Clostridium chauvoei vaccine and method of production

Abstract

The present invention provides a vaccine against C chauvoei, which comprises a C chauvoei component and an additional cctA protein. Methods of making and using the vaccine are also provided.

Description

[Technical field]

[0001] The present invention is in the field of Clostridium vaccines for ruminants. [Background technology]

[0002] Clostridial myositis (blackleg disease), caused by Clostridium chauvoei, is a disease of great economic importance 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 cross the intestinal mucosa after ingestion, enter the bloodstream and be carried to skeletal muscles. The spores lie dormant until local trauma to the muscle, which in cattle is most often caused by bruising during handling in the chute or from trauma in crowded feedlots, causing muscle damage and local hypoxia and anoxia.

[0003] Current knowledge on the pathogenicity of C. chauvoei reveals that toxins, highly active DNAses, hyaluronidases, sialidases, and flagella represent the main virulence factors. Among the putative toxins, C. chauvoei toxin A (CctA) has been shown to represent the major cytotoxic and hemolytic activity of C. chauvoei.

[0004] In addition to the toxin CctA, the sialidase NanA and hyaluronidase NagH appear to allow C. chauvoei to migrate from the initial site of infection, which is generally thought to be the oral or respiratory tract or occasionally a cutaneous lesion, and access muscle tissue where the bacteria can replicate and cause muscle necrosis.

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

[0006] The vaccine against blackleg disease consists of chemically inactivated bacteria providing bacterial culture supernatants expected to contain the outer membrane and flagellar proteins proposed as immunogens, as well as the major toxins. Recently, it has been shown that the CctA toxin alone, which is conserved among C. chauvoei strains isolated worldwide and prepared by recombinant gene technology, provided effective protection in a guinea pig infection model, which serves as a biological test for efficacy testing in batch release procedures of commercial vaccines.

[0007] Although the literature on blackleg disease is abundant, there is little scientific evidence on the effectiveness of vaccination against C chauvoei to prevent disease and mortality in cattle. Uzal et al. found that the evidence of efficacy of C chauvoei vaccines to prevent infection with this microorganism in cattle is low to moderate. See Vet Clin North Am Food Anim Pract. 2012 Mar; 28 (1): 71-7, viii. Thus, there is a need in the art for additional vaccines that can protect ruminants against C chauvoei infection. Summary of the Invention

[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 different embodiments of this first aspect, a dose of the vaccine contains at least about 3.5 μg of the additional cctA protein and 0.025-0.148 RU of flagella. In certain preferred embodiments, the vaccine according to the first aspect of the invention contains about 0.074 RU of the flagella and about 5 μg of the additional cctA protein. In vaccines 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 a bacterium, a whole cell extract, a partial cell extract, or a combination thereof.

[0010] In a second aspect, the disclosure provides a method of preparing a vaccine against C chauvoei infection, the method comprising: Cultivating aC chauvoei; b. collecting the medium; c. enriching C. chauvoei from the culture medium; d. enriching cctA from the medium; and e. combining the enriched cctA with C chauvoei.

[0011] In certain embodiments, the method includes microfiltering the medium to obtain a microfiltered retentate and a microfiltered permeate.

[0012] In certain embodiments according to this second aspect of the invention, the step of culturing C chauvoei may be carried out in a medium comprising a plant-derived peptone, an animal-derived peptone, or a combination thereof.

[0013] In certain embodiments, the step of concentrating the cctA from the medium comprises: a. ultrafiltration of the microfiltered permeate to obtain an ultrafiltered retentate; and b. Diafiltration to obtain a diafiltered retentate; and c. Obtaining a sterile solution of the additional cctA protein by sterile filtration.

[0014] In certain embodiments, ultrafiltration comprises passing the microfiltered permeate through a 10 kDa cassette, in certain embodiments, diafiltration comprises passing the ultrafiltered retentate through a 10 kDa cassette, and / or diafiltration is followed by sterile filtration of the diafiltered retentate through a 0.2 μm filter to prepare a sterile solution of additional cctA protein.

[0015] Preferably, in the method according to any of the embodiments of this second aspect of the invention, the microfiltered retentate is diafiltered to obtain the C chauvoei component.

[0016] In a 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 present disclosure provides a vaccine prepared according to any embodiment of the second aspect of the invention, wherein a single dose of the vaccine contains at least 3.5 μg of the concentrated cctA protein.

[0018] In certain embodiments of the third aspect, a single dose of the vaccine contains about 0.074-0.148 RU of the flagellum. Preferably, a single dose contains about 5 μg of the concentrated cctA, and even 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 toxoid. In any embodiment of this third aspect, the vaccine may be unadjuvanted or may include an adjuvant.

[0020] In a fourth aspect, the 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 aspects of the invention. In certain embodiments of this fourth aspect, the subject is a bovine or ovine subject. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram of a process for preparing C chauvoei components and concentrated cctA for the preparation of a vaccine according to certain embodiments of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022]

[0022] In order to better understand the present invention, the following non-limiting definitions are provided.

[0023] The phrase "vaccine with 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 with additional cctA protein, the ratio of cctA to flagella (a proxy for the amount of C chauvoei) is greater than in a vaccine lacking this 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" applied to a reference figure refers to the reference figure to plus or minus ten 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 grown in the absence of other species or types.

[0027]

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

[0028]

[0029] "Lysate" refers to a lysis product, which is the disruption of cells by rupture of the cell wall or membrane. Disruption can be accomplished 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, which extract is capable of eliciting an immune response that is approximately the same as the immune response elicited by a whole cell extract. Preferably, the partial cell extract comprises a cell wall and / or cell membrane fraction. For the avoidance of doubt, the partial cell extract does not comprise purified and / or concentrated cctA.

[0030]

[0031] As used herein with respect to a vaccine or other composition, "protection," "protecting," "protective immunity," and the like mean that the vaccine or composition prevents or reduces the symptoms of a disease caused by the organism from which the antigen(s) used in the vaccine or composition are derived. The terms "protection," "protecting," and the like also mean that the vaccine or composition can be used to "treat" a disease, or one or more symptoms of a disease, that is already present in a subject.

[0031]

[0032] "Therapeutically effective amount" refers to an amount of an antigen or vaccine that will induce an immune response in a subject receiving the antigen or vaccine that is sufficient to prevent or reduce signs or symptoms of disease, including adverse health effects or complications, resulting from infection with a pathogen, such as a virus or bacteria. Humoral or cell-mediated immunity, or both humoral and cell-mediated immunity, may be induced. The immunogenic response of an animal to a vaccine may be assessed indirectly, for example, via measurement of antibody titers, lymphocyte proliferation assays, or directly, via monitoring signs and symptoms after challenge with a wild-type strain. Protective immunity conferred by a vaccine may be assessed, for example, by measuring reductions in clinical signs, such as mortality, morbidity, body temperature, overall physical condition, and overall health and performance of the subject. The amount of a vaccine that is therapeutically effective may vary depending on the particular adjuvant used, the particular antigen used, or the condition of the subject, and may be determined by one of skill in the art.

[0032]

[0033] "Treating" refers to preventing the disorder, condition, or disease to which such term applies, or preventing or alleviating one or more symptoms of such disorder, condition, or disease.

[0033]

[0034] "Vaccine" or "vaccine composition" as used herein refers to an immunogenic composition that contains an antigen and can induce a specific immune response against the antigen. Administration of the vaccine to a subject results in a protective immune response, which can be fully protective or partially protective. The vaccine can be directly introduced into the subject by any known route of administration, including parenterally, orally, etc. These terms refer to a composition that prevents or reduces infection, or prevents or reduces one or more signs or symptoms of infection. The protective effect of a vaccine composition against a pathogen is usually achieved by inducing an immune response in the subject. In general, elimination or reduction of the incidence of infection, improvement of signs or symptoms, or accelerated clearance of the microorganism from an infected subject indicates the protective effect of the vaccine composition.

[0034]

[0035] "Veterinarily acceptable," as used herein, refers to materials that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of veterinary subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio, and are effective for their intended use.

[0035]

[0036] As used herein, "veterinarily acceptable carrier" refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and that 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, for example, C chauvoei cell lysates.

[0037]

[0038] The present disclosure is based on the unexpected discoveries that it is possible to concentrate cctA 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] Thus, in a first aspect, the present invention provides a vaccine comprising a 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, the C chauvoei is a whole cell extract. In yet other embodiments, the C chauvoei is a partial cell extract. Preferably, the partial cell extract comprises 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 the present disclosure, the amount of C chauveoei components is quantified using flagellar relative units (RU). Thus, the use of flagellar RU is a proxy for the amount of C chauveoei. However, it should be understood that other protein(s) can be used as a proxy for quantifying the amount of C chauveoei components.

[0041]

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

[0042]

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

[0043]

[0045] Non-limiting examples of suitable preparations of C. chauvoei include products currently available on the market as components of commercial vaccines, including, but not limited to, 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 then inactivating it with formalin. The suspension containing the inactivated C chauvoei is then microfiltered through a 0.2 μm cassette and then diafiltered with saline through a 0.2 μm microfiltration cassette.

[0045]

[0047] Additional cctA protein

[0048] The cctA protein according to the present invention has the sequence represented by SEQ ID NO:1: [Table A] or a protein that is at least 90% identical to SEQ ID NO: 1, and may be 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, more preferably at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or all 100% of these substitutions are conservative substitutions.

[0046]

[0049] Those skilled in the art will recognize that changes in a nucleic acid sequence that result in modifications of the amino acid sequence of the encoded protein will have little, if any, effect on the resulting three-dimensional structure of the protein. For example, a codon for the amino acid alanine, a hydrophobic amino acid, can be replaced by a codon that codes for another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes that result in the replacement 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 for one another: [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] The C chauvoei cctA protein can be prepared by a number of methods, including purification of the cctA protein from the supernatant of cultured C chauvoei, and / or genetic engineering, and / or chemical synthesis, which methods are well known in the art.

[0049]

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

[0050]

[0053] In vaccines disclosed herein that include an additional cctA protein, the ratio of cctA to flagella is greater than that in vaccines that lack the 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 that includes a C. chauvoei component but lacks added cctA protein.

[0051]

[0054] In certain embodiments, the vaccine according to the invention contains about 3.5 μg or more of the additional cctA protein per dose, and in different embodiments may contain up to 30 μg of the additional cctA protein per dose, and the vaccine contains 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. The additional cctA protein 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 dosage of the component can be reduced from about 0.148 RU of flagella to about 0.1 RU of flagella, or further reduced, for example, to about 0.074 RU of flagella, or about 0.05 RU of flagella.

[0053]

[0056] It will be appreciated that the greater the amount of added cctA, the greater the reduction in the amount of C chauvoei components. In practical terms, the addition of 0.1 μg of cctA can potentially reduce the amount of C chauvoei components by about 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 per dose, or 0.65 RU or more per dose, or 0.070 RU or more per dose, 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 of preparing a vaccine against C chauvoei infection. Generally, when culturing C chauvoei for the production of a vaccine, the cctA-containing supernatant is discarded. However, the present inventors have discovered that a vaccine can be made by a method that utilizes enrichment of cctA from the supernatant, the method comprising: Cultivating aC chauvoei; b. collecting the medium; c. enriching C. chauvoei from the culture medium; d. enriching cctA from the medium; and e. combining the enriched cctA with C chauvoei.

[0055]

[0059] In certain embodiments, C. chauvoei is grown in a medium containing a peptone that is a product of protein hydrolysis. In certain embodiments, the peptone can be of plant origin (e.g., soy peptone) or animal origin (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 concentration of peptone in the medium is about 20 g / L to about 100 g / L, for example, about 30 g / L, or about 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 accomplished by passing the solution through a 100,000 kDa to 0.45 μm cassette followed by diafiltration into a 100,000 kDa to 0.45 μm cassette to prepare the C chauvoei component. For example, the cassettes used for 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 the concentrated (or enriched) cctA solution can start via a general step of microfiltration, preferably a 0.2 μm cassette. After the step of microfiltration, the microfiltered retentate is diafiltered to prepare the C chauvoei component. The microfiltered permeate is then ultrafiltered through a cassette to obtain an ultrafiltered retentate, and then the ultrafiltered retentate is diafiltered as described above to obtain a diafiltered retentate, thus preparing the cctA solution. See FIG. 1.

[0059]

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

[0060]

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

[0061]

[0065] Ultrafiltration is a variety of membrane filtration in which forces such as pressure or a concentration gradient result in separation across a semipermeable membrane. High molecular weight suspended solids and solutes are retained (retentate), while water and low molecular weight solutes pass through the membrane (permeate). This particular process is carried out over a 10 3 ~10 6 It is used to purify and concentrate compounds, especially proteins, with molecular weights of 10 Da. Ultrafiltration is generally defined by the molecular weight cut-off of the membrane used in the process.

[0062]

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

[0063]

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

[0064]

[0068] Vaccines and immunogenic compositions

[0069] Vaccines and immunogenic compositions according to the invention may contain other antigens in addition to the combination of C chauvoei components and additional cctA proteins. Particularly preferred antigens are derived from pathogens affecting ruminants. In certain embodiments, the antigen may be derived from an 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.

[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, water-in-oil, and water-in-oil-in-water emulsions), aluminum double salt compounds, including both aluminum hydroxide and aluminum phosphate, triterpenoid saponins, immune stimulating complexes, quaternary amine compounds (e.g., dimethyldioctadecylammonium salts, e.g., bromides), CpG-containing oligonucleotides, oligoribonucleotides, glycolipids such as N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanamide, also known as BAY® 1005, and its salts (e.g., acetates), DEAE dextran, and combinations thereof.

[0067]

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

[0068]

[0073] The immunogenic compositions and vaccines of the present invention may include one or more veterinarily acceptable carriers, such as solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonicity agents, adsorption retarding agents, etc. Diluents include water, saline, dextrose, ethanol, glycerol, etc. Isotonicity agents include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others known to those of skill in the art. Stabilizers include albumin, among others known to those of skill in the art. Preservatives include merthiolate, among others known to those of skill in the art. 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 and route of administration

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

[0070]

[0076] The immunogenic composition and vaccine of the present invention can be prepared in various forms depending on the route of administration. For example, the immunogenic composition and vaccine can be prepared in the form of a sterile aqueous solution or dispersion suitable for injection. The immunogenic composition and vaccine can also be prepared in the form of a suspension or emulsion.

[0071]

[0077] The immunogenic compositions and vaccines generally comprise 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 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 the immunogenic compositions and vaccines may be any of those described above.

[0072]

[0078] According to the methods of the present invention, a single dose can be administered to an animal, or alternatively, two or more inoculations can be administered at intervals of about 2 weeks to about 26 weeks, e.g., 3 weeks, 4 weeks, 5 weeks, 6 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks. Boosting regimens may be required, and administration regimens can be adjusted to provide optimal immunity. One of skill in the art can readily determine the optimal administration regime.

[0073]

[0079] Immunogenic compositions and vaccines may be administered directly into the bloodstream, muscle, or an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle (including microscopic needle) injectors, needle-free injectors, and injection techniques.

[0074]

[0080] Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates, and buffering agents (preferably 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), although for some applications they may be more suitably formulated as sterile nonaqueous solutions or as a dry form for use 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 modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release. Thus, the immunogenic composition and / or vaccine of the present invention can be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot that provides modified release of active compound. Examples of such formulations include drug-coated stents and poly(dl-lactic-coglycolic)acid (PGLA) microparticles.

[0076]

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

[0077] Example 1: Production method of Clostridium chauvoei for flagellar and ccctA antigens

[0083] Four-fold strength medium for seed scale and antigen production was prepared as follows: Soy peptone 200 g / L and yeast extract 20 g / L were added to distilled water at 50°C ± 5°C and mixed until dissolved. A 400 g / L magnesium sulfate solution was prepared and added to the dissolved peptone and yeast extract solution in sufficient volume to give a magnesium sulfate concentration of 2.0 g / L. Four-fold strength medium was made up to single strength with distilled water. The pH was adjusted to 7.4-7.5 with sodium hydroxide solution. The medium was 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 give a final concentration of 0.05% cysteine ​​HCl and 0.025% ascorbic acid, as well as sufficient 50% sterile glucose solution to give a final glucose concentration of 0.2%.

[0079]

[0085] A freeze-dried ampoule of Clostridium chauvoei (strain CH3) was 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. The seed stage 1 culture was incubated until very turbid and 2% v / v inoculum was inoculated into PYE medium (seed stage 2). Seed stage 2 was incubated until very turbid. The fermenter was then inoculated with 5% v / v of culture from the seed stage 2 culture. The temperature was held at 37°C. The pH was lowered to pH 6.5 and the controller was reset to pH 6.8. After the pH reset, a 50% glucose feed was added at a rate of 7.2 mL / L of culture / hour for the next 5 hours. The culture was completed when the glucose level was zero or when the culture reached stationary phase. Cultures were inactivated by the addition of 0.8% v / v formalin. The pH was adjusted to 6.8-6.9 and the resulting cultures were incubated at 37°C for 7 days. The inactivated cultures were kept at 2-8°C until further processing.

[0080]

[0086] Another preparation of the vaccine was made as above, but using 200 g / L meat peptone instead of 200 g / L soybean peptone.

[0081] Example 2: Downstream processing of C. chauvoei antigens:

[0087] For each group indicated, inactivated fermentation cultures (prepared as described above in Example 1) were treated as follows: These treated C. chauvoei antigens were used in Example 3 below. T01: (whole bulk) not treated. T02: (15x MF concentrated) Inactivated culture concentrated 30x via tangential flow filtration through a 0.2 μm molecular weight cut-off membrane cassette. The cell concentrate was harvested from the system with a saline wash, resulting in a final volume reduction equal to 15x. T03: (15x MF Concentration and Diafiltration) Inactivated culture concentrated 30x via tangential flow filtration through a 0.2 μm molecular weight cut-off membrane cassette. The cell concentrate was diafiltered with saline (4x volumetric wash) and the diafiltered cell concentrate was withdrawn from the system with a saline wash, resulting in a final volume reduction equal to 15x. T04: (15x UF cell-free permeate) The permeate collected during cell concentration was concentrated 30x 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 permeate concentrate was recovered from the system with a saline wash, resulting in a final volume reduction equal to 15x. T05: (Diafiltration of 15x UF cell-free permeate) The permeate collected during cell concentration was concentrated 30x via tangential flow filtration using a 10 kDa molecular weight cut-off membrane cassette. The concentrated permeate was diafiltered with saline (4 metered washes) and sterilized through a 0.2 μm filter. The permeate concentrate was recovered from the system with a saline wash, resulting in a final volume reduction equal to 15x.

[0082] Analytical testing of C. chauvoei downstream processed antigens.

[0088] Downstream processed Clostridium chauvoei antigen (prepared as described above) was tested for ccctA antigen and flagella, and the results are presented in Table 1. [Table 1]

[0083] Example 3: Preparation of experimental clostridial vaccine formulations:

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

[0084]

[0090] All experimental multivalent clostridial 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 antigens are shown in Table 4. [Table 4]

[0085] Example 4: Guinea pig protection:

[0091] For each set of vaccines, the guinea pigs were randomly divided into groups of 5 animals. One group of 5 guinea pigs per set of vaccines was left as a control. In the vaccinated group, each animal received one dose of the test vaccine (monovalent vaccine at a dose of 1 mL and ULTRAVAC® 7 In 1 vaccine at a dose of 1.25 mL) subcutaneously in the flank. 28 days after the first injection, each animal in the test group received a second injection of the test vaccine subcutaneously in the opposite flank. 14 days after the second injection, the vaccinated and control animals were challenged with a suspension of viable Clostridium chauvoei spores strain CH4 (0.5 ml) in 5% CaCl2 solution, injected intramuscularly into the thigh muscle of the right hind leg.

[0086]

[0092] The challenged guinea pigs were examined daily for the next 5 days and any mortality that occurred was recorded on a worksheet. For mortality to occur, the obvious symptoms of blackleg disease edema must be present. At the end of the challenge on day 5, all surviving animals were euthanized. For ethical reasons, moribund animals were euthanized and considered to have died from C. chauvoei infection.

[0087]

[0093] In the first experiment, a monovalent C. chauvoei vaccine containing different ratios of flagella and cctA was tested for efficacy by virulent challenge in guinea pigs. By increasing the level of cctA in the vaccine in the absence of cells, 100% protection was achieved (see Table 5). Interestingly, the addition of cells (flagellum) significantly reduced the level of cctA required to achieve full protection.

[0088]

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

[0089]

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

[0090]

[0096] All publications, patents and non-patent publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications are herein fully incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0091]

[0097] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is thus to be understood that numerous modifications can be made to the exemplary embodiments and that 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. 1. A vaccine against Clostridium chauvoei (C. chauvoei), the vaccine comprising a C. chauvoei component and an additional cctA protein, the C. chauvoei component being selected from a whole cell extract, a C. chauvoei membrane fraction, and / or a C. chauvoei cell wall fraction.

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

3. 2. The vaccine of claim 1, wherein one dose contains 0.025 to 0.148 RU of flagella.

4. 10. The vaccine of claim 1, wherein one dose contains about 0.074 RU of said flagella.

5. 10. The vaccine of claim 1, wherein one dose contains about 5 μg of the additional cctA protein.

6. 2. The vaccine of claim 1, wherein the cctA protein is a recombinantly produced cctA protein.

7. 2. The vaccine of claim 1, wherein the cctA protein is produced from the supernatant of cultured C. chauvoei.

8. 10. The vaccine of claim 1, wherein the C. chauvoei component is a bacterin, a whole cell extract, a partial cell extract, or a combination thereof.

9. The vaccine of claim 8, wherein the C. chauvoei component is the partial cell membrane comprising a membrane fraction.

10. A method for preparing a vaccine against C. chauvoei infection, said method comprising: a. Cultivating C. chauvoei; b. collecting the medium; c. Enriching C. chauvoei from the culture medium; d. Enriching cctA from the culture medium; e. combining the enriched cctA with C. chauvoei.

11. 11. The method of claim 10, comprising microfiltering the culture medium to obtain a microfiltered retentate and a microfiltered permeate.

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

13. concentrating the cctA from the culture medium, a. ultrafiltration of the microfiltered permeate to obtain an ultrafiltered retentate; and b. Diafiltration to obtain a diafiltered retentate, followed by c) obtaining a sterile solution of the additional cctA protein by sterile filtration.

14. 14. The method of claim 13, wherein the ultrafiltration comprises passing the microfiltered permeate through a 10 kDa cassette.

15. 14. The method of claim 13, wherein the diafiltration comprises passing the ultrafiltered retentate through a 10 kDa cassette.

16. 13. The method of claim 12, wherein the diafiltration is followed by sterile filtration of the diafiltered retentate through a 0.2 μm filter to prepare a sterile solution of the additional cctA protein.

17. 12. The method of claim 11, wherein the microfiltered retentate is diafiltered to obtain the C. chauvoei component.

18. 18. The method of claim 10, wherein at least a portion of the C. chauvoei component of claim 17 and at least a portion of the sterile solution of the additional cctA protein are combined.

19. 10. The vaccine of claim 1, further comprising 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.

20. 20. The vaccine of claim 19, wherein the at least one additional antigen is an inactivated culture or a toxoid.

21. The vaccine of any one of claims 1 to 9, further comprising an adjuvant.

22. A method of preventing C. chauvoei infection in a non-human subject, comprising administering to the subject the vaccine of any one of claims 1 to 9.

23. 23. The method of claim 22, wherein the subject is a bovine or ovine subject.

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