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WO1998032461A1 - Enterotoxine mutante utile en tant qu'adjuvant non toxique pour le vih - Google Patents

Enterotoxine mutante utile en tant qu'adjuvant non toxique pour le vih Download PDF

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Publication number
WO1998032461A1
WO1998032461A1 PCT/US1998/001546 US9801546W WO9832461A1 WO 1998032461 A1 WO1998032461 A1 WO 1998032461A1 US 9801546 W US9801546 W US 9801546W WO 9832461 A1 WO9832461 A1 WO 9832461A1
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Prior art keywords
hiv
mlt
adjuvant
mucosal
antigen
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Ceased
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PCT/US1998/001546
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English (en)
Inventor
John D. Clements
Bonny L. Dickinson
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Tulane University
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Tulane University
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Priority to AU60445/98A priority Critical patent/AU6044598A/en
Publication of WO1998032461A1 publication Critical patent/WO1998032461A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55544—Bacterial toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is directed towards a genetically distinct mutant of E. coli heat-labile enterotoxin (LT) and its use as an adjuvant to induce mucosal and serum antibodies and cell mediated immune responses.
  • LT heat-labile enterotoxin
  • the mutant LT is modified by a single amino acid substitution that reduces its inherent toxicity but leaves intact the adjuvant properties of the molecule.
  • Microbial pathogens can infect a host by one of several mechanisms. They may enter through a break in the integument induced by trauma, they may be introduced by vector transmission, or they may interact with a mucosal surface. The majority of human pathogens initiate disease by the last mechanism, i.e., following interaction with mucosal surfaces. Bacterial and viral pathogens that act through this mechanism first make contact with the mucosal surface where they may attach and then colonize, or be taken up by specialized absorptive cells (M cells) in the epithelium that overlay Peyer ' s patches and other lymphoid follicles [Bockman and Cooper, 1973, Am. J. Anat.
  • M cells absorptive cells
  • Organisms that enter the lymphoid tissues may be readily killed within the lymphoid follicles, thereby provoking a potentially protective immunological response as antigens are delivered to immune cells within the follicles (e.g., Vibrio cholerae) .
  • pathogenic organisms capable of surviving local defense mechanisms may spread from the follicles and subsequently cause local or systemic disease (i.e., Salmonella spp. , poliovirus, rotavirus in immunocompromised hosts) .
  • Secretory IgA antibodies directed against specific virulence determinants of infecting organisms play an important role in overall mucosal immunity [Cebra et al., 1986, In: Vaccines 86, Brown et al. (ed.), Cold Spring Harbor Laboratory, New York. p.p. 129-133]. In many cases, it is possible to prevent the initial infection of mucosal surfaces by stimulating production of mucosal slgA levels directed against relevant virulence determinants of an infecting organism. Secretory IgA may prevent the initial interaction of the pathogen with the mucosal surface by blocking attachment and/or colonization, neutralizing surface acting toxins, or preventing invasion of the host cells.
  • Mucosal immunization can be effective for induction of specific slgA responses if the antigens are presented to the T and B lymphocytes and accessory cells contained within the Peyer ' s patches where preferential IgA B-cell development is initiated.
  • the Peyer ' s patches contain helper T (TH) -cells that mediate B-cell isotype switching directly from IgM cells to IgA B-cells.
  • the patches also contain T-cells that initiate terminal B-cell differentiation.
  • the primed B-cells then migrate to the mesenteric lymph nodes and undergo differentiation, enter the thoracic duct, then the general circulation, and subsequently seed all of the secretory tissues of the body, including the lamina intestinal of the gut and respiratory tract.
  • IgA is then produced by the mature plasma cells, complexed with membrane-bound Secretory Component, and transported onto the mucosal surface where it is available to interact with invading pathogens [Strober and Jacobs, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 1-30; Tomasi and Plaut, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 31-61].
  • CT cholera toxin
  • LT heat-labile enterotoxin
  • the 56,000 dalton region or choleragenoid, is responsible for binding of the toxin to the host cell membrane receptor, G M1 (galactosyl- N-acetylgalactosaminyl- (sialyl)- galactosyl-glucosyl ceramide) , which is found on the surface of essentially all eukaryotic cells.
  • Choleragenoid is composed of five non-covalently associated monomers, while the A region (27,000 daltons) is responsible for the diverse biological effects of the toxin.
  • CT is not subject to oral tolerance [Elson and Ealding, 1984, J. Immunol. 133: 2892-2897]. • CT-B is not subject to oral tolerance [Elson and Ealding, 1984, J. Immunol. 133: 2892-2897].
  • CT can act as an adjuvant for CT-B [Elson and Ealding, 1984, J. Immunol. 133: 2892-2897]. • Heat aggregated CT has little toxicity but is a potent oral immunogen [Pierce et al., 1983, Infect. Immun. 40: 1112-1118] .
  • CT-B can serve as an immunologic "carrier" in a traditional hapten-carrier configuration [Cebra et al., 1986, In: Vaccines 86, Brown et al. (ed.), Cold Spring Harbor
  • B-subunit a traditional method of preparing B-subunit has been to subject holotoxin to 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
  • the isolated subunits are then pooled and the dissociating agent removed.
  • 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 reconstituted.
  • the second reason has to do with the definition of an immunologic carrier.
  • B-subunit can serve as an immunologic vehicle for presentation of antigens to the immune system.
  • the immunologically relevant sites for initiation of an immune response are the Peyer ' s patches, especially for antigen-specific T cell-dependent B cell activation [Strober and Jacobs, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 1-30; Tomasi and Plaut, 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 31-61; Brandtzaeg, 1989, Curr. Top. Microbiol. Immunol. 146: 13-25].
  • Antigens localized on the epithelial cell surface may contribute to antigen induced B cell proliferation in that the class II positive epithelial cells may act as antigen presenting cells for T cell activation at the secretory site, thereby increasing cytokine production, terminal B cell differentiation, increased expression of secretory component, and increased external transport of antigen specific IgA [Tomasi, T. B., and A.G. Plaut. 1985, In: Advances in host defense mechanisms. Vol. 4. Mucosal Immunity, Gallin and Fauci (ed.), Raven Press, New York. p.p. 31-61].
  • B-subunit As a carrier of other antigens and use of the term "adjuvant" would seem inappropriate for such an effect. It is clear that the adjuvant property of the molecule resides in the holotoxin in which B-subunit is required for receptor recognition and to facilitate penetration of the A-subunit into the cell. The A-subunit is also required for adjuvant activity, presumably as a function of its ADP-ribosylating enzymatic activity and ability to increase intracellular levels of cAMP (see below) .
  • the B-subunit alone may act as a carrier of other antigens in that when conjugated to those antigens they can be immobilized for processing by the mucosal associated lymphoid tissues.
  • LT and CT have many features in common, these are clearly distinct molecules with biochemical and immunologic differences which make them unique, including a 20% difference in nucleotide and amino acid sequence homology [Dallas and Falkow, 1980, Nature 288: 499-501].
  • the two toxins have the same subunit number and arrangement, same biological mechanism of action, and the same specific activity in many in vitro assays [Clements and Finkelstein, 1979, Infect. Immun. 24:760-769; Clements et al., 1980, Infect. Immun. 24: 91-97].
  • LT remains cell associated and is only released from E. coli during cell lysis [Clements and Finkelstein, 1979, Infect. Immun. 24:760-769].
  • CT is secreted from the vibrio as soon as it is synthesized and can be readily identified in, and purified from, culture supernatants . Consequently, in contrast to CT, LT is not fully biologically active when first isolated from the cell. Consistent with the A-B model for bacterial toxins, LT requires proteolysis and disulfide reduction to be fully active.
  • the enzymatically active A t moiety is unable to dissociate from the A 2 component and cannot reach its target substrate (adenylate cyclase) on the basolateral surface of the intestinal epithelial cell.
  • target substrate adenylate cyclase
  • proteases in the culture supernatant, to which the toxin is exposed during purification perform the proteolysis. Since LT is not fully biologically active, it is difficult to identify during purification using in vitro biological assays such as the Y-l adrenal cell assay or permeability factor assay.
  • LT has an unusual affinity for carbohydrate containing matrices. Specifically, LT, with a molecular weight of 90,000, elutes from Sephadex columns (glucose) with an apparent molecular weight of 45,000 and from Agarose columns (galactose) with an apparent molecular weight of 0. That is, it binds to galactose containing matrices and can be eluted from those matrices in pure form by application of galactose. LT binds not only to agarose in columns used for purification, but more importantly, to other biological molecules containing galactose, including glycoproteins and lipopolysaccharides.
  • This lectin-like binding property of LT results in a broader receptor distribution on mammalian cells for LT than for CT which binds only to G M1 . This may account in part for the reported differences in the abilities of these two molecules to induce different helper T lymphocyte responses [McGhee et al., 1994, Mucosal Immunology Update, Spring 1994, Raven Press, New York. p. 21].
  • LT induces both T H 1 and T H 2 cells and predominantly antigen-specific IgA responses without IgE responses when used as an orally administered mucosal adjuvant.
  • the two molecules also have many immunologic differences, as demonstrated by immunodiffusion studies [Clements and Finkelstein, 1978, Infect. Immun. 21: 1036-1039; Clements and Finkelstein, 1978, Infect. Immun. 22: 709-713], in vitro neutralization studies, and the partial protection against LT associated E. coli diarrhea in volunteers receiving B-subunit whole cell cholera vaccine [Clemens et al., 1988, J. Infect. Dis. 158: 372-377].
  • the present inventors examined a number of parameters, including the effect of LT on oral tolerance to ovalbumin (OVA) and the role of the two subunits of LT in the observed response, the effect of varying the timing and route of delivery of LT, the effect of prior exposure to OVA on the ability of LT to influence tolerance to OVA, the use of LT as an adjuvant with two unrelated antigens, and the effect of route of immunization on anti-OVA responses.
  • OVA ovalbumin
  • the results obtained from these studies [Clements et al., 1988, Vaccine 6:269-277; Clements et al., 1988, Abstract No. B91, 88th Ann. Meet. Am. Soc. Microbiol.] are summarized below: 1.
  • Tamura et al. Studies by Tamura et al., [Tamura et al., U.S. Patent No. 5,182,109] demonstrated that LT and/or CT administered intranasally (i.n.) enhanced the antibody titer against a co- administered antigen. However, nowhere in Tamura et al. is it taught that these toxins can induce a protective immune response when administered orally.
  • LT has significant immunoregulatory potential, both as a means of preventing the induction of tolerance to specific antigens and as an adjuvant for mucosally administered antigens and it elicits the production of both serum IgG and mucosal IgA against antigens with which it is delivered. This raises the possibility of an effective immunization program against a variety of pathogens involving the mucosal administration of killed or attenuated agents or relevant virulence determinants of specific agents.
  • 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.
  • 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 egg, or a protective antigen obtained by genetic engineering techniques or chemical synthesis.
  • Table I demonstrates the unexpected finding that mLT retained a basal level of activity in the Y-l adrenal cell assay even though it could not be proteolytically processed.
  • CT and native LT treated with trypsin have the same level of activity (15 pg) on Y-l adrenal cells.
  • mLT (48,000 pg) was >1, 000-fold less active than CT or native LT and could not be activated by trypsin.
  • the residual basal activity undoubtedly reflects a different and heretofore unknown pathway of adrenal cell activation than that requiring separation of the A [ - A 2 linkage.
  • Figure 7 shows that animals immunized i.n. or s.c. with gpl20 in conjunction with mLT (LT(R192G)) had significantly higher anti-gpl20 serum IgG antibody titers than did animals immunized i.n. with gpl20 alone.
  • Figure 8 shows that the highest vaginal anti-gpl20 IgA responses were observed in animals immunized i.n. with gpl20 in conjunction with mLT and boosted i.p. with gpl20.
  • Figure 9 shows that there was a limited cytokine response by spleen mononuclear cells from animals immunized i.n. or s.c. with gpl20 alone.
  • mononuclear cells from animals immunized i.n. or s.c. with gpl20 in conjunction with mLT had significantly increased production of antigen-specific IFN-7, IL-2 , and IL-10.

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Abstract

L'invention concerne des compositions et des procédés d'utilisation d'une nouvelle forme mutante d'entérotoxine thermolabile E. coli qui a perdu sa toxicité mais qui a conservé son activité immunologique. Cette entérotoxine est utilisée en association avec un antigène étranger afin d'améliorer la réponse immunitaire à cet antigène lorsque ladite entérotoxine est administrée en tant que composante d'une préparation vaccinale.
PCT/US1998/001546 1997-01-29 1998-01-26 Enterotoxine mutante utile en tant qu'adjuvant non toxique pour le vih Ceased WO1998032461A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60445/98A AU6044598A (en) 1997-01-29 1998-01-26 Mutant enterotoxin effective as a non-toxic adjuvant for hiv

Applications Claiming Priority (2)

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US79069197A 1997-01-29 1997-01-29
US08/790,691 1997-01-29

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AR (1) AR011592A1 (fr)
AU (1) AU6044598A (fr)
CO (1) CO4810235A1 (fr)
WO (1) WO1998032461A1 (fr)
ZA (1) ZA98738B (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7285281B2 (en) 2001-06-07 2007-10-23 Wyeth Holdings Corporation Mutant forms of cholera holotoxin as an adjuvant
US7384640B1 (en) 1999-09-30 2008-06-10 Wyeth Holdings Corporation Mutant cholera holotoxin as an adjuvant
US7658931B2 (en) 2001-06-07 2010-02-09 Wyeth Holdings Corporation Mutant forms of cholera holotoxin as an adjuvant

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5182109A (en) * 1988-04-08 1993-01-26 National Institute Of Health Vaccine preparation comprising a bacterial toxin adjuvant
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5182109A (en) * 1988-04-08 1993-01-26 National Institute Of Health Vaccine preparation comprising a bacterial toxin adjuvant
US5182109C1 (en) * 1988-04-08 2001-10-02 Nat Inst Health Vaccine preparation comprising a bacterial toxin adjuvant
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

Non-Patent Citations (4)

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Title
BAECKSTROEM M., ET AL.: "INSERTION OF A HIV-1-NEUTRALIZING EPITOPE IN A SURFACE-EXPOSED INTERNAL REGION OF THE CHOLERA TOXIN B-SUBUNIT.", GENE., ELSEVIER, AMSTERDAM., NL, vol. 149., 1 January 1994 (1994-01-01), NL, pages 211 - 217., XP002910754, ISSN: 0378-1119, DOI: 10.1016/0378-1119(94)90152-X *
HOLMGREN J., LYCKE N., CZERKINSKY C.: "CHOLERA TOXIN AND CHOLERA B SUBUNIT AS ORAL-MUCOSAL ADJUVANT AND ANTIGEN VECTOR SYSTEMS.", VACCINE, ELSEVIER LTD, GB, vol. 11., no. 12., 1 January 1993 (1993-01-01), GB, pages 1179 - 1184., XP002910755, ISSN: 0264-410X, DOI: 10.1016/0264-410X(93)90039-Z *
LOOSMORE S. M., ET AL.: "ENGINEERING OF GENETICALLY DETOXIFIED PERTUSSIS TOXIN ANALOGS FOR DEVELOPMENT OF A RECOMBINANT WHOOPING COUGH VACCINE.", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 58., no. 11., 1 November 1990 (1990-11-01), US, pages 3653 - 3662., XP002910752, ISSN: 0019-9567 *
WILLIAMS D. P., ET AL.: "CELLULAR PROCESSING OF THE INTERLEUKIN-2 FUSION TOXIN DAB 486-IL-2 AND EFFICIENT DELIVERY OF DIPHTHERIA FRAGMENT A TO THE CYTOSOL OF TARGET CELLS REQUIRES ARG 194.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 265., no. 33., 25 November 1990 (1990-11-25), US, pages 20673 - 20677., XP002910753, ISSN: 0021-9258 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7384640B1 (en) 1999-09-30 2008-06-10 Wyeth Holdings Corporation Mutant cholera holotoxin as an adjuvant
US7285281B2 (en) 2001-06-07 2007-10-23 Wyeth Holdings Corporation Mutant forms of cholera holotoxin as an adjuvant
US7361355B2 (en) 2001-06-07 2008-04-22 Wyeth Holdings Corporation Mutant forms of cholera holotoxin as an adjuvant
US7658931B2 (en) 2001-06-07 2010-02-09 Wyeth Holdings Corporation Mutant forms of cholera holotoxin as an adjuvant

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AU6044598A (en) 1998-08-18
AR011592A1 (es) 2000-08-30
CO4810235A1 (es) 1999-06-30
ZA98738B (en) 1998-08-04

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