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
  • A61K39/02—Bacterial antigens
  • A61K39/105—Delta proteobacteriales, e.g. Lawsonia; Epsilon proteobacteriales, e.g. campylobacter, helicobacter
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine

Definitions

• This invention relates to methods of inducing an immune response in an avian species against Campylobacter by administering, in ovo, live Campylobacter cells.
• Campylobacters typically grow and colonize in the avian gut environment. Colonization has been reported in chickens, ducks, pigeons, quail, ostriches and turkeys. Colonization of poultry does not result in disease but is commensal in nature.
• the use of a competitive exclusion culture to exclude Salmonella or Campylobacter from the digestive tract of a bird has been described in U.S. Patent No. 6,491 ,910. The efficacy of this method against Campylobacter appears variable.
• the present invention provides a method of inducing an immune response in an avian species against Campylobacter by administering, in ovo, live cells of Campylobacter.
• live cells of Campylobacter can be safely administered to any avian species for the purpose of inducing an immune response against Campylobacter, especially a domesticated avian species such as chicken, turkey, duck, goose and quail.
• Campylobacter species suitable for use in the present method include, but are not limited to, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, or a combination thereof.
• the cells of Campylobacter can be wild type cells or cells of Campylobacter that have been genetically modified to contain one or more mutations in the genome, or to contain a desirable heterologous sequence.
• the cells are combined with a veterinary-acceptable carrier prior to administration and are administered in an amount that is effective to induce an immune response in the avian species developed from the treated egg, preferably in an amount of at least about 5 x 10 5 , and more preferably, of at least about 1 x 10 6 live cells.
• the present invention provides a method of inducing an immune response in an avian species against Campylobacter by in ovo administration of live cells of Campylobacter.
• avian species or "bird” is meant to include any avian species, including a domestic or a game bird, e.g., chicken, turkey, duck, goose, or quail.
• live cells of Campylobacter are administered to eggs of a domesticated bird raised for commercial production of eggs or meat, such as a domesticated chicken, turkey, duck, goose and quail.
• the term "Campylobactei” refers to any Campylobacter species or any strain of a Campylobacter species, including Campylobacter jejuni, Campylobacter coli, Campylobacter lari.
• Campylobacter strains suitable for use in the present method include C. jejuni UA535 and C. jejuni 81 -176, as well as the following mutant strains: C. jejuni CsrA (mutated within gene CJ1103, a csrA homolog), C.
• the Campylobacter strain employed in the method of the present invention has been genetically engineered to contain a heterologous polynucleotide sequence.
• the heterologous sequence can be transferred into the Campylobacter strain via a plasmid, phage or cosmid vector by various means such as conjugation, electroporation, or transformation.
• Other methods such as transduction are also suitable, wherein the recombinant DNA in the form of a transducing phage or cosmid vector is packaged within a phage.
• the heterologous polynucleotide sequence can encode an antigen from an organism, such as a virus, bacterium or parasite, that causes disease in bird or that causes food-borne illness in humans upon consumption of a bird contaminated with the organism.
• Administration of live cells of genetically engineered Campylobacter containing such heterologous sequence can induce an immune response in the avian species against both Campylobacter and the pathogenic organism.
• heterologous polynucleotide sequence can also encode a protein essential in colonization of domesticated birds by Campylobacter. Proteins known to be essential in colonization of Campylobacter include, e.g., the gene products of dnaJ and cadF. Additionally, the heterologous polynucleotide sequence can encode a protein or peptide that stimulates the immune system of bird. Examples of proteins or peptides that stimulate the immune system of birds include, e.g., cholera toxin or E. coli beat labile toxin.
• the heterologous polynucleotide sequence can itself enhance the growth or feed efficiency of a domesticated bird, or can encode a protein or peptide that enhances the growth or feed efficiency of a domesticated bird.
• examples of such molecules or proteins include epidermal growth factor, insulin-like growth factor, interleukins, and antimicrobial peptides.
• a Campylobacter species is cultured by standard methods known to those skilled in the art and live cells of such Campylobacter are collected for in ovo administration to eggs of an avian species. Live cells of more than one Campylobacter species can be combined for in ovo administration.
• a veterinary-acceptable carrier can also administered.
• Campylobacter cells and a veterinary-acceptable carrier are both administered in ovo, either together or separately.
• a veterinary-acceptable carrier can be administered to the bird any time post-hatch in feed or water, or by aerosol spray.
• the live Campylobacter cells administered, whether with or without a veterinary- acceptable carrier are free of neutralizing factors, such as neutralizing antibodies or fragments of antibodies, as described in U.S. Patent 6,440,408.
• the term "a veterinary-acceptable carrier” includes solvents, dispersion media, coatings, adjuvants, stabilizing agents, diluents, preservatives, antifungal agents, isotonic agents, adsorption delaying agents, and the like.
• Diluents can include water, saline, dextrose, ethanol, glycerol, and the like.
• Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others.
• Stabilizers include albumin, among others.
• Adjuvants suitable for use in the present method include but are not limited to: mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin; glycosides, e.g., saponin derivatives such as Quil A or GPI-0100 (United States Patent No.
• cationic surfactants such as DDA, pluronic polyols; polyanions; non-ionic block polymers, e.g., Pluronic F-127 (B.A.S.F., USA); peptides; mineral oils, e.g. Montanide ISA-50 (Seppic, Paris, France), carbopol, Amphigen (Hydronics, Omaha, NE USA), Alhydrogel (Superfos Biosector, Frederikssund, Denmark) oil emulsions, e.g.
• an emulsion of mineral oil such as BayolF/Arlacel A and water, or an emulsion of vegetable oil, water and an emulsifier such as lecithin; alum, cholesterol, rmLT, cytokines and combinations thereof.
• the immunogenic component may also be incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation. Additional substances that can be included in a product for use in the present methods include, but are not limited to one or more preservatives such as disodium or tetrasodium salt of ethylenediaminetetracetic acid (EDTA), merthiolate, and the like.
• EDTA ethylenediaminetetracetic acid
• Immunostimulants which enhance the immune system's response to antigens may also be included in a product.
• suitable immunostimulants include cytokines such as IL-12 or lL-2, or stimulatory molecules such as muramyl dipeptide, aminoquinolones, lipopolysaccharide, and the like.
• the adjuvant can be combined with live cells of Campylobacter prior to in ovo administration, or can be administered independently any time after hatching by way of feed, water or aersol spray, provided to the bird.
• the live cells are administered at a dose effective to induce an immune response against Campylobacter in the avian species developed from the treated egg.
• the amount of live cells of Campylobacter that is effective to induce an immune response may vary, depending on the particular species or strains of Campylobacter used in the administration and the species of bird being immunized. Generally speaking, at least about 5 x 10 5 live cells should be administered to be effective in inducing an immune response. More preferably, at least about 1 x 10 6 live cells of Campylobacter are administered.
• an immune response is meant that the live cells of Campylobacter administered to an egg induce an immune response in the bird developed from the egg, which in turn provides some degree of protection to the bird against colonization of a Campylobacter species, which can be the same as, or different from, the Campylobacter species used in the in ovo administration.
• An immune response induced by in ovo administration of live cells of Campylobacter can be a cellular immune response mediated primarily by cytotoxic T-cells, or a humoral immune response mediated primarily by helper T-cells, which in turn activates B-cells leading to antibody production, or the combination of a cellular immune response and a humoral immune response.
• one or more additional immunogens can be included in the administration in ovo.
• immunogens include antigens derived from viruses, e.g. avian infectious bronchitis virus, avian infectious bursal disease virus, avian encephalomyelitis virus, egg drop syndrome virus, influenza virus, reovirus, adenovirus, hydropericardium syndrome virus, among others; antigens derived from bacteria, e.g., Haemophilus paragallinarum, Salmonella typhimurium, S. enteritidis, S. pullorl, S. gallinarum, S. choleraesuis, E.
• in ovo administration is meant administration to eggs of an avian species, preferably eggs in the fourth quarter of incubation. That is, for chicken eggs, the administration is conducted preferably on about the fifteenth to nineteenth day of incubation, and more preferably on about the eighteenth day of incubation.
• the administration is conducted preferably on about the twenty-first to twenty-sixth day of incubation, and more preferably on about the twenty-fifth day of incubation.
• the administration can be conducted by any method which results in the introduction of live cells of Campylobacter into an egg through the shell.
• a preferred method of administration is by injection.
• the injection can be made at any site of an egg, so long as the injection does not damage the tissues or organs of the embryo, or the extraembryonic membranes surrounding the embryo.
• the injection can be achieved by using any one of the well-known egg injection devices, such as a conventional hypodermic syringe fitted with a needle of about 18 to 22 gauge, or a high speed automated egg injection system as described in U.S. Patent No. 4,681 ,063, 4,040,388, 4,469,047, and 4,593,646.
• the present invention is further illustrated by the following non-limiting examples.
• EXAMPLE 1 Broiler chicken eggs were injected in ovo on day 18 of incubation with 10 6 colony forming units (CFU) of Campylobacter jejuni UA535. This is a wild type, unmodified strain of C. jejuni, originally isolated from a human clinical case of campylobacteriosis. A second group of eggs remained uninoculated until the day of hatch, at which time the chicks in this group were inoculated per os with 10 6 CFU C. jejuni UA535. At hatch, the number of live chicks and unhatched eggs in each treatment were recorded and all chicks were placed on litter with 3 replicate pens of twenty birds per treatment.
• CFU colony forming units
• the geometric mean antibody titer in response to in ovo inoculation was at least as great as, if not greater than, the titer resulting from peros inoculation at day of hatch. Therefore, not only can wild type C. jejuni UA535 be safely administered in ovo to broiler chicks, but administration by this route results in robust colonization and immune response.
• EXAMPLE 2 To demonstrate that colonization after in ovo inoculation with C. jejuni is not unique to strain UA535, colonization of birds by another human clinical isolate, C. jejuni 81 -176, was evaluated. Groups of broiler eggs were injected on Day 18 of embryo incubation with 1.8 x 10 5 or 1.4 x 10 7 CFU of C. jejuni 81-176. A third group of eggs remained uninoculated. At hatch, a group of chicks from the uninoculated eggs were retained as uninoculated control birds, and a second group of chicks from these eggs were given 8.3 x 10 6 CFU of C. jejuni 81 - 176 orally. The data set obtained demonstrated that in ovo administration of C. jejuni strain 81 -
• 176 is at least as effective at colonizing broiler chicks as oral administration on the day of hatch.
• EXAMPLE 3 On day 18 of embryo incubation, a group of broiler chicken eggs were inoculated in ovo with 7 x 10 7 CFU of C. jejuni CsrA. This strain has a mutation in CsrA, a protein involved in global regulation of carbon storage. A second group of eggs were retained as uninoculated controls. At hatch, a group of chicks from the uninoculated eggs were given 3 x 10 8 CFU of C. jejuni CsrA orally. Another group of chicks hatched from uninoculated eggs were retained as controls. The results indicated that the mutant C. jejuni strain colonized all of the birds when administered in ovo, yet colonized only 3 out of 6 birds when given orally. Also, the mutant was able to fully colonize birds and induce an antibody response when administered in ovo, but appeared to be less effective at colonizing birds when given by the oral route on the day of hatch.
• EXAMPLE 4 On day 18 of embryo incubation, groups of broiler chicken eggs were inoculated in ovo with 9 x 10 6 CFU of C. jejuni HspR, 8 x 10 7 CFU of C. jejuni HtrA, or 6 x 10 7 CFU of C. jejuni Dps.
• the HspR strain has a mutation in a heat shock protein; the HtrA strain has a mutation in a serine protease gene; and the Dps strain is mutated in a gene involved in iron acquisition.
• Another group of eggs was retained as uninoculated controls. At hatch, groups of chicks from the uninoculated eggs were given 4 x 10 6 CFU of C.
• jejuni HspR 1 x 10 8 CFU of C. jejuni HtrA, or 1 x 10 8 CFU of C. jejuni Ops orally.
• Another group of chicks hatched from uninoculated eggs was retained as controls.
• the HspR mutant was undetectable in samples from birds inoculated per os, but half the birds given this strain in ovo were colonized with C. jejuni. The results indicate that this mutant was able to colonize broiler chicks after in ovo administration, but not after oral administration on the day of hatch. The other mutants readily colonized birds and induced an immunological response regardless of route of administration.

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• 2004
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