Campylobacter polypeptides and methods of use

ES 2 395 022 T3

DESCRIPTION

BACKGROUND

[0001] Campylobacter spp. it is part of the normal intestinal flora of a wide variety of wild and domestic animals with a particular niche for the avian host. Campylobacter spp. it appears to have a limited ability to be pathogenic in wild and domestic animals. In cattle, C. fetal subsp. jejuni and C. fetus subsp. intestinalis have been isolated from intestines and experimentally transmitted to ruminant and preruminant calves that developed clinical signs of fever, diarrhea and sporadic dysentery (Dannenberg et al. Am. J. Pathol. 34: 1099 (1958) and Thomas et al. Aust. Vet . J. 36: 146 (1981)). A syndrome of profuse watery diarrhea with fever, anorexia and depression has also been reported in lambs with Campylobacter fetus as the causative agent. Campylobacter spp. causes clinical manifestations of dysentery, intestinal adenomatosis, and hemorrhagic enteritis in pigs and horses, and mastitis in commercial dairy cattle.

[0002] In humans, Campylobacter is the most commonly reported bacterial cause of endemic diarrheal disease worldwide. In the United States, it is becoming the most widespread cause of foodborne infection, affecting more than 2 million people annually. In England and Wales, around 50,000 cases of campylobacter are reported annually with no signs of declining incidence. It is estimated that for each case reported for laboratory surveillance, another seven unreported cases occur. C. jejuni and C. coli are the two most common isolated species responsible for human Campylobacteriosis with C. jejuni now being the most frequently isolatable species.

[0003] The incubation period after ingestion of C. jejuni has been shown to be approximately 24-72 hours. The inoculum size required to elicit clinical symptoms is as low as 800 organisms. The disease rate increases with larger numbers ingested by the body. Commonly reported symptoms of human Campylobacteriosis include diarrhea, fever, and abdominal cramps. Less commonly, Campylobacter, particularly C. jejuni, can cause secondary sequelae after an acute infection, including reactive arthritis, kidney failure, Guillian-Barre, Reiter syndrome, and other extra-intestinal symptoms.

[0004] The transmission of Campylobacter spp. Human populations are primarily through environmental contamination and contaminated food, including poultry and poultry products such as eggs. Campylobacter spp. 30-100% of birds can be isolated from many wild and domestic avian species at any one time. In children, contact with puppies and kittens with diarrhea has been shown to be an important additional risk factor. Additional sources of infection have resulted from drinking raw milk derived from cows with clinical mastitis caused by Campylobacter. All milkborne outbreaks have been associated with raw or improperly pasteurized milk.

[0005] The virulence and pathogenesis of Campylobacter spp. it involves both host and pathogen specific factors. Many pathogen-specific virulence determinants contribute to the pathogenesis of these bacteria. The bacterial virulence of these bacteria is the result of many different attributes, which often contribute to different stages in the complicated series of events recognized as an infection. Exposure occurs primarily through consumption of contaminated water, food, or direct person-to-person contact. Once ingested, stages of infection common to these bacteria include attachment, colonization, proliferation, tissue damage, invasion, and spread.

[0006] The first host barrier that Campylobacter must normally overcome is the mucosal surface. A single epithelial cell layer separates the host from the lumen of the gastrointestinal tract. This barrier and a plethora of other host antimicrobial mechanisms deter commensal, pathogenic, and opportunistic organisms from establishing infection. Adherence to mucosal surfaces is a prerequisite for this pathogen to establish infection. One of the most marked clinical manifestations of intestinal colonization is diarrhea. It has been proposed that this clinical syndrome is produced by the synthesis and excretion of enterotoxins that cause a net secretion of fluid and electrolytes (diarrhea). Other specific virulence factors include flagella, which help the bacteria overcome the compensatory movement of peristalsis and allow the organism to enter and cross the mucous layer that covers the epithelium (Black et al., J. Infect. Dis. 157: 472-479 (1988), Caldwell et al., Infect Immun. 50: 941-943 (1985), Morooka et al., J. Gen. Micro. 131: 1973-1980 (1980) and Newell et al. J. Hyg. Camb. 95: 217-227 (1985)). Other suspected pathogenicity determinants include chemotaxis, iron acquisition, host cell invasion, inflammation and active secretion and epithelial disruption with serous fluid drip (Black et al. J. Infect. Dis. 157: 472-479 (1988)).

[0007] Divalent metal ions such as iron, cobalt, copper, magnesium, manganese, molybdenum, nickel, selenium, and zinc are trace elements often required for the survival of bacteria that infect both animal and human hosts. These metallic trace elements are used by bacteria as cofactors for enzymes that catalyze biochemical reactions for various metabolic pathways and transport systems required by the body. The metals iron, zinc and manganese are the three most important metals required for the survival of the bacteria. Zinc ions are essential for DNA and RNA polymerase activity, while manganese is required for mitochondrial superoxide dismutase activity. Iron is the most thoroughly studied of all metal ions with direct correlations in virulence and pathogenesis of bacteria. The

ES 2 395 022 T3 Iron is essential for all life and is required for metabolic and enzymatic pathways of organisms at all phylogenic levels.

[0008] The ability of Campylobacter to evade the natural defense mechanisms of the vertebrate host depends in part on its ability to obtain host iron, which in turn directly influences the pathogen-host interaction. Because of the essential nature of iron, vertebrate hosts have developed elaborate mechanisms to bind iron to body fluids (eg, transferrin in blood and lymphatic fluids and lactoferrin in external secretions). These high-affinity iron-binding proteins create an iron-restricted environment within the host reducing the iron level to approximately 10^-18 molar, too low a concentration to support the growth of most bacteria. These host iron sequestering mechanisms act as a natural defense mechanism to combat bacterial invasion. To circumvent these iron restrictive conditions, many bacterial species have evolved mechanisms for obtaining iron. The most common mechanisms include diffusion of soluble iron through porins and specialized transport systems that mediate iron intake by siderophores. The latter system is by far the most widespread or ubiquitous mechanism for iron acquisition and involves the specific chelation of ferric iron by siderophores and the synthesis of its cognate transport systems, allowing bacteria to continue to replicate and overcome the mechanisms. non-host specific defense. Continued replication, and hence each step in the infectious process, ultimately depends on the body's ability to obtain iron from its host.

[0009] With so many basic functions relying on the availability of iron, bacteria have evolved a complex regulatory network to acquire iron under varying physiological conditions. Iron is a divalent cation that exists both in the ferrous state (Fe²⁺) and the iron state (Fe³⁺). Under anaerobic conditions, iron is present in the soluble ferrous form (Fe²⁺) and can freely diffuse through the porins of the outer membrane into the periplasm. For example, in E. coli the FeoAB transport system present in the cytoplasmic membrane will transport the ferrous iron molecules within the cell cytoplasm. Under aerobic conditions and neutral pH, iron is mainly present in the insoluble form ferric (Fe³⁺) and cannot pass through the porins of the outer membrane by passive diffusion. Instead, molecules called siderophores are secreted by bacteria, which have a high affinity for ferric iron. Ferri-siderophore complexes are recognized by receptors on the outer membrane, collectively referred to as the TonB-dependent receptors. These receptors, once attached to charged siderophores, are believed to interact with TonB and its associated proteins located in the periplasm and cytoplasmic membrane. These protein-protein interactions, although poorly understood, serve to provide the energy necessary to transport the ferri-siderophore complexes across the outer membrane and through the periplasmic space. The ABC transport systems present in the cytoplasmic membrane serve to transport the iron-siderophore complexes across the cytoplasmic membrane. The reductase enzymes then serve to reduce ferric iron to its ferrous form, dissociating it from the siderophore and releasing iron into the cell.

[0010] Various species of pathogenic bacteria use additional mechanisms to obtain iron from mammalian hosts, including direct binding of heme and hemoglobin. The receptor proteins that bind these iron-containing molecules most likely rely on the TonB complex for the energy required to transport heme along the outer membrane, similar to iron-siderophore complexes. The specialized ABC carriers are then used to transport the heme across the cytoplasmic membrane. In addition, some bacteria secrete hemophores, small molecules that can bind heme and present it to receptors on the bacterial cell surface. Several pathogenic species also produce hemolysins, which are toxins that lyse red blood cells, releasing heme and hemoglobin for ingestion by the bacteria.

[0011] The outer membrane proteins of gram-negative bacteria control the selective permeability of many nutrients essential for the survival of the bacteria, including all pathogenic bacteria that cause disease in animals and humans. This selective permeability of nutrients is controlled by a class of membrane proteins called porins. It now appears that most of the outer membrane proteins on the surface of gran-negative bacteria are porins, identified as the general porins (e.g. OmpF), monomeric porins (e.g. OmpA), the specific porins (e.g. for example, maltose-specific porin LamB) and TonB-dependent gate porins (for example, siderophore receptor FepA). The porin class of proteins generally share structural characteristics, including the presence of beta barrels that span the outer membrane.

Little is known about the acquisition of iron by Campylobacter spp. Studies indicate that C. jejuni does not synthesize siderophores (Field et al. Infect. Immun. 54: 126-132 (1986) and Picket et al. Infect Immun. 60: 3872-3877 (1992)). These data have been confirmed by sequence analysis of the C. jejuni genome in which no homologues of common siderophore synthesis genes were identified. C. jejuni is limited in the iron compounds he can use as shown by various feeding trials. These assays demonstrated that C. jejuni can use the siderophores enterochelin and ferrichrome but not aerobactin, desferal, ferritin, lactoferrin, or transferrin. Therefore, it has been suggested that other iron compounds are required to support the growth of Campylobacter spp. such as heme compounds such as hemin and hemoglobin, ferric iron, and ferrous iron. The fact that Campylobacter has known transport systems for siderophores, although it is unable to synthesize them, suggests

ES 2 395 022 T3 that these bacteria recover siderophores produced by other enteric pathogens (Arnoud et al. FEMS Microbiol. Rev. 26: 173-186 (2002)).

ABSTRACT

[0013] The present invention provides a composition according to claim 1. The disclosure provides an isolated metal-regulated polypeptide obtainable from a Campylobacter spp., Wherein the polypeptide is expressed by a Campylobacter spp. at a detectable level during growth under low metallic conditions and is not expressed by Campylobacter spp. at a detectable level during growth under high metallic conditions, and a composition including the polypeptide. The isolated metal-regulated polypeptide can have a molecular weight between 150 kDa and 152 kDa, between 143 kDa and 145 kDa, between 123 kDa and 125 kDa, between 92 kDa and 94 kDa, between 88 kDa and 90 kDa, between 73 kDa and 75 kDa, between 69 kDa and 71 kDa, between 51 kDa and 53 kDa, between 50 kDa and 52 kDa, or between 38 kDa and 40 kDa. The composition may further include a second metal-regulated polypeptide with a molecular weight between 57 and 59 kDa, between 54 kDa, and 56 kDa, between 47 and 49 kDa, between 42 and 44 kDa, between 37 and 39 kDa. kDa, or between 28 kDa and 30 kDa, where the second polypeptide is expressed by a Campylobacter spp. during growth under high metallic conditions and expressed at an increased level during growth under low metallic conditions.

[0014] The disclosure also provides an isolated metal-regulated polypeptide obtainable from a Campylobacter spp., Wherein the polypeptide is expressed by a Campylobacter spp. during growth under low metallic conditions, and a composition including the polypeptide. The metal-regulated polypeptide can have a molecular weight of between 57 and 59 kDa, between 54 and 56 kDa, between 47 and 49 kDa, between 42 and 44 kDa, between 37 and 39 kDa, or between 28 kDa. and 30 kDa.

The composition may further include a second metal-regulated polypeptide with a molecular weight between 150 kDa and 152 kDa, between 143 kDa and 145 kDa, between 123 kDa and 125 kDa, between 92 kDa and 94 kDa, between 88 kDa and 90 kDa, between 73 kDa and 75 kDa, between 69 kDa and 71 kDa, between 51 kDa and 53 kDa, between 50 kDa and 52 kDa, or between 38 kDa and 40 kDa, where the second polypeptide is expressed by a Campylobacter spp . at a detectable level during growth under low metallic conditions and is not expressed by Campylobacter spp. to a detectable level during growth under high metallic conditions.

[0016] The disclosure also includes methods for using the compositions disclosed herein, including methods for treating an infection in a subject, for treating a disease caused by a Campylobacter spp., To decrease colonization of an animal. The methods include administering an effective amount of a composition to an animal, where the composition includes an isolated metal-regulated polypeptide obtainable from a Campylobacter spp.

[0017] The present invention includes a composition according to claim 3.

[0018] The present invention includes a composition according to claim 4.

Also included in this disclosure is a composition including an isolated whole cell preparation of a Campylobacter spp., Where the cells either include a metal-regulated polypeptide expressed by Campylobacter spp. during growth under low metallic conditions and not expressed during growth under high metallic conditions, a metal-regulated polypeptide expressed by Campylobacter spp. during growth under high metallic conditions and expressed at an increased level during growth under low metallic conditions, or the combination thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0020]

Figure 1. Gel image of the extracted membrane protein profile of Campylobacter jejuni expressed under growth conditions of iron saturation and iron deficiency.

Figure 2. The difference in fecal deposition between vaccinated and unvaccinated mice after oral challenge with Campylobacter jejuni. Log 10 CFU, mean number of bacteria in the stool sample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0021] The disclosure provides polypeptides and compositions including polypeptides. As used herein, "polypeptide" refers to a polymer of amino acids linked by peptide linkages. Thus, for example, the terms peptide, oligopeptide, protein, and above are included within the definition of polypeptide. This term also includes post-expression modifications of the polypeptide, eg, glycosylations, acetylations, phosphorylations, and the like. The term polypeptide does not connote a specific length of an amino acid polymer. A polypeptide can be directly obtainable from a natural origin, or it can be prepared with the aid of chemical, enzymatic, or recombinant techniques. In the case of a naturally occurring polypeptide or polynucleotide, such a polypeptide or polynucleotide is normally isolated. An "isolated" polypeptide is one that has been removed from its natural environment. For example, an "isolated" polypeptide is a polypeptide that has been removed from the cytoplasm or outer membrane of a

ES 2 395 022 T3 cell, and many of the polypeptides, nucleic acids, and other cellular matter from its natural environment are no longer present. A "purified" polypeptide is one that is at least 60% free, preferably 75% free, and more preferably 90% free of other components with which it is naturally associated. Polypeptides that are produced outside of the organism in which it occurs naturally, for example, by recombinant or chemical means, are considered isolated and purified by definition, since they were never present in a natural environment. Unless otherwise specified, a, the, and at least one are used interchangeably and mean one or more than one.

[0022] The polypeptides of the disclosure are obtainable from a member of the Campylobacteriaceae family, (Vandamme et al. Int. J. Syst. Bacteriol. 41: 451-455 (1991)), preferably the genus Campylobacter. A member of the genus Campylobacter is also referred to herein as Campylobacter spp. Examples of Campylobacter spp. of those obtainable polypeptides from the disclosure include C. hyointestinalis, C. mucosalis, C. concisus, C. sputorum, C. jejuni, C. coli, C. lari, C. upsaliensis, C. rectus, C. curvus, C. hominis, C. fetus, C. intestinalis, and C. doylei. Campylobacter spp. from which the compositions of the present invention are obtained is C. jejuni. These microbes are commercially available from a repository such as the American Type Culture Collection (ATCC). Furthermore, such microbes are readily obtainable by isolation techniques known and used in the art. For example, a microbe can be derived from an infected animal as a field isolate, and used to obtain polypeptides of the disclosure as described herein, or stored for future use, for example, in a container frozen at about -20 ° C. up to about -95 ° C, in an appropriate bacteriological medium containing 20% ​​glycerol, and other similar media. The methods for obtaining the polypeptides of Campylobacter spp. are described here.

[0023] A polypeptide of the disclosure can be characterized by molecular weight. The molecular weight of a polypeptide, usually expressed in kilodaltons (kDa), can be determined using routine methods including, for example, gel filtration, gel electrophoresis including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS), capillary electrophoresis. , mass spectrometry, and liquid chromatography including HPLC. The polypeptides of the disclosure can be metal-regulated polypeptides. As used herein, a metal-regulated polypeptide is a polypeptide that is expressed by a member of the genus Campylobacter at a higher level when the microbe grows under low metallic conditions compared to the growth of the same microbes under high metallic conditions. Metals are those present in the periodic table in Groups 1 to 17 (IUPAC notation; also referred to as Groups IA, II-A, III-B, IV-B, VB, VI-B, VII-B, VIII, IB , II-B, III-A, IV-A, VA, VI-A, and VII-A, respectively, under the CAS notation). Preferably, the metals are those in Groups 2 to 12, more preferably Groups 3-12. Still more preferably, the metal is iron, zinc, copper, magnesium, nickel, cobalt, manganese, molybdenum, or selenium, more preferably iron.

[0024] For example, a type of metal-regulated polypeptide produced by Campylobacter spp. it is not expressed at detectable levels during growth of the microbe under high metallic conditions but expressed at detectable levels during growth under low metallic conditions. Low metallic conditions and high metallic conditions are described in greater detail here. Examples of such metal-regulated polypeptides obtainable from a Campylobacter spp. have molecular weights (as determined by separating the polypeptides using a stacking gel of about 4% on a gel of about 10% resolution under reducing and denaturing conditions of between 150 kDa and 152 kDa, between 143 kDa and 145 kDa , between 123 kDa and 125 kDa, between 92 kDa and 94 kDa, between 88 kDa and 90 kDa, between 73 kDa and 75 kDa, between 69 kDa and 71 kDa, between 50 kDa and 53 kDa, or between 38 kDa and 40 kDa. Preferably, the metal-regulated polypeptides have molecular weights of 151 kDa, 144 kDa, 124 kDa, 93 kDa, 89 kDa, 74 kDa, 70 kDa, 52 kDa, 51 kDa, or 39 kDa.

[0025] Another type of metal-regulated polypeptide produced by Campylobacter spp. it is expressed at detectable levels during growth of microbes under high metallic conditions but is expressed at high levels during growth under low metallic conditions. Expression of such polypeptides is referred to herein as enhanced during growth under low metallic conditions. Examples of metal-regulated polypeptides showing improved expression and obtainable from Campylobacter spp. have molecular weights (determined by separation of the polypeptides using an SDS-PAGE gel of about 10% under denaturing and reducing conditions) of between 57 kDa and 59 kDa, between 54 kDa and 56 kDa, between 47 kDa and 49 kDa, between 42 kDa and 44 kDa, between 37 kDa and 39 kDa, or between 28 kDa and 30 kDa. Preferably, the metal-regulated polypeptides with enhanced expression have molecular weights of 58 kDa, 55 kDa, 48 kDa, 43 kDa, 38 kDa, or 29 kDa.

[0026] Whether a metal-regulated polypeptide is expressed at a detectable level or has enhanced expression during growth under low metal conditions can be determined by useful methods to compare the presence of polypeptides, including, for example, gel filtration, electrophoresis of gel including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS), capillary electrophoresis, mass spectrometry, and liquid chromatography including HPLC. Separate cultures of a Campylobacter spp. are grown under high metal conditions and under low metal conditions, polypeptides of the disclosure are isolated as described herein, and the polypeptides present in each culture are resolved and compared. Typically, an equal amount of polypeptide from each culture is used. For example, when using SDS polyacrylamide gel electrophoresis to compare polypeptides, about 30 pg micrograms of polypeptide from each culture is loaded into one well. After running the gel and staining the polypeptides, the two lanes can be compared.

ES 2 395 022 T3

[0027] Preferably, the polypeptides of the disclosure have innumogenic activity. Immunogenic activity refers to the ability of a polypeptide to elicit an immune response in an animal. An immune response to a polypeptide is the development in an animal of a cellular and / or antibody-mediated immune response to the polypeptide. Typically, an immune response includes but is not limited to one or more of the following: production of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells, targeting an epitope or epitopes of the polypeptide. Epitope refers to the site on an antigen to which specific B cells and / or T cells respond such that antibodies and / or a cellular immune response are produced.

Also provided by the disclosure are whole cell preparations of a microbe, where the microbe expresses one or more of the polypeptides of the disclosure. The cells present in a whole cell preparation are preferably inactivated so that the cells cannot replicate, but the immunogenicity of the polypeptides of the disclosure expressed by the microbe is maintained. Typically, cells are killed by exposure to agents such as glutaraldehyde, formalin, or formaldehyde. The present invention includes a composition of claim 3.

[0029] Compositions. The disclosure also provides compositions including at least about 1 of the disclosure polypeptides, more preferably at least about 2, at least about 3, at least about 4, and so on, up to at least about 8 disclosure polypeptides. A composition may include polypeptides obtainable from 1 species of Campylobacter, or they may be obtainable from a combination of 2 or more species of Campylobacter, for example C. jejuni and a second Campylobacter other than C. jejuni. In addition, a composition can include polypeptides obtainable from 2 or more strains of the same Campylobacter species. For example, a composition can include polypeptides obtainable from 2 different isolates of C. jejuni.

[0030] Optionally, a polypeptide of the disclosure can be covalently linked to a carrier polypeptide to enhance the immunological properties of the polypeptide. Useful carrier polypeptides are known in the art. Chemical coupling of a polypeptide of the disclosure can be carried out using known and routine methods. For example, various homobifunctional and / or heterobifunctional crosslinking reagents can be used such as bis (sulfosuccinimidyl) suberate, bis (diazobenzidine), dimethyl adipimidate, dimethyl pimellimidate, d-dimethyl superimidate, disuccinimidyl suberate, glutaraldehydebenzopho-maleimido-sulphidoxysuccinimido-sulphidoxysil-maleimide. mmaleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 4- (p-maleimido-phenyl) butyrate and (1-ethyl-3- (dimethylaminopropyl) carbodiimide (Harlow and Lane, Antibodies, A Laboratory Manual, generally and Chapter 5, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York , NY (1988)).

[0031] Preferably, such compositions of the disclosure include low concentrations of lipopolysaccharide (LPS). LPS is a component of the outer membrane of most gram-negative microbes (see, for example, Nikaido and Vaara, Outer Membrane, In: Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, Neidhardt et al., (Eds .) American Society for Microbiology, Washington, DC, pp. 7-22 (1987), and typically includes polysaccharides (specific O chain, the inner and outer core) and the A region of lipid. LPS lipid component A is the most biologically active component of the LPS framework and together induces a broad spectrum of pathophysiological effects in mammals. The most important effects are fever, disseminated intravascular coagulation, complement activation, hypotensive shock, and death. The non-specific immunostimulatory activity of LPS can enhance granuloma formation at the site of administration of compositions including LPS. Such reactions can result in undue stress on the animal whereby the animal may give up food or water for a period of time, and exasperate infectious diseases in the animal. Additionally, the formation of a granuloma at the injection site can increase the likelihood of carcass degradation due to scarring or tissue damage at the injection site (see, for example, Rae, Injection Site Reactions, available at www. animal.ufl.edu/short94/rae.htm).

[0032] The LPS concentration can be determined using routine methods known in the art. Such methods typically include the measurement of dye binding by LPS (see, for example, Keler and Nowotny, Analyt. Biochem., 156, 189 (1986)) or the use of a Limulus Amebocyte Lysate (LAL) assay ( see, for example, Endotoxins and Their Detection With the Limulus Amebocyte Lystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY (1982)). There are four basic commercially available methods that are commonly used with an LAL assay: the gel clot assay; the turbidimetric test (spectrophotometric); the colorimetric test; and the chromogenic assay. An example of a clot gel assay is available under the trademark E-TOXATE (Sigma Chemical Co., St. Louis, MO; see Sigma Technical Bulletin No. 210), and PYROTELL (Associates of Cape Cod, Inc., East Falmouth , MA). Typically, the test conditions include contacting the composition with a preparation containing a lysate of the circulating amoebocytes of the caderola crab, Limulus polifemus. When exposed to LPS, the lysate increases in opacity as well as viscosity and can gel. About 0.1 milliliters of the composition is added to the lysate. Typically the pH of the composition is between 6 and 8, preferably between 6.8 and 7.5. The composition and lysate mixture is incubated for about 1 hour uninterrupted at about 37 ° C. After incubation, the mixture is observed to determine if there was gelation of the mixture. Gelation indicates the presence of endotoxin. To determine the amount of endotoxin present in the composition, dilutions of a standard endotoxin solution are made and tested at the same time that the composition is tested. Standardized solutions

ES 2 395 022 T3 endotoxin are commercially available from, for example, Sigma Chemical (Catalog No. 210-SE), US Pharmacopeia (Rockville, MD, Catalog No. 235503), and Associates of Cape Cod, Inc., (Catalog No. E0005). In general, when a composition of the disclosure is prepared by isolating polypeptides from a microbe by a method as described herein (eg, a method that includes disrupting and solubilizing cells, and collecting insoluble polypeptides), the amount of LPS in a The composition of the present invention is less than the amount of LPS present in a mixture of the same amount of the microbe that has been affected by it but not solubilized. Typically, the level of LPS in a composition of the disclosure is reduced by, in increasing order of preference, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% relative to the LPS level in a composition prepared by breaking down, but not solubilizing, the same microbe.

The disclosure also provides compositions that include a whole cell preparation of at least 1, at least 2, at least 3, at least 4, at least 5, or 6 Campylobacter spp.

[0034] The compositions of the present invention and disclosure optionally further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable refers to a diluent, carrier, excipient, salt, etc., which is compatible with the other ingredients of the composition, and not deleterious to the container thereof. Typically, the composition includes a pharmaceutically acceptable carrier when the composition is used as described herein. The compositions of the present invention and disclosure can be formulated into pharmaceutical preparations in a variety of forms adapted to the chosen route of administration, including routes suitable for stimulating an immune response to an antigen. Thus, a composition of the present invention and disclosure can be administered via routes including, for example, oral; parental including intradermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc., and topically, such as, intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, and rectally etc. It is envisioned that a composition can be administered to a mucosal surface, such as by administration to the respiratory or nasal mucosa (for example spray or aerosol), to stimulate mucosal immunity, such as the production of IgA secreting antibodies, throughout the body of the animal.

[0035] A composition of the present invention and disclosure can also be administered by means of a sustained or delayed release implant. Suitable implants for use according to the invention are known and include, for example, those disclosed in Emery and Straub (WO 01/37810 (2001)), and Emery et al. (WO 96/01620 (1996)). The implants can be produced in sizes small enough to be administered by aerosol or spray. Implants also include nanospheres and microspheres.

[0036] A composition of the present invention and disclosure is administered in an amount sufficient to treat certain diseases as described herein. The amount of polypeptides or whole cells present in a composition of the present invention and disclosure can vary. For example, the dose of polypeptides can be between 0.01 micrograms (pg) and 300 mg, usually between 0.1 mg and 10 mg. When the composition is a whole cell preparation, the cells may be present in a concentration of, for example, 10⁶ bacteria / ml, 10⁷bacteria / ml, 10⁸ bacteria / ml, or 10⁹ bacteria / ml. For an injectable composition (eg subcutaneous, intramuscular, etc.) the polypeptides can be present in the composition in an amount such that the total volume of the composition administered is 0.5 ml to 5.0 ml, usually 1.0-2.0 ml. When the composition is a whole cell preparation, the cells are preferably present in the composition in an amount such that the total volume of the composition administered is 0.5 ml to 5.0 ml, usually 1.0-2.0 ml. The amount administered varies depending on various factors including, but not limited to, the specific polypeptides chosen, the weight, physical condition and age of the animal, and the route of administration. Thus, the absolute weight of the polypeptide included in a given unit dose form can vary widely, and depends on factors such as the species, age, weight and physical condition of the animal, as well as the method of administration. Such factors can be determined by one of skill in the art. Other examples of suitable doses for the invention are disclosed in Emery et al. (US Patent 6,027,736).

[0037] The formulations may be conveniently presented in unit dose forms and may be prepared by methods well known in the art of pharmacy. All methods of preparing a composition including a pharmaceutically acceptable carrier include the step of associating the active compound (eg, a polypeptide or whole cell of the present invention) with a carrier that constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately associating the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

[0038] A composition that includes a pharmaceutically acceptable carrier may also include an adjuvant. An adjuvant refers to an agent that can act in a non-specific way to enhance an immune response to a particular antigen, thus potentially reducing the amount of antigen required in a given immunizing composition, and / or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. Adjuvants can include, for example, IL-1, IL-2, emulsifiers, muramil dipeptides, dimethyldiocradecylammonium bromide (DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol, polysaccharides, emulsified paraffins (including by for example, those available under the trademark EMULSIGEN from MVP Laboratories, Ralston, Nebraska), ISA-70, RIBI, and other substances known in the art.

ES 2 395 022 T3

[0039] In another embodiment, a composition of the invention or disclosure including a pharmaceutically acceptable carrier may further include a biological response modifier, such as, for example, IL-2, IL-4 and / or IL-6, TNF, IFN-alpha, IFN-gamma, and other cytokines that affect immune cells. An immunizing composition can also include other components known in the art such as an antibiotic, a preservative, an antioxidant, or a chelating agent.

Elaboration Methods

[0040] The polypeptides and whole cell preparations of the disclosure can be obtained by incubating a member of the genus Campylobacter under conditions that promote the expression of one or more of the polypeptides described herein. The disclosure also includes compositions prepared by the processes disclosed herein. Typically such conditions are low metallic conditions. As used herein, the phrase "low metallic conditions" refers to an environment, typically bacteriological media, that contains amounts of a free metal that causes a microbe to express metal-regulated polypeptides. As used herein, the phrase "high metallic conditions" refers to an environment that contains amounts of a free metal that causes a microbe to either not express one or more of the metal-regulated polypeptides described herein, or to reduce the expression of such a polypeptide. . Low metallic conditions are generally the result of the addition of a metal chelating compound to a bacteriological medium. High metallic conditions are generally present when a chelator is not present in the medium, and / or a metal is added to the medium. Examples of metal chelators include synthetic and natural compounds. Examples of natural compounds include phenolic compounds from plants, such as flavonoids. Examples of flavonoids include the copper chelators catechinqa and naringenin, and the iron chelators myricetin and quercetin. Examples of synthetic copper chelators include, for example, tetrathiomolybdate, and examples of synthetic zinc chelators include, for example, N, N, N ', N'-Tetrakis (2-pyridylmethyl) -ethylene diamine. Examples of synthetic iron chelators include 2,2'-dipyridyl (also referred to in the art as α, α'-bipyridyl), 8-bidroxyquinoline, ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxamine methanesulfonate (desferol), transferrin , lactoferrin, ovotransferrin, biological siderophores, such as, the catecholates and hydroxamates, and citrate. Preferably 2,2'-dipyridyl is used for iron chelation. Typically 2,2'-dipyridyl is added to the medium at a concentration of at least 0.0025 micrograms / milliliter (pg / ml), at least 0.025 pg / ml, or at least 0.25 pg / ml. High 2,2'-dipyridyl levels can be 10 pg / ml, 20 pg / ml, or 30 pg / ml.

[0041] It is expected that a Campylobacter spp. a mutation in a fur gene will result in the constitutive expression of many, if not all, of the metal-regulated polypeptides of the disclosure. A fur gene has been identified in a C. jejuni (van VLiet et al., J. Bacteriol., 180, 5291-5298, (1998)). The production of a fur mutation in a Campylobacter spp. it can be produced using routine methods including, for example, electroporation and genetic constructs useful for gene blocking in large negative bacteria.

[0042] Many Campylobacter spp. they are able to grow under low metallic conditions in vitro on artificial media only after adaptation. For example, a Campylobacter spp. it can be adapted to low iron conditions in vitro by growing in the presence of low concentrations of an iron chelator and, after growth in a medium containing the chelator, gradually increasing the concentration of the chelator. For example, a Campylobacter spp. It can be adapted to growth under low iron conditions by adding 10 pg / ml 2,2'-dipyridyl to a medium, and gradually increasing the concentration of the chelator to a higher concentration, eg, 20 pg / ml.

[0043] The medium used to incubate the microbe and the volume of the media used to incubate the microbe can vary. When a Campylobacter spp. being evaluated on the ability to produce the polypeptides described herein, the microbe can be cultured in a suitable volume, for example, 10 milliliters to 1 liter of the medium. When a microbe is being cultured to obtain polypeptides for use in, for example, administration to animals, the microbe can be cultured in a fermentation medium to allow the isolation of larger amounts of polypeptides. Methods for growing microbes in a fermentation medium are routine and known in the art. Conditions used to grow a microbe preferably include a metal chelator, more preferably an iron chelator, for example 2,2'-dipyridyl, a pH of between about 6.5 and about 7.5, preferably between about 6.9 and 7.1, and a temperature of about 37 ° C. When a fermentation medium is used, the culture can be purged with an appropriate gas, for example carbon dioxide, to maintain microaerophilic conditions. Elements of the genus Campylobacter are microaerophilic organisms, so growth conditions do not include oxygen levels that impede growth.

[0044] A Campylobacter spp. can be collected after growth. Harvesting includes concentrating the microbe to a smaller volume and suspending in a medium other than culture media. Methods for concentrating a microbe are routine and known in the art, and include, for example, filtration and / or centrifugation. Normally, the concentrated microbe is suspended in decreasing amounts of buffer. Preferably, the final buffer includes a metal chelator, preferably ethylenediaminetetraacetic acid (EDTA). An example of a buffer that can be used contains Tris-base (7.3 grams / liter) and EDTA (0.9 grams / liter), at a pH of 8.5. Optionally, the final buffer also minimizes proteolytic degradation. This can be achieved with the final buffer at a pH greater than 8.0, preferably 8.5, and / or by including one or more proteinase inhibitors (eg, phenylmethanesulfonyl fluoride). Optionally and preferably, the concentrated microbe is frozen at -20 ° C or below until broken.

ES 2 395 022 T3

[0045] When Campylobacter spp. To be used as a whole cell preparation, the harvested cells can be processed using known and routine methods to inactivate the cells. Alternatively, when a Campylobacter spp. to be used to prepare polypeptides of the disclosure, Campylobacter spp. It can be broken using routine mechanical, physical, or chemical methods known in the art, including, for example, French press, sonication, or homogenization. Preferably, homogenization is used. As used herein, "breakdown" refers to the breakdown of the cell. The breakdown of a microbe can be measured by methods that are routine and known in the art, including, for example, changes in optical density. Typically, a microbe is disrupted until the percent transmittance increases by 20% when a 1: 100 dilution is measured. The temperature during breakage is normally kept low, preferably 4 ° C, to further minimize proteolytic degradation.

[0046] The broken microbe is solubilized in a detergent, for example an anionic, zwitterionic, non-ionic, or cationic detergent. Preferably the detergent is sarcosine, more preferably sodium lauroyl sarcosinate. As used herein, the term "solubilize" refers to dissolving cellular matter (eg, polypeptides, nucleic acids, carbohydrates) in the aqueous phase of the buffer in which the microbe was broken down, and the formation of insoluble cellular matter aggregates. . Conditions for solubilization preferably result in the aggregation of polypeptides of the present invention into soluble aggregates that are large enough to allow easy isolation by, for example, centrifugation.

[0047] Preferably, the sarcosine is added such that the final ratio of sarcorsin to the gram weight of the broken microbe is between 1.0 grams of sarcosine per 4.5 grams of granulated mass and 6.0 grams of sarcosine per 4.5 grams of granulated mass, preferably , 4.5 grams of sarcosine per 4.5 grams of granulated mass. Solubilization of the microbe can be measured by methods that are routine and known in the art, including, for example, changes in optical density. Typically, solubilization is allowed to take place for at least 24 hours, more preferably at least 48 hours, still more preferably at least 60 hours. The temperature during breakage is normally kept low, preferably 4 ° C.

[0048] Insoluble aggregates including the polypeptides of the disclosure can be isolated by methods that are routine and known in the art. Preferably, the insoluble aggregates are isolated by centrifugation. Typically, centrifugation of outer membrane polypeptides that are insoluble in detergents require centrifugal forces of at least 50,000 xg, typically 100,000 x g. The use of such centrifugal forces requires the use of ultracentrifuges, and scaling up to process large sample volumes is often difficult and uneconomical with these types of centrifuges. The methods described herein provide for the production of insoluble aggregates large enough to allow the use of significantly lower centrifugal forces (eg, 46,000 xg). Methods for processing large volumes at these lower centrifugal forces are available and known in the art. Thus, insoluble aggregates can be isolated at significantly lower cost.

[0049] Optionally and preferably, sarcosine is extracted from the isolated polypeptides. Methods for extracting sarcosine from isolated polypeptides are known in the art, and include, for example, diafiltration, precipitation, hydrophobic chromatography, ion exchange chromatography, and / or affinity chromatography, and ultrafiltration and washing of the polypeptides in alcohol. by diafiltration. After isolation, the polypeptides are suspended in buffer and stored at low temperature, -20 ° C or lower.

The polypeptides of the disclosure can also be isolated from Campylobacter spp. using methods that are known in the art. Isolation of polypeptides can be achieved as described in, for example, Emery et al., (US Patent 5,830,479) and Emery et al., (US Patent Application 20030036639 A1).

[0051] In those aspects of the present invention where a whole cell preparation must be made, after culturing a Campylobacter jejuni the microbe can be killed with the addition of an agent such as glutaraldehyde, formalin, or formaldehyde, in a sufficient concentration To deactivate cells in culture, For example, formalin can be added in a concentration of almost 3% (vol: vol). After a period of time sufficient to inactivate the cells, the cells can be harvested by, for example, diafiltration and / or centrifugation, and washed.

Methods of Use

[0052] The disclosure is further directed to methods of using the compositions of the present invention and disclosure. The methods include administering to an animal an effective amount of a composition of the present invention and disclosure. Preferably, the composition further includes a pharmaceutically acceptable carrier. The composition can be administered at a time when maternal antibody may be present, for example, as early as one day of age, or at a later time during the life of the animal. The animal can be, for example, an ungulate, a bird, a human, or a companion animal. Examples of birds include poultry such as turkeys, chickens, ducks, pheasants, and ostriches. Examples of ungulates include animals that are bovine (including, for example, cattle), goats (including, for example, goats), sheep (including, for example, sheep), porcine (including, for example, avian pigs), equine (including, for example, horses), members of the family Cervidae (including, for example, roe deer, elk, deer, caribou, and reindeer), and Bison (including, for example, buffalo). Examples of companion animals include cats and dogs.

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[0053] The methods may further include additional administrations (eg, one or more booster administrations) of the composition to the animal to increase or stimulate a secondary immune response. A booster can be administered at a time after the first administration, for example, 1 to 8 weeks, preferably 2 to 4 weeks, after the first administration of the composition. Subsequent boosters can be administered one, two, three, four, or more times annually. Without attempting to be bound by theory, it is expected that annual boosters will not be necessary, as an animal will be affected in the field by exposure to members of the genus Campylobacter that express polypeptides with epitopes that are identical or structurally related to epitopes present on the polypeptides. present in the composition administered to the animal.

[0054] The disclosure is directed to methods of inducing antibody production in an animal or by recombinant techniques. The antibody produced includes antibodies that specifically bind at least one polypeptide present in the composition. In this part of the disclosure, an "effective amount" is an amount effective to result in the production of antibodies in the animal. Methods for determining whether or not an animal has produced antibodies that specifically bind polypeptides present in a composition of the disclosure can be determined as described herein.

[0055] The method can be used to produce antibodies that specifically bind polypeptides expressed by a microbe other than the microbe from which the polypeptides of the composition were isolated. As used herein, an antibody that can specifically bind a polypeptide is an antibody that interacts with the epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope. At least some of the polypeptides present in the compositions of the present invention typically include epitopes that are conserved in the polypeptides of different species and different genera of microbes. Therefore, antibodies produced using a composition derived from one microbe are expected to bind to polypeptides expressed by other microbes and provide broad spectrum protection against gram negative organisms. Examples of gram negative microbes to which the antibodies specifically bind are enteropathogens, for example, members of the Enterobacteriaceae family.

[0056] The disclosure is also directed to treating an infection in an animal caused by a member of the genus Campylobacter. The method includes administering an effective amount of the composition of the present invention or disclosure to an animal with an infection caused by a member of the genus Campylobacter, and determining whether Campylobacter spp. that causes the infection has decreased or not. Methods for determining whether or not an infection is caused by a member of the genus Campylobacter are routine and known in the art.

[0057] The disclosure is also directed to methods of treating one or more symptoms of certain diseases in animals, preferably humans, that may be caused by, or associated with, infection by a member of the genus Campylobacter. Examples of diseases caused by infections with Campylobacter spp. include diarrhea, fever, and abdominal cramps, as well as symptoms such as bacteremia, septic arthritis, Guilain-Barre syndrome Reiter syndrome (Peterson et al. Wes. J. Med. 161: 148-152 (1994) and Allos et al. J. Infest Dis. 176: S125-128 (1997)). Treatment of these diseases can be prophylactic or, alternatively, it can be started after the development of a disease described herein. Treatment that is prophylactic, eg, initiated before a subject manifests symptoms of a disease caused by Campylobacter spp., Is referred to herein as treatment of a subject who is "at risk" for developing the disease. Normally, an animal at risk ”of developing a disease is an animal with probable exposure to Campylobacter spp. that causes the disease. For example, the animal is present in an area where the disease has been diagnosed in at least one other animal, or is being transported to an area where a Campylobacter spp. is endemic, and / or where diseases caused by Campylobacter spp. are prevalent. Therefore, the administration of a composition can be carried out before, during or after the appearance of the diseases described herein. Treatment started after the development of a disease can reduce the severity of the symptoms of one of the diseases, or eliminate the symptoms completely. In this part of the disclosure, an effective amount is an amount effective to prevent the manifestation of the symptoms of a disease, decrease the severity of the symptoms of a disease, and / or completely eliminate the symptoms. The potency of a composition of the present invention or disclosure can be tested according to routine methods (see, for example, Stanfield et al., Microb Pathog., 3: 155-165 (1987), Fox et al., Am. J Vet Res., 48: 85-90 (1987), Ruiz-Palacios, Infect. Immun., 34: 250-255 (1981), and Humphrey et al., J. Infect. Dis., 151: 485-493 (1985)). Methods for determining whether an animal has the diseases disclosed herein and the symptoms associated with the diseases are routine and known in the art.

[0058] The disclosure is also directed to reduce colonization of the intestinal tract or reproductive tract of an animal by a Campylobacter spp. The method includes administering an effective amount of a composition of the present invention or disclosure to an animal colonized by, or at risk of being colonized by, a gram-negative microbe, preferably a Campylobacter spp. In this part of the disclosure, an effective amount is an amount effective to decrease colonization of the animal by the microbe. Colonization of an animal's intestinal tract by a microbe can be determined by measuring the presence of the microbe in the animal's feces. Methods for evaluating the colonization of an animal reproductive tract by a microbe are routine and known in the art. It is expected to reduce the colonization of an animal by Campylobacter spp. it will reduce the transmission of Campylobacter spp. to the humans.

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[0059] A composition of the invention and disclosure can be used to provide passive immunization against infection by Campylobacter spp. For example, the composition can be administered to an animal to induce the production of immune products, such as antibodies, which can be collected from the producing animal and administered to another animal to provide passive immunity. Immune components, such as antibodies, can be collected to prepare antibody compositions of serum, plasma, blood, colostrum, etc. for passive immunization therapies. Antibody compositions including monoclonal antibodies, anti-idiotypes, and / or recombinant antibodies can also be prepared using known methods. Passive antibody compositions and fragments thereof, for example scfv, Fab, F (ab ') 2 or Fv or other modified forms thereof, can be administered to a recipient in the form of serum, plasma, blood, colostrum. , and the like. However, the antibodies can be isolated from serum, plasma, blood, colostrum and the like, using known methods and spray dried or lyophilized for later use in reconstituted or concentrated form. Passive immunizing preparations may be particularly advantageous for the treatment of acute systemic disease, or passive immunization of young animals that failed to receive adequate levels of passive immunity through maternal colostrum.

The disclosure also provides methods for detecting antibodies that specifically bind polypeptides of the disclosure. These methods are useful in, for example, detecting whether an animal has antibodies that specifically bind polypeptides of the disclosure, and diagnosing whether an animal may have an infection caused by Campylobacter spp. Preferably, such diagnostic systems are in kit form. The methods include contacting an antibody with a preparation that includes at least one polypeptide of the disclosure result in a mixture. Preferably, the antibody is present in a biological sample, more preferably blood, serum, milk, mucous secretions, or colostrum. The method further includes incubating the mixture under conditions that allow the antibody to specifically bind a polypeptide to form a polypeptide: antibody complex. As used herein, the term "polypeptide complex: antibody" refers to the complex that results when an antibody specifically binds to a polypeptide. The preparation that includes the polypeptides present in a composition of the present invention or disclosure may also include reagents, for example a buffer, that provide appropriate conditions for the formation of the polypeptide: antibody complex. The polypeptide: antibody complex is then detected. Detection of antibodies is known in the art and can include, for example, immunofluorescence and peroxidase. The methods for detecting the presence of antibodies that specifically bind polypeptides of the disclosure can be used in various formats that have been used to detect antibodies, including radioimmune assay and enzyme-linked immunosorbent assay.

The disclosure also provides a kit for detecting antibodies that specifically bind polypeptides of the disclosure. The kit includes at least one polypeptide of the disclosure in suitable packaging material in an amount sufficient for at least one assay. Optionally, other reagents such as buffers and solutions needed to practice the invention are also included. Instructions for use of the packaged polypeptides are also typically included.

[0062] As used herein, the phrase packaging material refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label indicating that the polypeptides can be used to detect antibodies induced by infection with Campylobacter spp. In addition, the packaging material contains instructions indicating how the materials within the kit are used to detect such antibodies. As used herein, the term "packaging" refers to a matrix or solid matter such as glass, plastic, paper, aluminum, and the like, capable of holding polypeptides within fixed limits. Thus, for example, a container may be a well of microtiter plates to which microgram quantities of polypeptides have been attached. Instructions for use typically include a tangible expression describing the concentration of the reagent or at least one parameter of the assay method, such as the relative amounts of reagent and sample to be mixed, holding time periods for the reagent / sample mixtures, temperature , buffer conditions, and the like.

EXAMPLE

Example 1

Production and Isolation of Metal-Regulated Proteins

[0063] Campylobacter spp. jejuni can be grown under controlled fermentation conditions so that it expresses proteins, including proteins associated with the outer membrane. Bacteria can be harvested and proteins can then be isolated and used as immunogens in a composition described in detail in the following example. The microaerophilic conditions for the culture of C. jejuni on plates and in small liquid cultures were established by incubation in an anaerobic flask containing a CampyPak gas generator system (BBL, Sparks, MD). A master seed stock of Campylobacter jejuni originating from a turkey was prepared by inoculating the isolate into 200 ml of Porcine Brain Heart Infusion Broth (P-BHI, Difco) containing 0.025% metabisulfite (Sigma) and containing 10 to 20 micrograms per milliliter (pg / ml) of 2,2-dipyridyl (Sigma-Aldrich St. Louis, MO). The culture was continued without shaking for 16 hours at 37 ° C under microaerophilic conditions. Before development in a starter culture, C. jejuni was adapted to grow on the 2,2-dipyridyl iron chelator

ES 2 395 022 T3 by repeatedly subculturing the isolate in increasing concentrations of the iron chelator, starting at 10 pg / ml, and increasing to 20 pg / ml. The bacteria were collected by centrifugation at 10,000 x g. The bacterial pellet was resuspended in 20 ml P-BHI containing 20% ​​glycerol, and dispensed sterile in 2 ml cryogenic vials (1 ml per vial) and stored at -90 ° C. The isolate was given the Campy-1 identification, and established as a master seed. The master seed was expanded into an active seed which was then used for the production of metal-regulated proteins. This strain was deposited with the American Type Culture Collection, PO Box 1549, Manassas, Va., 20108, USA, on September 20, 2004. The deposit was made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of the Patent Procedure.

Example 2

Production of metal-regulated proteins

[0064] Fermentation: A cryogenic vial of the active seed (1 ml to 10⁹ CFU / ml) was used to inoculate 130 ml of P-BHI or Soy T at 37 ° C containing 15-20 micrograms (pg) of 2,2-dipyridyl and 0.025% metabisulfite (Sigma) and it was incubated in an anaerobic flask containing a Campy-Pak gas generator system (BBL, Sparks, MD). The culture was incubated at 37 ° C for 12-24 hours at which point it was sterilely transferred to 1.3 liters of the previous medium. This second culture was allowed to grow for an additional 10 hours at 37 ° C. This culture was used to inoculate a 20-liter Bioflo IV benchtop fermenter, (New Brunswick Scientific Co, Edison NJ) loaded with 13 liters of the above-described medium. The pH was kept constant between 6.9 and 7.1 by automatic titration with NaOH 30% and HCl 10%. The stirring speed was adjusted to 100 revolutions per minute (rev / minute), and the culture was maintained under microaerophilic conditions. The culture was allowed to develop continuously under these conditions for 24 hours at which point the fermentation was terminated by reducing the fermentation temperature to 10 ° C.

[0065] Harvest: Bacterial fermentation was concentrated and washed using a Millipore Pellicon Tangential Flow Filter set (Millipore Corporation, Bedford, MA), equipped with a 25 ft screen channel Alpha 300K series Centrasette filter.² (Pall Filtron). The original culture volume of 13 liters was reduced to 2.5 liters. The bacterial concentrate was then adjusted to 25 liters using physiological saline (0.85%) and then concentrated again to 2.5 liters to help remove any non-cell associated contaminants, eg secreted proteins. The concentrate (2.5 liters) was adjusted to 15 liters using Osmotic Shock Buffer (OMS) containing 7.26 grams / liter Tris-base and 0.93 grams / liter EDTA adjusted to a pH of 8.5. The concentrate was mixed thoroughly and evenly dispensed (3.0 liters each) into 5 sterile four liter Nalgene containers and placed in a -20 ° C freezer for storage. The granulated mass was calculated by centrifugation of 30 ml samples of the fermented culture and final harvest. Briefly, pre-weighed 50 ml Nalgene conical tubes were centrifuged at 39,000 xg for 90 minutes in a Beckman J2-21 centrifuge using a 21-JA rotor (Beckman Instruments, Palo Alto CA). At the end of the run, the supernatant was poured out and the tubes were weighed again. The granulated mass was calculated for each stage.

[0066] Breakdown (Homogenization): Three liters of bacterial cell suspension frozen in OMS were thawed at 4 ° C (180g of granulated mass). The liquid culture suspension was aseptically transferred to a 50 liter jacketed process tank containing 44 liters of WHO pH 8.5 containing 0.1 grams thimerosal / liter as preservative. The bulk bacterial suspension was cooled to 4 ° C with continuous mixing for 18 hours at 200 rpm at which time it was rotated by homogenization. Briefly, the 50 liter tank containing the bacterial suspension was connected to a Rannie Homogenizer model 12.51 H, (APV Systems, Rosemont, IL). A second 50 liter jacketed process tank (empty) was connected to the homogenizer so that the fluid in the process tank could be passed through the homogenizer, into the empty tank and back again, allowing multiple homogenizer passes while still maintaining a closed system. The temperature during homogenization was kept at 4 ° C. At the beginning of each pass, 70 psi fluid was circulated through the homogenizer and back to the source tank, while the homogenizer pressure was adjusted to 13,500 psi. Before the first pass, two pre-homogenization samples were removed from the homogenizer to establish a baseline to determine the degree of breakdown and monitoring of pH. The degree of breakage was monitored by transmittance (% T at 540nm in 1: 100 dilution) compared to the non-homogenized sample. The bacterial suspension was passed 3 times through the homogenizer to give a final transmittance percentage between 78-83% T at a dilution of 1: 100.

[0067] After homogenization, sodium lauroyl sarcosinate (Hamptosyl L-30, Chem / Serv, Minneapolis, MN) was added aseptically to the homogenized bacterial suspension for solubilization. The amount of Sarcosine (30%) added was 0.0664 times the solubilization volume, in liters, (1.0 gram of sarcosine / 4.5 grams of granulated mass). The process tank was removed from the homogenizer and kept at 4 ° C while stirring at 240 rpm for 60-70 hours.

[0068] Protein Harvest: Proteins within the solubilized process fluid were harvested by centrifugation using T-1 Sharples, (Alfa Laval Seperations, Warminster, PA). Briefly, the homogenate was fed at 6 Sharples with a feed rate of 250 ml / minute at 17 psi at a centrifugal force of 60,000 x g. The temperature during centrifugation was kept at 4 ° C. the solubilized homogenate was passed through the centrifuges twice. The protein was collected, resuspended and dispensed in 10 liters of Tris-buffer pH 8.5 containing 0.3% formalin (Sigma) as a preservative.

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[0069] Diafiltration: The protein suspension (10 liters) was adjusted to 60 liters using sterile Tris-buffer, pH 8.5. The suspension was washed and dialyzed using a Millipore Pellicon Tangential Flow Filter set (Millipore Corporation), equipped with a 25 ft screen channel Alpha 300K series Centrasette filter.² (Pall Filtron) to remove residual sarcosine. The protein solution was concentrated by filtration to a target volume of 10 liters at which point 50 liters of Tris-liter pH 7.4 containing 5% isopropyl alcohol was slowly added to the concentrate from a second process tank. Isopropyl alcohol is thought to cause a slight splitting of the protein structure allowing the removal of bound sarcosine without compromising the immunogenicity of the proteins. Diafiltration continued until the pH stabilized at 7.4 at which point 50 liters of Tris-liter pH 7.4 was slowly added by diafiltration to remove residual alcohol. The protein suspension was then likewise concentrated to approximately 5 liters. The protein concentrate was dispensed equally (500 ml) into ten sterile 1 liter Nalgene containers and stored at -20 ° C until use.

Example 3

Protein Analysis.

[0070] The protein profile of isolated C. jejuni grown in iron saturation and / or iron deficiency media was examined by SDS-PAGE. Briefly, the organism was developed from a stock of frozen master seeds by subculturing in 25 ml of P-BHI containing 0.025% metabisulfite and 15 to 20 micrograms per milliliter (pg / ml) of 2,2-dipyridyl (Sigma-Aldrich St Louis, MO) and / or P-BHI with metabisulfite containing 200 uM of ferric chloride incubated for 18 hours at 37 ° C while shaking at 100 rpm. At 18 hours of incubation, 5 ml of each culture were transferred to 500 ml of pre-incubated media (37 ° C) with iron saturation and / or iron deficiency. The cultures were allowed to grow for 18 hours at 37 ° C while shaking at 100 rpm. At 18 hours after incubation, each culture was centrifuged at 10,000 xg for 20 minutes. The bacterial pellet was resuspended in about 100 ml of tris-buffered saline and centrifuged at 10,000 xg for 10 minutes to remove any contaminating proteins from the medium. The bacterial granule of the media with iron saturation and / or iron deficiency was resuspended in 40 ml tris-buffered saline solution of pH 7.2 and rotated by sonication. The broken bacterial suspension was clarified by centrifugation at 32,000 xg for 12 minutes. The supernatant was collected and solubilized by the addition of sodium lauroyl sarcosinate 4% vol / vol at 4 ° C for 24 hours. The fraction enriched in detergent insoluble OMP was collected by centrifugation at 32,000 xg for 2.5 hours at 4 ° C. The OMP granule was resuspended in 200 µl tris-buffer at pH 7.2 and stored at -90 ° C. A sample of each extract was redissolved on a 10% SDSPAGE gel to compare the protein profile obtained from cells grown in media with iron saturation and / or iron deficiency. The gel was scanned using a BioRad GS-800 densitometer to compare the difference in the protein profile of C. jejuni developed under conditions of iron saturation and / or iron deficiency.

Example 4

Preparation of immunizing compositions derived from C. jejuni

[0071] The composition made from C. jejuni as described in Example 2 was used to prepare a vaccine. A stock vaccine was prepared from the composition by diluting the antigen in phosphate buffered saline (PBS) containing 8.0 g / l of NaCl, 0.2 g / l of KCl, 1.44g / l of Na2HPO4 and 0.24g / l of KH2HPO4 pH 7.4 containing 10% aluminum hydroxide (Rehydrogel, Reheis Chemical Company Berkeley Heights, NJ). The aluminum hydroxide suspension (500 pg total protein / ml) was then emulsified in the commercial adjuvant, EMULSIGEN, (MVP Laboratories, Ralston, Nebraska) using an IKA Ultra Turrax T-50 homogenizing container (IKA, Cincinnati, OH). A mouse dose was administered to give a final dose of 50 pg of total protein in an injectable volume of 0.1 ml with an adjuvant concentration of 22.5% vol / vol. A placebo was prepared by replacing the antigen with physiological saline in the previous formulation and emulsifying the suspension in EMULSIGEN to give an adjuvant concentration of 22.5%.

Example 5

Preparation of the affected organism

[0072] The C. jejuni isolate as described above was used for the affectation. Briefly, the isolate from a frozen stock (Example 1) was rapidly passed onto a blood agar plate and incubated at 37 ° C for 18 hours. Several colonies were cultured in 50 ml P-BHI containing 15 pg / ml of 2,2 'dipyridyl and 0.025% metabisulfite. The culture was incubated at 37 ° C for 16 hours, and then centrifuged at 10,000 xg for 10 minutes at 4 ° C to granulate the bacteria. The bacterial pellet was washed once by centrifugation (10,000 xg for 15 minutes) at 4 ° C. The final granule was resuspended in 25 ml of P-BHI without dipyridyl. Just before involvement, 1 ml of the above bacterial suspension was serially diluted ten times to list the number of CFU / dose.

Example 6

Mice vaccination and oral challenge study with Camplylobacter jejuni. (Fecal Stool Evaluation)

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[0073] In this experiment the efficacy of the C. jejuni vaccine was carried out against a live oral disease in mice. The outcome parameters used to evaluate the efficacy of the vaccine in this experiment were 1) individual mortality of the mice, and 2) differences in the concentration of Campylobacter deposited between treatment groups after involvement. Twenty (N = 20) female CF-1 mice obtained from Harlan Breeding Laboratories (Indianapolis, IN) weighing 16-22 grams were distributed equally in two groups (10 mice / group). Mice were housed in polycarbonate mouse cages (Ancore Corporation, Bellmore, NY). Two cages were used, one for each treatment group. The groups were designated as placebo, unvaccinated (Group 1), and vaccinated (Group 2). Food and water were supplied discretely to all mice.

[0074] Mice were vaccinated three times at 14-day intervals subcutaneously with the placebo and / or the C. jejuni vaccines described in Example 4. The volume of vaccine administered was 0.1 ml / mouse. Fourteen days after the third vaccination, mice in groups 1 and 2 were orally affected with C. jejuni at 4.05 x 10⁹ colony forming units (CFU) in a volume of 0.2 cc. The target organism was prepared as described in Example 5.

[0075] To enumerate the difference in fecal stool between the control and vaccinated groups, the mouse droppings were collected at 12 hours after challenge. The droppings were collected by placing a sterile pad on the floor of each cage 1 hour before collection. At each time period the pad was removed and placed on a laminar flow cover. Using fire sterilized forceps, twenty individual droppings were collected at random. The forceps were flamed between each collection to avoid cross-contamination of the samples. Individual droppings were placed in sterile saline dilution blankets (0.9 ml), two droppings per tube, to give ten tubes. Each sample was macerated using a sterile 1 ml pipet and serially diluted 10 times. The dilutions were plated on Campylobacter Agar (Difco Laboratories, Detroit, MI) incubated at 37 C CO2 5% for 72 hours. The number of bacteria was enumerated for each sample and the log10 of the colony forming units was averaged for each treatment group in each time period.

[0076] Table 1 shows the difference in fecal stool between vaccinated and unvaccinated mice after oral involvement with C. jejuni at each time period. There was a large difference between treatment groups in the amount of Campylobacter deposition in feces after involvement. The dose of affectation represented as time 0 in Table 1 shows the initial inoculum given to each mouse. In twelve hours after the affectation there was a drastic decrease in the amount of Campylobacter deposited from the vaccinated group compared to the Placebo group. Averaged over the study period and counting with repeated estimates, the vaccinated deposited less Campylobacter in each sampling period when compared to the unvaccinated, with a degree of importance of P = 0.005. The amount of Campylobacter deposited in the vaccinated group decreased dramatically with each sampling period compared to the non-vaccinated placebo group (Figure 2). In 12 hours after the affectation, the difference in the amount of Campylobacter deposited between the vaccinated and non-vaccinated groups was greater than 3 logs (Table 1, Figure 2).

TABLE 1

<td colspan="3">Table 1. Difference in Deposition of Campylobacter jejuni Between the Treatment Groups of Unvaccinated and Vaccinated After Oral Infection.</td>

<td></td><td colspan="2">Mean log10 Colony Forming Units</td>

<td>Sampling Times</td><td>Group 1 (not vaccinated)</td><td>Group 2 (vaccinated)</td>

<td>Dose of affectation (moment 0)</td><td> 9.607</td><td> 9.607</td>

<td>12 hours</td><td> 3·6</td><td>0 (a)</td>

<td colspan="3">(a) The detection limit of the assay was 10 '¹</td>

[0077] The experiment was terminated at 12 hours due to contamination with a Pseudomonas aeruginosa that developed by selective antibiotics on Campylobacter agar in all subsequent samplings. No mortality was observed in any mouse after involvement. The results clearly demonstrate that subcutaneous vaccination with the composition results in a significant difference (P = 0.005) in Campylobacter colonization compared to an unvaccinated Placebo group.

[0078] The above detailed description and examples have been provided for clarity of understanding only. This should not be understood as unnecessary limitations. The invention is not limited to the exact details shown and described, since variations obvious to one skilled in the art will be included within the invention defined by the claims.

ES 2 395 022 T3

[0079] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the headline, unless otherwise specified.