EP0575517A1 - Konjugate aus lipid a-analog und immunogenem träger, deren verwendung als impfstoffe und polyvalente hyperimmungammaglobuline - Google Patents

Konjugate aus lipid a-analog und immunogenem träger, deren verwendung als impfstoffe und polyvalente hyperimmungammaglobuline

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Publication number
EP0575517A1
EP0575517A1 EP92908659A EP92908659A EP0575517A1 EP 0575517 A1 EP0575517 A1 EP 0575517A1 EP 92908659 A EP92908659 A EP 92908659A EP 92908659 A EP92908659 A EP 92908659A EP 0575517 A1 EP0575517 A1 EP 0575517A1
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EP
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Prior art keywords
group
acyl groups
lipid
immunogenic carrier
analog
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EP92908659A
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English (en)
French (fr)
Inventor
Nelson Teng
Jerald Sadoff
Apurba Bhattacharjee
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Univax Biologics Inc
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Univax Biologics Inc
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Publication of EP0575517A1 publication Critical patent/EP0575517A1/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • 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
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • 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
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (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
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • 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
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • 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
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]

Definitions

  • This invention is in the field of medicinal chemistry.
  • this invention relates to pharmaceutical compositions and the use thereof as vaccines and polyvalent hyperimmune gammaglobulins.
  • BACKGROUND OF THE INVENTION Each year approximately 194,000 patients in U.S. hospitals develop bacteremia; of these, about 75,000 die. Maki, D.G., in Nosocomial Infections, R.E. Dixon (ed.), pages 183-196, Yorke Medical Books, New York
  • LPS lipopolysaccharide
  • antiserum may also facilitate the removal of Gram-negative bacteria by the reticuloendothelial system.
  • Several workers have demonstrated successful broad-spectrum protection against challenge by several Gram-negative organisms or endotoxins in different animal models by using rough mutants of E. coli or S. minnesota as .Immunizing agents to generate antisera. Braude, A.I., et al., J. Immunol. 108:505-512 (1972); Braude, A.I., et al . , J. Infect. Dis.
  • Typical Gram-negative bacterial LPS has three major structural regions: the O-polysaccharide, the R-core oligosaccharide, and lipid A.
  • the structure of the O-polysaccharide is highly variable between organisms, even in the same species. Its antigenicity serves as a basis for serotyping the bacteria.
  • the R region is a bridge between the O-antigen and lipid A; its structure is similar in most Gram-negative bacteria.
  • Antibodies to LPS may promote phagocytosis or the death of the bacteria.
  • the O-antigen is the most antigenic component of the LPS, yet it has little known toxicity.
  • Lipid A in contrast, contains the toxic center of the molecule and is remarkably similar in structure across a wide range of bacterial genera.
  • lipid A region of LPS is responsible for a complex array of inflammatory responses to tissue invasion by Gram-negative bacteria by directly stimulating host cells such as macrophages, neutrophils, and endothelial cells which mediate the inflammatory changes. Responses are both toxic (hypotension, coagulation disturbances, death) and beneficial to the infected host (enhancement of antibody for ⁇ vation, mobilization of phagocytes, acute phase protein synthesis).
  • lipid A The structure of lipid A is depicted as Formula (I) below:
  • Lipid A is a glucosamine disaccharide that is phosphorylated at positions 1 and 4' and has six or seven esterified fatty acids.
  • Four molecules of 3- hydroxytetradecanoate are attached to the glucosamine disaccharide at positions 2, 3, 2', and 3'; the hydroxyl groups of the 3'-OH-14:0 residues at positions 2 ' and 3' (and sometimes 2) are substituted with normal fatty acids (dodecanoate, tetradecanoate, hexadecanoate) to form acyloxyacyl groups.
  • lipid A analogs and biosynthetic precursors of lipid A have been examined.
  • Luderitz, O., et al . , Rev. Infect. Dis. 6:428-431 (1984) disclose the preparation of lipid A analogs and the relationship thereof between the chemical structure and biologic activity.
  • the authors report that phosphate substitution of the glucosamine disaccharide is not essential for the expression of antigenicity, and that the amine-bound 3-hydroxyacyl residues can be replaced by non-hydroxylated fatty acids without reduction of activity.
  • the immunodeterminant structure comprises the linkage region of the amide-linked fatty acids and glucosamine.
  • Antibodies were produced in titers comparable to those obtained by immunization under similar conditions with acid-treated bacteria coated with lipid A.
  • This altered bacterial LPS is therapeutically useful as a vaccine to prevent Gram-negative bacterial diseases by inducing antibodies to LPS, as an antidote to treat or prevent Gram-negative bacterial sepsis, or as an adjuvant to enhance formation of antibodies to other antigens.
  • the acyloxyacyl hydrolase itself may also be prophylactically or therapeutically useful to detoxify endogenous LPS in patients with Gram-negative bacterial diseases. This enzyme may also be used to remove toxic LPS from therapeutic injectants.
  • capsular polysaccharides from Gram negative and Gram positive microorganisms for use as vaccines and to raise antibodies in animals which can be used for passive immunization. Where the capsular polysaccharides are poorly immunogenic, they may be linked to an immunogenic polypeptide to improve the immune response of the immunized animal.
  • the induced serum antibodies recognized the polysaccharides capsule of S. pneumonia type 6A and 6B strains and killed the N. meningitidis group C bacteria by complement-mediated bacterial lysis.
  • Iinuma H. and Okinaga, K., J. Infect. Dis. 160:66-75 (1989), disclose the prevention of pneumococcal bacteremia by immunization with type 6 Streptococcus pneumonlae polysaccharide vaccine in rats.
  • the polysaccharide vaccine elicited significantly higher serum opsonic activity and increased antibody titer to the type 6 polysaccharide compared to controls.
  • Prakobphol A. et al . , Infect. Immun. 27:150-157 (1980), disclose the preparation of a polysaccharide from Streptococcus mutans. This polysaccharide was immunogenic in rabbits which were immunized with whole cells or cell walls.
  • Fattom A. et al . , Infect. Immun. 58:2367-2374 (1990), disclose amethod of preparing a Staphylococcua aureus CPS/immunogenic polypeptide conjugate and the use thereof as a vaccine.
  • lipid A analogs as immunogens for induction of active immunity to LPS
  • a need continues to exist for new compounds which can be used in the treatment or prevention of septic shock in animals.
  • a need continues to exist for the development of multivalent hyperimmune gammaglobulins specific for lipid A, Gram negative and Gram positive microorganisms.
  • the invention relates to a lipid A analog/immunogenic carrier conjugate, wherein said lipid A analog the following Formula (III):
  • n 1 or 2;
  • R 1 and R 3 are the same or different and selected from the gtoup consisting of hydrogen, a C 2 -C 18 acyl group, a 3-hydroxy C 3 -C 18 acyl groups, a 3-(C 2 -C 12 -acyloxy)-C 3 -C 18 acyl group and a linkage to an immunogenic carrier;
  • R 2 is selected from the group consisting of C 2 -C 18 acyl groups, 3-hydroxy C 3 -C 18 acyl groups, and 3-(C 2 -C 12 - acyloxy)-C 3 -C 18 acyl groups;
  • R 4 is hydrogen, a C 2 -C 18 acyl group, a phosphate group or a linkage to an immunogenic carrier
  • R 1 , R 3 or R 4 is a linkage to an immunogenic carrier, wherein said linkage does not interfere substantially with the ability of the lipid A analog to stimulate an immune response in an animal.
  • the invention relates to a lipid A analog/immunogenic carrier conjugate having the following Formula (IV):
  • n, R 1 , R 2 , R 3 and R 4 are defined above;
  • A is an immunogenic carrier
  • n 0 or 1
  • p 1 to 200
  • L is a linker group which does not interfere substantially with the characteristic ability of the lipid A analog to stimulate an immune response in an animal
  • R 1 and R 3 are the same or different and selected from the group consisting of hydrogen, C 2 -C 18 acyl groups, 3-hydroxy C 3 -C 18 acyl groups, and 3-(C 2 -C 12 -acyloxy)-C 3 -C 18 acyl groups;
  • R 2 is selected from the group consisting of C 2 -C 18 acyl groups, 3-hydroxy C 3 -C 18 acyl groups, and 3-(C 2 -C 12 - acyloxy)-C 3 -C 18 acyl groups;
  • R 4 is hydrogen, a C 2 -C 18 acyl group or a phosphate group.
  • the invention also relates to a lipid A analog/- immunogenic carrier conjugate having the following Formula (V):
  • n, m, p, L, R 1 , R 2 , R 3 and R 4 are defined above.
  • the invention also relates to a vaccine for the prophylaxis of septic shock comprising a lipid A analog/immunogenic carrier conjugate of the present invention together with a pharmaceutically acceptable carrier or adjuvant.
  • the invention also relates to a method for treating or preventing septic shock in an animal comprising administering a pharmaceutical composition to an animal comprising a lipid A analog/immunogenic carrier conjugate of the invention;
  • lipid A analog/immunogenic carrier conjugate is present in an amount effective to induce active immunity to LPS in an animal.
  • the invention also relates to intermediates useful for the preparation of the lipid A analog/immunogenic carrier conjugates of the invention.
  • This invention also provides for a polyvalent vaccine comprising
  • lipid A analog/immunogenic carrier conjugate of the invention (b) at least one Gram negative and/or Gram positive bacteria, or antigenic determinant thereof, optionally conjugated with an immunogenic carrier,
  • This invention also provides for a polyvalent hyperimmune gammaglobulin comprising an effective neutralizing amount of a gammaglobulin specific to the lipid A/immunogenic carrier conjugate of this invention and an effective opsonizing amount of a Gram negative and/or Gram positive bateria-specific gammaglobulin.
  • This invention also concerns a method of preparing a polyvalent hyperimmune gammaglobulin comprising (a) immunizing an animal with the lipid A analog/immunogenic carrier of this invention and at least one Gram negative and/or Gram positive bacteria, or antigenic determinant thereof, optionally conjugated with an immunogenic carrier,
  • step (b) optionally boosting said animal with the immunizing step of step (a),
  • This invention also provides a polyvalent hyperimmune gammaglobulin obtained by
  • step (b) optionally boosting said animal with the immunizing step of step (a),
  • This invention further provides for a method of treating Gram negative and/or Gram positive bacterial sepsis comprising administering an effective neutralizing amount of a gammaglobulin specific to the lipid A/immunogenic carrier conjugate of this invention and an effective opsonizing amount of Gram negative and/or Gram positive-specific polyvalent hyperimmune gammaglobulin to an animal suffering from a Gram negative and/or Gram positive bacterial infection.
  • This invention also provides for a kit for treating Gram negative and/or Gram positive bacterial sepsis comprising a carrier compartmentalized to receive in close confinement therein one or more container means wherein a first container contains a lipid A analog/immunogenic carrier conjugate of the invention.
  • This invention also provides for a kit for treating Gram negative and/or Gram positive bacterial sepsis comprising a carrier compartmentalized to receive in close confinement therein one or more container means wherein a first container contains a hyperimmune gammaglobulin specific for the lipid A analog/.Immunogenic conjugate of this invention.
  • the kit may further comprise gammaglobulins specific for at least one Gram negative and/or Gram positive bacteria.
  • Figure 1 depicts a graph showing the elution profile of FD-4 PS from a G75 gel filtration column.
  • Figure 2 depicts a graph showing the elution profile of FD-7 PS from a G75 gel filtration column.
  • Figure 3 depicts a graph showing the elution profile of INT-3 from a G75 gel filtration column.
  • Figure 4 depicts a graph showing the elution profile of INT-4 PS from a G75 gel filtration column.
  • Figure 5 depicts a graph showing the elution profile of KPO-2 PS from a G75 gel filtration column.
  • Figure 6 depicts a graph showing the elution profile of the FD4-PS-DT conjugate from a CL-6B Sepharose column.
  • Figure 7 depicts a graph showing the elution profile of the FD5-PS-DT conjugate from a CL-6B Sepharose column.
  • Figured 8 depicts a graph showing the elution profile of the FD7-PS-DT conjugate from a CL-6B Sepharose column.
  • Figure 9 depicts a graph showing the elution profile of the INT4-PS-DT conjugate from a CL-6B Sepharose column.
  • Figure 10 depicts a graph showing the elution profile of the KPO2-PS-DT conjugate from a CL-6B Sepharose column.
  • Figure 11 depicts a graph showing the elution profile of the high molecular weight (HMW) and low molecular weight (LMW) KPO2-PS-DT conjugates from a CL-6B Sepharose column.
  • HMW high molecular weight
  • LMW low molecular weight
  • the invention relates to lipid A analog/immunogenic carrier conjugates comprising glucose or a polyglucose molecule such as gentiobiose linked to an immunogenic carrier, wherein at least C 3 position of the glucose or gentiobiose moieties are substituted with an acyl group (R 2 ). In another embodiment, the C 2 position of the glucose or gentiobiose moieties is not substituted by hydroxy (R 1 ⁇ H).
  • the immunogenic carrier may be linked to any free hydroxyl group on the Lipid A analog molecule, so long as the conjugate induces active immunity to LPS when administered to an animal.
  • the immunogenic carrier is linked to the C 1 or C 6 position of the glucose or polyglucose moiety.
  • lipid A analog/immunogenic carrier conjugates of the present invention include all non-amino saccharides and polysaccharides acylated at least at the C 3 position of at least one of the saccharide moieties and linked to an immunogenic carrier, wherein the conjugate induces active immunity to LPS when administered to an animal.
  • a lipid A analog/immunogenic carrier conjugate of the inventfbn is considered to induce active immunity to LPS wherein when the conjugate is administered to an animal, anti-LPS antibodies are produced by the animal.
  • the efficacy of the anti-LPS antibodies can be tested, for example, by administering the sera of an .Immunized animal to a second animal followed by challenge of the second animal with LPS-producing bacteria (see Example 7, below). Where administration of the anti-LPS sera results in enhanced survival of the second animal, the conjugate is considered to induce immunity to LPS.
  • Anti-LPS antibodies can also be tested by inhibition of the Shwartzman reaction, a well-known biological response to LPS (Lee, L. et al . , In: Zweifach, B.W. et al . (eds.) THE INFLAMMATORY PROCESS, Academic Press, NY, 1965, p. 791). Animals, particularly rabbits, actively immunized with LPS or with one of the compositions of the present invention are given an appropriate LPS challenge to induce a localized Shwartzman reaction. Prevention or amelioration of the erythema, hemorrhage or necrotic reaction is evidence for the presence of anti-LPS antibodies.
  • serum from an animal immunized with LPS or with one of the compositions of the present invention is transferred to a naive rabbit, which is then tested for a Shwartzman reaction. Inhibition of the reaction is a measure of antibody activity.
  • the vaccines of the present invention are useful for the prophylaxis of septic shock .in an animal which is due to the release of LPS endotoxin by Gram negative microorganisms.
  • Gram negative microorganisms include, but are not limited to Salmonella, Eacherichia, Hemophilua and Neiaaeria, Klebaiella, Shigella, Paeudomonaa, Enterobacter, Acinetobacter, and Bacteroidea. See Bergey's Manual of Syatematic Microbiology.
  • the conjugates of the invention are useful as vaccines which induce active .Immunity toward LPS in animals.
  • animals are humans, however the invention is not intended to be so limiting. Any animal which may experience the beneficial effects of the vaccines of the invention are within the scope of animals which may be treated according to the claimed invention.
  • the invention relates to lipid A analog/immunogenic carrier conjugates having the Formula (IV):
  • A is an immunogenic carrier
  • L is a linker group which does not interfere substantially with the characteristic ability of the lipid A analog to stimulate an immune response in an animal
  • R 1 and R 3 are selected from the group consisting of hydrogen', C 2 -C 18 acyl groups, 3-hydroxy C 3 -C 18 acyl groups, and 3-(C 2 -C 12 -acyloxy)-C 3 -C 18 acyl groups;
  • n 0 or 1
  • n 1 or 2;
  • p 1 to 200
  • R 2 is selected from the group consisting of C 2 -C 18 acyl groups, 3-hydroxy C 3 -C 18 acyl groups, and 3-(C 2 -C 12 -acyloxy)-C 3 -C 18 acyl groups;
  • R 4 is hydrogen, a C 2 -C 18 acyl group or a phosphate group.
  • the invention also relates to a lipid A analog/-immunogenic carrier conjugate having the following Formula (V):
  • n, m, p, L, R 1 , R 2 , R 3 and R 4 are defined above.
  • immunogenic carrier any macromolecule which is capable of inducing an immunogenic reaction in an animal. Since many small molecules such as the lipid A analogs of the invention do not induce active immunity by themselves, it is necessary to conjugate the analog to an immunogenic carrier to induce production of antibodies which are specific for the small molecule.
  • Such immunogenic carriers include, but are not limited to, proteins such as bovine serum albumin, diphtheria toxoid, tetanus toxoid, edestin, keyhole-limpet hemocyanin, Pseudomonal Toxin A, choleragenoid, cholera toxin, pertussis toxin, viral proteins, and eukaryotic proteins such as interferons, interleukins, or tumor necrosis factor.
  • proteins may be obtained from natural or recombinant sources according to methods known to those skilled in the art.
  • the immunogenic carrier may comprise a protein fragment comprising at least the immunogenic portion of the molecule.
  • immunogenic macromolecules which may be used in the practice of the invention include, but are not limited to, polysaccharides, tRNA, nonmetabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4'4'-diaminodiphenyl-methane-3,3'-dicarboxylic acid and 4-nitro-2-aminobenzoic acid (See Sela, M., Science 166:1365-1374 (1969)) or glycolipids, lipids or carbohydrates.
  • the immunogenic carrier is a protein.
  • linker group is intended one or more bifunctional molecules which can be used to link the immunogenic carrier to the lipid A analog and which do not interfere with the production of antilipid A antibodies in vivo.
  • the linker group may be attached to any part of the glucose or gentiobiose moiety so long as the point of attachment does not interfere with the production of anti-lipid A antibodies in vivo and thus interfere with the induction of active immunity.
  • Typical acyl groups which can be substituted on the carbohydrate moiety include, but are not limited to, acetate, propionate, butanoate, pentanoate, hexanoate, heptanoate, octanoate, nonanoate, decanoate, palmitoyl, olelyl, myristoyl, stearoyl, 3-hydroxybutanoate, 3-hydroxypentanoate, 3-hydroxyhexanoate, 3-hydroxyheptanoate, 3-hydroxyoctanoate, 3-hydroxynonanoate, 3 -hydroxydecanoate, 3-hydroxydecanoate, 3-hydroxypalmitoyl, 3-hydroxyoleyl, 3-hydroxymyristoyl, and 3-hydroxystearoyl groups.
  • R groups include the 3- (C 2 -C 12 acyloxy)-substituted aforementioned C 3 -C 18 acyl groups wherein the C 2 -C 12 acyloxy groups include, but are not limited to, acetate, propanoate, butanoate, pentanoate, hexanoate, heptanoate, octanoate, nonanoate, decanoate, and dodecanoate groups.
  • Preferred lipid A analog/immunogenic carrier conjugates are derived from gentiobiose and have the following formula (IX):
  • a preferred lipid A analog/immunogenic carrier conjugate is gentiobiose peracetate linked at the C 1 -position to an immunogenic carrier having the following Formula (X):
  • Another preferred lipid A analog/immunogenic carrier conjugate is an acyl glucose molecule linked at the C 1 -position having the Formula (XI):
  • Another preferred lipid A analog/immunogenic carrier conjugate is an acyl gentiobiose molecule linked at the C 6' -position to an immunogenic carrier having the Formula (XII):
  • Another preferred lipid A analog/immunogenic carrier conjugate is an acyl glucose molecule linked at the C 6' -position to an immunogenic carrier having the Formula (XIII):
  • the lipid A analog/immunogenic carrier conjugates linked at the exposition may be prepared (see Scheme I) by treatment of an appropriately substituted and protected carbohydrate with a reagent capable of linking the carbohydrate to the immunogenic carrier having Formula (XIV) either directly or through a linker group.
  • a reagent capable of linking the carbohydrate to the immunogenic carrier having Formula (XIV) either directly or through a linker group.
  • the acetobromo derivative (XVI) may then be reacted with a linker such as 2-aminoethanol (XVII), 3-aminopropanol, 4-aminobutanol, or 5-aminopentanol, to give the aminoethyl peracetate saccharide derivative (XVIII) which may be coupled to the immunogenic carrier (XIV) with, for example, 1-ethyl-3-(3- dimethyl-aminopropyl) carbodiimide (EDAC) to give (IV).
  • EDAC 1-ethyl-3-(3- dimethyl-aminopropyl) carbodiimide
  • the immunogenic peptide may be derivatized with a second bifunctional spacer group, such as succinic acid or any of the other linker groups having two carboxyl groups, to give the derivative (XIX).
  • a second bifunctional spacer group such as succinic acid or any of the other linker groups having two carboxyl groups
  • the derivative having Formula (XIX) may then be condensed with (XVIII) in the presence of EDAC to give (IV).
  • R 4 is a phosphate group
  • the hydroxy group may be phosphorylated prior to preparation of the conjugate Iby reacting an appropriately protected glucose or gentiobiose compound with the product of phosphorous acid and N-methylimidazole in the presence of HgCl 2 . This reaction allows the preparation of the phosphate ester at the free hydroxyl group.
  • the ratio of lipid A analog molecules per immunogenie carrier molecule may vary considerably according to the molecular weight of the immunogenic carrier, the number of binding sites on the immunogenic carrier capable of being coupled to the lipid A molecule, and the antigenic characteristics of the particular lipid A molecule.
  • the ratio of lipid A analog molecules to immunogenic carrier molecules may be about 1:1 to about 200:1.
  • the ratio may range from about 5:1 to about 100:1.
  • the immunogenic carrier is diphtheria toxoid
  • the ratio of lipid A analog molecules to diphtheria toxoid molecules may range from about 5:1 to about 40:1.
  • the conjugates may be prepared according to scheme II by the selective tritylation of, for example, the C 6' -position of gentiobiose (XX) with trityl chloride (XXI) to give the C 6' -trityl gentiobiose derivative (XXII).
  • XXI gentiobiose
  • XXII trityl chloride
  • XXIII 6'-O-tritylgentiobiose heptaacetate
  • the invention also relates to intermediates useful for the preparation of the lipid A analog/immunogenic carriers of the invention.
  • the invention relates to a 1-bromo-saccharide having the Formula (XXVI):
  • the invention also relates to the intermediate having Formula (XXVII):
  • the invention also relates to lipid A analog/linker conjugates having the Formula (XXVIII):
  • the invention relates to a lipid A analog/linker conjugate having the Formula (XXIX) :
  • the invention also relates to a lipid A analog/linker conjugate having the Formula (XXX):
  • the invention also relates to a lipid A analog/linker conjugate having the Formula (XXXI):
  • the invention also relates to a lipid A analog/linker conjugate having the Formula (XXXII):
  • the conjugates of the invention may be purified by any method known to those of ordinary skill in the art.
  • the conjugates may be purified by reverse phase chromatography, ion exchange chromatography, size exclusion chromatography, or by dialyzing the reaction product against water followed by freeze-drying.
  • the conjugates may be purified by passing a solution of the conjugate through a column having anti-lipid A antibodies immobilized on a solid phase support. (See the Examples section of the application for a method of preparing antibodies to lipid A.)
  • the invention also provides for a polyvalent vaccine comprising
  • Preferred Gram negative bacteria are the Pseudomonadales and Eubacteriales. As Pseudomonadales, included are the genera Paeudomonas, Xantohmonas, Zoogloea, and Gluconobacter. Preferred are the Pseudomonas which include bacteria of the species Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas pseudomallei, Pseudomonas mallei, and Pseudomonas saccharophilia. Most preferred are the bacteria of the species Pseudomonas aeruginosa.
  • Enterobacteriaceae and Brucellaceae.
  • Eachericbia, Edwardaiella, Citrobacter, Salmonella, and Shigella are preferred. Included as preferred in the genus Eacherichia are the bacteria of the species Eacherichia coli . Among the Klebsielleae, the genera Klebsiella, Enterobacter, Hafnia, and Serratia are preferred. Included as preferred among the genus
  • Klebsiella are the bacteria of the species Kebsiella pneumoniae, Klebsiella ozaenae, Klebaiella oxytoca, and Klebsiella rhinoacleromatia.
  • Preferred Gram positive bacteria are the Micrococcaceae and the Streptococcaceae. Included in the Micrococcaceae are the genera Micrococcus, Staphylcoccus, and Planococcus. Among the Staphylococci, bacteria of the species Staphlycoccus aureus and Staphlycoccus epidermidis are preferred. Included in the Streptococcaceae are the genera Streptococcus, Leuconostoc, Pediococcus, Aerococcus, and Gemella. Preferred are bacteria of the species Streptococcus faecalis, Streptococcus faecium, Streptococcus mitis, and Streptococcus pyogenes.
  • This invention also provides for a polyvalent hyperimmune gammaglobulin comprising an effective opsonizing and neutralizing amount of a gammaglobulin specific to the lipid A/immunogenic carrier conjugate of this invention and an effective opsonizing amount of an O antigen specific gammaglobulin.
  • the polyvalent hyperimmune gammaglobulin can further comprise at least one antigenic determinant of a Gram positive bacteria.
  • the hyperimmune gammaglobulin can be separated from ⁇ animals which have been hyperimmunized.
  • An animal can be hyperimmunized by administration, preferably by intramuscular or subcutaneous injection, of one or more conjugates of this invention.
  • the conjugates can be effectively administered in a range of between about 10 and 100 ⁇ g of polysaccharide as a component of each conjugate.
  • the animal can be reinjected with between about 10 and 100 ⁇ g of polysaccharide as a component of each conjugate.
  • the animal receives an injection of between about 12.5 and 50 ⁇ g of polysaccharide, optionally as a component of each conjugate.
  • a second injection exponentially increases the number of B cells bearing antigen-specific cell-surface antibodies.
  • a third or subsequent injection can also be administered which duplicates the second injection. As is well known in the art, this repeated boosting of antigen results in a hyperimmunized animal.
  • An adjuvant suitable for injection may also be used to enhance the immune response to the conjugates.
  • Plasma separated from an immunized animal contains a relatively large concentration of antibody to the injected antigens and is appropriately called a hyperimmune serum.
  • Methods for separating the hyperimmune serum from a hyperimmunized animal are known in the art as exemplified by Harlow et al., Antibodies, Cold Spring Harbor Laboratory, 1988.
  • Hyperimmune gammaglobulin preparations can be prepared from the hyperimmune serum using various methods known in the art such as the Cohn fraction method See, Conn, E.J. et al . , J. Amer. Chem. Soc. 68: 4.59 (1946) and U.S. Patent No. 4,965,068.
  • plasma may be separated into fractions which are immunologically active (immunoglobulins) and immunologically inactive (albumins and clotting factors) by fractional precipitation using ammonium sulfate.
  • immunologically active fraction is then briefly digested with pepsin to remove the complementactivating component of the molecules to render them less immunogenic.
  • the immunologically active component then is recovered by dialysis and fractional precipitation or centrifugation. See Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, (1990).
  • opsonizing and neutralizing concern activity which relate to both humoral and cellular immunity.
  • humoral immunity lipid A-specific antibodies "neutralize” lipid A, but not any other type of toxin.
  • cellular immunity is not dependent entirely upon circulating antibody.
  • Streptococcus pneumoniae an encapsulated coccus
  • S. pneumoniae an encapsulated coccus
  • death will not generally occur. This result is due to the antibody binding to and altering the surface of the challenging organism, thereby promoting phagocytosis. This activity is typically referred to as "opsonizing" activity.
  • Rat Neutropenic Model These models are well known to those of ordinary skill in the art.
  • the Murine Hog Mucin Model as described by Teng, N.N.H. et al . , Proc. Natl . Acad. Sci . USA 82: 1790-1794 (1985), can be used to measure opsonic activity.
  • mice are inoculated with graded doses of an 18-hour culture of Pseudomonas aeruginoaa 134 VA (Fisher Devlon type I), Paeudomonaaa aeruginoaa Fisher Devlon type VI, Klebaialla pneumoniae, Eacherichia coli 0111:B 4 , Escherichia coli 017 or Streptococcus pneumoniae.
  • Graded doses of one or more of the 18-hour cultures are mixed with equal volumes of 14% hog gastric mucin, and 0.5 ml of the bacterial suspension is injected into the mouse peritoneum.
  • the hyperimmune gammaglubulin of this invention Prior to this injection, the hyperimmune gammaglubulin of this invention is injected into the mice, with controls not receiving this injection. Protection against lethal bacteremia can then be determined by calculating the protection ratio.
  • Tli ⁇ s ratio is defined as the ratio of LD 50 between the hyperimmune gammaglubulin treated group and the non-treated control group. A protection ration of at least 5:1 is considered to indicate positive opsonic activity. A 10:1 ratio is preferred, with a ratio of at least 15:1 being most preferred.
  • the gammaglobulins specific for the Grama negative and Gram positive bacterial conjugates of this invention will show opsonic activity.
  • neutralizing activity can be screened by the Dermal Schwartzman Reaction as also described by Teng, N.N.H. et al . , supra.
  • rabbits are inoculated in the ear vein with the hyperimmune gammaglubulin of this invention.
  • the rabbits are given challenge doses of lipopolysaccharide or Iscove's medium as a control.
  • Neutralizing activity is evident when there is a reduction of hemorrhagic necrosis at the site of challenge injection.
  • neutralizing activity will be indicated when the incidence of positive reactions is less than about 90%. It is preferred that the incidence of positive reactions will be less than about 15%, with an incidence of less than about 12% being most preferred.
  • Preferred O antigen specific gammaglobulins are gammaglobulins which are specific to the O antigens of the Gram negative bacteria of this invention. Especially preferred are gammaglobulins specific to the O antigens of Eacherichia coli, Pseudomonas aeruginoaa, Klebaiella pneumoniae, Klebaiella oxytoca, and Klebai ella ozanae.
  • This invention also concerns a method of preparing a polyvalent hyperimmune gammaglobulin comprising (a) Immunizing an animal with the lipid A analog/immunogenic carrier of this invention and at least one Gram negative and/or Gram positive bacteria, or antigenic determinant thereof, optionally conjugated with an immunogenic carrier,
  • step (b) optionally boosting said animal with the immunizing step of step (a),
  • This invention further provides for a method of treating Gram negative bacterial sepsis comprising administering an effective neutralizing amount of a gammaglobulin specific to the lipid A/immunogenic carrier conjugate of this invention and an effective opsonizing amount of 0 antigen specific polyvalent hyperimmune gammaglobulin to an animal in need of such treatment.
  • Treatment can be prophylactic, or it can be to animals actually suffering from a bacterial infection.
  • an effective neutralizing amount is considered to be between 50 and 1000 mg/kg of gammaglobulin to body weight.
  • Preferably, between 100 and 500 mg/kg of gammaglobulin to body weight is to be administered.
  • opsonic activity a range of between 50 and 1000 mg/kg body weight is considered to be effective.
  • between 100 and 500 mg/kg of gammaglobulin to body weight may be administered.
  • kits for treating Gram negative bacterial sepsis comprising a carrier compartmentalized to receive in close confinement therein one or more container means wherein a first container contains a lipid A analog/immunogenic carrier of the invention.
  • the kit can also include at least one other container means which contains at least one Gram negative and/or Gram positive bacteria, or antigenic determinant thereof, conjugated with an immunogenic carrier.
  • the Gram negative and Gram positive bacteria may be killed or attenuated bacteria.
  • the preferred Gram negative and positive bacteria are those which are preferred in the polyvalent vaccine.
  • an additional container means may be present which contains an adjuvant.
  • This invention also provides for a kit wherein the first container contains gammaglobulins specific for the lipid A analog/immunogenic carrier of this invention.
  • a second container can be provided which contains gammaglobulins specific for said Gram negative or Gram positive bacteria, or antigenic determinant thereof.
  • the preferred Gram negative and positive bacteria are those which are preferred in the polyvalent vaccine.
  • Administration of the vaccines or hyperimmune gammaglobulin may be parenteral, intravenous, intramuscular, subcutaneous, intranasal, or any other suitable means. Preferably, administration is by subcutaneous or intramuscular means.
  • the dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered.
  • the conjugates may be administered at a dosage of 0.0001 to 25.0 mg/kg of conjugate per animal weight.
  • the initial dose may be followed up with a booster dosage after a period of 4 weeks to enhance the immunogenic response. Further booster doses every 4-6 weeks may be administered for as long as the risk of infection and septic shock exists.
  • the lipid A analog/immunogenic carrier conjugates and other antigenic conjugates of this invention useful in the method of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions.
  • Any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties for use in the method of the present invention.
  • the lipid A analog/immunogenic carrier conjugates are in lyophilized form or in a weakly acidic buffer solution.
  • the Gram negative and Gram positive conjugations are preferably stored in a weakly basic solution.
  • the vaccines may be in the form of single dose preparations or in multi-dose flasks which can be used for mass vaccination proGrams.
  • the vaccines of the invention comprising the lipid A analog/immunogenic carrier conjugates of the invention may further comprise adjuvants which enhance production of lipid A analog-specific antibodies.
  • adjuvants include, but are not limited to, various oil formulations such as Freund's complete adjuvant, the dipeptide known as MDP, saponin, aluminum hydroxide, Bordetella pertussis, dephosphorylated lipid A, or an interferon.
  • Freund's adjuvant is an emulsion of mineral oil and water which is mixed with the immunogenic substance. Although Freund's adjuvant is powerful, it is usually not administered to humans. Instead, the adjuvant alum (aluminum hydroxide) may be used for administration to a human. The conjugate may be absorbed onto the aluminum hydroxide from which it is slowly released after injection.
  • alum aluminum hydroxide
  • the lipid A analog/immunogenic peptide conjugate may also be encapsulated within liposomes according to Fullerton, U.S. Patent No. 4,235,877.
  • Gentiobiose octaaeetate obtained from Sigma Chemical Co. (St. Louis, MO), was converted into the acetobromo derivative by dissolution in dichloromethane (1 gm in 5 ml) and treatment with 33% HBr in glacial acetic acid (10 ml) (Fluka) at 5°C for 45 minutes. A volume of dichloromethane equal to the total volume of the reaction mixture was added and the mixture transferred to a separatory funnel. The organic phase was washed with an equal volume of saturated sodium bicarbonate solution (3-4 times), followed by two portions of water. The organic layer was then dried over MgSO 4 , filtered, and the solvent evaporated to give a syrup.
  • the acetobromogentiobiose was crystallized from dichloromethane and diethyl ether, filtered and dried in a vacuum desiccator. The crystalline compound gave a single spot on T.L.C. and the H-1 NMR spectrum was correct.
  • Acetobromogentiobiose (5 gm in 5 ml chloroform) was coupled to 2-aminoethanol (0.5 ml, approx. 0.5 gm, in 1 ml chloroform) in the presence of drierite (1 gm;
  • AGH aminoethyl gentiobiose heptaacetate
  • BSA bovine serum albumin
  • Dioxane was the solvent found capable of dissolving both the protein carriers and the AGH ligand.
  • Suc-BSA (10 mg) was coupled to AGH in 20% 1,4-dioxane
  • DT Diphtheria Toxoid
  • AGH obtained according to Example 1
  • 1,4-dioxane was coupled to AGH in 25% 1,4-dioxane and 0.05M NaCl in the presence of EDAC.
  • the resulting conjugate (DT-AGH) was dialyzed against water and freeze-dried. Analysis demonstrated that 16% of the conjugate consisted of AGH.
  • Example 3 Preparation of Gentiobiose Heptaacetate C1 Conjugate Vaccine (Succinyl-DT-APGH)
  • the resultant reaction mixture was then neutralized to pH 7.0 with 1.0 M HCl and was dialyzed against three changes of pyrogen free water (3 ⁇ 10 L) in 48 hours.
  • the dialyzed solution was distributed into 50 sterile bottles and lyophilized under sterile conditions.
  • Crystalline gentiobiose octaacetate (2.0 g, Sigma) is dissolved into dichloromethane (99+%, 5 ml), and to this solution is added 33% HBr in glacial acetic acid (8 ml) in a 100 ml Round Bottom Flask. The flask is placed on an ice/water bath for 45 min. While stirring the reaction mixture, 30 ml of dichloromethane was added, and the mixture was washed successively with cold water (30 ml), saturated sodium bicarbonate solution (2 ⁇ 40 ml), and cold water (40 ml), using a separating funnel.
  • the dichloromethane phase was dried with anhydrous sodium sulfate (2.0 g) and evaporated to dryness in a rotary evaporator at or below 40°C.
  • the dry product was dissolved into diethyl ether (5 ml) and allowed to crystallize for one h at room temperature and then left overnight at 5°C.
  • the crystalline compound was filtered, washed with cold ether and dried under vacuum in a dessieator at room temperature. The yield was 1.4 g (70%).
  • Crystalline acetobromo gentiobiose (1.0 g) was mixed with dry chloroform (5 ml, 99.9+%) and to this solution was added dry Drierite (1.0 g). The mixture was stirred, and 3-amino propanol (400 ⁇ l, 98%) was added. The reaction vial was wrapped with aluminum foil and stirred for 16 h at room temperature. The reaction mixture was filtered on a sintered glass funnel, and the filtrate was washed once with cold water (10 ml), and then the chloroform phase was dried with anhydrous sodium sulfate and filtered again.
  • the dry product was dissolved into ethanol (10 ml) that had been warmed to 50°C. The mixture was then allowed to slowly cool to room temperature for 2 h.
  • the aminopropyl gentiobiose heptaacetate (APGH) crystallized under these conditions.
  • the crystalline material was filtered, washed with cold ethanol (5 ml) and dried under vacuum in a desiccator at room temperature. The yield was 350 mg (36%), m.p. 164- 165°C.
  • the analysis calculated for C 29 H 42 O 18 N was: C, 50.25%; H, 6.06%; N, 2.02%. Found C, 49.50%; H, 6.12%; N, 1.98%.
  • the final compound was aminopropyl gentiobiose heptaacetate (APGH).
  • Aminopropyl gentiobiose heptaacetate (APGH, 100 mg) was dissolved into 1,4-dioxane (4.0 ml), and to this solution was added pyrogen free water (16 ml) to make 20% dioxane (20 ml). This solution was sterile filtered through 0.22 ⁇ m membrane and added to the solution of Suc.DT. Combined volume was 42.5 ml. The pH of this mixture was adjusted to 5.8 (autoclaved probe used) with sterile 0.1 N HCl.
  • the hapten content of this conjugate was determined by the phenol sulfuric acid method (Dubois et al . , Anal . Chem. 28:350 (1956)) and was found to be
  • Example 4 Binding of Gentiobiose-Protein Carrier Conjugates to LPS specific antibodies Although gentiobiose octaacetate is insoluble in water, the protein conjugates dissolve easily. These soluble conjugates were tested for their ability to bind anti-lipid A human monoclonal antibody (obtained by fusion of the heteromyeloma SHMA6(H4) with Epstein Barr virus (EBV) trinsformed lymphocytes from the spleen of an animal immunized with the J5 mutant of E.
  • EBV Epstein Barr virus
  • the alum used was a sterile suspension called "Rehsorptar” and was obtained from Armour
  • This preparation has 2% Al 2 O 3 (w/v) as specified by the manufacturer.
  • Rehsorptar suspension (104 ml) was diluted with sterile 0.9% NaCl (221 ml) to give 325 ml of diluted alum suspension.
  • the diluted alum suspension was distributed into sterile vials (5.0 ml in each) and was autoclaved. This formulation contains 6.4 mg Al 2 O 3 (w/v) or 3.38 mg of aluminum metal per ml of suspension.
  • the Suc.DT-APGH lipid A analog C1-conjugate vaccine has the following composition:
  • Each bottle of lyophilized vaccine will come with a bottle of diluted alum suspension (5 ml) that has been prepared with water for injection and a bottle of sterile 0.9% NaCl (5 ml) for reconstitution of the vaccine.
  • the lyophilized vaccine will be reconstituted with 4.0 ml of sterile 0.9% NaCl.
  • This solution will be mixed with 3.5 ml of diluted alum suspension to give a final volume of 7.5 ml of vaccine formulation.
  • This formulation will contain 200 ⁇ g of vaccine/ml and 1.57 mg of aluminum/ml.
  • the human dose will be 0.5 ml of this vaccine formulation. Therefore, one human dose will contain 100 ⁇ g vaccine and 785 ⁇ g of aluminum.
  • the aluminum to protein ratio will be 7.8:1.
  • the reconstituted vaccine will be used within 2 h. Any remaining vaccine in the bottle will be discarded.
  • the molar ratio of hapten to protein must be 4.0:1 or higher.
  • a 50 ⁇ g dose of the vaccine administered to rabbits with alum as adjuvant should give at least a 20 fold rise in titer against the hapten, after two immunizations four weeks apart, as determined by ELISA using the BSA conjugate of the hapten as the plate antigen.
  • the following five tables are concerned with the stability of the gentiobiose-toxoid conjugate.
  • the vaccine is stored at minus (-) 70°C. It is used within 2 hours of thawing and is disposed of after that time. It contains no preservative.
  • Table 2 clearly shows that the amount of free hapten which has dissociated from the toxoid is constant at about 5% over 35 months.
  • Table 3 evidence is presented for the maintenance of strong immunogenic potential.
  • Tables 4, 5, and 6 show that the succinylated but unconjugated toxoid does not induce an immune response to the hapten (Table 4), but that the lipid A conjugated vaccine is very immunogenic after 7 months of storage (Table 5).
  • Table 6 presents data from a dose response assay in rabbits. This experiment was performed after over 1 year storage at minus (-) 70°C, the required temperature for storage. The data show that the 1 meg dose is not effective; the 10 meg dose is more immunogenic; the 50 and the 100 meg doses, which include the dose to be used clinically, are very immunogenic.
  • the gentiobiose octaacetate protein/diphtheria toxoid conjugate was administered to rabbits according to the immunization schedule outlined in Table 7.
  • Antibody titers were then determined by ELISA.
  • Sera from rabbits 1 and 2 was then assayed for antibodies specific for a series of antigens.
  • Table 8 shows that the rabbit sera has reactivity with LPS and diphtheria toxin, but little reactivity with APGH.
  • TT-APGH tetanus toxoid-APGH conjugate
  • Antibody specificity for LPS on boiled J5 was then determined by specific adsorption experiments.
  • the sera of rabbits was diluted 1:500 and adsorbed twice with boiled J5 organisms at 10% volume of packed cells. The first adsorption was for 2 hours followed by a second overnight adsorption (both at 4°C). The J5 organisms were analyzed by GLC and found to be free of galactose.
  • the sera from the final bleeds of rabbits 1 and 5 were then assayed pre- and post-adsorption by ELISA using TT-APGH. The results are shown in Table 10.
  • Gentiobiose (4 gm, Sigma) was added slowly with stirring to pyridine (60 ml) and to the resulting suspension was added with stirring triphenylmethylchloride (4 gm, Aldrich). The mixture was stirred overnight at room temperature.
  • the aqueous solution was extracted with dichloromethane (50 ml) and the organic phase was washed with saturated NaHCO 3 solution, cold water, dried with anhydrous sodium sulfate and evaporated to dryness.
  • the solid product was crystallized from diethyl ether.
  • the pH was maintained between 5.7 and 6.0 with 0.1N
  • the vaccine was prepared for immunization studies by dissolving the lyophilized conjugate in sterile phosphate buffered saline to a final concentration of
  • A. Active Immunization Three rabbits were immunized twice with Suc-DT-APGH (see Example 3) in aluminum hydroxide gel (20:1, w/w) and subsequently received 4 injections of the antigen in saline. To test development of a localized Shwartzman reaction, rabbits each received an intracutaneous injection of 50 ⁇ g E. coli 06 LPS followed 23 hours later with 25 ⁇ g LPS in 0.1 ml saline, intravenously. Only 1 of 3 immunized rabbits showed a positive erythema, although no hemorrhagic reaction, whereas 3 of 3 non-immune rabbits were positive for erythema and 2 had hemorrhagic reactions. No necrosis was observed.
  • Test rabbits were injected intracutaneously with 100 ⁇ g E. coli 06 LPS in 0.2ml saline. After 21 hours, rabbits received 15 ml of the hyperimmune serum or normal serum intravenously (in the ear vein) and 2 hours later were challenged by intravenous injection of 20 ⁇ g LPS in 0.2 ml. In the control rabbits receiving normal serum, 4 of 5 showed hemorrhage and 1 had tissue necrosis. Only 1 of 5 rabbits receiving hyperimmune serum showed a positive hemorrhagic reaction and none had any signs of necrosis.
  • E. coli 018 is obtained and grown in Trypticase soy broth. The cells are washed by centrifugation.
  • Lipopolysaceharide (LPS) is prepared by phenol extraction from the cell paste according to a modification of the phenol-water method of Westphal et al . , Naturforscher 7: 148-155 (1952), and then sequentially treated with DNase, RNase, and pronase, followed by a second phenol extraction and ethanol precification dialysis and by lyophilization.
  • Toxin A is purified from the supernatant of a culture of Pseudomonaa aeruginoaa 103 as described by Shiloach, J. et al., in Yu, P.L., ed., Fermentation Technologiea: Induatrial Appliationa.
  • Example 9 The E. coli-Toxin A conjugate of Example 9 is prepared for immunization studies as in Example 7. The results will show that the vaccine induced ligand-specific antibodies in each rabbit that is immunized. To determine the protective efficacy of these antibodies, their ability to protect mice in a hog mucin challenge model is assayed as in Example 7. The results will indicate substantial opsonic activity.
  • Unencapsulated Klebaiella pneumoniae 01 is grown and prepared as in Example 9 and the LPS is recovered. Diphtheria Toxoid (DT) from SSVI (Bern, Switzerland) is coupled, to the LPS as in Example 9 to form a conjugate. The conjugate is then injected into 10-12 week old mice and antibodies specific to the conjugate are recovered as in Example 7.
  • DT Diphtheria Toxoid
  • the conjugate is also separated by electrophoresis using nitrocellulose paper and serum extracted from each mouse is tested for antibody binding according to the Western Blot method. Approximately 70% of the mice tested are found to have antibody specific for the conjugate.
  • Staphylococcua aureus type 5 and 8 are grown as described by Fattom, Ali et al . , Infection and Immuni ty 58: 2367-2374 (1990), the disclosure of which is incorporated by reference herein.
  • Capsular polysaccharide is separated and conjugated to Toxin A as in Example 7 to form Staphylococcus aureus-Toxin A Conjugate.
  • the conjugate is injected into mice and the antibody specific to the conjugate is recovered and immunoiogically tested as in Example 11. The results will indicate substantial opsonic activity.
  • the conjugates of Examples 6, 9 and 11-13 comprising 50 ⁇ g of each polysaccharide and lipid A analog, is injected into human volunteers. Approximately 4 to 6 weeks, later the volunteers are reinjected with 50 ⁇ g of polysaccharide (in each conjugate). Plasma is recovered from each volunteer and antibodies are screened by means of the passive hemagglutination test or by other appropriate means such as by ELISA. A volunteer generating a minimum antibody titer of 5 ⁇ g/ml is considered a responding donor. Plasma from responding donors is pooled and stored. This plasma will contain a relatively large concentration of antibody to the injected antigen and is appropriately called a hyperimmune serum.
  • the plasma is also screened for effective LPS neutralizing and bacteria opsonizing activity in the Swartzman and Hog mucin models, respectively.
  • Hyperimmune gammaglobulin preparations are subsequently made using the Conn fraction method. See Conn, E.J. et al . , J. Amer. Chem. Soc. 68:459 (1946). If necessary, donors are reinjected with the conjugates to achieve the minimum antibody level.
  • Blood is collected from patients exhibiting bacterimic symptoms. The blood is tested for presence of a high concentration of bacteria using the direct
  • Gram stain procedure Gram negative and Gram positive bacteria are counted.
  • the patients identified as suffering from bacteremia are then injected intramuscularly with 250 mg/kg of the hyperimmune gammaglobu-k'in per body weight of Example 14.
  • blood is collected from the injected patients and Gram negative and Gram positive bacteria are recounted. It will be found that after each interval of collection, the presence of both Gram negative and positive bacteria will be significantly decreased. In addition, the symptoms of septic shock will be reduced or eliminated.
  • PA ED-5 PA FD-7, PA INT-3, PA INT-4), Klebsiella
  • Figures 1-5 depict graphs showing the elution profile by gel filtration of the polysaccarides obtained from P. aeroginosa (FD-4, FD-7, INT-3 and INT-4), and Klebaiella (KPO-2), respectively.
  • Purified pseudomonas polysaccharides were derivatized by EDAC Reaction and purified klebsiella polysaccharides were derivatized by CNBR Reaction using the method described by Chu et al . , Inf. & Immun. 59 : 4450-4458 (Dec. 1991). According to this method, the purified pseudonomas polysaccharides (5.0 mg/ml of H 2 O) were brought to pH 11.0 with 1 N NaOH in a pH stat, and an equal weight of CNBr (1 g/ml of acetonitrile) was added. The pH was maintained at 11.0 with 1.0 N NaOH for 6 min at 4°C.
  • Kp01 dLPS conjugated to DT These materials were prepared according to Chu, Infect. Immun . 59:4450-4458 (1991) except for KpO1 dLPS, where detoxified LPS was employed.
  • HMW and LMW represent high and low molecular weight polysaccharide conjugates obtained by gel filtration (see Figure 11.
  • the detoxified LPS-DT conjugates were obtained according to the following procedure.
  • Table 15 represents the ELISA units obtained for animals immunized with the conjugates. The higher the number, the higher the immunogenicity. Table 15
  • mice immunized with HMW conjugate 5ug Ps/injection three time two weeks apart. The sera were screened and the high responders (20) were pooled and assigned 100 Elisa units and used on each plate as a reference. Results show:
  • Ps-DT conjugates are potent, regardless of their molecular size and heterogenicity.
  • O serotypes of E. coli are the most prevalent isolates from extra-testinal locations, mainly blood: 01, 02, 04, 06, 07, 08, 09, 011, 018, 022, 025, 075.
  • Step 1
  • mice per group were immunized with 2.5 ug PS alone or conjugated to DT. The animals were immunized once, twice or three times two weeks apart, and bled one week following the last immunization.
  • Reference sera were obtained by immunizing 10-20 animals with 5 ug/dose X3 two weeks apart. The sera from these mice were pooled and assigned 100 ELISA units.
  • Table 20 shows the results of a study to determine the extent of cross reactivity of E. coli O-specific PS (antibodies specific for each serotype were tested for crossreactivity with the other serotype).
  • Conjugates elicit serotype-specific antibodies.
  • a tetravalent vaccine comprising 4 E. coli PSA-Conjugates, compared to PS alone, were injected into mice.
  • the immunogenic response for each serotype was then determined (Table 21).
  • the PS alone are not immunogenic.
  • Conjugates are: (a) immunogenic; (b) demonstrate booster response; (c) demonstrate carrier priming.
  • Multivalent PS vaccine still not immunogenic.
  • conjugates demonstrated the same potency as in the case of monovalent vaccines.

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EP92908659A 1991-03-15 1992-03-16 Konjugate aus lipid a-analog und immunogenem träger, deren verwendung als impfstoffe und polyvalente hyperimmungammaglobuline Withdrawn EP0575517A1 (de)

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US5439808A (en) * 1993-07-23 1995-08-08 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US5747287A (en) * 1995-04-28 1998-05-05 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
EP1001805A1 (de) * 1997-06-18 2000-05-24 The Rockefeller University Verfahren zur identifizierung von antikörpern und peptiden für die behandlung von septischen schock und experimenteller arthritis und verwendungen
CN107531736B (zh) 2015-01-06 2022-04-15 免疫疫苗科技公司 脂质a模拟物、其制备方法和用途

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DE3516119A1 (de) * 1985-05-04 1986-11-06 Biotest Pharma GmbH, 6000 Frankfurt Polyvalentes hyperimmunglobulin-praeparat
US4693891A (en) * 1985-09-09 1987-09-15 Miles Laboratories, Inc. Vaccine for Pseudomonas aeruginosa
US4789544A (en) * 1986-05-23 1988-12-06 Midcon Labs. Inc. Co-vaccination using non-O-carbohydrate side-chain gram-negative bacteria preparation

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CA2105965A1 (en) 1992-09-16
JPH06508606A (ja) 1994-09-29
AU1570792A (en) 1992-10-21
WO1992016230A1 (en) 1992-10-01

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