WO2024086846A1 - Procédés et compositions pour immuniser contre campylobactor hepaticus - Google Patents

Procédés et compositions pour immuniser contre campylobactor hepaticus Download PDF

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WO2024086846A1
WO2024086846A1 PCT/US2023/077530 US2023077530W WO2024086846A1 WO 2024086846 A1 WO2024086846 A1 WO 2024086846A1 US 2023077530 W US2023077530 W US 2023077530W WO 2024086846 A1 WO2024086846 A1 WO 2024086846A1
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hepaticus
composition
vector
strain
chickens
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Subhashinie KARIYAWASAM
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University Of Florida Research Foundation, Incorporated
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

  • SLD Spotty Liver Disease
  • the disease is controlled by treating the affected flocks with antibiotics. Regardless of antibiotic therapy, the disease can occur multiple times in the same flock. Considering the negative consequences of antibiotic use, such as the emergence of antibiotic resistant bacteria, consumer perceptions, and the economic impact on antibiotic-free management practices, alternative approaches for SLD control are necessary.
  • C. hepaticus is a slow-growing, fastidious bacterium, which does not grow in the media that support the growth of other Campylobacter species. Therefore, growing C. Attorney Docket No. 10457-543PC0 hepaticus in large volumes for live (e.g., autogenous) or killed vaccine formulations can be difficult and prohibitive. Nevertheless, the genomes of C. hepaticus, C. jejuni, and C. coli are closely related, suggesting a possible zoonotic potential of C. hepaticus, though it is only speculative at this stage. This will further limit the use of live vaccines. It has been determined that these limitations can be overcome by using subunit vaccines containing protective antigen/s of C.
  • a live-vectored vaccine carrying and expressing C. hepaticus antigens has been developed to vaccinate commercial layer chickens against SLD.
  • Suitable immunogenic mimotopes have been identified, which can be used to develop an APEC-vectored C. hepaticus vaccine for commercial layer use.
  • Such vectored vaccine will protect commercial layer chickens not only from SLD but also from E. coli egg peritonitis, another economically important disease to the table egg industry.
  • Figure 1 A C. hepaticus UF2019SK1 colonies on sheep blood agar.
  • Figure 1 B Characteristic multiple greyish white or cream 1 -2 mm foci of necrosis lesions of the liver of a chicken that died of C. hepaticus-'mduceti SLD.
  • FIG. 2 Detection of C. hepaticus DNA in the liver (lanes 1 and 2) and cecum (lanes 3 and 4) of an SLD affected hen using conventional PCR.
  • M denotes the 100 bp ladder.
  • Both 463 bp and 308 bp amplicons correspond to two specific regions of glycerol kinase (G) gene of C. hepaticus (40).
  • Figure 3A Gel electrophoresis of the N-lauroylsarcosine-derived OMP fractions. Molecular masses of marker proteins (lane M), OMP from C. hepaticus strain UF2019SK1 , C. hepaticus strain HV10 - NCTC 13823 (lane 2). Proteins were stained with Coomassie blue. . , as it has been proven to simulate natural infection, in terms of age at infection, clinical manifestations, gross and macroscopic lesions, and bacterial recovery from organs subsequent to infection. Attorney Docket No. 10457-543PC0
  • adjuvant means a pharmacological or immunological agent that modifies the effect of other agents, such as a drug or immunogenic composition.
  • adjuvants are often included in immunogenic compositions to enhance the recipient's immune response to a supplied antigen. See below for a further description of adjuvants.
  • antibody or “antibodies”, as used herein, mean an immunoglobulin molecule able to bind to an antigen by means of recognition of an epitope.
  • Immunoglobulins are serum proteins composed of “light” and “heavy” polypeptide chains, which have “constant” and “variable” regions, and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.
  • An antibody that is “specific” for a given antigen indicates that the variable regions of the antibody recognize and bind a particular antigen exclusively.
  • Antibodies can be a polyclonal mixture, or monoclonal. They can be intact immunoglobulins derived from natural or recombinant sources, or can be immunoreactive portions of intact immunoglobulins.
  • Antibodies can exist in a variety of forms, including Fv, Fab', F(ab')2, Fc, as well as single chain.
  • An antibody can be converted to an antigen-binding protein, which includes, but is not limited to, antibody fragments.
  • antigen binding protein As used herein, the term “antigen binding protein”, “antibody” and the like, which may be used interchangeably, refer to a polypeptide or polypeptides, or fragment(s) thereof, comprising an antigen binding site.
  • the term “antigen binding protein” or “antibody” preferably refers to monoclonal antibodies and fragments thereof, and immunologic-binding equivalents thereof that can bind to a particular protein and fragments thereof.
  • antibody and “antigen binding protein” also includes antibody fragments, unless otherwise stated.
  • Exemplary antibody fragments include Fab, Fab', F(ab')2, Fv, scFv, Fd, dAb, diabodies, their antigen-recognizing fragments, small modular immunopharmaceuticals (SMIPs) nanobodies and the like, all recognized by one of skill in the art to be an antigen binding protein or antibody fragment, and any of above-mentioned fragments and their chemically or genetically manipulated counterparts, as well as other antibody fragments Attorney Docket No.
  • Antibodies and antigen binding proteins can be made, for example, via traditional hybridoma techniques (Kohler et al., Nature 256:495-499 (1975)), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display techniques using antibody libraries (Clackson et al., Nature 352:624-628 (1991 ); Marks et al., J. Mol. Biol. 222:581 -597 (1991 )).
  • Antibodies A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988 as well as other techniques that are well known to those skilled in the art.
  • Antigen means a molecule that contains one or more epitopes (linear, conformational or both), that upon exposure to a subject, will induce an immune response that is specific for that antigen.
  • An epitope is the specific site of the antigen which binds to a T-cell receptor or specific B-cell antibody, and typically comprises about 3 to about 20 amino acid residues.
  • the term “antigen” can also refer to subunit antigens — antigens separate and discrete from a whole organism with which the antigen is associated in nature — as well as killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes.
  • antigen also refers to antibodies, such as anti-idiotype antibodies or fragments thereof, and to synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).
  • the term “antigen” also refers to an oligonucleotide or polynucleotide that expresses an antigen or antigenic determinant in vivo, such as in DNA immunization applications.
  • An “antigen”, as used herein, is a molecule or a portion of a molecule capable of being specifically bound by an antibody or antigen binding protein. In particular, an antibody, or antigen binding protein, will bind to epitopes of the antigen.
  • An epitope refers to the antigenic determinant recognized by the hypervariable region, or Complementarity Determining Region (CDR), of the variable region of an antibody or antigen binding to protein.
  • CDR Complementarity Determining Region
  • administering a vaccine mucosally includes both directly administering the vaccine to the avian, such as by directly administering the vaccine to the avian's mouth, eye, harderian gland, choanal cleft or anus, and providing the Attorney Docket No. 10457-543PC0 vaccine such that the avian ingests the vaccine, such as providing a vaccine for the birds to eat, drink or peck off of each other.
  • Exemplary methods of providing the vaccine include, but are not limited to, oral administration or spraying the vaccine on the birds, and/or otherwise applying the vaccine to the feathers, such that the vaccine is ingested as the birds peck at each other's feathers, and/or preen themselves, or providing the vaccine in a form that the birds will ingest, such as in a gel that birds will peck at, or a liquid that the birds will drink.
  • spraying may also facilitate direct administration because spray droplets may directly enter the mouth, choanal cleft and/or eye of a bird.
  • Spraying may be performed by any suitable technique, such as a backpack sprayer or a spray cabinet. Typically, a suitable spraying technique will spray the composition onto the upper exposed surfaces of the bird as possible, such as the bird's back, head, face, tail feathers, etc.
  • Attenuated refers to a strain of a microorganism whose pathogenicity has been reduced so that it will generally initiate an immune response but without producing disease.
  • An attenuated strain is less virulent than the parental strain from which it was derived.
  • Attenuated microorganisms can be screened in vitro or in vivo to confirm that they are less pathogenic than its parental strain.
  • Conventional means are used to introduce attenuating mutations, such as in vitro passaging, as well as chemical mutagenesis.
  • An alternative means of attenuating comprises making pre-determined mutations using site-directed mutagenesis, where one or more mutations may be introduced.
  • ''Infection or “challenge” means that the subject has been exposed to live disease-causing organisms that may result in the subject exhibiting one or more clinical signs of the disease.
  • immunogenic composition means a composition that generates an effective immune response (i.e. , has effective and/or at least partially protecting immunogenic activity) when administered alone, or with a pharmaceutically-acceptable carrier, to an animal.
  • the immune response 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 activate B-cells, Attorney Docket No. 10457-543PC0 leading to antibody production.
  • specific T-lymphocytes or antibodies can be generated to allow for the future protection of an immunized host.
  • mutant means an individual or organism arising or resulting from an instance of mutation, which is a base-pair sequence change within the nucleic acid or chromosome of an organism, and results in the creation of a new character or trait not found in the wild-type individual or organism.
  • prevent means to inhibit the replication of a microorganism, to inhibit transmission of a microorganism, or to inhibit a microorganism from establishing itself in its host. These terms, and the like, can also mean to inhibit or block one or more signs or symptoms of infection.
  • avian and “avian subjects” or “bird” and “bird subjects” as used herein are intended to include males and females of any avian or bird species, and in particular are intended to encompass poultry which are commercially raised for eggs, meat or as pets. Accordingly, the terms “avian” and “avian subject” or “bird” and “bird subject” encompass chickens, turkeys, ducks, geese, quail, pheasant, parakeets, parrots, cockatoos, cockatiels, ostriches, emus and the like. In particular embodiments, the subject is a chicken or a turkey. Commercial poultry includes broilers and layers, which are raised for meat and egg production, respectively.
  • the avian to be innoculated can be an in ovo, live fetus or embryo or may be a hatched bird, including newly- hatched (i.e., about the first one, two or three days after hatch), adolescent, and adult birds.
  • terapéuticaally effective amount means an amount of an active ingredient, e.g., an agent according to the invention, with or without an adjuvant, as appropriate under the circumstances, provided in a single or multiple doses as appropriate, sufficient to effect beneficial or desired results when administered to a subject or patient.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a composition according to the invention may be readily determined Attorney Docket No. 10457-543PC0 by one of ordinary skill in the art, and provides a measurable benefit to a patient, such as protecting the animal from subsequent challenge with a similar pathogen.
  • a “therapeutic” agent of the invention may act in a manner, or a treatment may result in an effect, that is prophylactic or preventive, including those that incorporate procedures designed to target animals that can be identified as being at risk (pharmacogenetics); or in a manner that is ameliorative or curative in nature; or may act to slow the rate or extent of the progression of at least one symptom of a disease or disorder being treated.
  • compositions comprising one or more peptide antigens of SEQ ID NOs 1 -16, or a vector for expressing the one or more peptides, in vivo, and, optionally, at least one adjuvant, where the composition is formulated for administration to an avian.
  • the composition may further comprise a vehicle, surfactant, inactivating agent, neutralizing agent, cell fragments, or a combination thereof.
  • the inactivating agent may be formaldehyde, formalin, binary ethyleneimine, thimerosal, beta propiolactone, a detergent, or a combination thereof.
  • the composition does not comprise saline solution.
  • the composition may have an osmolarity of from greater than zero to 2% (w/v) sodium chloride solution, and/or may have a substantially isotonic osmolarity. Additionally, or alternatively, the composition may have a viscosity of from greater than zero to 6 mPa-s, such as from 2 mPa-s to 5 mPa-s. And/or in some embodiments, the composition is formulated as a liquid or a suspension of particles within an aqueous base, but in other embodiments, the composition may be formulated as a gel.
  • the gel may comprise a gelling agent, such as carboxymethyl cellulose, carboxymethyl chitosan, chitosan, sodium hyaluronate, polyethylene glycol, xantham gum, starches, pectins, gelatin, polysaccharides and oligopolysaccharides, carrageenan, derivatives Attorney Docket No. 10457-543PC0 and combinations thereof.
  • the composition may include added immunostimulants, such as a saponin, Quil A, dimethyldioctadecyl ammonium bromide, dimethyldioctadecyl ammonium chloride, poloxamer, polyethylene maleic anhydride, or a combination thereof.
  • the adjuvant may have an adjuvant concentration in the composition of from greater than zero to 80% (v/v) , such as from 5% to 80%, or from greater than zero to 50%, from 0.5% to 50%, from 1% to 50% or from 5% to 50%.
  • the composition is formulated for addition to drinking water and the adjuvant concentration is from greater than zero to less than 100%, such as from 15% to 80%.
  • the composition may be formulated for spray administration and have an adjuvant concentration of from greater than zero to 80%, such as from 0.5% to 80%, from 0.5% to 50%, from 1 % to 50%, from 5% to 50% or from 15% to 40%.
  • the composition may be formulated for administration to the eye and the adjuvant concentration may be from 5% to 25%.
  • the composition is formulated for gel administration and the adjuvant concentration is from 15% to 40% prior to mixing with a gel composition, such as a composition comprising a gelling agent.
  • a drinking water composition comprising water and from greater than zero to less than 100% (v/v), such as from greater than zero to less than 80% (v/v), or from greater than zero to 50% (v/v) of the disclosed composition
  • a gel composition comprising a first composition comprising the disclosed composition, and a second composition comprising a gelling agent.
  • the gel composition may have a ratio between the first composition to the second composition of from 25:75 to 75:25, such as 50:50.
  • Additional embodiments concern a method comprising administering a composition according to any of the disclosed embodiments to an avian.
  • the method may comprise administering to an avian mucosal membrane.
  • the avian is a chicken, turkey, goose, duck, Cornish game hen, quail, partridge, pheasant, guinea-fowl, ostrich, emu, swan, or pigeon, preferably a chicken or a turkey.
  • the composition may be delivered by any suitable route, such as orally, parenterally, Attorney Docket No. 10457-543PC0 ocularly or topically. Application may be made by spraying, misting, eye dropper, drinking water, feed and combinations thereof.
  • Administering the composition may comprise a first administration of the composition followed by a second administration subsequent to the first administration, or there may be just a single application or multiple re-administrations in long-lived avians.
  • the first administration may occur when the avian is from greater than zero to 14 days of age.
  • the second administration is from greater than zero to 6 weeks after the first administration, such as from 1 day to 4 weeks after the first administration, or from 3 days to 10 days after the first administration.
  • the method may comprise administering a composition comprising on or more peptides of SEQ ID NOs 1 -16.
  • administering to the avian may comprise spraying the composition onto the avian and/or administering the composition to the avian's eye.
  • administering to the avian may comprise providing to the avian a drinking water composition or a gel composition that comprises the composition.
  • the drinking water composition may comprise from greater than zero to less than 100% (v/v) of the composition, such as from 80% to less than 100%, or 70% to less than 100%.
  • the method further comprises mixing the composition and water to form the drinking water composition. Also, the method
  • a method of inducing an immune response in an avian species is also disclosed.
  • the method may comprise administrating to the avian a composition according to any of the disclosed embodiments.
  • Inducing an immune response may comprise inducing an IgA response and optionally both IgA and IgY responses.
  • a method of treating or preventing SLD in an avian where the method comprises administering to the avian a composition according to any of the disclosed embodiments, wherein the composition comprises one or more peptides of SEQ ID NOs 1 -16, or a vector for expressing one or more of SEQ ID NOs 1 -16 in vivo.
  • a method comprising mucosally administering a composition described herein and at least one mucosal adjuvant to an avian.
  • Administering the composition may comprise spraying the composition on to the avian, Attorney Docket No. 10457-543PC0 and/or may comprise administering the composition ocularly, nasally and/or orally.
  • the composition may comprise from 80% to less than 100% water and/or comprise an aqueous-based adjuvant.
  • the composition comprises from 0.5% to 50% adjuvant, such as from 1 % to 50% adjuvant or from 5% to 50% adjuvant, and/or the adjuvant may be a polyacrylic acid adjuvant.
  • the composition is a suspension, and may be an aqueous suspension comprising polyacrylic acid particles, such as polyacrylic acid particles having particle size of from 250 nm to 10 microns.
  • the composition is administered mucosally, such as orally, topically or ocularly, to an avian.
  • avians include, but are not limited to, chickens, including laying hens, breeders and broilers, turkey, goose, duck, Cornish game hen, quail, partridge, pheasant, guinea-fowl, ostrich, emu, swan, or pigeon.
  • the avian is a chicken or turkey.
  • the composition may be administered to an avian of any age.
  • the initial administration is to avians of from day of hatching to 14 days of age, such as from day of hatching to 10 days, from day of hatching to 5 days, or from day of hatching to 3 days.
  • the composition may be administered at day 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in some embodiments, the composition is administered at the day of hatching, for example, by spraying. It may also be administered up to within 7 days of processing the carcass. In addition, it may be administered daily in the drinking water throughout the life of the avian.
  • Administering the composition mucosally to the avian typically results in an immune response, such as an IgA immune response and/or an IgY response.
  • the immune response may help to overcome maternal antibodies that the avian may receive from a hen that is positive for a bacteria or virus. Such maternal antibodies may be passed to the embryo in ovo. If the avian receives maternal antibodies while in ovo and then receives a parenteral vaccination after hatching, the maternal antibodies often block the subject from developing its own protective antibodies.
  • the method of mucosal vaccination such as the administration routes described herein, may produce an IgA Attorney Docket No. 10457-543PC0 immune response that can help overcome the effect of the maternal antibodies transferred to the bird.
  • Immunogenic compositions for mucosal administration have several advantages over compositions that are administered by other routes, such as the intramuscular or subcutaneous routes.
  • the advantages include, but are not limited to: 1 ) protecting the juvenile birds using humane techniques; 2) not exposing juvenile birds to stressful needle injections; 3) not involving injecting birds with live or modified live organisms that can shed and spread disease; 4) allowing the juvenile birds to develop IgA antibodies that can overcome the effect of any maternal IgY antibodies transferred from the parent; 5) not leaving any injection site lesions and thus allowing a zero day withdrawal time; 6) being easier to administer and reducing the workload; 7) reducing or substantially eliminating the risk of accidental selfinjection of the worker; and/or 8) being able to be administered in the face of an outbreak to stop disease spread.
  • Other advantages may be apparent from the description and the example section below.
  • the disclosed composition is formulated for mucosal administration to avians.
  • Mucosal administration includes, but is not limited to, oral, ocular, nasal, topical, and/or anal administration.
  • the composition is delivered ocularly, such as by eye drops or by spray.
  • the composition is administered orally, and may be administered by spraying the bird or providing a gel comprising the composition such that the birds peck at and thereby eat the gel/composition mixture.
  • the gel is administered topically to the back of the birds, such that other birds peck at the gel spot.
  • the gel may be colored to attract pecking, such as with a red or blue color.
  • the adjuvanted immunogenic composition is mixed with a gelling composition comprising a gelling agent in a ratio suitable to provide an effective amount of the adjuvanted immunogenic composition to the bird(s).
  • the ratio may be from 25:75 (immunogenic compositiomgelling composition) to 75:25, such as from 34:66 to 66:34, or from 40:60 to 60:40, and in some embodiments, the ratio is 50:50.
  • the composition may be provided as a liquid or suspension for the birds to ingest.
  • the liquid or suspension may be provided as a drinking liquid, or it may be sprayed as a liquid or suspension onto the birds.
  • spraying the birds may administer the composition ocularly, nasally, topically, and/or orally, either directly to the eye, nasal cavity, chloanal crest, and/or mouth, or indirectly, such as by birds ingesting the composition as they peck at each other and/or preen their feathers.
  • spraying the composition administers the composition ocularly, nasally and optionally orally.
  • a dose of the composition suitable for mucosal administration is from 0.15 mL or less per bird to 0.35 mL or more per bird, such as from 0.2 mL per bird to 0.3 mL per bird, or about 0.25 mL per bird.
  • the composition may be administered in an amount of from 15 mL or more/100 birds to 35 mL or more/100 birds, such as from 20 mL/100 birds to 30 mL/100 birds, or about 25 mL/100 birds.
  • each bird theoretically receives about 80% of a dose, such as a 0.25 mL dose, of the composition.
  • the disclosed composition are able to induce protective levels of antibodies as measured by the ELISA in >50%, typically >80%, >85%, >90%, or >95% of the individuals that are administered the composition.
  • the mucoadhesive composition also is able to maintain protective levels of antibodies against strains of bacteria and/or viruses, such as Clostridium perfringens type A, Salmonella Kentucky, Salmonella typhimurium, Salmonella enteriditis and/or E. coli, throughout the early growth phase of an avian after hatching.
  • the composition can produce a persistent immune response against C. hepaticus in avians.
  • a "persistent immune response” refers to a protective antibody immune response which is capable of protecting avians throughout their growth period from hatching to adult.
  • Example 1 Attorney Docket No. 10457-543PC0
  • C. hepaticus isolation and identification protocols In our laboratory, we have established the protocols for C. hepaticus isolation and identification from various specimen types, including bile, liver, small intestines, cecum, and cloacal swabs. We have modified the published bacterial culture protocols to optimize bacterial recovery ( Figure 1 A; Page 4). Molecular identification protocols also have been refined in the laboratory. We are providing C. hepaticus isolation and detection service to Florida commercial egg producers, as needed. Figure 1A shows C. hepaticus strain UF2019SK1 grown on sheep blood agar (Remel, Lenexa, KS) under microaerophilic conditions at 37°C for 3 days following pre-enrichment in modified Preston broth under the same conditions. C.
  • C. hepaticus colonies appear as nonhemolytic, thinly spreading, irregular, flat films.
  • C. hepaticus can be grown on nonselective agar, such as sheep blood agar or Brucella agar with 5% horse blood (HBA).
  • C. hepaticus strain UF2019SK1 was isolated from the liver ( Figure 1 B; Page 4) of a hen with SLD. Affected liver showed the presence of a multifocal hepatitis characterized by small white necrotic foci on the surface.
  • C. hepaticus PCR ⁇ Ne have also optimized the protocols for the detection of C. hepaticus DNA from various specimen types (bile, liver, small intestines, cecum, and cloaca) ( Figure 2; Page 4). Recently, we developed a real-time PCR for the quantification of C. hepaticus from tissue specimens using primers and a Taqman probe targeting the same glycerol kinase gene.
  • C. hepaticus genome Three strains of C. hepaticus (named UF2019SK1 , UF2019SK2, andUF2019SK3) have been sequenced in the Pi’s laboratory. The sequence of UF2019SK1 has already been published (1 ) (GenBank accession # i c
  • APEC vector strain The vector strain of APEC (PSUO78) has been sequenced and already deposited in the GenBank under the accession number CP0121 12.1 . This APEC was isolated from the oviduct of a hen having E. coll-induced salpingitis/peritonitis ( Figure 4). We have already constructed the aroA-deleted mutant strain of PSUO78 (named PSUO78.1 ) using the phage Red recombinase system as described previously.
  • Bacterial vaccine strains delivering heterologous antigens The Curtiss lab has been working on vaccine development and bacterial heterologous antigen delivery Attorney Docket No. 10457-543PC0 systems for many decades. Similar approaches are implemented for the construction of APEC-vectored C. hepaticus mimotope expressing vaccine as explained in the Examples below.
  • the Asd+ recombinant expression plasmid pYA4515 is used as the vector for C. hepaticus antigen. It is an improved version of pYA3493 (25) ( Figure 5; Page 6) containing a modified beta-lactamase signal sequence (bla SS) as a type 2 secretion system (T2SS).
  • the promotor Ptrc directs the constitutive expression of the cloned recombinant antigen and asd encodes diaminopimelic acid (DAP), which is an essential component of the cell wall peptidoglycan.
  • DAP diaminopimelic acid
  • the signal peptide targets the protein for secretion by the T2SS pathway into the periplasm, with subsequent release into the culture supernatant.
  • the Curtiss lab has used this strategy to deliver vaccine antigens of various pathogens via live oral recombinant attenuated Salmonella enterica serovar Typhimurium lacking asd.
  • DAP is an essential component of the cell wall peptidoglycan, and, therefore the survival of the mutant Salmonella totally depends on exogenously supplied DAP (in this case Asd expressing pYA4515 plasmid). This ensures the stable maintenance of the Asd-i- plasmid used to express and deliver the antigen via attenuated Salmonella not producing DAP.
  • DAP exogenously supplied DAP
  • Asd+ plasmid vector in trans complementation
  • Glycosylation of C. hepaticus antigens A pgl operon from Campylobacter is introduced into the chromosome of the APEC vector by replacing the chromosomal cysG gene with the pgl operon. See Figure 6 for genomic map and location of insertion site. This strategy is implemented to deliver glycosylated antigens of C. hepaticus by both the peptide-expressing E. coli GI826 and APEC vector.
  • Campylobacter hepaticus strain FL2019SK1 isolated from the liver of a layer chicken affected with SLD is used to Attorney Docket No. 10457-543PC0 generate hyperimmune sera, to prepare the heat-killed vaccine, and in challenge experiments.
  • the genome of FL2019SK1 has already been sequenced by our laboratory and the annotation and the assembly of the genome is currently in progress.
  • the Ph.D.-12 Phage Display Peptide Library Kit (New England BioLabs Inc., Ipswich, MA) and the host strain Escherichia coli ER2537 are used for the phage library (New England BioLabs).
  • E. coli GI826 (Invitrogen, Carlsbad, CA) will be used to express recombinant epitopes of C. hepaticus. Strain maintenance, phage tittering, and storage will be carried out according to manufacturer instructions.
  • the sequencing primer and horseradish peroxidase (HRP)-labeled anti-M13 pill monoclonal antibody will also from New England BioLabs.
  • Chickens Specific pathogen-free Leghorn female chickens purchased from Charles River Laboratories (Wilmington, MA) are used to raise antibodies.
  • Commercial Hy-Line layer chickens obtained from a known SLD-free flock with no history of past SLD will be used in vaccine immunogenicity and protection studies. The SLD-free status of Hy-Line chickens will be confirmed by testing cloacal swabs for C. hepaticus DNA prior to purchase. Age of the chickens at purchase will depend on the experiment.
  • Chickens will be fed an antibiotic-free corn- based starter diet or a wheat/barley-based grower's diet (Purina Mills, St. Louis, MO). Feed and water will be provided ad libitum. All chicken experiments will be performed according to the protocols approved by the Institutional Animal Care and Use Committee of University of Florida (IACUC).
  • IACUC Institutional Animal Care and Use Committee of University of Florida
  • the polyclonal antibody is purified according the method described previously (3). Briefly, the chicken serum is mixed with caprylic acid (2.5%, Attorney Docket No. 10457-543PC0 v/v), precipitated with ammonium sulfate and dialyzed against PBS overnight. The purity of the antibody is assessed by running the purified antibodies on an SDS- polyacrylamide gel (SDS-PAGE) gel. The antibody titers are determined in an ELISA after coating microtiter plates with the whole cell C. hepaticus FL2019SK1 .
  • Example 2 Identify immunogenic mimotopes of C. hepaticus as a vaccine candidate antigen using a phage display library and reverse vaccinology strategy
  • phage display panning: For the identification of peptide mimotopes of C. hepaticus FL2019SK1 , the Ph.D.-12 Phage Display Peptide Library purchased from New England BioLabs (Ipswich, MA) are screened using polyclonal antibodies raised against C. hepaticus FL2019SK1. This phage display peptide library contains linear 12-mer peptides fused to the outer minor phage coat protein (pill) of M13 phage. Library screening is performed according to manufacturer instructions.
  • Purified polyclonal antibodies are added to microtiter plates (1.0 mg/well) and incubated overnight at 4°C. Unbound antibodies are removed by washing with PBST (PBS plus 0.1%, v/v; Tween-20), blocking the wells with PBS containing 3% bovine serum albumin (BSA) and incubating at 37°C for 1 h. The selected phage clones (1 x10 9 virions/well, in triplicate) are added and incubated for 1 h at 37°C. Plates are washed with PBST five times and peroxidase- conjugated anti-phage M13 antibody diluted in PBS 1 :5000 is added and incubated for 1 h at 37°C.
  • PBST PBS plus 0.1%, v/v; Tween-20
  • BSA bovine serum albumin
  • TMB 3, 3D ,5,5D-tetramethyl benzidine dihydrochloride
  • OD optical density
  • hepaticus antibody-binding phage Each assay will be carried out in triplicate. The best clones will be selected for more detailed testing with another round of competitive ELISA but this time using serial dilutions of sonicated whole cell C. hepaticus. All clones are tested in triplicates.
  • DNA sequencing DNA templates of the positive clones confirmed in the competition- inhibition ELISA will be prepared according to the manual of the Ph.D.-12 phage display peptide library kit and sequenced with the sequencing primer provided in Attorney Docket No. 10457-543PC0 the kit.
  • the DNA sequences are translated into peptide sequences with the ExPaSy Translate tool software (www.expasy.ch/tools/dna.html).
  • the DNA sequences and the peptides are compared with our sequenced strain of C. hepaticus FL2019SK1.
  • the peptides are searched by BLASTP and PHI-BLAST in the National Center for Biotechnology Information C. hepaticus-encoded ORF database. Proteins with high similarity to each mimotope are subjected for subcellular localization prediction for further selection.
  • CLC Genomics Workbench (v20), Geneious software, and the resources from Basic Local Alignment Search Tool (BLAST; blast.ncbi.nlm.nih.gov/Blast.cgi) to perform these analyses.
  • BLAST Basic Local Alignment Search Tool
  • Epitopes corresponding to proteins with more than one TMH are discarded because they are unlikely to be transported beyond the bacterial inner membrane.
  • Proteins that will be identified as being located on the cell surface and showing high similarity to the mimotopes (> five continuous amino acid residues) or lower similarity to Attorney Docket No. 10457-543PC0 the mimotopes (> three but ⁇ five continuous amino acid residues) are evaluated for surface probability using Protean software, with corresponding amino acids showing the characteristics of functional epitopes determined based on the surface probability plot (DNASTAR, Madison, Wl, USA).
  • Peptide synthesis The epitopes of C. hepaticus for synthesis is selected based on strong, reproducible and specific reaction of phage clones with anti-C. hepaticus antibodies in ELISA, prediction of peptide hydrophilicity and subcellular localization of the corresponding protein, and common motive groups in the sequences.
  • the 12-mer linear peptides will be synthesized at >98% purity at GenScript (Piscataway, NJ).
  • GenScript Procataway, NJ
  • the C-terminus is elongated with a GGGS-CONH2 tail to mimic the GGGS-peptide spacer between the random peptide sequence and the phage protein pill and to block the negative charge of the carboxyl terminus. All synthetic peptides will be reconstituted in sterile deionized water.
  • E. coli GI826Gly to aid glycosylation of C. hepaticus mimotopes: E. coli GI826 is constructed for the expression of glycosylated mimotopes of C. hepaticus using a suicide vector carrying the pgl operon of C. jejuni or C. hepaticus (pgl operon of C. jejuni is essentially identical to that of C. hepaticus). Specifically, the pgl operon in a suicide vector is introduced into the cysG gene of E. coli via allelic exchange. This strain designated GI826Gly is used along with the E. coli GI826 in immunogenicity and efficacy assays.
  • the two complementary oligodeoxynucleotides coding for the positive epitopes identified above are synthesized with Apal and Xhol overhangs at the 5’- and 3’-ends, respectively (Invitrogen) and annealed to generate synthetic DNA fragments.
  • the synthetic DNA fragments are subcloned into the Apal and Xhol cloning sites of the pFlitrx plasmid and the resultant recombinant plasmid will be transferred into the E. coli GI826 (Invitrogen) and GI826Gly by electroporation.
  • the pFliTrx vector allows the display of peptides on the surface of E. coli b using the major bacterial flagellar protein (FliC) and thioredoxin (TrxA).
  • the transconjugants are selected on RM medium (1 X M9 Salts, 2% casamino acids, 0.5% Attorney Docket No. 10457-543PC0 glucose, 1 mM MgCI2, 100 mg/ml ampicillin, 1.5% agar) to purify the plasmids. After restriction digestion of the purified plasmids, the inserts of the positive clones are sequenced to verify the cloned epitopes. The correct epitope-expressing E.
  • coli GI826 and GI826Gly are induced according to the instructions provided by the manufacturer (Invitrogen).
  • the induced bacterial cells is harvested by centrifugation (6000 g, 15 min) and resuspended in PBS containing 0.01% thiomersal, then analyzed by SDS-PAGE gel and immunoblotting using antibodies raised against the respective epitope expressed by each recombinant E. coli GI826 and GI826Gly.
  • immunoblotting goat anti-chicken IgG-HRP conjugate (Sigma-Aldrich) and 3,3'-Diaminobenzidine (Sigma- Aldrich) are used as the secondary antibody and the substrate for detection, respectively.
  • the parent GI826 is included as a negative control.
  • Example 3 Assess the immunogenicity and efficacy of selected positive phage clones against a challenge of C. hepaticus.
  • E. coli GI826 and E. coli GI826Gly expressing C. hepaticus mimotopes selected in Examples above is evaluated in an established chicken model of C. hepaticus SLD. Up to seven clones of unique epitopes expressing E. co// clones are tested at this stage. Chicken experiments will be duplicated or repeated.
  • coli GI826Gly expressing each of the epitopes are administered without any adjuvants. Seven chickens from each group are euthanized to collect intestinal washings for intestinal secretory IgA (slgA) ELISA and remaining seven chickens are challenged with C. hepaticus strain FL2019SK1 by administering 10 10 CFU/ml/bird of bacteria in Preston broth by direct oral gavage at 26 weeks of age (40). Attorney Docket No. 10457-543PC0
  • Chickens are monitored daily for clinical signs and euthanized two weeks post-infection. At necropsy, macroscopic lesions will be recorded, and samples (liver, gall bladder, small intestines, ceca, colon, and cloaca) are taken for bacterial isolation and identification, PCR, and histopathology (liver). Unchallenged negative control groups receive Preston broth only. Bacterial culture is performed according to the methods described by Van et al. 2016 (39) and standardized in our laboratory. Briefly, the macerated tissues and aliquots of bile will be inoculated into modified Preston broth and grown for 3 days under microaerophilic conditions at 37°C.
  • samples are inoculated onto HBA or sheep blood agar and incubated under the same conditions for 7 days.
  • a 20 pl aliquot of bile sample is directly insolated onto HBA plates and incubated at 37°C in microaerophilic conditions for three days.
  • Bacteria are confirmed as C. hepaticus by performing 16S rRNA gene sequencing and glycerol kinase PCR on few selected isolates.
  • inhouse quantitative real-time PCR will be performed on bacterial DNA extracted from liver and bile to ascertain the bacterial load.
  • Enzyme-linked immunosorbent assay The ELISAs are carried out according to the methods optimized in the Pi’s prior work with peptide antigens (27,28). Briefly, 96-well microtiter plates are coated overnight at 4°C with 0.2 pg of the synthetic epitope per well. To measure serum IgG antibodies, the plates are washed three times with PBS containing Tween 20, blocked for 1 h with the same buffer, and then incubated for 1 h at 37°C with 100 pl of serum (1 :10 in PBS) per well. After stringent washing steps, alkaline phosphate-conjugated goat anti-chicken immunoglobulin IgG (Fc fragment) will be added to each well. The plates are incubated for 1 h at room temperature and washed three times prior to adding the enzyme substrate solution.
  • OD After incubation for 1 h, OD will be measured. The OD values are recorded as the mean of duplicate wells on each plate. Positive and negative control sera and blank wells with no sera will be used as controls.
  • intestinal washings from live birds are collected according to a previous protocol. Briefly, the birds are administered a lavage solution containing polyethylene glycol, Na2SO4, NaHCO3, KCI, and NaCI, orally at 5 ml/kg of body weight. The birds are individually placed in clean empty buckets and a Attorney Docket No. 10457-543PC0 sterile solution of 5% pilocarpine in water is injected intramuscularly (50 mg/kg body weight) when the birds begin to excrete feces. At this point, birds will pass intestinal fluid, which is collected into a clean container.
  • Intestinal fluid is centrifuged to collect the supernatant, which is stored at -20 °C after adding phenylmethanesulfonyl fluoride, sodium azide, and bovine serum albumin.
  • the secretory IgA (sig A) is measured as above, except that alkaline phosphate- conjugated goat anti-chicken IgA (a chain) will be used in place of goat anti-chicken IgG.
  • Example 4 Construct and assess the immunogenicity and efficacy of an avian E. co//-vectored vaccine expressing the selected immunogenic mimotope/s of C. hepaticus.
  • an APEC-vectored vaccine is constructed carrying the selected mimotopes of C. hepaticus. Specifically, an aroA mutant of APEC (PSUO78.1 ) is used as the vector. We have already constructed PSUO78.1 attenuated strain and demonstrated its ability to protect chickens from egg layer peritonitis under experimental conditions. This APEC-vectored vaccine is expected to protect chickens from E. co//-associated peritonitis in addition to SLD. A maximum of two mimotopes selected according to the Examples above will be used at this step.
  • the following are performed: (i) construct the AaroA asd double mutant strain designated PSUO78.2 by deleting the asd from PSUO78.1 harboring AaroA via Red recombination, (ii) create PSUO78.3 by replacing its chromosomal cysG gene with the Campylobacter pgl operon via allelic exchange using a suicide vector harboring the pgl operon, (iii) construct the expression plasmid containing C. hepaticus epitope (one or two epitopes) identified in Examples above, and (iv). introduce the expression recombinant plasmid into E. coli PSUO78.3.
  • the resultant PSUO78.4 strain is tested for epitope expression and its immunogenicity and efficacy against both C. hepaticus-mduced SLD and E. coli-mduced egg peritonitis. Chicken experiments will be duplicated or repeated.
  • Plasmid and APEC strain designations Attorney Docket No. 10457-543PC0 p4515: Asd+ plasmid p4516: p4515 containing the cloned C. hepaticus mimotopes
  • PSUO78 Wild type 078 strain isolated from a chicken with egg peritonitis
  • PSUO78.1 AaroA
  • PSUO78.2 AaroA Aasd
  • PSUO78.3 AaroA Aasd cysG pgl
  • PSUO78.4 AaroA Aasd cysG:: pgl containing p4516 (expresses C. hepaticus mimotopes)
  • PSU02 Wild type 02 strain isolated from a chicken with egg peritonitis
  • E. col i PSU078.2 double mutant (AaroA Aasd) vaccine vector strain This is achieved by the Red recombinase-mediated one step inactivation approach, which has been standardized in our laboratory. In brief, this method relies on the overproduction of A-derived recombination proteins encoded by pKD46 and PGR amplification of a chloramphenicol (Cm)-resistant cassette in pKD3 flanked by 5' and 3' sequences of the gene or region targeted for deletion. Cells are electro-transformed, and Cm (25 pg/ml) resistant derivatives are identified. After electroporation and Cm selection, the expected deletions is verified by PCR targeting the deleted gene and the new CmR junction.
  • Cm chloramphenicol
  • the epitopes are PCR amplified and cloned into the multicloning site of pYA4515 ( Figure 4). This plasmid is designated pYA4516.
  • E. coli vector vaccine expressing C. hepaticus epitopes First, the plasmid pYA4516 carrying the epitope/s is electroporated into E. coli BL2 to propagate the plasmid. Expression of the cloned epitope is confirmed by SDS PAGE and immunoblotting. Then, the plasmid is purified from E. coli BL2 and introduced to electrocompetent APEC PSUO78.3 mutant strain to create the APEC Attorney Docket No. 10457-543PC0
  • the vaccine strain harboring the expression plasmid (PSUO78.4) is grown for more than 50 generations in the presence of DAP without antibiotics.
  • the DAP dependency is tested by growing the strain on medium without DAP.
  • the PSUO78.4 strain carrying the epitope/s of C. hepaticus are grown overnight in LB broth, inoculated into fresh LB broth Attorney Docket No. 10457-543PC0 at 1 :100 dilution the next day, and grown until the O.D.600 reaches 0.8.
  • three sample types are collected for SDS PAGE and Western blot analysis using antiepitope antibodies mentioned under Objective 2 (ELISA). Bacterial culture supernatant collected after sonication are tested for expression of the peptide as a cytoplasmic soluble protein.
  • the protein localization in the periplasm and secretion into culture supernatant is assessed by testing the subcellular fractionation of periplasm contents using the lysozyme digestion of the bacterial pellet by osmotic shock with sucrose. In addition, the culture supernatant ise tested after filtering through a 0.22 pm filter and precipitating with 10% trichloroacetic acid solution overnight at 4°C.
  • APEC strain PSUO78.3 and PSUO78.3 transformed with the parent plasmid pYA4515 used to construct pYA4516 is used as negative controls.
  • Samples for bacteriologic examination are collected from the air sacs, pericardial sac, bile, intestines, cloaca, and liver with sterile cotton applicators. Remaining chickens will be booster vaccinated/treated at week 12 by the same route and kept for 30 weeks. Blood will be collected every 10 days to monitor the antibody response. At the time of necropsy, macroscopic lesions are recorded, and air sac and intestinal washings are collected. Serum IgG and intestinal slgA antibodies against C. hepaticus antigen are measured by ELISA after coating microtiter plates with the respective C. hepaticus antigen as described above. Serum IgG and air sac slgA is monitored by ELISA after coating microtiter plates with APEC PSUO78. Air sac wash samples and intestinal wash samples is collected as described previously.
  • Chickens (7 chickens/group) are vaccinated twice by administering the vaccine strain at a dose of 10 7 CFU/ml in drinking water at day 1 and 12 weeks of age as described above.
  • Chickens are challenged with C. hepaticus strain UF2019SK1 by administering 1 O 10 CFU/ml/bird of bacteria in Preston broth by direct oral gavage at 26 weeks of age as described above.
  • Chickens are vaccinated as described above. Challenged groups will be Attorney Docket No. 10457-543PC0 infected either with PSUO78 or PSUO2 wild-type strains by depositing 4x10 9 CFU/ml of bacteria mixed with egg yolk in the oviduct, using an artificial insemination gun at 26 weeks of age. Surviving chickens are euthanatized two weeks post-infection and necropsied to record macroscopic lesions characteristic to APEC peritonitis. Appropriate controls (vaccinated/unchallenged, unvaccinated/challenged and unvaccinated/unchallenged) are included.
  • peritoneum and oviduct Lesions are scored from 0 to 4 for peritoneum and oviduct, separately. If septicemic signs (perihepatitis and pericarditis) are observed, a score of 4 will be assigned. The maximum score will thus be 12/bird. Samples will be taken from the peritoneum, ovaries, oviduct, air sacs, liver and pericardium for bacterial isolation.
  • the two-tailed t-test will be employed to compare IgA and IgG mean antibody concentrations in serum, intestinal wash samples, air sac washings and lesion scores between different experimental groups. Chi-square test will be used to analyze numbers of dead/sick chickens and the presence or absence of bacteria, in different groups. When the experiments are duplicated, and the results are not significantly different between the experiments, the data will be pooled and analyzed as one set of data. A P ⁇ 0.05 is considered significant.

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Abstract

L'invention concerne des compositions comprenant un ou plusieurs antigènes peptidiques pour immuniser un animal aviaire contre C. hepaticus, ou un vecteur pour exprimer le ou les antigènes peptidiques in vivo. L'invention concerne également des procédés d'immunisation d'un animal aviaire contre C. hepaticus.
PCT/US2023/077530 2022-10-21 2023-10-23 Procédés et compositions pour immuniser contre campylobactor hepaticus WO2024086846A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020142008A1 (en) * 2000-04-12 2002-10-03 O'hanley Peter Immunogenic pili presenting foreign peptides, their production and use
US20210177955A1 (en) * 2017-11-08 2021-06-17 Advaxis, Inc. Immunogenic heteroclitic peptides from cancer-associated proteins and methods of use thereof
WO2021177979A1 (fr) * 2020-03-06 2021-09-10 The Royal Institution For The Advancement Of Learning/Mcgill University Vaccin à vecteur bactérien contre des vers parasites et procédé de vaccination

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020142008A1 (en) * 2000-04-12 2002-10-03 O'hanley Peter Immunogenic pili presenting foreign peptides, their production and use
US20210177955A1 (en) * 2017-11-08 2021-06-17 Advaxis, Inc. Immunogenic heteroclitic peptides from cancer-associated proteins and methods of use thereof
WO2021177979A1 (fr) * 2020-03-06 2021-09-10 The Royal Institution For The Advancement Of Learning/Mcgill University Vaccin à vecteur bactérien contre des vers parasites et procédé de vaccination

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