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WO1991012819A1 - Compositions immunogeniques ameliorees - Google Patents

Compositions immunogeniques ameliorees Download PDF

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Publication number
WO1991012819A1
WO1991012819A1 PCT/CA1991/000063 CA9100063W WO9112819A1 WO 1991012819 A1 WO1991012819 A1 WO 1991012819A1 CA 9100063 W CA9100063 W CA 9100063W WO 9112819 A1 WO9112819 A1 WO 9112819A1
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WO
WIPO (PCT)
Prior art keywords
epitope
composition
layer
response
bearing moiety
Prior art date
Application number
PCT/CA1991/000063
Other languages
English (en)
Inventor
Uwe B. Sleytr
Wolfgang Mundt
Paul Messner
Richard H. Smith
Frank M. Unger
Andrew Malcolm
Original Assignee
Alberta Research Council
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US07/487,729 external-priority patent/US5043158A/en
Application filed by Alberta Research Council filed Critical Alberta Research Council
Publication of WO1991012819A1 publication Critical patent/WO1991012819A1/fr

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Classifications

    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • 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
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP

Definitions

  • the invention relates to improved immunogenic compositions in which haptens to which an immune response is desired are conjugated to crystalline or
  • para-crystalline carriers especially those represented by bacterial surface layers, or "S-layers.”
  • hapten it may be necessary to supplement the administration of the hapten with a material which enhances its ability to elicit either or both a B-cell mediated or T-cell mediated response.
  • a material which enhances its ability to elicit either or both a B-cell mediated or T-cell mediated response has generally been classified as adjuvants. These are materials administered along with the hapten which seem to aid in securing the desired response. The use of killed
  • Naked or acid-treated bacteria have also been shown to act as adjuvants to induce humoral immunity to
  • European patent application publication no. 180,564 describes the preparation of a complex from bacterial substrates designated "Iscom.” This complex is prepared by solubilizing the hydrophobic peptides from bacteria into detergent, removing the detergent from the solubilized material and replacing it with glycosides, such as saponin.
  • Haptens which are contained in small molecules, especially carbohydrates, are also rendered more immunogenic by conjugation to a carrier such as bovine serum albumin, keyhole limpet hemocyanin, diphtheria or tetanus toxoids and the like.
  • a carrier such as bovine serum albumin, keyhole limpet hemocyanin, diphtheria or tetanus toxoids and the like.
  • vaccination antigens possess increased immunogenic potential with respect to the low-molecular weight haptens.
  • vaccines which are administered orally are those containing live attenuated organisms such as the attenuated polio virus vaccine.
  • Vaccines which consist of killed organisms or subunit vaccines generally are not administered orally because this route of administration does not induce a strong systemic immune response.
  • extensive research has demonstrated that oral ingestion of an antigen usually results in an induced state cf systemic
  • oral tolerance hyporesponsiveness referred to as oral tolerance. It is believed that active immunosuppression by suppressor T cells is the mechanism behind the induction of oral tolerance. Another important factor in the induction of oral tolerance is thought to be the processing of the antigens by the mucosal reticuloendothelial system. 2 Both cholera toxin (CT) and cationized bovine serum albumin (cBSA) have been shown to generate systemic responses after oral immunization. 3 ' 4 Neither CT nor cBSA are suitable for use as vaccine carriers in humans.
  • CT cholera toxin
  • cBSA cationized bovine serum albumin
  • the immune response to weak immunogens is usually an antibody response, mediated by B-lymphocytes, whereas effective protection through vaccination would generally require participation of T-lymphocytes in the immune response.
  • the invention provides two-dimensional crystalline carriers which permit definition of these parameters so that an ordered structure bearing the
  • immunostimulatory or immunoregulatory substance can be obtained, and problems associated with toxicity are minimized.
  • the invention also demonstrates that S-layers can also generate a strong systemic immune response after an oral immunization.
  • the problems of low or lacking immunogenicity, lack of T- lymphocyte-mediated response, ill-defined (poorly reproducible) coupling of haptens or immunoactive substances, and residual toxicity of antigen carriers is solved by using as the carrier to which the immunoactive substances are bound two-dimensional crystalline arrays of proteins or glycoproteins.
  • the carrier molecules display a constant, precisely defined spatial orientation with respect to each other; thus both the nature of the linkages and the number and spatial orientation of the bound haptens or immunoactive substances and the distance between the binding sites which can carry the haptens or immunoactive substances are always precisely defined.
  • the invention is directed to pharmaceutical compositions comprising a
  • the two-dimensional crystalline carrier coupled to a moiety which bears an epitope, e.g., a hapten, to which an immunological response is desired.
  • the two-dimensional crystalline array may optionally be stabilized by
  • crystalline carrier is obtained from isolation of the S-layers of microbial cell walls.
  • the invention is directed to methods to prepare the pharmaceutical compositions of the invention which comprises coupling the epitope-bearing moieties to the above carriers.
  • the method is directed to methods to elicit a T-cell mediated or B-cell mediated immune response to an epitope of interest, which method comprises administration of the compositions of the invention to a vertebrate subject in an amount effective to elicit the desired response.
  • the administration of compositions may be done orally to provide oral immunizations.
  • the present invention further provides a systemic cell-mediated immune response.
  • Figure 1 is an illustration which shows the three stages of purification of S-layers from intact bacteria.
  • Figure 2 is a graph illustrating one of the analytical procedures enabling the precise coupling methods of this invention.
  • Figure 3 is a graph illustrating the dose response of the T-disaccharide linked S-layer needed to prime for a delayed-type-hypersensitivity (DTH) response.
  • DTH delayed-type-hypersensitivity
  • Figure 4 is a graph illustrating the amount of T-disaccharide needed to elicit a DTH response in primed mice.
  • Figure 5 is a graph illustrating the effect adjuvants have on the T-disaccharide S-layer response.
  • DTH delayed-type-hypersensitivity reaction
  • FIG. 7 shows data indicating that T-cells stimulated by immunization with the invention
  • compositions can transfer the capacity to exhibit DTH.
  • Figure 8 shows two bar graphs comparing oral and intramuscular immunization with S-layer conjugates.
  • Figure 9 shows two charts showing the tertiary antibody response to Y hapten coupled to unfixed L-111 S- Layers, antibody response to the Y hapten negative control and tertiary antibody responses to unfixed L111 S-Layer coupled with Y hapten, and antibody response to unfixed L111, negative control.
  • Figure 10 is a bar graph showing immunoglobulin isotyping of tertiary antibody response to unfixed Y- L111/FA, isotypic antibody response to the Y hapten, and isotypic antibody response to unfixed L111.
  • Figure 11 is a bar graph comparing tertiary antibody response to T-disaccharide coupled by EDC to fixed L111 S-Layer; antibody response to T-disaccharide at different molar hapten rations, and antibody response to the L111 S-Layer carrier.
  • Figure 12 is a graph showing tertiary antibody response to Streptococcus pneumoniae capsular
  • CPS polysaccharide
  • Figure 13 is a bar graph showing immunoglobulin isotyping of tertiary antibody response to S. pneumoniae CPS type 3-L111 S-Layer; isotypic antibody response to CPS type 3 when mice were immunized with 3-L111
  • Figure 14 is a graph showing secondary antibody response to Streptococcus pneumoniae oligosaccharide type 3.
  • the invention provides pharmaceutical compositions which are capable of enhancing the immune
  • compositions may be used both for stimulation of B-cell and T-cell responses.
  • the two-dimensional crystalline arrays may consist of proteins or glycoproteins whereby the structure of the carriers may further approximate the shape of a bacterium.
  • the appropriate arrays may be derived from one or several microbial cell wall layers.
  • the proteins or glycoproteins composing these arrays are obtained in an especially simple manner.
  • Such suitable arrays may contain other adhering cell wall components.
  • microbial cell wall fragments as such can be used to carry the immunoactive substances.
  • Particularly suitable as sources of this type of carrier are those microorganisms which, aside from the crystalline surface layer proteins, contain additional rigid layers such as those composed of peptidoglycan or pseudo-murein.
  • FIG. 1 shows several stages of purification of typical bacterial cell wall arrangements.
  • Section a shows a section of intact bacterial cell wall wherein S represents the S-layer; PG represents peptidoglycan and CM represents the cytoplasmic membrane. As shown, the S- layer is associated with the proteoglycan and cytoplasmic membrane in a layered structure.
  • Section b shows the empty peptidoglycan sacculus after glutaraldehyde fixation.
  • the cytoplasmic membrane is removed, and the fixed segment contains a double S-layer separated by the peptidoglycan.
  • Section c shows the glutaraldehyde fixed
  • the two-dimensional crystalline arrays are derived from microbial cell walls as S-layers with or without additional peptidoglycan layers.
  • bacteria are incubated with detergent at elevated temperatures of 30-60°C, preferably around 50oC, for a suitable time period to disintegrate the cytoplasm and plasma membrane of the organism.
  • Suitable detergents include, for example Triton X-100, various alkylpolyoxyethylene ethers, various
  • acylpolyoxyethylene sorbitol esters such as the Tweens, and the like.
  • Intact bacteria may be treated with the detergent, or the culture ay be sonicated or otherwise disrupted in the presence of buffer when the cellular shape of the microorganism does not need to be conserved.
  • the nonsolubilized components are recovered, preferably through centrifugation, and the pellets are washed. It is desirable to further remove cytoplasmic components and nucleic acids by treatment with suitable enzymes
  • the washed and pelleted aggregates containing the S-layers are then utilized as carriers, preferably after treatment with a cross-linking agent such as glutaraldehyde.
  • a cross-linking agent such as glutaraldehyde.
  • cross-linker is added in an amount which stabilizes the ordered structure of the S-layer.
  • the residual peptidoglycan can be removed from the recovered S-layers by treatment with a peptidoglycan-degrading enzyme, for example lysozyme.
  • a peptidoglycan-degrading enzyme for example lysozyme.
  • the recovered pellet is treated with the enzyme at about 37oC in buffer for a time suitable to remove the peptidoglycan.
  • the two-dimensional crystalline array provided as the S-layer from bacterial cells can be recovered, in general, by extraction of nonordered components using detergent; optionally this structure can be cross-linked, and optionally the peptidoglycan may be removed.
  • the preparation can be conducted either on whole cells or on a disruptate, such as a sonicate.
  • a disruptate such as a sonicate.
  • lipopolysaccharides can be removed by filtration of the derivatized carrier.
  • Carriers comprising immunoactive substances may be combined with other substances and compositions. It is thus possible to achieve various functions of the pharmaceutical composition. For example, strongly hydro- phobic carrier molecules can be mixed with aggregates carrying the immunoactive substances causing increased uptake of the pharmaceutical structure by accessory cells.
  • Carriers comprising different immunoactive substances can be attached to an auxiliary matrix which can be cross-linked to the carriers. Thereby, a pharmaceutical preparation can be generated that would combine different types of carrier molecules such as those differing in their crystalline structures.
  • carrier aggregates comprising more than one hapten can be used so that the profile of activity of the pharmaceutical composition can be precisely controlled.
  • subunits of the carrier aggregate can be separated, conjugated separately to the desired haptens, and then reconstituted to obtain a carrier aggregate with a multiplicity of haptens.
  • separated portions of the S-layer sample can be
  • a chaotropic agent such as guanidine hydrochloride or a detergent.
  • the protomers originating from the different portions will self-assemble into an S-layer derivatized with different haptens or immunoactive substances.
  • epitope-bearing moiety refers to a substance which contains a specific determinant to which the immune response is desired.
  • epitope-bearing moiety may itself be a hapten--i.e., a simple moiety which, when rendered immunogenic, behaves as an antigen, or may be a more complex moiety, only portions of which are responsible for the
  • the need for an appropriate carrier is, of course, greater when the epitope-bearing moiety is a hapten, since these simple and low molecular weight substances are only weakly immunogenic.
  • the use of the carriers of the invention is applicable to any moiety which contains an epitope to which an immune response is desired.
  • the two-dimensional crystalline arrays frequently contain glycoprotein and thus the hapten or immunoactive/immunoregulative substances may be linked to either or both the protein or the carbohydrate portions of the carrier.
  • linkages to the protein per se must of course be employed.
  • a mixed mode of attachment can be
  • the immunoactive substances can be attached to the respective carrier molecules directly or by way of bridging molecules such as homo- or heterobifunctional cross-linking agents or peptide chains (e.g.,
  • preferred sites of cleavage of the immunogenic aggregates may also be introduced.
  • Commercially available homobifunctional or heterobifunctional linkers may be obtained, for example, from Pierce Chemical Co., Rockford, IL.
  • binding sites within the glycoproteins may be generated by oxidation, e.g., using periodate. Binding sites on the protein portions can also be generated by reacting with glutaraldehyde, the reagent used for cross-linking and activation. Formation of binding sites can also occur by the introduction of active groups, whereby a precise control of the number and kind of binding sites can be achieved.
  • the haptens can be attached by amide linkages to the carboxyl groups of the protein portion of the carrier. Attachment of the haptens can also be through aldehydes in the form of Schiff bases. The Schiff bases can be reduced to secondary amines.
  • Binding sites on the immunoactive substances can be activated and the immunoactive substances attached by means of these activated binding sites, to the
  • compositions containing carrier coupled with one or more epitope-bearing moieties are then useful in eliciting an immune response.
  • Suitable epitope-bearing moieties include those conventionally employed including polypeptides, carbohydrates, nucleic acids and lipids.
  • the proteins, glycoproteins and peptides can include cytokines, hormones, glucagon, insulin-like growth factors,
  • thyroid-stimulating hormone prolactin, inhibin
  • somastatin thymic hormones, various releasing factors, as well as antigenic fragments of proteins characteristic of viruses and other infective agents.
  • carbohydrates and carbohydrate complexes can be used as well, including bacterial capsules or exopolysaccharides, for example, from Hemophilus influenza B, blood group antigens, and the like.
  • lipid materials can be used such as the steroids or prostaglandins, as well as glycolipids.
  • Other molecules of interest include
  • alkaloids such as vindoline, serpentine, or any other hapten-containing material to which an immune response is sought.
  • the immune compositions of the invention can be utilized to elicit a T-cell response, as can be verified, as described below, by the ability to elicit a delayed-type hypersensitive (DTH) response in inoculated subjects.
  • DTH delayed-type hypersensitive
  • T-cells obtained from animals immunized with the compositions of the invention can be transplanted into other animal subjects, resulting in the transfer of immunity to these subjects.
  • compositions of the present invention are particularly suitable as immunizing
  • anti-idiotypic antibodies may be prepared by this method.
  • compositions can be used to advantage for primary immunization and boosting when one and the same immunoactive substance is bound to S-layers derived from two different bacterial strains.
  • Various regimens can be used in administering the compositions of the invention.
  • a series of injections is employed wherein subsequent injections boost the immune response obtained from the initially injected compositions.
  • the availability of a multiplicity of two-dimensional crystalline carriers permits the use of analogous but different carriers in the series.
  • the problem of inducing carrier typic
  • epitope-bearing moiety is injected first conjugated to S- layer prepared from a first microorganism and followed by injection of the epitope-bearing moiety coupled to a carrier which is composed of an S-layer derived from a second microorganism.
  • carrier-epitope moiety complexes are applicable also for use as immunosorbents or as affinity matrices, e.g., for diagnostic kits or extracorporeal depletion of undesirable antibodies from human blood.
  • L111-69 (2.5 g) are suspended in 50 mM Tris-HCl buffer, pH 7.1, and sonicated briefly (about 1 minute).
  • the suspension is incubated at 50oC for 15 minutes.
  • S-Layer two-dimensional crystalline protein-containing cell wall layer
  • the underlying peptidoglycan layer are conserved as fragments.
  • the mixture is centrifuged at 20,000 x g and the pellets washed three times to remove detergent.
  • the pellets are then suspended in 5 mM magnesium chloride solution (25 ml).
  • DNAse 125 ug
  • RNAse 500 ug
  • the suspension is then centrifuged at 20,000 x g and washed three times with water.
  • the pellet is then suspended in 1.1M cacodylate buffer (pH 7.2; 20 ml) and a 50% aqueous solution of glutaraldehyde is added at 4oC to a final concentration of 0.5%.
  • the s pension is well stirred at 4oC for a few minutes, centrifuged, and washed with water.
  • the pellet is then suspended in water (25 ml) and Tris-hydroxymethylaminomethane ("Tris”) is added.
  • Tris Tris-hydroxymethylaminomethane
  • Ultrasonic treatment is omitted when the cellular shape of the microorganisms is to be conserved and only the cytoplasmic constituents are to be removed.
  • the underlying peptidoglycan layer remains associated with the protein-containing cell wall layer. With numerous organisms, this may result in the formation of an
  • peptidoglycan-degrading enzyme e.g., lysozyme
  • the material produced as under this section A is treated for 1 h at 36oC with a solution of lysozyme (0.5 mg lysozyme per ml of a 50 mM solution of Tris-HCl buffer, pH 7.2).
  • 10 ml of lysozyme solution is added per 0.5 g wet pellet.
  • the S-Layer Depending on the microorganism used, the S-Layer
  • fragments obtained consist of a single or double S-Layer. Following ultrasonic treatment of cells, open fragments are formed whereas in the absence of ultrasonic
  • the cellular shape i.e., the crystalline two- dimensional array is preserved intact.
  • the pellet prepared according to A is suspended in water (5 ml) and a 0.1M solution of sodium periodate (5 ml) added, the suspension is allowed to stand for 24 h with exclusion of light to allow oxidation and give rise to the formation of aldehyde functions as binding sites. Subsequently, the suspension is centrifuged and the pellet washed with 10 mM sodium chloride solution, to remove the iodine-containing salts.
  • the pellet of oxidized material obtained according to B (about 0.2 g) is suspended in water (1 ml) and the suspension is mixed with a solution (1 ml) of bovine serum albumin (50 mg) in water (10 ml). This solution is allowed to stand at room temperature (60 minutes) and is then centrifuged.
  • Cells of Bacillus stearothermophilus PV72 (2.5 g) are suspended in 50 ⁇ M Tris-HCl buffer, pH 7.2, and sonicated for about 1 minute. Following addition of 2% Triton X-100 (12.5 ml), the suspension is incubated for 15 min. at 50oC. By means of this treatment, the
  • fragments are formed which correspond in shape more or less to the original shape of the bacterial cell (so called "ghosts").
  • the suspension is centrifuged at 20,000 x g and the pellet washed three times with water to remove the detergent.
  • the pellet is then suspended in 5 mM magnesium chloride solution (25 ml), DNAse
  • RNAse ribonuclease, 500 ⁇ g
  • the pellet is washed three times with water, centrifugation in between being at 20,000 x g.
  • the pellet is then suspended in 0.1M cacodylate buffer (pH 7.2) and the suspension mixed with a 50% solution of glutaraldehyde in water at 4oC to a final concentration of 0.5%.
  • the suspension is then vigorously stirred at 4°C for a few minutes, centrifuged, and the pellet washed with water.
  • the modified S-Layers prepared in section 2A are mixed with a solution of bovine serum albumin as in Example 1C, and the amount of protein bound is determined as described there.
  • Cell walls of Clostridium thermohvdrosulfuricum Llll-69 are treated with glutaraldehyde (0.5% in 0.1M sodium cacodylate buffer, pH 7.2) for 20 minutes at 20oC, so as to stabilize the outermost cell wall layer (S- layer).
  • the reaction is terminated by the addition of excess ethanolamine.
  • the cell wall fragments may be either in suspension or attached to a porous surface (e.g., an S-Layer ultrafiltration
  • the cell wall fragments are then washed with distilled water to remove the reagent mixture.
  • the pellet of a cross-linked preparation as under A above is suspended in distilled water (30 ml) and to the suspension is added 1-ethyl-3,3- (dimethyl- aminopropyl) carbodiimide (EDAC; 60 mg) maintaining a pH of 4.75.
  • EDAC 1-ethyl-3,3- (dimethyl- aminopropyl) carbodiimide
  • This step activates the exposed carboxyl groups of the S-Layer.
  • an excess of hexamethyl- enediamine 0.5 g
  • the pH kept at 8.0 for 60 minutes Subsequently, the reaction is terminated by addition of acetic acid.
  • the suspension is centrifuged at 20,000 x g and the pellet washed three times with distilled water.
  • the wet pellet (100 mg) is suspended in 50 mM phosphate buffer, pH 7 (9 ml) and a solution of meta-maleimidobenzoyl-N-hydroxysuccinimide ester (50 mg per ml of tetrahydrofuran; 1 ml) is added. The mixture is then incubated for 30 minutes at 20°C.
  • the pellet is suspended in 50 mM phosphate buffer (pH 7.0), ⁇ -galactosidase (20 mg) is added and the mixture is incubated for 2 h at 20oC. After centrifugation at 20,000 x g and repeated washing with phosphate buffer, the activity of the ⁇ -galactosidase covalently linked to the protein matrix is determined.
  • the vicinal diol groupings of the carbohydrate portion (glycan) of S- Layer glycoprotein are utilized.
  • Cell wall fragments are treated with glutaraldehyde, as described in Section A of Example 3, to stabilize the outermost cell surface.
  • the cell wall fragments from Section A (100 mg) are suspended in anhydrous tetrahydrofuran (THF), incubated at 20oC for 10 minutes, centrifuged at 20,000 x g and suspended again in a 2.5% solution of cyanogen bromide in anhydrous tetrahydrofuran (10 ml). Following incubation for 2 h, the cell wall fragments are separated by centrifugation at 20,000 x g and washed with THF for removal of residual reagent.
  • THF tetrahydrofuran
  • the pellet is suspended in 50 mM phosphate buffer (pH 8.0 (10 ml), containing invertase (20 mg) and incubated for 18 h at 4oC. Following centrifugation at 20,000 x g, the pellet is washed twice with phosphate buffer and the enzyme activity of the invertase bound to the protein matrix determined.
  • 50 mM phosphate buffer pH 8.0 (10 ml)
  • invertase 20 mg
  • thermohydrosulfuricum L111-69 are cross-linked with glutaraldehyde as described in Example 3, section A.
  • Cell wall fragments (0.1 g) are suspended in anhydrous dimethylformamide (DMF, 20 ml) and EDC (60 mg) and N-hydroxysuccinimide (0.5 g) are added to the suspension. Following incubation for 1 h, the suspension is centrifuged at 20,000 x g and washed twice with DMF.
  • DMF dimethylformamide
  • EDC 60 mg
  • N-hydroxysuccinimide 0.5 g
  • the pellet obtained as under 5B is suspended in 0.1M sodium hydrogencarbonate (pH 8.8) containing dissolved dextranase (20 mg) and the reaction mixture is incubated at 4°C for 18 h.
  • the cell wall fragments containing the bound dextranase are obtained by
  • the oxidized (polyaldehyde) derivative of the S-Layer prepared in Section A is incubated with the 3- (2-aminoethyl)thiopropyl glycoside of a disaccharide whereby Schiff base formation occurs.
  • This step can also be performed with any other saccharide attached to an aglycone that contains amino groups.
  • a solution of the allyl glycoside (5 mM) in a solution of cysteamine hydrochloride (15 milliequivalents of SH-groups in 10 ml) is allowed to stand for 1.5 h at room temperature. The duration of this reaction may vary.
  • the reaction mixture is subsequently separated over a column of cation exchange resin (e.g., Rexyn 101, ammonium form, 200-400 mesh).
  • the column is eluted with water, 0.5 M ammonia, and 1.0 M ammonia. Unreacted allyl glycoside appears in the aqueous eluate, and the 3-(2- aminoethylthio) propyl glycoside is eluted in the
  • the Schiff base derivative of the S-Layer as obtained by binding of the 3-(2-aminoethylthio)propyl glycoside can be used directly for binding of antibodies. These can be assayed directly if they are labelled with ferritin, horseradish peroxidase, 125 I (iodine-125) or in any other appropriate manner.
  • the bound antibodies can also be assayed via a so-called "sandwich” method by binding of labelled antibodies directed against the first, hapten-bound antibodies.
  • the Schiff base derivative of the S-Layers as obtained by binding of the 3-(2-aminoethylthio)propyl glycoside may be converted into a secondary amine
  • the secondary amine derivative of the S-Layer would be more stable to acid than the Schiff base derivative.
  • Suitable carbohydrate-containing S-Layers are oxidized with sodium metaperiodate as described in
  • Example 1 sections A and B. Iodine-containing salts are removed by dialysis against water. Subsequently, a solution of the corresponding hydrazine reagent in 10% acetic acid is added and the mixture is allowed to react for 1 h. Then the excess reagent is removed by dialysis and the amount of hydrazone groups determined by
  • a cross-linked S-Layer preparation from cell wall fragments of Clostridium thermohydrosulfuricum L111- 69 (hereinafter L111) and Bacillus stearothermophilus NRS2004/3A (hereinafter 3A) were prepared using the procedure set forth in Example 1A. Electron microscopic studies of these compositions show that both the "L111" and "3A” preparations exhibit an S-Layer lattice on either face of a peptidoglycan sacculus ( Figure 1).
  • the peptidoglycan of the foregoing preparations was removed by digestion with lysozyme as described in Example 1A to provide a double layer of S-Layer
  • Bacillus alvei CCM2051 (hereinafter 2051), prepared from whole cell walls as described in Example 3, part A, cross-linked with glutaraldehyde and digested with lysozyme as described in Example 1A also showed two identical subunits.
  • the glycan chains of the L111 preparation are known to consist of approximately 60 disaccharide repeats of 4(- ⁇ -D-Man ⁇ -(1-3)- ⁇ -L-Rhap-(1, which comprises about 10% of the S-Layer glycoprotein.
  • the mannose residues were oxidized completely as estimated by the phenol-sulfuric acid assay.
  • Decreasing the pH value to 4.5 shortens the oxidation times and up to 23 molecules of a carbohydrate hapten linked to a spacer terminating in a primary amino function (for example, A- trisaccharide) per S-Layer protomer was achieved.
  • the 3A S-Layer protomers have two different glycan chains and the total carbohydrate content is about 7.5%. These glycans have the structures:
  • Glutaraldehyde-fixed S-Layer fragments (2-3 mg) were suspended in 2.5 mL of either 0.2 M sodium
  • Glutaraldehyde-fixed S-Layer fragments (2-3 mg) were suspended in 2 mL of appropriate buffer (see epoxy activation, pH 9.1 - 11.4) and then divinyl sulfone (0.2 mL) was added to the suspensions. Treatment of blanks, estimation of shedded S-Layer glycan, incubation times with hapten solutions, and blocking of unreacted vinyl groups was performed exactly as described for the epoxy activation.
  • T-disaccharide is a tumor marker of the formula BGalp(1-3)aGalNAc.
  • a composition containing this disaccharide as a hapten was prepared by coupling the spacer-linked disaccharide of the formula ⁇ Gal-(1- 3) ⁇ GalNAc-O(CH 2 ) 8 CONHNH 2 to S-Layers by either of the general procedures described under Example 7.B.4.
  • T-disaccharide (hydrazide form, 5 mg) is added to one of the oxidized samples, while one oxidized and one unoxidized sample are
  • the preparations are incubated at 37° for 24 h with stirring (500 rpm).
  • the S-Layers are washed once with 0.2 M borate buffer, once with 0.2 M sodium chloride solution, and twice with distilled water.
  • the centrifugation pellets of the respective samples may then be frozen for storage, or processed further.
  • the immobilization of the haptens occurs by way of reductive amination on oxidized, unfixed S-Layers.
  • the presence of the peptidoglycan layer provides for a stabilizing influence similar to the one exerted by glutaraldehyde fixation of the S-Layer protein.
  • ultrafiltration membrane e.g. BIOFIL TM produced from S- Layers of Bacillus stearothermophilus PV72.
  • the suspension is mixed with lysozyme (5 mg) and incubated at room temperature for 90 min with stirring (500 rpm).
  • the enriched suspension in the ultrafiltration cell (ca. 1 mL) is mixed with 5 M guanidine hydrochloride (5 mL) and incubated for 1 h at room temperature with stirring.
  • 5 M guanidine hydrochloride 5 mL
  • the process is accompanied by a clearing of the suspension.
  • the protein-containing solution is enriched by the application of pressure (0.2 MPa) with stirring (500 rpm) and the guanidine
  • hydrochloride washed out with water until extinction at 280 nm is no longer detectable.
  • the clear washes were combined, dialyzed and analyzed for S-Layer fragments by SDS-PAGE.
  • S-Layer protein reaggregates.
  • the suspension is removed from the cell, lyophilized and examined by SDS-PAGE.
  • the aggregates consist only of S- Layer protein containing bound hapten. The quantity of bound hapten is estimated by the difference in
  • S-Layer-hapten-conjugates are produced aseptically in a sterilizable reaction vessel.
  • the starting material S-Layer-containing cell walls
  • LPS lipopolysaccharide
  • the S-Layer hapten conjugates thus obtained can be considered pyrogen-free.
  • mice were pretreated with cyclophosphamide (CP) and two days later were immunized with the T-disaccharide coupled, 3a-S-Layer preparations described in Example 8. Injection was made
  • mice were footpad-challenged with the T- disaccharide-coupled L111 S-Layer preparation, also prepared according to the procedure outlined in Example 8. Changes in footpad thickness were determined 24 h later using a Mitutoyo Engineering micrometer.
  • mice were immunized with 10 ⁇ g of T-disaccharide-3a-S-Layer, and seven days later were footpad-challenged with
  • T-disaccharide L111 S-Layer varying amounts of T-disaccharide L111 S-Layer. Maximum response (4.5 mm -1 ) was obtained using 20 ⁇ g of T- disaccharide L111 S-Layer ( Figure 4). The foregoing results show that the T- disaccharide S-Layer preparations induced a hapten- specific cellular response to the carbohydrate hapten, and give results comparable to the response seen with strong immunogens such as complex viral antigens.
  • DDA dimethyl- dioctadecyl ammonium bromide
  • results show an absence of cross-reactions between different S-Layer preparations and make it possible to utilize different S-Layers for multiple immunization with the same hapten.
  • mice were immunized with antigen mixed with 100 ⁇ g DDA.
  • L111 was sham-treated with EDAC to control for the possible modification of L111 by the EDAC reagent.
  • c Significantly different from all other groups
  • Example 10 The immunization protocols of Example 10 were followed where mice were CP treated or not, then
  • mice were immunized as outlined in example 10 using T-disaccharide-3a-S-Layers, T-disaccharide bovine serum albumin (T-disaccharide BSA) or 8- methyloxycarbonyloctanol (the linker arm attached to the T-disaccharide) linked to BSA. Seven days after T-disaccharide-3a-S-Layers, T-disaccharide bovine serum albumin (T-disaccharide BSA) or 8- methyloxycarbonyloctanol (the linker arm attached to the T-disaccharide) linked to BSA. Seven days after T-disaccharide-3a-S-Layers, T-disaccharide bovine serum albumin (T-disaccharide BSA) or 8- methyloxycarbonyloctanol (the linker arm attached to the T-disaccharide) linked to BSA. Seven days after T-disaccharide-3a-S-La
  • mice were footpad challenged with T- disaccharide-L111 according to example 10.
  • the results in Figure 6 demonstrates the immunopotentiating
  • T-disaccharide-KLH or T-disaccharide-BSA The failure of T-disaccharide-KLH or T-disaccharide-BSA to elicit a blastogenic response may be explained by the notion that the S-Layer conjugate may be taken up preferentially by the macrophages because S- Layers take on the shape of bacteria.
  • b 2051 is the unbound S-Layer isolated from B.
  • T-disaccharide haptenated with the T-disaccharide was a significant DTH response observed. Also, when the primed lymphocytes were depleted of specific T-cell populations, it became evident that the primed cells involved in the DTH response were helper T-cells.
  • the pharmaceutical structures constituting the embodiment of the present invention are particularly suitable as immunizing antigens for achieving high antibody titres and protective isotypes.
  • anti-idiotypic antibodies may be prepared by this method.
  • the pharmaceutical structures can be used to advantage for primary immunization and boosting when one and the same immunoactive substance is bound to S-Layer proteins or glycoproteins derived from two different strains.
  • the structures are applicable also as immune sorbents or affinity matrices, e.g., for diagnostic kits or
  • mice were sham-immunized or immunized with 10 ⁇ g of a preparation of T-disaccharide bound to 2051, or 10 ⁇ g of the S . pneumoniae type 8 CPS antigen bound to Llll, either intramuscularly or orally.
  • the oral immunization was carried out as described by Smith et al., Infect. Immun. 31:129 (1980). Seven days later each group of mice was footpad-challenged with 10 ⁇ g of a preparation of T-disaccharide bound to L111, or 8 CPS coupled to PV72. Footpad swelling was measured 24 h later with a Mitutoyo Engineering micrometer.
  • mice were immunized with T-S-Layer 2051 alone, either intramuscularly or orally. Seven days after immunization the groups of mice were subsequently challenged with T-S-Layer L111. Twenty-four hours later the DTH swelling response was measured. The results are shown in Figure 8a. Both routes of immunization were able to prime for the development of an antigen-specific DTH response. Interestingly, the oral immunization appears to elicit a slightly stronger DTH response than does the IM immunization. This result demonstrates the efficacy of S-Layer conjugates for induction of a systemic immune response after oral administration.
  • mice were immunized with type 8 polysaccharide linked to the S-Layer L111, either intramuscularly or orally. Seven days after immunization the groups of mice were challenged with type 8
  • Example 17 (predominantly IgG class response) and an anamnestic response to a booster vaccination. Included here are some examples of T and Y carbohydrate S-Layer conjugates and a Streptococcus pneumoniae S-Layer conjugate which meet these criteria.
  • Example 17
  • Different hapten molar ratios were produced by varying the time S-Layer pellets were incubated with the hapten solution.
  • Fixed L111 S-Layers with 3.6, 12.7, 23.8 or 37.6 moles of coupled T hapten were injected intraperitoneally at 20 ⁇ g/mouse with Adjuvax (Alfa-Beta Technology).
  • Adjuvax Alfa-Beta Technology
  • the antibody response to the T hapten and to the L111 carrier, assayed by ELISA, is shown in Figure 11.
  • Antibody responses to the T disaccharide were directly proportional to the amount of haptenation.
  • Type 3 S . pneumoniae CPS (ATCC #169-X) was activated by EDC and coupled to glutaraldehyde fixed L111 as in Example 7B. (Specific methods for attachment of haptens). Mice were injected with Type 3 -L111
  • Oligosaccharides were prepared from Type 8 S. pneumoniae CPS using methods modified from Albersheim et al . , Carbohydrate Research 5:340, 1967. Oligosaccharide preparations were coupled to unfixed PV72, or 2051 S- Layers and tetanus toxoid (TT) as described in the previous example.
  • mice were immunized intraperitoneally with oligo 8-PV72 (8 ⁇ g 8/20 ⁇ g PV72) in PBS, oligo 8-2051 (10 ⁇ g 8/20 ⁇ g 2051) in PBS, oligo 8-TT (4 ⁇ g 8/20 ⁇ g TT), oligo 8 (9 ⁇ g) in PBS or PBS alone.
  • mice were bled on day 21 and an ELISA was performed to compare the different anti-oligo responses.
  • the antibody response to the 8 oligo as assayed by ELISA was greater when 8-PV72 or 8-2051 were the immunogens than when uncoupled 8 or 8-TT were injected.
  • ELISA plates (Maxisorb, NUNC #4-39450) were coated with 1 ⁇ g/well of antigen diluted in 0.05 M carbonate-bicarbonate coating buffer (pH 9.6). Plates were incubated overnight at 4°C and then blocked with 1% BSA (Sigma A-7030) in PBS (pH 7.2). After 1 hour
  • Streptococcus pneumoniae CPS type specific
  • ELISA plates were first coated with 1/250 rabbit anti-CPS (type specific; Statens Seruminstitut), diluted in coating buffer and incubated at room
  • FIG. 1 Preparation scheme of glutaraldehyde-fixed S- Layer fragments; (a) intact bacterial cell; (b) empty peptidoglycan sacculus after glutaraldehyde fixation. A second S-Layer has been formed on the inner surface; (c) glutaraldehyde-fixed S-Layer fragments after lysozyme treatment consisting of two S-layers.
  • PG Peptidoglycan
  • CM cytoplasmic membrane
  • S S-layer - is and oS, inner and outer S-Layer (adapted from Sara and Sleytr).
  • FIG. 3 Effect of the dose of T-disaccharide-linked to B. stearothermophilus S-Layer (T-3a) on the immunological priming for DTH to the T-hapten.
  • Mice were treated with cyclophosphamide (200 mg/kg) and immunized two days later with varying concentrations of the T-3a. Seven days later all groups of mice were footpad-challenged with 10 ⁇ g of a preparation of T-disaccharide bound to C.
  • thermohydrosulfuricum (T-L111) S-layer. Footpad swelling was measured 24 h later. The increase in footpad
  • mice were footpad challenged with 10 ⁇ g of a preparation of T-disaccharide bound to B. stearothermophilus (T-3a) S-Layers, mixed with PBS, DDA or FIA. Seven days later each group of mice were footpad challenged with 10 ⁇ g of a preparation of T-disaccharide bound to C. thermohydrosulfuricum (T- L111) S-Layers. Footpad swelling was measured 24 h later . The increase in footpad thickness is expressed in mm x 10 -1 . Figure 6. Groups of 10 mice were treated, or not, with cyclophosphamide and subsequently immunized with 10 ⁇ g of a preparation of T-disaccharide bound to B.
  • T-3a stearothermophilus
  • T- L111 thermohydrosulfuricum
  • T-3a stearothermophilus
  • BSA bovine serum albumin
  • G-BSA bovine serum albumin
  • T-L111 thermohydrosulfuricum
  • FIG. 7 Transfer of the lymphocytes mediating DTH response against the T-disaccharide.
  • a group of 10 mice were pretreated with cyclophosphamide and subsequently immunized with 10 ⁇ g of a preparation of T-disaccharide- 3a-S-Layer (T-3a). Seven days later mice were sacrificed and draining lymph nodes and spleens were isolated.
  • T-disaccharide-3a primed lymphocytes were bulk stimulated with either PBS, L111 S-Layers, or T-disaccharide-L111-S-Layer conjugates (T-L111). Four days later the lymphocytes were collected and washed in PBS.
  • Primed lymphocytes stimulated with T- disaccharide-L111 were then divided into 4 groups and sham treated or treated with monoclonal antibodies L3T4, (specific for helper T-cells), Ly 2.2 (specific for suppressor T-cells) and Thy 1.2 (specific for all T- cells). The 4 groups cells were then treated with complement and then washed and counted.
  • the primed cells were then treated with complement and then washed and counted.
  • mice (5 x 10 5) were then mi.xed wi.th ei.ther PBS or T- disaccharide 2051 S-Layer (T-2051) and then injected into naive mice. Footpad swelling was measured 24 h later. The increase in footpad thickness is expressed in mm x 10 -1 .
  • mice were immunized with 10 ⁇ g of a preparation of T-disaccharide bound to L111 S-layers (T-L111), orally or intramuscularly. Seven days later, each group of mice were footpad challenged with 10 ⁇ g of a preparation of T-disaccharide bound to 2-51 S-Layers (T-2051). Footpad swelling was measured 24 h later. The increase in footpad thickness is expressed in mm -1 .
  • mice polysaccharide bound to L111 S-Layers (8CPS-L111), orally or intramuscularly. Seven days later each group of mice were footpad challenged with 10 ⁇ g of a preparation of S. puemoniae type 8 capsular polysaccharide bound PV72 S- Layers (8 CPS-PV72). Footpad swelling was measured 24 h later. The increase in footpad thickness is expressed in mm -1 .

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Abstract

On décrit des compositions pharmaceutiques supérieures qui comprennent des porteurs associés à des fragments portant des déterminants antigéniques. Les porteurs sont constitués de rangées de protéines ou glycoprotéines, dérivées d'enveloppes de cellules. Ces conjugués sont capables de favoriser la formation d'anticorps aussi bien qu'une réponse immunitaire à médiation lymphocyte suite à une administration parentérale ou orale.
PCT/CA1991/000063 1990-03-02 1991-03-04 Compositions immunogeniques ameliorees WO1991012819A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO1995019371A3 (fr) * 1994-01-14 1995-11-16 Solvay Expression de proteines de couches superficielles
US9107906B1 (en) 2014-10-28 2015-08-18 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection

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KR20070089231A (ko) * 2004-12-14 2007-08-30 알크-아벨로 에이/에스 이종 단백질성 화합물을 제시하는 박테리아 세포를포함하는 약제학적 조성물

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EP0306473A1 (fr) * 1987-08-21 1989-03-08 Sleytr, Uwe B., Dipl.-Ing., Dr. Structure pharmaceutique avec agents liés à des supports protéiniques

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EP0306473A1 (fr) * 1987-08-21 1989-03-08 Sleytr, Uwe B., Dipl.-Ing., Dr. Structure pharmaceutique avec agents liés à des supports protéiniques

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Title
Carbohydrate Research, volume 93, 1981, Elsevier Scientific Publishing Company, (Amsterdam, NL), R.M. Ratcliffe et al.: "Synthesis of T(B-D-Gal-(1-3)-X-D-GalNAc)-antigenic determinant in a form useful for the preparation of an effective artificial antigen and the corresponding immunoadsorbent", pages 35-41 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019371A3 (fr) * 1994-01-14 1995-11-16 Solvay Expression de proteines de couches superficielles
US5874267A (en) * 1994-01-14 1999-02-23 Solvay (Societe Anonyme) Expression of surface layer proteins
US9107906B1 (en) 2014-10-28 2015-08-18 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9714283B2 (en) 2014-10-28 2017-07-25 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9815886B2 (en) 2014-10-28 2017-11-14 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US9969793B2 (en) 2014-10-28 2018-05-15 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US10683343B2 (en) 2014-10-28 2020-06-16 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US11339206B2 (en) 2014-10-28 2022-05-24 Adma Biomanufacturing, Llc Compositions and methods for the treatment of immunodeficiency
US11780906B2 (en) 2014-10-28 2023-10-10 Adma Biomanufacturing, Llc Compositions and methods for the treatment of immunodeficiency
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
US11084870B2 (en) 2017-03-15 2021-08-10 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
US11897943B2 (en) 2017-03-15 2024-02-13 Adma Biomanufacturing, Llc Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection

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