Clostridium chauvoei vaccine and method of preparation

Abstract

We provide a vaccine against C. chauvoei. [Solution] A vaccine comprising the C chauvoei component and additional cctA protein is provided. Methods for producing and using the vaccine are also provided.

Description

Technical Field

[0001] The present invention relates to the field of Clostridium vaccines for ruminants.

Background Art

[0002] Clostridium myositis (blackleg) caused by Clostridium chauvoei is a very economically important disease in sheep and cattle. Clostridium chauvoei is a gram-positive anaerobic bacillus that forms spores. The spores are ubiquitous in soil and manure and can pass through the intestinal mucosa after ingestion, enter the bloodstream, and be carried to skeletal muscle. The spores remain dormant until local trauma to the muscle, which in cattle is most often caused by a blow during handling of shoots or by trauma in a crowded feedlot, causing muscle damage and local hypoxia and anoxia.

[0003] Current knowledge about the pathogenicity of C. chauvoei has revealed that toxins, highly active DNase, hyaluronidase, sialidase, and flagella represent the main pathogenic factors. Among the putative toxins, C. chauvoei toxin A (CctA) has been shown to represent the major cytotoxic and hemolytic activities of C. chauvoei.

[0004] In addition to toxin CctA, sialidase NanA and hyaluronidase NagH appear to enable C. chauvoei to move from the initial site of infection, which is generally considered to be the oral cavity or respiratory tract or occasionally skin lesions, to the muscle tissue where the bacteria can replicate and cause muscle necrosis.

[0005] Generally, there is no effective treatment for cattle with blackleg, and death occurs rapidly. Prevention is the best approach. Vaccination against Clostridium toxins and maintenance of a safe environment are important.

[0006] Vaccines for black leg disease consist of chemically inactivated bacteria that provide the bacterial culture supernatant expected to contain the outer membrane and flagellar proteins proposed as immunogens, as well as the main toxin. Recently, it has been shown that CctA toxin alone, conserved among C. chauvoei strains isolated worldwide and prepared by recombinant gene technology, provided effective protection in a guinea pig infection model serving as a biological test for efficacy testing in batch release procedures for commercially available vaccines.

[0007] While there is ample literature on black leg disease, there is limited scientific evidence regarding the effectiveness of C. chauvoei vaccination in preventing the disease and lethality in cattle. Uzal et al. found the evidence for the effectiveness of C. chauvoei vaccine in preventing infection by this microorganism in cattle to be low to moderate. See Vet Clin North Am Food Anim Pract. 2012 Mar;28(1):71-7,viii. Therefore, there is a need in this field for an additional vaccine that can protect ruminants from C. chauvoei infection. [Overview of the project]

[0008] In a first aspect, the Disclosure provides a vaccine against Clostridium chauvoei, the vaccine comprising a C chauvoei component and an additional cctA protein.

[0009] In a different embodiment of this first aspect, a single dose of the vaccine is at least about It contains 3.5 μg of the additional cctA protein and 0.025 to 0.148 RU of flagella. In a particular preferred embodiment, the vaccine according to the first aspect of the present invention contains about 0.074 RU of flagella and about 5 μg of the additional cctA protein. In the vaccine according to any embodiment of the first aspect, the cctA protein is produced from the supernatant of cultured C. chauvoei, and / or the C. chauvoei component is bacteria, whole cell extract, partial cell extract, or a combination thereof.

[0010] In a second aspect, the Disclosure provides a method for preparing a vaccine against C chauvoei infection, the method being: Culturing aC chauvoei and b. Collecting the culture medium, c. Concentrate C chauvoei from the culture medium, d. Concentrating cctA from the culture medium, e. This includes combining the concentrated cctA with C chauvoei.

[0011] In a particular embodiment, this method includes the step of microfiltration the culture medium to obtain a microfiltered retention solution and a microfiltered permeate.

[0012] In a particular embodiment of this second aspect of the present invention, the step of culturing C chauvoei may be carried out in a culture medium containing plant-derived peptone, animal-derived peptone, or a combination thereof.

[0013] In a particular embodiment, the step of concentrating the cctA from the culture medium is: a. Obtain an ultrafiltered retaining solution by ultrafiltration of the precisely filtered permeate, and subsequently, b. Obtain the retained solution filtered by dialysis filtration, and subsequently c. Obtaining a sterile solution of the additional cctA protein by sterile filtration, the method comprises these steps.

[0014] In certain embodiments, ultrafiltration includes passing the microfiltered permeate through a 10 kDa cassette. In certain embodiments, diafiltration includes passing the ultrafiltered retained solution through a 10 kDa cassette, and / or the diafiltration is subsequently followed by the preparation of an additional sterile solution of cctA protein by sterile filtration of the dialyzed retained solution through a 0.2 μm filter.

[0015] Preferably, in a method according to any of the embodiments of this second aspect of the present invention, the microfiltered retaining solution is dialyzed to obtain the C chauvoei component.

[0016] In the most preferred embodiment, at least a portion of the C chauvoei component is combined with at least a portion of a sterile solution of additional cctA protein.

[0017] In a third aspect, the Disclosure provides a vaccine prepared according to any embodiment of a second aspect of the Invention, wherein a single dose of the vaccine contains at least 3.5 μg of concentrated cctA protein.

[0018] In certain embodiments of the third aspect, a single dose of the vaccine contains about 0.074 to 0.148 RU of the flagellum. Preferably, a single dose contains about 5 μg of the concentrated cctA, and more preferably, a single dose contains about 5 μg of the concentrated cctA and about 0.074 RU of the flagellum.

[0019] In certain embodiments, the vaccine may contain at least one additional antigen selected from the group consisting of Clostridium septicum, Clostridium haemolyticum, Clostridium novyi, Clostridium sordellii, Clostridium tetani, Clostridium perfringens types C and D, Erysipelothrix rhusiopathiae, Leptospira borgpetersenii serovar Hardjo, Leptospira interrogans serovar Pomona, and Mannheimia haemolytica antigens. In certain embodiments, the at least one additional antigen is an inactivated culture or a toxoid. In any embodiment of this third aspect, the vaccine may be non - adjuvant or may contain an adjuvant.

[0020] In a fourth aspect, the present disclosure provides a method of preventing C chauvoei infection in a subject, the method comprising administering to the subject a vaccine according to any one of the embodiments of the first or third aspect of the present invention. In certain embodiments of this fourth aspect, the subject is a bovine or ovine subject.

Brief Description of the Drawings

[0021] [Figure 1] Schematic diagram of a process for preparing a C chauvoei component and concentrated cctA for the preparation of a vaccine according to certain embodiments of the present invention.

Modes for Carrying Out the Invention

[0022]

[0022] For a better understanding of the present invention, the following non - limiting definitions are provided.

[0023] The phrase "vaccine containing additional cctA protein" refers to a vaccine containing a C chauvoei component spiked with additional purified or partially purified cctA protein. Thus, in a vaccine containing additional cctA protein, the ratio of cctA to flagella (substituting for the amount of C chauvoei) will be greater than that ratio in a vaccine lacking such additional cctA. In certain embodiments, the ratio of cctA to flagella is at least 50% greater, or at least 100% greater, or at least 300% greater, or at least 500% greater, or at least 1000% greater, or at least 1500% greater, or at least 2000% greater.

[0023]

[0024] The term "about" as applied to a reference numeral refers to a reference numeral that is plus or minus 10 percent of that value.

[0024]

[0025] The term "bacterin" refers to a suspension of killed bacteria.

[0025]

[0026] The term "C chauvoei component" refers to C chauvoei bacteria, C chauvoei whole cell extract, C chauvoei partial cell extract, or any combination thereof.

[0026]

[0027] The term "culture", as used herein, means a population of cells or microorganisms that grow in the absence of other species or types.

[0027]

[0028] "Dose" refers to a vaccine or immunogenic composition administered to a subject. "First dose" or "priming dose" refers to the dose of such composition administered on day 0. "Second dose" or "third dose" or "annual dose" refers to the amount of such composition administered following the first dose, which may be the same vaccine or immunogenic composition as the first dose, but is not required to be. be.

[0028]

[0029] "Lysate" refers to lysate products, which are the breakdown of cells due to the rupture of the cell wall or membrane. Breakdown can be achieved by mechanical or chemical means known in the art.

[0029]

[0030] The term "partial cell extract" refers to a fraction of a whole cell extract, and the extract can induce an immune response that is substantially the same as the immune response induced by the whole cell extract. Preferably, the partial cell extract includes the cell wall and / or cell membrane fraction. To avoid doubt, the partial cell extract does not contain purified and / or concentrated cctA.

[0030]

[0031] When used herein in relation to vaccines or other compositions, “protection,” “protection,” “protective immunity,” etc., means that the vaccine or composition prevents or reduces the symptoms of a disease caused by an organism from which the antigen(s) used in the vaccine or composition are derived. The terms “protection,” “protection,” etc., also mean that the vaccine or composition can be used to “treat” a disease or one or more symptoms of a disease that already exists in a subject.

[0031]

[0032] "Therapeutic dose" refers to the amount of antigen or vaccine that would induce an immune response in a subject receiving the antigen or vaccine, sufficient to prevent or mitigate signs or symptoms of a disease, including adverse health effects or complications resulting from infection by a pathogen such as a virus or bacteria. Humoral immunity, cell-mediated immunity, or both may be induced. The immunogenic response of an animal to a vaccine can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly, by monitoring signs and symptoms after challenge with a wild-type strain. The protective immunity conferred by a vaccine can be assessed by measuring, for example, a reduction in clinical signs such as mortality, morbidity, body temperature, overall physical condition, and the overall health and capacity of the subject. The therapeutically effective dose of vaccine may vary depending on the specific adjuvant used, the specific antigen used, or the condition of the subject, and may be determined by those skilled in the art.

[0032]

[0033] "To treat" means to prevent a disorder, condition, or disease to which such term applies, or to prevent or alleviate one or more symptoms of such disorder, condition, or disease.

[0033]

[0034] When used herein, “vaccine” or “vaccine composition” refers to an immunogenic composition containing an antigen that can induce a specific immune response to the antigen. Administration of a vaccine to a subject results in a protective immune response, which may be fully protective or partially protective. Vaccines can be directly introduced to a subject by any known route of administration, including parenteral or oral. These terms mean a composition that prevents or reduces infection, or prevents or reduces the signs or symptoms of one or more infections. The protective effect of a vaccine composition against a pathogen is usually achieved by inducing an immune response in the subject. Generally, eliminating or reducing the incidence of infection, improving signs or symptoms, or accelerating the elimination of microorganisms from an infected subject demonstrates the protective effect of a vaccine composition.

[0034]

[0035] As used herein, "veterinary acceptable" means a substance that, within reasonable medical judgment, is suitable for use in contact with the tissues of a veterinary subject without causing excessive toxicity, irritation, allergic reactions, etc., is commensurate with a reasonable benefit-to-risk ratio, and is effective for its intended use.

[0035]

[0036] As used herein, “veterinary-acceptable carrier” refers to the biological carrier of the active ingredient. This refers to a carrier culture medium that does not interfere with the efficacy of the veterinary activity and is not toxic to the veterinary subject to which it is administered.

[0036]

[0037] Terms such as "whole cell extract" or "whole organism extract" refer to cell lysates, including bacterial cell lysates, such as C. chauvoei cell lysates.

[0037]

[0038] This disclosure is based on the unexpected discovery that cctA can be concentrated from the supernatant of cultured C chauvoei, that adding this concentrated cctA to the C chauvoei component provides a more efficient vaccine than C chauvoei alone, and that if the vaccine contains additional cctA protein, the amount of C chauvoei component per dose can be reduced without sacrificing vaccine efficiency.

[0038]

[0039] Accordingly, in a first aspect, the present invention provides a vaccine comprising Clostridium chauvoei component and further comprising an additional cctA protein.

[0039]

[0040] C chauvoei ingredients

[0041] In certain embodiments, the C chauvoei compound is a bacterium. In other embodiments, C chauvoei is a whole cell extract. In yet another embodiment, C chauvoei is a partial cell extract. Preferably, the partial cell extract includes a C chauvoei membrane fraction and / or a C chauvoei cell wall fraction. Different combinations of these embodiments are also possible.

[0040]

[0042] According to this disclosure, the amount of C chauveoei component is quantified using flagellar relative units (RUs). Therefore, the use of flagellar RUs is a substitute for the amount of C chauveoei. However, it should be understood that other proteins may be used as substitutes for quantifying the amount of C chauveoei component.

[0041]

[0043] Several methods are known for preparing the C chauvoei component. For example, cultured and harvested C chauvoei can be inactivated with formalin, betapropriolactone (BPL), or binary ethyleneimine (BEI), or by other methods known to those skilled in the art.

[0042]

[0044] Whole cell extracts can be prepared by rupturing the cell walls and membranes of bacteria. This can be achieved by a number of means, including chemical and physical methods. For example, bacteria can be dissolved by using hypotonic buffer and / or lyophilization and / or centrifugation. Partial cell extracts can be obtained, for example, by ultracentrifugation of the C. chauvoei preparation.

[0043]

[0045] Suitable preparations of C chauvoei include, but are not limited to, products currently marketed as components of commercial vaccines, including, ONE SHOT ULTRA® 8 or ULTRAVAC® 7 IN 1 by Zoetis.

[0044]

[0046] In certain embodiments, the C chauvoei component is a bacterium prepared by culturing C chauvoei and subsequently inactivating it with formalin. The suspension containing the inactivated C chauvoei is then microfiltered through a 0.2 μm cassette and subsequently dialyzed with physiological saline through a 0.2 μm microfiltration cassette.

[0045]

[0047] Additional cctA protein

[0048] The cctA protein according to the present invention is, SEQ ID NO: [Table A] Alternatively, the protein may be at least 90% identical to SEQ ID NO: 1 and at least 91%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to SEQ ID NO: 1. Preferably, the amino acids that differ between the sequence of cctA and SEQ ID NO: 1 are substitutions, and more preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of these substitutions are conservative substitutions.

[0046]

[0049] Those skilled in the art will recognize that changes in nucleic acid sequence resulting from modifications to the amino acid sequence of a encoded protein have little effect on the resulting three-dimensional structure of the protein. For example, the codon of the amino acid alanine, a hydrophobic amino acid, can be substituted with a codon encoding another less hydrophobic residue such as glycine, or a more hydrophobic residue such as valine, leucine, or isoleucine. Similarly, changes resulting from the substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a protein with substantially the same functional activity.

[0047]

[0050] The following six groups each contain amino acids that are typical conservative substitutions of each other: [1] alanine (A), serine (S), threonine (T), [2] aspartic acid (D), glutamic acid (E), [3] asparagine (N), glutamine (Q), [4] arginine (R), lysine (K), histidine (H), [5] isoleucine (I), leucine (L), methionine (M), valine (V), and [6] phenylalanine (F), tyrosine (Y), tryptophan (W) (see U.S. Patent Publication No. 20100291549).

[0048]

[0051] C chauvoei cctA protein can be prepared by a number of methods, including purification of cctA protein from the supernatant of cultured C chauvoei, and / or genetic manipulation, and / or chemical synthesis. These methods are well known in the art.

[0049]

[0052] For the purposes of this disclosure, the amount of the C chauvoei component in a single dose of the vaccine is determined by the amount of flagella measured in relative units, or RU. A single dose of the vaccine without additional cctA contains approximately 0.148 RU of flagella. However, it should be noted that other methods exist for determining the amount of the C chauvoei compound, and the final results of these methods can be converted to flagellar RU.

[0050]

[0053] In vaccines disclosed herein that include additional cctA protein, the ratio of cctA to flagella is greater than the ratio in vaccines lacking this additional cctA. In certain embodiments, the ratio of cctA to flagella is at least 50% greater, preferably at least 75% greater, at least 100% greater, at least 150% greater, at least 200% greater, at least 250% greater, at least 300% greater, at least 350% greater, etc., compared to a composition containing the C chauvoei component but lacking the added cctA protein.

[0051]

[0054] In certain embodiments, the vaccine according to the present invention contains about 3.5 μg or more of the additional cctA protein per dose, and in different embodiments, it may contain up to 30 μg of the additional cctA protein per dose. The vaccine may contain 3.5 to about 4 μg of the additional cctA protein per dose, or 3.5 to about 6 μg of the additional cctA protein per dose, or 3.5 to about 9 μg of the additional cctA protein per dose, or 3.5 to about 10 μg of the additional cctA protein per dose, or 3.5 to about 15 μg of the additional cctA protein per dose, or 3.5 to about 20 μg of the additional cctA protein per dose, or 3. It may contain 5 to about 25 μg of the additional cctA protein, or 3.5 to about 30 μg of the additional cctA protein per dose, or about 4 to about 30 μg of the additional cctA protein per dose, or about 4 to about 25 μg of the additional cctA protein per dose, or about 4 to about 20 μg of the additional cctA protein per dose, or about 4 to about 15 μg of the additional cctA protein per dose, or about 4 to about 10 μg of the additional cctA protein per dose, or about 4 to about 8 μg of the additional cctA protein per dose, or about 4 to about 6 μg of the additional cctA protein per dose, or about 5 μg of the additional cctA protein per dose.

[0052]

[0055] By adding cctA to the C chauvoei component, the dose of the component can be reduced from approximately 0.148 RU of flagella to approximately 0.1 RU of flagella, or further reduced to, for example, approximately 0.074 RU of flagella or approximately 0.05 RU of flagella.

[0053]

[0056] It will be understood that the greater the amount of additional cctA, the greater the reduction in the amount of C chauvoei component. In practice, adding 0.1 μg of cctA may reduce the amount of C chauvoei component by approximately 0.00175 RU of flagella. Preferably, the amount of flagella is about 0.025 RU or more per dose, more preferably 0.055 RU or more per dose, more preferably 0.060 RU or more, or 0.65 RU or more, or 0.070 RU or more, or 0.074 RU or more per dose.

[0054]

[0057] How to make a vaccine

[0058] In a second aspect, the present disclosure provides a method for preparing a vaccine against C. chauvoei infection. Generally, when C. chauvoei is cultured for vaccine production, the cctA-containing supernatant is discarded. However, the inventors have discovered that a vaccine can be produced by a method that utilizes the concentration of cctA from the supernatant, and this method is Culturing aC chauvoei and b. Collecting the culture medium, c. Concentrate C chauvoei from the culture medium, d. Concentrating cctA from the culture medium, e. This includes combining the concentrated cctA with C chauvoei.

[0055]

[0059] In certain embodiments, C chauvoei is grown in a culture medium containing peptone, which is a product of protein hydrolysis. In certain embodiments, the peptone may be plant-derived (e.g., soy peptone) or animal-derived (e.g., meat peptone or casein peptone), or any combination thereof. In a preferred set of embodiments, the peptone is soy peptone.

[0056]

[0060] In different embodiments, the peptone concentration in the culture medium is about 20 g / L to about 100 g / L, for example, about 30 g / L, or 40 g / L, or about 50 g / L, or about 60 g / L, or about 70 g / L, or about 80 g / L, or about 90 g / L. In certain embodiments, the concentration is about 25 to about 75 g / L, more preferably about 45 to about 55 g / L.

[0057]

[0061] C chauvoei may be concentrated by microfiltration followed by diafiltration. In certain embodiments, microfiltration is achieved by passing the solution through a 100,000 kDa to 0.45 μm cassette and then diafiltration to a 100,000 kDa to 0.45 μm cassette to prepare the C chauvoei component. For example, the cassettes used in the microfiltration and diafiltration steps may independently be 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, or 0.45 μm. In certain embodiments, both the microfiltration and diafiltration cassettes are 0.2 μm.

[0058]

[0062] Advantageously, the method for preparing the C chauvoei component and the method for preparing a concentrated (or enhanced) cctA solution can be initiated via a general step of microfiltration, preferably via a 0.2 μm cassette. After the microfiltration step, the microfiltered retained solution is dialyzed to prepare the C chauvoei component. The microfiltered permeate is then ultrafiltered through a cassette to obtain an ultrafiltered retained solution, and then the ultrafiltered retained solution is dialyzed as described above to obtain a dialyzed retained solution, thus preparing the cctA solution. See Figure 1.

[0059]

[0063] The size of cctA is approximately 32 kDa. Therefore, the cassette used to concentrate cctA should be less than 32 kDa. In certain embodiments, the cassette may be 30 kDa, 20 kDa, 10 kDa, or 5 kDa. In the method according to the present invention, a 10 kDa cassette is preferably used in this step.

[0060]

[0064] In certain embodiments, the step of concentrating the cctA from the culture medium includes ultrafiltration to obtain an ultrafiltered retention solution, followed by diafiltration to obtain a dialysfiltered retention solution, followed by sterile filtration to obtain a sterile solution. Such procedures are known in the art.

[0061]

[0065] Ultrafiltration is a type of membrane filtration in which forces such as pressure or concentration gradients result in separation across a semipermeable membrane. High molecular weight suspended solids and solutes are retained (retaining liquid), while water and low molecular weight solutes pass through the membrane (permeate). This particular process is 10 3 ~10 6 Ultrafiltration is used to purify and concentrate compounds with a molecular weight of Da, particularly proteins. Ultrafiltration is generally defined by the molecular weight cutoff of the membrane used in the process.

[0062]

[0066] Diafiltration is a special type of ultrafiltration process in which the retained fluid is diluted with a buffer such as saline, PBS, or water, and then ultrafiltered again to reduce the concentration of soluble permeate components while maintaining the concentration of retained components. For example, cassettes of the same size, such as 10 kDa, can be used for both ultrafiltration and diafiltration.

[0063]

[0067] In a particular embodiment, after the steps of ultrafiltration and diafiltration, the concentrated cctA is sterile filtered. The sterile filtered cctA solution can be added to the C chauvoei component to prepare the vaccine or immunogenic composition disclosed herein.

[0064]

[0068] Vaccines and immunogenic compositions

[0069] The vaccines and immunogenic compositions according to the present invention may contain other antigens in addition to the combination of the C chauvoei component and additional cctA proteins. Particularly preferred antigens are derived from pathogens that affect ruminants. In certain embodiments, the antigens may be derived from a selection of the group consisting of Clostridium septicum, Clostridium haemolyticum, Clostridium novyi, Clostridium sordellii, Clostridium tetani, Clostridium perfringens types C and D, Erysipelothrix rhusiopathiae, Leptospira borgpetersenii serovar Hardjo, Leptospira interrogans serovar Pomona, and Mannheimia haemolytica antigens.

[0065]

[0070] Preferably, these antigens are inactivated organisms, attenuated organisms, or bacterial toxins, or whole organism extracts, or partial organism extracts.

[0066]

[0071] Vaccines and immunogenic compositions may also contain adjuvants. Such agents are known in the art. Suitable adjuvants include oil emulsions (including oil-in-water emulsions and water-in-water emulsions), aluminum double salt compounds containing both aluminum hydroxide and aluminum phosphate, triterpenoid saponins, immunostimulatory complexes, quaternary amine compounds (e.g., dimethyldioctadecylammonium salts, e.g., bromides), CpG-containing oligonucleotides, oligoribonucleotides, glycolipids and their salts (e.g., acetates), such as N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanamide, also known as BAY® 1005, DEAE dextran, and combinations thereof.

[0067]

[0072] In certain embodiments, the adjuvant contains a complex formed by Quil A (triterpenoid saponin) and sterols (e.g., cholesterol) in an oil-in-water emulsion formed by mineral oil and lecithin known as PREZENT-A®. In other embodiments, the adjuvant contains a triterpenoid saponin and CpG. In yet another embodiment, the adjuvant contains a mixture of CpG oligonucleotide, triterpenoid saponin, and sterols.

[0068]

[0073] The immunogenic compositions and vaccines of the present invention may contain one or more veterinarily acceptable carriers, such as solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, and adsorption retarders. Examples of diluents include water, physiological saline, dextrose, ethanol, and glycerol. Examples of isotonic agents, known to those skilled in the art, include sodium chloride, dextrose, mannitol, sorbitol, and lactose. Examples of stabilizers, known to those skilled in the art, include albumin. Examples of preservatives, known to those skilled in the art, include melthiolate. Examples of antibiotics include, but are not limited to, those from the classes of aminoglycosides, carbapenems, cephalosporins, glycopeptides, macrolides, penicillins, polypeptides, quinolones, sulfonamides, and tetracyclines.

[0069]

[0074] Form, dosage, route of administration

[0075] The immunogenic compositions and vaccines of the present invention can be administered to animals to induce an effective immune response against C. chauvoei infection. Accordingly, the present invention provides a method for stimulating an effective immune response against C. chauvoei infection by administering to animals a therapeutically effective amount of the immunogenic compositions or vaccines of the present invention described herein.

[0070]

[0076] The immunogenic compositions and vaccines of the present invention can be prepared in various forms depending on the route of administration. For example, the immunogenic compositions and vaccines can be prepared in the form of sterile aqueous solutions or dispersions suitable for injection. The immunogenic compositions and vaccines can also be prepared in the form of suspensions or emulsions.

[0071]

[0077] Immunogenic compositions and vaccines generally contain a veterinarily acceptable carrier in a volume of about 0.5 ml to about 5 ml. In another embodiment, the volume of the carrier is about 1 ml to about 4 ml, or about 2 ml to about 3 ml. In yet another embodiment, the volume of the carrier is about 1 ml, or about 2 ml, or about 5 ml. Veterinarily acceptable carriers suitable for use in immunogenic compositions and vaccines may be any of those described above.

[0072]

[0078] According to the method of the present invention, a single dose can be administered to an animal, or alternatively, two or more doses can be administered at intervals of about 2 weeks to about 26 weeks, for example, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 10 weeks, 15 weeks, 20 weeks, and 25 weeks. An augmentation regimen may be required, and the administration regimen can be adjusted to provide optimal immunization. Those skilled in the art can easily determine the optimal administration regimen.

[0073]

[0079] Immunogenic compositions and vaccines may be administered directly into the bloodstream, muscles, or internal organs. Suitable means of parenteral administration include intravenous, intra-arterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous methods. Suitable devices for parenteral administration include needle syringes (including microscopic needles), needle-free syringes, and injection techniques.

[0074]

[0080] Parenteral formulations are typically aqueous solutions that may contain salts, carbohydrates, and excipients such as buffers (preferably up to a pH of about 3 to about 9, or about 4 to about 8, or about 5 to about 7.5, or about 6 to about 7.5, or about 7 to about 7.5), but for some applications they can be more preferably formulated as sterile non-aqueous solutions or as dry forms used with a suitable vehicle such as sterile, pyrogen-free water.

[0075]

[0081] Formulations for parenteral administration can be formulated to be immediate-release and / or regulated-release. Regulated-release formulations include delayed-release, sustained-release, pulsed-release, controlled-release, targeted-release, and programmed-release. Accordingly, the immunogenic compositions and / or vaccines of the present invention can be formulated as solid, semi-solid, or thixotropic liquids for administration as implanted depots that provide regulated release of the active compound. Examples of such formulations include drug-coated stents and poly(dl-lactic acid-coglycol) acid (PGLA) microparticles.

[0076]

[0082] The following embodiments are presented as illustrative examples and should not be construed as limiting the scope of the invention. Many variations, modifications, alterations, and other uses and applications of the invention will be apparent to those skilled in the art. [Examples]

[0077] Example 1: Method for producing Clostridium chauvoei for flagella and ccctA antigen

[0083] A four-fold intensity medium for seed scale and antigen production was prepared as follows: 200 g / L of soy peptone and 20 g / L of yeast extract were added to distilled water at 50°C ± 5°C. The mixture was stirred until dissolved. A 400 g / L magnesium sulfate solution was prepared and added in sufficient volume to the dissolved peptone and yeast extract solution to obtain a 2.0 g / L magnesium sulfate solution. Quadruple-strength culture media were prepared with distilled water to a single strength. The pH was adjusted to 7.4-7.5 using sodium hydroxide solution. The culture media were sterilized by heating to a minimum of 121°C for a minimum of 30 minutes.

[0078]

[0084] To prepare the final medium (PYE), a sterile solution of 10% cysteine ​​HCl and 5% ascorbic acid was added to the base medium to obtain a final concentration of 0.05% cysteine ​​HCl and 0.025% ascorbic acid, as well as a sufficient 50% sterile glucose solution, resulting in a final glucose concentration of 0.2%.

[0079]

[0085] Lyophilized ampoules of Clostridium chauvoei (CH3 strain) were resuspended in sterile Robertson's cooked meat medium and used to inoculate test tubes containing Robertson's cooked meat medium supplemented with 0.05% cysteine ​​HCl, 0.025% ascorbic acid, and 0.2% glucose. Seed stage 1 cultures were incubated until heavily turbid, and a 2% v / v inoculum was inoculated into PYE medium (seed stage 2). Seed stage 2 cultures were incubated until heavily turbid. Then, 5% v / v of the culture from the seed stage 2 cultures was inoculated into a fermenter. The temperature was maintained at 37°C. The pH was lowered to pH 6.5, and the controller was reset to pH 6.8. After the pH reset, 50% glucose feed was added at a rate of 7.2 mL / L culture / hour for the next 5 hours. Culturing was completed when the glucose level became zero or when the culture reached a quiescent phase. The culture was inactivated by adding 0.8% v / v formalin. The pH was adjusted to 6.8-6.9, and the resulting culture was incubated at 37°C for 7 days. The inactivated culture was kept at 2-8°C until further treatment.

[0080]

[0086] Another vaccine preparation was manufactured as described above, but 200 g / L of meat peptone was used instead of 200 g / L of soy peptone.

[0081] Example 2: Downstream treatment of C. chauvoei antigen:

[0087] For each group shown, the inactivated fermented culture (prepared as described above in Example 1) was treated as follows. These treated C. chauvoei antigens were used in Example 3 below. T01: (Entire bulk) Not processed. T02: (15×MF concentration) Inactivated culture concentrated 30-fold via tangential flow filtration through a 0.2 μm molecular weight cutoff membrane cassette. The cell concentrate was recovered from the system by washing with physiological saline, resulting in a final volume reduction equivalent to 15-fold. T03: (15×MF concentration and diafiltration) Inactivated cultures were concentrated 30-fold via tangential flow filtration through a 0.2 μm molecular weight cutoff membrane cassette. The cell concentrate was diafiltration with physiological saline (4x quantitative wash), and the dialysis-filtered cell concentrate was recovered from the system by physiological saline wash, resulting in a final volume reduction equivalent to 15-fold. T04: (15×UF cell-free permeate) Permeate collected during cell enrichment was concentrated 30-fold via tangential flow filtration using a 10 kDa molecular weight cutoff membrane cassette. The concentrated permeate was sterilized through a 0.2 μm filter. The concentrated permeate was recovered from the system by saline washing, resulting in a final volume reduction equivalent to 15-fold. T05: (15×UF Dialysis Filtration of Cell-Free Permeate) Permeate collected during cell enrichment was concentrated 30-fold via tangential flow filtration using a 10 kDa molecular weight cutoff membrane cassette. The concentrated permeate was dialyzed with physiological saline (4 quantitative washes) and sterilized through a 0.2 μm filter. The concentrated permeate was recovered from the system by physiological saline washing, resulting in a final volume reduction equivalent to 15-fold.

[0082] Analytical testing of C. chauvoei downstream-treated antigens

[0088] The downstream-treated Clostridium chauvoei antigen (prepared as described above) was tested against ccctA antigen and flagella. The results are shown in Table 1. [Table 1]

[0083] Example 3: Preparation of experimental Clostridium vaccine formulation:

[0089] All experimental monovalent Clostridial chauvoei vaccines were formulated with a final concentration of 1.6 mg / mL of aluminum hydroxide (as aluminum). Details of lot 370 C chauvoei antigen are shown in Table 2. [Table 2] [Table 3]

[0084]

[0090] All experimental polyvalent Clostridium vaccines (ULTRAVAC® 7 In 1) were formulated with aluminum hydroxide at a final concentration of 1.6 mg / mL (as aluminum). Details for lot 065 antigen are shown in Table 4. [Table 4]

[0085] Example 4: Guinea pig defense:

[0091] For each vaccine set, guinea pigs were randomly divided into groups of 5 animals. One group of 5 guinea pigs was kept as a control for each vaccine set. In the vaccinated groups, each animal received a single dose of the test vaccine (1 mL of monovalent vaccine and 1.25 mL of ULTRAVAC® 7 In 1 vaccine) subcutaneously in the flank. 28 days after the first injection, each animal in the test group received a second subcutaneous injection of the test vaccine in the opposite flank. 14 days after the second injection, the vaccinated and control animals were subjected to 5% CaC The challenge was performed with a suspension of viable Clostridium chauvoei spore strain CH4 (0.5 ml) in solution l2, and administered intramuscularly to the thigh muscle of the right hind limb.

[0086]

[0092] The challenged guinea pigs were examined daily for the next five days, and any deaths were recorded on a worksheet. If a death occurred, there had to be clear symptoms of black leg disease edema. At the end of the challenge on day five, all surviving animals were euthanized. For ethical reasons, animals that were near death were euthanized and presumed to have died from C. chauvoei infection.

[0087]

[0093] In the first experiment, monovalent C. chauvoei vaccines containing different proportions of flagella and cctA were tested for efficacy in guinea pig toxicity challenges. 100% protection was achieved by increasing the level of cctA in the vaccine in the absence of cells (see Table 5). Interestingly, the addition of cells (flagellars) significantly reduced the level of cctA required to achieve complete protection.

[0088]

[0094] In the second experiment, a polyvalent ULTRAVAC® 7-in-1 vaccine containing five Clostridium antigens and two Leptospira antigens was tested for efficacy in guinea pigs via toxicity challenges. Complete protection was achieved in the balance between flagella and cctA. The levels of cctA and flagella required to provide complete protection were higher than those required for a monovalent vaccine.

[0089]

[0095] In the third experiment, a polyvalent ULTRAVAC® 7-in-1 vaccine containing five Clostridium antigens and two Leptospira antigens was tested for efficacy in guinea pigs via a toxicity challenge. The difference from Experiment 2 was that the C chauvoei antigen used in this experiment was derived from soy peptone growth antigen. Again, complete protection was achieved with a balance of flagellar and cctA antigens. [Table 5] [Table 6] [Table 7]

[0090]

[0096] All publications, patents, and non-patent publications described herein represent the level of skill of those skilled in the art in which the present invention relates. All publications are incorporated herein by reference to the same extent as each individual publication is specifically and individually indicated as being incorporated by reference.

[0091]

[0097] While the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. Therefore, it should be understood that numerous modifications can be made to the exemplary embodiments and other configurations can be devised without departing from the spirit and scope of the invention as defined by the following claims. [Table B-1] [Table B-2] [Table B-3]

Claims

1. A vaccine against Clostridium chauvoei, wherein the vaccine comprises a C chauvoei component and an additional cctA protein.

2. The vaccine according to claim 1, wherein a single dose of the vaccine contains at least about 3.5 μg of the additional cctA protein.

3. The vaccine according to claim 1 or 2, wherein a single dose contains flagella of 0.025 to 0.148 RU.

4. The vaccine according to any one of claims 1 to 3, wherein a single dose contains approximately 0.074 RU of the flagella.

5. The vaccine according to any one of claims 1 to 4, wherein a single dose contains approximately 5 μg of the additional cctA protein.

6. The vaccine according to any one of claims 1 to 5, wherein the cctA protein is a recombinantly produced cctA protein.

7. The vaccine according to any one of claims 1 to 5, wherein the cctA protein is produced from the supernatant of cultured C chauvoei.

8. The vaccine according to any one of claims 1 to 7, wherein the C chauvoei component is a bacterium, a whole cell extract, a partial cell extract, or a combination thereof.

9. The vaccine according to claim 8, wherein the C chauvoei component is the partial cell membrane containing the membrane fraction.

10. A method for preparing a vaccine against C. chauvoei infection, wherein the method is a. Culturing C chauvoei, b. Collecting the culture medium, c. Concentrating C chauvoei from the culture medium, d. Concentrating cctA from the culture medium, e. A method comprising combining the concentrated cctA with C chauvoei.

11. The method according to claim 10, further comprising the step of microfiltration the culture medium to obtain a microfiltered retaining solution and a microfiltered permeate.

12. The method according to claim 10 or 11, wherein the step of culturing C chauvoei is carried out in a culture medium containing plant-derived peptone, animal-derived peptone, or a combination thereof.

13. The step of concentrating the cctA from the culture medium is a. Ultrafiltration of the microfiltered permeate to obtain an ultrafiltered retaining liquid, and subsequently b. Obtain the retained solution filtered by diafiltration, and subsequently c. The method according to claim 11 or 12, comprising obtaining a sterile solution of the additional cctA protein by sterile filtration.

14. The method according to claim 13, wherein the ultrafiltration includes passing the microfiltered permeate through a 10 kDa cassette.

15. The method according to claim 13 or 14, wherein the dialysis filtration includes passing the ultrafiltered retained liquid through a 10 kDa cassette.

16. The method according to any one of claims 12 to 15, wherein the dialysis filtration is subsequently followed by sterile filtration of the dialyzed retained solution through a 0.2 μm filter to prepare a sterile solution of the additional cctA protein.

17. The method according to any one of claims 11 to 16, wherein the microfiltered retaining liquid is subjected to dialysis filtration to obtain the C chauvoei component.

18. The method according to any one of claims 10 to 17, wherein at least a portion of the C chauvoei component described in claim 17 is combined with at least a portion of the sterile solution of the additional cctA protein.

19. A vaccine comprising the C chauvoei antigen, wherein the C chauvoei antigen is produced by the method according to any one of claims 7 to 18, and a single dose of the vaccine contains at least 3.5 μg of the concentrated cctA protein.

20. The vaccine according to claim 19, wherein a single dose contains approximately 0.074 to 0.148 RU of flagella.

21. The vaccine according to claim 19 or 20, wherein a single dose contains approximately 5 μg of the concentrated cctA.

22. The vaccine according to claim 21, wherein a single dose contains approximately 0.074 Ru of the flagella.

23. Clostridium septicum, Clostridium haemolyticum, Clostridium novyi, Clostridium sordellii, Clostridium tetani, Clostridium perfringens types C and D, Erysipelothrix rhusiopathiae, Leptospira borgpetersenii serovar Hardjo, Leptospira interrogans serovar Pomona and Mannheimia The vaccine according to any one of claims 1 to 6 or 19 to 22, further comprising at least one additional antigen selected from the group consisting of hemolytica antigens.

24. The vaccine according to claim 23, wherein the at least one additional antigen is an inactivated culture or toxoid.

25. The vaccine according to any one of claims 1 to 9 or 19 to 24, further comprising an adjuvant.

26. A method for preventing C. chauvoei infection in a subject, comprising administering to the subject a vaccine according to any one of claims 1 to 9 or 19 to 24.

27. The method according to claim 26, wherein the subject is a cattle or sheep.

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