WO2002004017A2 - Amelioration apportee a des vaccins oraux - Google Patents

Amelioration apportee a des vaccins oraux Download PDF

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WO2002004017A2
WO2002004017A2 PCT/EP2001/007784 EP0107784W WO0204017A2 WO 2002004017 A2 WO2002004017 A2 WO 2002004017A2 EP 0107784 W EP0107784 W EP 0107784W WO 0204017 A2 WO0204017 A2 WO 0204017A2
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polypeptide
epitope sequence
ovalbumin
sequence
immunogenic
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WO2002004017A3 (fr
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Dirk Bumann
Thomas F. Meyer
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal

Definitions

  • the present invention relates to a method for preparing and improving oral vaccines by modifying polypeptides which comprise at least one immunogenic epitope, particularly at least one immunogenic T cell epitope.
  • An orally administered soluble antigen can induce a specific peripheral immune tolerance ("oral tolerance") which prevents systemic immune responses against food-derived antigen and antigens of commensal microorganisms (Weiner, Immunol. Today 18 (1997), 335-343).
  • oral tolerance a specific peripheral immune tolerance
  • soluble factors produced by enteric pathogens can induce active mucosal and systemic immune responses that protect against a subsequent infection by the same or a related pathogen.
  • Pre-clinical studies using rodent models have shown that both types of immune responses can be induced experimentally, and that oral tolerance can be used to suppress autoimmune diseases (Weiner ( 1997), supra), whereas oral vaccination with soluble antigens mixed with a mucosal adjuvant protects against various infectious diseases (Shalaby, Clin.
  • ovalbumin For the most commonly used model antigen, ovalbumin, it is unknown to what extent gastrointestinal digestion occurs. Based on its very slow in vitro digestion by pepsin and trypsin it has been assumed that most oral ovalbumin remains essentially intact during its luminal passage through the stomach and the small intestine (Furrie et al., Immunology 86 (1995), 480- 486) but this has not been experimentally verified. Shortly after feeding, significant amounts of intact ovalbumin appears in the serum of mice but only very low amounts of smaller fragments (Bruce & Ferguson, Immunology 59 (1986), 295-300; Furrie et al. (1995), supra) which seems to support a minor role of luminal digestion. However, free serum levels might underrepresent the intestinal absorption of small fragments because of their rapid clearance (Bloch et al., Gastroenterology 95 (1988), 1272- 1278).
  • the present application is based on a study, wherein the extent, localization and kinetics of luminal digestion of orally administered ovalbumin in mice was analyzed by western blot of small intestinal contents.
  • a new method involving adoptive transfer of ovalbumin-specific transgenic T effector cells was developed.
  • the induction site of ovalbumin-specific naive T helper cells was determined.
  • the present invention relates to a method of preparing oral vaccines comprising the steps
  • a further aspect of the present invention are novel polypeptides comprising at least one immunogenic epitope sequence which has been modified by (i) removing cleavage sites for proteolytic digestion enzymes from the epitope sequence and/or
  • Still a further aspect of the present invention is a method for improving the efficacy of oral vaccines comprising the steps: (a) providing a polypeptide comprising at least one immunogenic epitope sequence and (b) modifying said polypeptide sequence by
  • the method of the present invention is particularly suitable for preparing T cell epitopes, e.g. peptide sequences which are capable of stimulating the cellular immune response.
  • These peptide sequences preferably are capable of binding to MHC molecules such as mammalian MHC class I or class II molecules, particularly human HLA alleles such as HLA-A, HLA-B, HLA-C, HLA-DR and HLA-DQ.
  • the immunogenic epitope sequence preferably has a length of from 10-30 amino acids, more preferably from 15-25 amino acids and most preferably from 17-22 amino acids.
  • the immune response may comprise an enhanced immunoreactivity directed against the peptide sequence which is administered, or alternatively an enhanced immunotolerance against the peptide which is administered.
  • the nature of the immune response depends on the peptide sequence and the type of administration. For example, oral administration of the immunogenic epitope sequence without an adjuvant may enhance the oral tolerance. In contrast thereto, oral administration with an adjuvant may lead to enhanced immunogenicity.
  • a further subject matter of the invention is a modified allergen which can be administered orally to hyposensibilize against this allergen during an existing allergy by inducing peripherial tolerance.
  • Current approaches induce peripherial tolerance by intravenous injection of allergens.
  • Oral administration greatly facilitates the treatment and increases compliance of patients.
  • Epitope improvement can be based on the extensive characterization of allergic epitopes.
  • the epitope sequence is included in a polypeptide carrier sequence.
  • the polypeptide carrier sequence may be a sequence which is homologous to the epitope sequence, i.e. the polypeptide sequence which naturally contains the immunogenic epitope sequence.
  • the polypeptide sequence may be heterologous to the immunogenic epitope sequence, i.e. 5 the polypeptide carrier may be a naturally occurring or synthetic polypeptide sequence which is foreign to the epitope sequence.
  • the immunogenic epitope sequence may be derived from a pathogen- associated polypeptide, e.g. a polypeptide which is derived from a virus, a o bacterium, a protozoan or a fungi.
  • viruses are HIV (proteins nef, mag).
  • bacteria are Helicobacter, Mycobacterium, Chlamydia, Streptococcus and Pseudomonas.
  • pathogenic and virulent factors such as urease A, urease B, catalase, vacA, cagA, HP0231 , HP0410 and HP1098 are especially 5 preferred.
  • protozoan organisms are Plasmodium falciparum.
  • Candida albicans is an example for a fungi.
  • the immunogenic epitope sequence may also be derived from an autoimmune disease-associated polypeptide such as collagen II being o associated with rheumatoid arthritis. Furthermore, the immunogenic epitope may be derived from a tumor-associated polypeptide such as MAGE1 , MAGE2 being associated with melanoma.
  • Possible allergens include pollen allergens (e.g. birch pollen allergen). Allergens that normally enter the body through the respiratory tract are preferred, since local adverse reaction in the gastrointestinal tract are less likely to occur following oral administration for such allergens.
  • the polypeptide which is used for preparing an oral vaccine and which contains at least one immunogenic epitope sequence has preferably a length of at least 50 amino acids and more preferably of at least 100 amino acids. It should be noted that the polypeptide sequence is modified in a way that (i) cleavage sites for proteolytic digestion enzymes are removed from within the epitope sequence, if necessary, and (ii) cleavage sites for proteolytic digestion enzymes are introduced adjacent the epitope sequence. The cleavage sites are recognized by proteolytic digestion enzymes which occur in the intestinal tract of the animal to be treated.
  • Preferred examples for proteolytic digestion enzymes are pepsin, in particular pepsin A and pepsin C, gastro-intestinal proteases such as trypsin and chymotrypsin.
  • Cleavage sites for these enzymes are for pepsin A and pepsin C at both sides of Tyr, Phe, for trypsin at C-side of Arg, Lys and for chymotrypsin at C-side of Tyr, Trp, Phe, Leu.
  • the polypeptide of the present invention may be prepared by recombinant DNA techniques, e.g. by expressing a nucleic acid encoding the polypeptide in a suitable host cell.
  • the nucleic acid encoding the polypeptide is preferably operatively linked to an expression control sequence which is active in a desired host cell, e.g. a bacterium such as E.coli or a eukaryotic cell such a yeast cell, an insect cell or a mammalian cell.
  • a desired host cell e.g. a bacterium such as E.coli or a eukaryotic cell such a yeast cell, an insect cell or a mammalian cell.
  • Suitable expression control sequences for desired host cells are known and described in numerous publications and textbooks such as Sambrook et al., Molecular Cloning, Second Edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  • the nucleic acid of the present invention is located on a recombinant vector.
  • the recombinant vector may be an extrachromosomal or chromosomal vector.
  • suitable vectors are e.g. plasmids, cosmids and viral, e.g. phage vectors.
  • the recombinant vector may be used for transforming a suitable host cell by known methods, whereby a recombinant cell containing a nucleic acid encoding the polypeptide of the invention is obtained.
  • the recombinant cell e.g.
  • a prokaryotic cell or a eukaryotic cell such as a yeast cell, an insect cell and, particularly, a mammalian cell, may be cultured under suitable conditions, wherein the polypeptide is expressed and the polypeptide may be obtained from the cell and/or the culture medium.
  • the polypeptide of the present invention may be formulated into an oral vaccine.
  • the oral vaccine may further comprise a pharmaceutically acceptable carrier or diluent and depending on the intended use an adjuvant.
  • the oral vaccine may comprise an adjuvant such as aluminum oxide or cholera toxin, particularly, detoxified cholera toxin.
  • an adjuvant such as aluminum oxide or cholera toxin, particularly, detoxified cholera toxin.
  • Particularly suitable are chemically or genetically detoxified variants of heat-labile toxin from E. coli (LT) or Choleratoxin (from Vibrio cholerae).
  • the oral vaccine may be administered in any convenient form such as a liquid, e.g. a solution or dispersion, or as a solid such as tablet, coated tablet, capsule, powder, as it is known in the pharmacological art.
  • the oral vaccine may be administered together with other nutrients, or separately.
  • the administration protocol and the dosage greatly depend on the effect which is to be achieved. Suitable administration protocols are described, for example, in Michetti, P. et al., Gastroenterology 1 1 6 ( 1 999), 804, disclosing a study with Helicobacter urease using LT as adjuvants.
  • an oral vaccine comprising as an active ingredient at least one polypeptide as described above.
  • the oral vaccine may further comprise a pharmaceutically acceptable and particularly orally acceptable carrier or diluent.
  • the oral vaccine may be in the form of a drinkable liquid or a powder which is soluble or dispersible in a liquid.
  • the oral vaccine may be in the form of a tablet, a capsule, a coated tablet or an effervescent.
  • the oral vaccine is suitable for any application involving a stimulation of an immune response, particularly a cellular immune response.
  • the oral vaccine may be used for immunization against diseases mediated by pathogens such as viruses, bacteria or protozoans, against autoimmune diseases or against tumor diseases.
  • the dosage of the oral vaccine mainly depends on the type and severity of the disease to be prevented or treated. Typically a dosage of 1 ⁇ g to 1 mg per kilogram body weight is administered daily.
  • the administration protocol is e.g. as follows: peptide/protein is dissolved in 30 ml PBS and 3 doses are administered at weekly intervals.
  • the efficacy of oral vaccines may be greatly increased.
  • the mucosal uptake of immunogenic polypeptide fragments, particularly epitope sequences as described above is improved.
  • the present invention relates to a vaccination method, wherein a subject in need thereof, particularly a human, is immunized by orally administering a polypeptide in an amount sufficient to induce an immune response in said subject against an immunogenic epitope sequence.
  • Figure 1 shows the progressive in vivo digestion of ovalbumin in the small intestine of mice.
  • Figure 2 shows the progressive in vivo digestion of ovalbumin in the small intestine of mice.
  • Figure 2 shows the mucosal uptake of orally administered ovalbumin in the distal jejunum as revealed by an ovalbumin-specific accumulation of pre- stimulated transgenic T cells.
  • Figure 3 shows the mucosal uptake of orally administered ovalbumin along the intestine.
  • A Local accumuluation of ovalbumin-specific T cells.
  • B Mucosal distribution of transgenic ovalbumin-specific T cells after immunization.
  • Figure 4 shows the blast formation by naive ovalbumin-specific T helper cells in the mesenteric lymph nodes after an oral immunization with ovalbumin and the adjuvant cholera toxin.
  • Figure 5 shows the division and differentiation of in vivo stimulated transgenic ovalbumin-specific cells after oral immunization with ovalbumin.
  • Figure 6 shows the variation of specific blast formation after oral immunization for individual mesenteric lymph nodes.
  • mice and adoptive transfer Balb/c mice and DO1 1.10 mice were bred in the Bundesamt fur ??lichen Medeau für Veterinary Surgery, Berlin, Germany, and were kept at specific pathogen free conditions in full accordance to the german guidelines for animal care. All experiments were approved by the Berlin Animal Welfare Comittee.
  • Recipients of unstimulated cells were immunized one day after transfer while recipients of prestimulated T cells were immunized four days after transfer.
  • mice or normal Balb/c mice were intragastrically immunized with 5 mg ovalbumin in 100 ⁇ l phosphate-buffered saline containing 3% sodium bicarbonate with or without 10.000 U cholera toxin (equivalent to 10 ⁇ g fully active toxin). After immunization, the mice were given free access to drinking water and food pellets. Control mice were immunized with 5 mg bovine serum albumin instead of ovalbumin.
  • mice were sacrificed under anesthesia and various small intestinal segments were flushed with ice-cold phosphate-buffered saline.
  • the samples were separated on 16.5 % polyacrylamid gels using a tricine-buffer system with high resolution for small peptides (Schagger & von Jagow, Anal.Biochem.166 (1987), 368- 379), blotted onto nitrocellulose, and detected with a polyclonal rabbit antibody to ovalbumin followed by a peroxidase chemoluminescence assay.
  • Cryostat cross sections (7 ⁇ m) were treated with 0.3 % hydrogen peroxide in methanol to block endogenous peroxidase activity, stained with biotinylated KJ1 -26 clonotypic monoclonal antibody (Haskins et al., J.Exp.Med.1 57 (1983), 1 149-1 169) followed by avidin-biotin-complex, developed with diaminobenzidine, and counterstained with hematoxylin. The number of KJ1-26 positive cells per intestinal cross section was determined.
  • chimeric mice were sacrificed and single cell suspensions of Peyer's patches, mesenteric lymph nodes, and the spleen were prepared.
  • Cells were stained with biotinylated anti-tgTCR antibody KJ1-26, anti-CD4-fluorescein, anti-CD69-phycoerythrin, and anti-B220-allophycocyanin, followed by streptavidin-FarRed (Gibco), and analyzed with a Calibur flow cytometer (Beckton&Dickinson).
  • CD4 + tgTCR + B220 ' lymphocytes were analyzed for their forward scatter and CD69-expression.
  • cells were stained with biotinylated anti-tgTCR antibody KJ1-26, anti-CD4-fluorescein, anti-CD69-phycoerythrin, and anti-B220-allophycocyanin, followed by streptavidin-FarRed (Gibco), and CD4 + tgTCR + B220 ' lymphocytes were analyzed for CD45RB-expression.
  • mice were orally immunized with ovalbumin and small intestinal luminal contents were analyzed after various time intervals by protein immunoblotting with a polyclonal antibody to ovalbumin.
  • most ovalbumin was rapidly digested along its passage through the small intestine (Fig. 1 ) .
  • Undigested ovalbumin and large fragments appeared mainly in the proximal small intestine, whereas ovalbumin-derived peptides in the size range of a few kDa were most abundant in the distal part.
  • Fig. 1 demonstrates that progressive in vivo digestion of ovalbumin in the small intestine of mice rapidly generates ovalbumin fragments with a regionally varying size distribution. Intact ovalbumin and large fragments are most abundant in the proximal small intestine whereas small ovalbumin-derived peptides are most prominent in the distal small intestine.
  • This method is based on the rapid accumulation of ovalbumin-specific T effector cells in tissues in which immunogenic ovalbumin or ovalbumin-derived peptides are present (Hurst & Barrett, Semin.Gastrointest.Dis.7 (1996), 1 18-123) (Kearney et al., Immunity 1 (1994), 327-339).
  • Do1 1.10 mice that are transgenic for a class ll-restricted T cell receptor specific for ovalbumin (Murphy et al. (1990), supra) were used as a source of ovalbumin-specific T helper cells.
  • the transgenic T cells were first pre-stimulated in vitro by co-incubation with the recognized ovalbumin epitope which enabled them to populate non-lymphoid tissues including the intestinal mucosa (Hurst et al., J. Immunol.163 (1999), 5937-5945). After stimulation, a few transgenic effector cells were transferred into non-transgenic syngenic Balb/c mice (Pape et al., Immunol. Rev.156 (1997), 67-78) and traced in vivo using a clonotypic monoclonal antibody (Haskins et al. (1983), supra). The chimeric mice were orally immunized and sacrificed 6 h later.
  • ovalbumin-specific transgenic T cells Various small intestinal regions were sectioned and immunostained for ovalbumin-specific transgenic T cells. In mice that had been orally immunized with ovalbumin but not in control mice, many pre-stimulated ovalbumin-specific T cells accumulated in the small intestinal mucosa (Fig. 2, Fig. 3A) indicating mucosal uptake of ovalbumin or immunogenic ovalbumin-derived peptides that contain the recognized epitope.
  • Fig. 2 shows the mucosal uptake of orally administered ovalbumin in the distal jejunum as revealed by an ovalbumin-specific accumulation of pre-stimulated transgenic T cells. Most ovalbumin-specific cells accumulate in the crypt area. Ovalbumin-specific transgenic T cells were pre-stimulated in vitro and then transferred into non-transgenic mice. The chimeric mice were orally immunized with ovalbumin and sacrificed 6 h later. Sections from the distal jejunum were immunostained with a clonotypic monoclonal antibody that specifically recognizes the transgenic cells.
  • proximal-distal accumulation gradient with no detectable ovalbumin-specific T cell accumulation in the duodenum, little accumulation in the proximal jejunum, and strong accumulation in the distal jejunum and the ileum (Fig. 3A) indicating a corresponding proximal-distal gradient in ovalbumin uptake.
  • Fig. 1 shows that most ovalbumin uptake occurred in regions with abundant small ovalbumin fragments whereas regions exposed mainly to undigested ovalbumin or large fragments showed little uptake. This could indicate that small fragments of ovalbumin permeate the intestinal mucosa in contrast to undigested ovalbumin.
  • Fig. 3 shows a variation of the mucosal uptake of orally administered ovalbumin along the small intestine and is restricted to the crypt area.
  • Ovalbumin uptake was indirectly detected by the accumulation of transgenic ovalbumin-specific T cells as shown in Fig. 2.
  • the data represent averages and SEMs of the number of ovalbumin-specific T cells per cross section of 5 mice per group.
  • the intestinal epithelium which has low permeation rates for macromolecules has recently been shown to have high permeation rates for small peptides up to a molecular weight of a few kDa (Pappenheimer et al. ( 1 997), supra) which is compatible with the proposed differential uptake of ovalbumin fragments depending on their size.
  • Permeation of molecules in the size range of a few kDa has been proposed to occur mainly through a paracellular pathway between the immature crypt epithelial cells (Bjarnason et al., Gastroenterology 108 ( 1 995), 1 566-1 581 ; Ma et al., J.Lab.Clin. Med.1 20 ( 1 992), 329-341 ).
  • ovalbumin-specific T cells in the intestinal mucosa was largely restricted to the crypt area with very few ovalbumin-specific T cells directly interacting with the enterocytes of the villus epithelium (Fig. 2, Fig. 3B) indicating a preferential uptake of ovalbumin-derived peptides in the crypts.
  • This is in agreement with the proposed permeation pathway for small immunogenic ovalbumin fragments.
  • ovalbumin that might have been endocytosed by the enterocytes is apparently not directly presented to cognate T cells in vivo as has been proposed previously (Kaiserlian & Etchart (1 999), supra) since the necessary physical cell-cell interaction is not detectable. This supports that gastrointestinal digestion generates small immunogenic ovalbumin fragments that penetrate the intestinal mucosa whereas intact ovalbumin and large fragments are largely excluded by the intestinal epithelium.
  • ovalbumin-derived peptides must be presented to immune cells.
  • the activation of specific T helper cells is especially important for protective immune responses against many pathogens and for the induction of oral tolerance (McGhee et al., Vaccine 1 0 ( 1 992), 75-88; Mowat & Viney (1 997), supra; Weiner ( 1 997), supra) .
  • the accumulation of pre-stimulated T effector cells indicates that presentation of orally administered ovalbumin and cognate interactions can occur directly in the intestinal mucosa.
  • the important naive T helper cells rarely migrate through the intestinal mucosa (Hurst et al.
  • transgenic ovalbumin-specific T helper cells in the Peyer's patches, mesenteric lymph nodes, and the spleen upregulated the very early activation marker CD69 (Fig. 4) in agreement with previous studies (Gutgemann et al., Immunity 8 (1 998), 667-673; Sun et al., J.Immunol.162 (1 999), 5868-5875; Williamson et al., Immunology 97 (1 999), 565-572). Subsequently, many of the activated ovalbumin-specific cells in the mesenteric lymph nodes and some in the Peyer's patches became larger indicating blast formation. In the spleen, little blast formation was observed but large cells that had already downregulated CD69 migrated through the spleen at day 3.
  • Fig. 4 shows that many naive ovalbumin-specific T helper cells form blasts in the mesenteric lymph nodes after an oral immunization with ovalbumin and the adjuvant cholera toxin.
  • Logarithmic probability plots (50% levels) indicating early activation (measured as CD69 upregulation) and blast formation (measured as an increase in forward scatter) of transgenic ovalbumin-specific T helper cells at various time intervals after oral immunization in Peyer's patches (PP), mesenteric lymph nodes (mLN), and the spleen are shown.
  • ovalbumin-specific T cells activated only ovalbumin-specific T cells but not detectable numbers of non-transgenic bystander T helper cells (data not shown) and immunization with cholera toxin alone led to no detectable activation of ovalbumin-specific T cells (data not shown).
  • Fig. 5 demonstrates that in vivo stimulated transgenic ovalbumin-specific cells divide and differentiate after oral immunization with ovalbumin as indicated by a downregulation of CD45RB.
  • Transgenic cells were analyzed with flow cytometry 7 days after oral immunization of chimerical mice with ovalbumin and cholera toxin (solid line) or bovine serum albumin and cholera toxin (shadowed).
  • 2.5 Differences in T cell activation for individual mesenteric lymph nodes After oral immunization with ovalbumin and cholera toxin, blast formation by ovalbumin-specific T helper cells occurred mainly in the mesenteric lymph nodes (Fig. 4).
  • mice like other mammals have several mesenteric lymph nodes that drain sequential parts of the small intestine.
  • the different mesenteric lymph nodes have generally been pooled (Chen et al., Cell Immunol.178 (1997), 62-68; Gutgemann et al. (1998), supra; Sun et al. (1999), supra; Williamson et al. (1999), supra) which neglects regional differences among the various parts of the small intestine.
  • some regional variation in the immune response might be expected because of the observed regional differences in ovalbumin digestion and uptake (Fig. 1 , Fig. 3).
  • Fig. 6 shows that specific blast formation after oral immunization varies for individual mesenteric lymph nodes that drain different small intestinal regions. This regional heterogeneity is not due to the mucosal adjuvant cholera toxin or an intrinsically different immune responsiveness of various intestinal regions.
  • A Regional responses after overnight fasting before immunization. Ovalbumin-specific transgenic T cells in individual mesenteric lymph nodes along the small intestine (numbers 1 -4 in the proximal-distal direction) of chimeric mice were analyzed for blast formation 2 days after oral immunization with buffer ( +), ovalbumin (o), or ovalbumin and cholera toxin (•). The data represent averages and SEMs for groups of 3-7 mice.
  • ovalbumin-specific immune response The extensive intestinal digestion of ovalbumin could have important consequences for the induction of an ovalbumin-specific immune response. Cleavage of important epitopes would decrease the immunogenicity but digestion could also generate small immunogenic fragments that efficiently penetrate the intestinal epithelium. If epitope destruction is the dominant effect of digestion, the ovalbumin-specific immune response should regionally decrease with progressive digestion along the small intestine. In contrast, if generation of small immunogenic fragments is more important, antigen uptake and immune responses should increase with the progressive generation of small peptides along the small intestine.
  • ovalbumin-specific T cells were adoptively transferred and traced in the recipient mice following oral administration of ovalbumin.
  • Mesenteric lymph nodes draining the distal small intestine were the most important inductive site for a ovalbumin specific T cell induction and the proximal-distal response gradient closely correlated with the differential uptake of ovalbumin.
  • the combined data indicate that gastrointestinal digestion yields small immunogenic ovalbumin-fragments that efficiently permeate the epithelial barrier of the small intestinal mucosa and are transported by afferent lymphatic drainage to the regional mesenteric lymph nodes where they induce strong T helper cell responses.
  • Enterocytes have been shown to take up oral ovalbumin and to target it to MHC class ll-containing vesicles suggesting that enterocytes themselves present ovalbumin-derived epitopes to cognate T helper cells (Kaiserlian & Etchart (1999), supra; Zimmer et al. (2000), supra).
  • the data obtained here indicate that presentation by enterocytes to cognate T helper cells does rarely occur in vivo (Fig. 2, 3b).
  • Transcytosis by enterocytes could participate in oral antigen entry but probably makes only a weak contribution since it would not explain the observed strong proximal-distal uptake gradient as large fragments are at least as likely to be endocytozed as compared to small fragments.
  • Dendritic cells that directly sample the intestinal lumen.
  • Dendritic cells might, however, be involved in presentation to cognate T effector cells in the mucosa and the transport from the mucosa to the draining mesenteric lymph nodes (Liu & MacPherson, J.Exp.Med.177 (1993), 1299-1307; Williamson et al. (1999), supra).
  • passive permeation of small peptides is the most likely entry pathway for immunogenic ovalbumin fragments.
  • site-directed mutagenesis of the antigen could be used to introduce additional protease cleavage sites around protective epitopes that would facilitate the productive digestion to appropriate small peptides, and conversely, to delete existing protease cleavage sites within major T cell epitopes which would inhibit non-productive digestion.
  • Improved vaccines could thus be obtained that yield high efficacies using the attractive oral delivery route.
  • Oral administration of antigen without an adjuvant usually leads to specific oral tolerance instead of an active immune response against the antigen. This effect is currently under intense preclinical and clinical evaluation as a therapy against autoimmune diseases.

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Abstract

Cette invention se rapporte à un procédé servant à préparer et à améliorer des vaccins oraux en modifiant des polypeptides qui comprennent au moins un épitope immunogène, en particulier au moins un épitope de lymphocytes T immunogène.
PCT/EP2001/007784 2000-07-07 2001-07-06 Amelioration apportee a des vaccins oraux Ceased WO2002004017A2 (fr)

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EP2902037A1 (fr) * 2014-01-31 2015-08-05 Universität Zürich Compositions tolérogènes et utilisations associées
US11166986B2 (en) 2014-01-31 2021-11-09 Universitat Zurich Use of heliocbacter pylori extract for treating or preventing inflammatory bowel diseases and coeliac disease

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EP0654273A1 (fr) * 1993-11-18 1995-05-24 Harry H. Leveen Produit pharmaceutique et méthode pour traitement
US5780040A (en) * 1994-06-08 1998-07-14 Tufts University School Of Medicine Hospital, Inc. Helicobacter pylori nickel binding protein
AUPQ347199A0 (en) * 1999-10-15 1999-11-11 Csl Limited Novel polypeptide fragments

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EP2902037A1 (fr) * 2014-01-31 2015-08-05 Universität Zürich Compositions tolérogènes et utilisations associées
WO2015114575A1 (fr) * 2014-01-31 2015-08-06 Universität Zürich Compositions tolérogènes et leurs utilisations
US9999660B2 (en) 2014-01-31 2018-06-19 Universitat Zurich Tolerogenic compositions comprising and uses thereof
AU2015212357B2 (en) * 2014-01-31 2020-05-28 Leiden University Medical Center Tolerogenic compositions comprising and uses thereof
US11166986B2 (en) 2014-01-31 2021-11-09 Universitat Zurich Use of heliocbacter pylori extract for treating or preventing inflammatory bowel diseases and coeliac disease

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