formulation contenant l'holotoxine mutante sans excès de sous-unité B.
PCT/US1999/005622 1998-03-18 1999-03-17 Utilisation de l'enterotoxine mutante avec un exces de sous-unite b comme adjuvant WO1999047164A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000027205A1 (fr) * 1998-11-12 2000-05-18 United States Government As Represented By The Secretary Of The Navy Polypeptide recombine destine a etre utilise pour la fabrication de vaccins contre la diarrhee induite par des campylobacteries, et pour la reduction de la colonisation
US7037499B1 (en) 1996-11-14 2006-05-02 The United States Of America As Represented By The Secretary Of The Army Adjuvant for transcutaneous immunization
US7294118B2 (en) 2001-10-24 2007-11-13 Becton, Dickinson And Company Retractable needle assembly
US7378097B2 (en) 1996-11-14 2008-05-27 The United States Of America As Represented By The Secretary Of The Army Use of penetration enhancers and barrier disruption methods to enhance the immune response of antigen and adjuvant
US7527802B2 (en) 2001-02-13 2009-05-05 The United States Of America As Represented By The Secretary Of The Army Vaccine for transcutaneous immunization
CN101968485A (zh) * 2010-04-09 2011-02-09 中国疾病预防控制中心寄生虫病预防控制所 检测血吸虫循环抗原的方法及其酶联免疫试剂盒
WO2011119174A1 (fr) 2010-03-23 2011-09-29 Development Center For Biotechnology Traitement de l'allergie utilisant une entérotoxine thermolabile d'e. coli détoxifiée
CN102279257A (zh) * 2011-06-27 2011-12-14 刘永庆 一种用于诊断人或动物的免疫相关性疾病的试剂盒
US8088394B2 (en) 2006-10-27 2012-01-03 Development Center For Biotechnology Mutated E. coli heat-labile enterotoxin
US8110197B2 (en) * 2006-10-27 2012-02-07 Development Center For Biotechnology Mutated E. coli heat-labile enterotoxin
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
CN109781996A (zh) * 2019-01-09 2019-05-21 吉林特研生物技术有限责任公司 一种肺炎克雷伯菌抗体elisa快速检测试剂盒及检测方法

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US4808700A (en) * 1984-07-09 1989-02-28 Praxis Biologics, Inc. Immunogenic conjugates of non-toxic E. coli LT-B enterotoxin subunit and capsular polymers
US5308835A (en) * 1984-07-09 1994-05-03 Praxis Biologics, Inc. Production of the E. coli LT-B enterotoxin subunit

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7037499B1 (en) 1996-11-14 2006-05-02 The United States Of America As Represented By The Secretary Of The Army Adjuvant for transcutaneous immunization
US7378097B2 (en) 1996-11-14 2008-05-27 The United States Of America As Represented By The Secretary Of The Army Use of penetration enhancers and barrier disruption methods to enhance the immune response of antigen and adjuvant
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
WO2000027205A1 (fr) * 1998-11-12 2000-05-18 United States Government As Represented By The Secretary Of The Navy Polypeptide recombine destine a etre utilise pour la fabrication de vaccins contre la diarrhee induite par des campylobacteries, et pour la reduction de la colonisation
US7527802B2 (en) 2001-02-13 2009-05-05 The United States Of America As Represented By The Secretary Of The Army Vaccine for transcutaneous immunization
US7294118B2 (en) 2001-10-24 2007-11-13 Becton, Dickinson And Company Retractable needle assembly
US8110197B2 (en) * 2006-10-27 2012-02-07 Development Center For Biotechnology Mutated E. coli heat-labile enterotoxin
US8088394B2 (en) 2006-10-27 2012-01-03 Development Center For Biotechnology Mutated E. coli heat-labile enterotoxin
WO2011119174A1 (fr) 2010-03-23 2011-09-29 Development Center For Biotechnology Traitement de l'allergie utilisant une entérotoxine thermolabile d'e. coli détoxifiée
CN101968485A (zh) * 2010-04-09 2011-02-09 中国疾病预防控制中心寄生虫病预防控制所 检测血吸虫循环抗原的方法及其酶联免疫试剂盒
CN102279257A (zh) * 2011-06-27 2011-12-14 刘永庆 一种用于诊断人或动物的免疫相关性疾病的试剂盒
CN102279257B (zh) * 2011-06-27 2014-02-19 刘永庆 一种用于诊断人或动物的免疫相关性疾病的试剂盒
CN109781996A (zh) * 2019-01-09 2019-05-21 吉林特研生物技术有限责任公司 一种肺炎克雷伯菌抗体elisa快速检测试剂盒及检测方法

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