EP1769065A1 - Vaccine composition against rhodococcus equi - Google Patents
Vaccine composition against rhodococcus equiInfo
- Publication number
- EP1769065A1 EP1769065A1 EP05788683A EP05788683A EP1769065A1 EP 1769065 A1 EP1769065 A1 EP 1769065A1 EP 05788683 A EP05788683 A EP 05788683A EP 05788683 A EP05788683 A EP 05788683A EP 1769065 A1 EP1769065 A1 EP 1769065A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- bacteria
- polyoxyethylene sorbitan
- equi
- extract
- rhodococcus equi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
Definitions
- the invention relates to a method for preparing a soluble antigenic extract obtained from Rhodococcus egui, comprising a step of solubilizing the membranes of said bacteria by contacting these bacteria with an extraction solution containing a nonionic detergent of polyoxyethylene sorbitan fatty acid ester type.
- the subject of the invention is also a soluble antigenic extract obtainable from the preparation process, said extract comprising membrane antigens of Rhodococcus equi in solution in the extraction solution.
- the invention finally relates to a composition
- a composition comprising the drug-soluble antigenic extract, preferably comprising a nanoparticulate adjuvant or oil-in-water emulsion, and the use of the soluble antigenic extract for the preparation of a pharmaceutical composition for preventing or treating Rhodococcus equi-induced rhodococcosis in a mammal.
- Rhodococcus equi causes pyo- granulomatous bronchopneumonia or other rarer clinical forms (digestive, osteo-articular) in foals.
- the morbidity rate varies from 5 to 17% in the different breeding regions of the world, and the case-fatality rate from 40 to 80%.
- Rhodococcus equi infections account for approximately 10% of mortality causes in foals aged 24 hours to 6 months, with annual variations related to climatic conditions (15% in 1988) and 70% of cases of bronchopneumonia in animals aged 1 to 6 months.
- Rhodococcus equi bacterium is a real economic plague because of the mortality it causes, but also because of the consequences for the horse's career and the high cost of treatment.
- the insidious nature of the disease and the appearance of resistant strains make prevention through the development of an effective vaccine a priority in the fight against pathology.
- Carrying is particularly important in the gut of horses, especially in the intestine of foals, where the insufficient development of the anaerobic flora encourages multiplication.
- Rhodococcus equi is a normal host of the digestive tract of equines. The germ survives in the soil and can multiply in feces. This contamination of the external environment seems proportional to the density of the equidae and it is very important in farms maintaining horses for many years.
- Rhodococcus equi infections have been reported in other animal species: in cattle and pigs, Rhodococcus equi is mainly isolated from normal lymph nodes or lymph nodes with lesions suggestive of tuberculosis. At the slaughterhouse, the existence of these adenitis leads to confusion with tuberculosis. In cattle, Rhodococcus equi is also responsible for pyometers and pneumonia. In piglets, the germ can cause the formation of abscesses in the oral cavity causing anorexia. In the goat, we note the formation of pulmonary, splenic or hepatic abscesses that can evoke Corynebacterium pseudotuberculosis infections.
- abscesses can be accompanied by the development of vertebral osteomyelitis.
- sheep some cases of pneumonia as well as abortions and neonatal mortalities have been described.
- Rhodococcus equi causes adenitis of the lymphatic nodes of the anterior mediastinum and mesenteric lymph nodes, subcutaneous abscesses and the
- Rhodococcus equi belong to the genus Rhodococcus, in the class Actinobacteria, subclass of Actinobacteridae, order Actinomycetales, suborder Corynebacterineae, family Nocardiaceae.
- the term Coiynebacterium equi is sometimes also used to refer to Rhodococcus equi bacteria.
- Rhodococcus equi is an optional Gram-positive intracellular bacterium that inhibits phagosome-lysosome fusion and is able to survive and multiply in phagocytic cells.
- the virulence factors are not fully known but they appear to be related to the capsule, equi factor synthesis, mycolic acids, 15-17 kDa molecular weight proteins and a 20 kDa protein.
- vap proteins of 15 to 17 kDa are encoded by vap genes carried by plasmids of 85 to 90 kb.
- the analysis of the restriction fragments makes it possible to recognize at least 10 distinct plasmids whose distribution is linked to the geographical origin of the strains. In France, the virulent strains have an 85-kb type I plasmid or an 87-kb type I plasmid or an 85-kb type II plasmid that has only been found in French strains. These plasmids are only present in virulent strains and the strains of their plasmids survive less in phagocytes and are less pathogenic for the mouse or for the horse.
- vapA The 7 identified vap genes (yapA, vapC, vapD, vapE, vapF, vapG and vapH), are located on a pathogenicity island and they code for surface proteins, essential for survival in macrophages, expressed at acid pH and at 37 ° C. but not at 30 ° C.
- the sequence of the vapA gene appears very well preserved, so that a PCR test amplifying this gene makes it possible to diagnose virulent strains.
- Prescott et al. (Am J Vet Res, 1997 Apr; 58 (4): 356-9) used a VapA protein-rich fraction as the antigen. Vaccination elicits the production of immunoglobulin G (IgG) with opsonizing properties, so that the plasma of vaccinated animals can protect foals against experimental infection. However, vaccination of mothers followed by vaccination of foals does not provide any protection and could even increase the severity of infections.
- IgG immunoglobulin G
- Immunol Med Microbiol. 2002 Dec 13; 34 (4): 299-306) have been designated as having potential as vaccines against R. equi.
- the article published on February 26, 2003 by the National Studs (“Immunization of pregnant mares against R. equi antigens: evaluation of the immune response and passive transfer in different Norman farms", Cauchard et al.) Describes a vaccine consisting of antigenic extracts of a virulent strain of R. equi (85Fp + ) combined with a nanoparticle-based adjuvant (MONTANIDE IMS3012 ® ).
- this vaccine induces inflammatory reactions at the site of the injection.
- the subject of the invention is a process for the preparation of a soluble antigenic extract obtained from Rhodococcus equi bacteria, characterized in that the said process comprises the following steps: a) the solubilization of the membranes of said bacteria by contacting these bacteria with an extraction solution containing a polyoxyethylene sorbitan fatty acid ester nonionic detergent, and b) recovering the solubilized antigenic extract in step a).
- Rhodococcus equi Information about Rhodococcus equi, namely systematics, bacteriological characteristics, habitat and pathogenicity, power pathogenicity, bacteriological and serological diagnosis, antibiotic susceptibility, and prophylaxis are widely described in the veterinary bacteriology dictionary available on the World Wide Web. address http://www.bacterio.cict.fi:/bacdico/garde.html.
- the R. equi bacteria used in the process according to the invention are obtained by culturing in a medium and under conditions suitable for carrying out the invention; preferably, said bacteria are suspended in the culture medium ("wet bacteria").
- a suitable culture medium is the brain-heart medium (BHI for "Brain Heart”).
- Infusion at a pH of between 5 and 7, preferably between 6 and 7, and particularly preferably of 6.5.
- the appropriate culture conditions are generally room temperature, preferably 37 ° C, with stirring, for example 150 rpm (revolutions per minute), for one to several days, preferably for 48 to 72 hours, so that said bacteria are in the exponential phase of growth.
- said bacteria may be from the same strain or different strains, the bacteria of different strains being, in the latter case, mixed in the medium.
- All R. equi strains can be used for carrying out the present invention.
- R. equi strains mention may be made of, but not limited to, strains ATCC33701, ATCC6939, ATCC2572, and strain 85F deposited at the CNCM (National Collection of Culture of Microorganisms, Institut Pasteur, France). July 2, 2004 under the number 1-3250. All R. equi strains possessing a virulence plasmid can be used in the context of the present invention. Nevertheless, any R. equi strain, cured of its plasmid or naturally deprived, to which an expression vector containing one or more genes of the pathogenicity island of the natural plasmid is added, can also be used in the of the present invention.
- the membranes of these bacteria are solubilized by contacting said bacteria, which can to be suspended in an extraction solution containing a polyoxyethylene sorbitan fatty acid ester, which is a nonionic detergent (however, it should be noted that the antigenic extract of R. equi obtained is likely to contain R. equi antigens other than membrane antigens).
- a nonionic detergent is in particular described in the book "Handbook of pharmaceutical excipients", 2nd edition, "A seal publication of the American Pharmaceutical Association and the Royal Pharmaceutical Society of Great Britain,” The Pharmaceutical Press, Published by A WADE and PJ. WELLER, 1994.
- polyoxyethylene sorbitan fatty acid esters which can be used in step a) of solubilization of R. equi bacteria membranes of the process according to the invention include, but are not limited to limited to, the following compounds, commercially available: monolaurate polyoxyethylene 20 sorbitan, also called polysorbate 20 or Tween 20 ® (AMRESCO, Solon, OH), polyoxyethylene monolaurate (4) sorbitan, also known as polysorbate 21, or Tween 21® , the polyoxyethylene sorbitan monopalmitate, also called polysorbate 40, or Tween 40® ,
- - monostearate polyoxyethylene 20 sorbitan also called polysorbate 60 or Tween 60 ®, polyoxyethylene monostearate (4) sorbitan, also called polysorbate 61 or Tween 61 ®, sorbitan tristearate, polyoxyethylene 20 sorbitan, also known as polysorbate 65, or Tween 65 ®, - sorbitan monooleate polyoxyethylene 20 sorbitan, also called polysorbate 80 or Tween 80 ®, polyoxyethylene monooleate (5) sorbitan, also called polysorbate 81 or Tween ® 81,
- the polyoxyethylene sorbitan fatty acid ester is selected from polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, and polyoxyethylene sorbitan monooleate.
- the polyoxyethylene sorbitan fatty acid ester is polyoxyethylene sorbitan monolaurate.
- the preparation method according to the present invention is characterized in that said nonionic detergent is at a concentration of between 0.01% and 5% in said extraction solution.
- the concentration of the nonionic detergent is 0.1% in said extraction solution.
- step a) of solubilization of the preparation method according to the present invention the contacting of the bacteria with the extraction buffer is carried out at a proportion of 1 ml to 20 ml of said buffer per gram of wet bacteria. . Most preferably, this proportion is 5 ml of said buffer per gram of wet bacteria.
- wet bacteria bacteria suspended in a liquid medium.
- the preparation process according to the present invention is characterized in that the Rhodococcus equi bacteria are of the strain selected from strains ATCC33701, ATCC6939, ATCC2572, and strain 85F deposited at the CNCM on July 2, 2004. under the number 1-3250.
- the Rhodococcus equi bacteria are of 85F strain deposited at the CNCM July 2, 2004 under the number 1-3250.
- the preparation method according to the present invention is characterized in that said bacteria are in the form of a bacterial pellet obtained after centrifugation, before step a). This centrifugation step has the advantage of removing the impurities so as to recover only the bacterial cells.
- Bacteria in the form of a bacterial pellet are obtained by centrifugation under appropriate conditions known to those skilled in the art.
- the centrifugation making it possible to obtain the bacterial pellet before step a) is carried out at a speed of between 1000 g and 50000 g, preferably 10,000 g, for a period of 1 min to 45 min, preferably 20 min. preferably at a temperature of between 10 ° C. and 6 ° C., preferably 4 ° C.
- the bacterial pellet is washed at least once in a suitable washing buffer before step a).
- Tris acetate buffers are used at a suitable concentration and pH, for example at a concentration of 10 mM or
- wash buffers are filtered (for example using a 0.2 ⁇ m filter, .).
- step a) of solubilization is carried out with stirring, at a temperature in the range of 20 to 40 ° C, particularly preferably 37 0 C, for a period of 30 to 150 min, particularly preferably preferred for 90 minutes, preferably in the presence of microbeads.
- Step b) of recovery of the antigenic extract solubilized in step a) can be carried out according to various appropriate techniques, such as in particular centrifugation, filtration, chromatography.
- the preparation process according to the present invention is characterized in that in step b) the product is centrifuged. obtained in step a), and the centrifugation supernatant containing said solubilized antigenic extract is recovered.
- the centrifugation in step b) of the product obtained in step a) is carried out under appropriate conditions, known to those skilled in the art.
- this centrifugation is carried out at a speed of between 10,000 g and 150,000 g, preferably 100,000 g, for a period of 10 minutes to 120 minutes, preferably for 60 minutes, at a temperature of between 1 and 60 ° C. preferably at 40 ° C.
- the centrifugation supernatant containing said solubilized antigenic extract is stored at a temperature of between 1 and 60 ° C., preferably at 4 ° C.
- the preparation process according to the invention is characterized in that the Rhodococcus equi bacteria are of strain 85F deposited at the CNCM on July 2, 2004 under the number 1-3250, and in that the ester of The polyoxyethylene sorbitan fatty acid is polyoxyethylene sorbitan monolaurate.
- the subject of the present invention is a process for preparing a soluble antigenic extract obtained from bacteria
- Solubilization of the bacterial membranes of the washed bacterial pellet by contacting said pellet with an extraction solution containing polyoxyethylene sorbitan monolaurate at a concentration of 0.1% v / v in said extraction solution, the proportion of bacteria; extraction buffer being 5 ml / g of wet bacteria, said solubilization being carried out under stirring for 90 minutes at a temperature between 35 and 40 0 C, preferably in the presence of microbeads, and
- the product obtained in the preceding step is centrifuged at 100000 g for 1 hour, preferably at 4 ° C., and the centrifugation supernatant containing said solubilized antigenic extract is recovered.
- the R. equi bacteria used in the process according to the invention are obtained by culturing in a medium and under conditions suitable for carrying out the invention; preferably, said bacteria are suspended in the culture medium.
- a suitable culture medium is the Brain Heart Infusion (BHI) medium at a pH between 5 and 7, preferably between 6 and 7, and particularly preferably of 6.5.
- BHI Brain Heart Infusion
- the appropriate culture conditions are generally room temperature, preferably 37 ° C, with stirring, for example 150 rpm, for one to several days, preferably for 12 to 72 hours, so that said bacteria are in the exponential phase growth.
- the microbeads can be used to "break" the bacteria. It is possible to use, for example, glass beads with a diameter of 150 to 212 ⁇ m (SIGMA, Lyon, France).
- the subject of the invention is a soluble antigenic extract of Rhodococcus equi bacteria, obtainable from the process according to the present invention, characterized in that the said extract comprises R. equi membrane antigens in solution. in an extraction solution containing a polyoxyethylene sorbitan fatty acid ester nonionic detergent.
- the soluble antigenic extract of Rhodococcus equi bacteria according to the invention is obtained from the preparation method according to the present invention.
- the soluble antigenic extract of Rhodococcus equi bacteria according to the invention is characterized in that it comprises R. equi membrane antigens in solution in a solution containing a nonionic detergent of polyoxyethylene sorbitan fatty acid ester type.
- the R. equi membrane antigens included in the soluble antigenic extract according to the invention may be derived from the same strain R. equi or different R. equi strains, the R. equi bacteria of different strains being, in the latter case, mixed in the middle. All R. equi strains can be used for carrying out the present invention. Examples of R. equi strains include, but are not limited to, strains
- any R. equi strain cured of its plasmid or naturally deprived, to which an expression vector containing one or more genes of the pathogenicity island of the natural plasmid is added, can also be used in the of the present invention.
- the soluble antigenic extract of Rhodococcus equi bacteria according to the invention is characterized in that the polyoxyethylene sorbitan fatty acid ester is chosen from polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate and monostearate. polyoxyethylene sorbitan, and the polyoxyethylene sorbitan monooleate.
- the soluble antigenic extract of Rhodococcus equi bacteria according to the invention is characterized in that the polyoxyethylene sorbitan fatty acid ester is polyoxyethylene sorbitan monolaurate.
- the soluble antigenic extract of Rhodococcus equi bacteria according to the invention is characterized in that said nonionic detergent is at a concentration of between 0.01% and 5% in said extraction solution.
- the soluble antigenic extract of bacteria is characterized in that said nonionic detergent is at a concentration of between 0.01% and 5% in said extraction solution.
- Rhodococcus equi according to the invention is characterized in that the concentration of the nonionic detergent is 0.1% in said extraction solution.
- the soluble antigenic extract according to the invention is characterized in that the Rhodococcus equi bacteria are of strain 85F deposited at the CNCM on July 2, 2004 under the number 1-3250, and in that the ester
- the polyoxyethylene sorbitan fatty acid is polyoxyethylene sorbitan monolaurate.
- the subject of the invention is a composition comprising the soluble antigenic extract according to the present invention, as a medicament.
- the composition according to the invention as a medicament additionally comprises adjuvants of the immunity; these adjuvants may be of any type known to those skilled in the art, insofar as, when they are mixed with an effective amount of the soluble antigenic extract according to the present invention, they make it possible to increase the antigenicity of the composition and to promote a superior immune response.
- the composition according to the invention is characterized in that it additionally contains an immunity adjunct of the nanoparticulate type or of the oil-in-water emulsion type.
- adjuvants for oil-in-water emulsion immunity include vaccine adjuvants providing an oil-in-water emulsion and containing a non-mineral oil. Such adjuvants, very well tolerated, are adapted to induce short-term immunity, with a humoral meditation response.
- the adjuvant an oil-in-water emulsion containing a non-mineral oil adjuvant Montanide ISA 35 ® (SEPPIC, Paris, France).
- the adjuvant Montanide ISA 35 ® contains glycerol ester, squalane and Panhydromannitol octodecenoate ether, preferably in the following proportions: Glycerol ester: 50%, squalane: 10% ether and anhydromannitol octodecenoate: 40 %.
- the adjuvant composition for vaccines comprising a metabolizable oil and an emulsifying agent, in which the oil and the emulsifying agent are present in the form of an oil-in-water emulsion (oil droplets with a diameter of approximately 1 micron), as described in the international patent application published December 13, 1990 under the number WO 90/14837 (CHIRON Corp.); and
- the adjuvant for polysaccharide vaccines comprising an oil-in-water emulsion system containing a light non-biodegradable hydrocarbon oil or a biodegradable oil and a detergent, as well as a detoxified endotoxin, as described in the US patent published on February 7, 1989 under US 4,803,070 (RIBI ImmunoChem Research Inc.).
- adjuvants of the immunity of nanoparticulate type include adjuvants for vaccines based on liquid nanoparticles associated with an immunostimulant having GRAS status. Such adjuvants, well tolerated, are based on a new concept combining the adjuvant properties of nanoparticles and a new immunostimulant.
- the adjuvant of liquid nanoparticles associated with an immunostimulating having the GRAS status is the adjuvant MONTANIDE ® IMS 301x (SEPPIC, Paris, France 1), particularly preferably the IMS 3012 MONTANIDE ®. In general, this adjuvant
- MONTANIDE IMS 3012 ® contains modified corn oil, saline buffer solution and preservative, preferably in the following proportions: modified corn oil: 10%, saline buffer solution: 89.99% and preservative: 0 , 01%.
- nanoparticulate adjuvants the small particles (average diameter: 200 nm) used in particular for the administration of biologically active agents, described in the international patent application published October 23, 2003 under the number WO 03/087021 (GENESEGUES company).
- Such particles comprise a biologically active agent such as in particular an adjuvant, a surfactant, and a soluble polymer in aqueous solution;
- solid adjuvant composition of vaccine in the form of a powder or granules comprising an injectable solid support on which a liquid is capable of being absorbed, adsorbed, impregnated or anchored, described in the international patent application published on December 5, 2002 under the number WO 02/096386
- the liquid formulation comprising a discontinuous phase of microparticles in a nonaqueous liquid continuous phase, described in the international patent application published on 24 September 1998 under the number WO 98/41188 (EASTBRIDGE Limited).
- the microparticles described contain pulverized sugars carrying at least one biomolecular product such as a drug or other biologically active ingredients such as a protein, an antibody or an enzyme.
- Such a formulation can be used as a vaccine; surface-modified diamond nanoparticles as antigen delivery vehicles (adjuvants) described in Kossovsky et al., Bioconjug Chem 1995 Sep-Oct; 6 (5): 507-11; and the microspherical particles consisting of a continuous matrix of a biodegradable polymer, which matrix contains in particular an immunogen adsorbed on an aluminum salt adjuvant (aluminum hydroxide, aluminum phosphate, etc.), described in the application International patent published on July 21, 1994 under the number WO 94/15636 (CSL company
- the composition according to the invention is obtained by mixing the soluble antigenic extract according to the invention with the adjuvant of the immunity, preferably the adjuvant of the nanoparticle type immunity, under gentle stirring for a period of 1 minute to 5 hours, preferably 10 minutes, at a temperature between 1 and 6 ° C, preferably 4 ° C.
- said soluble antigenic extract is previously diluted to a concentration of between 0.5 and 10 mg / ml in the extraction buffer or any other buffer known to those skilled in the art to retain the structural properties and solubility of proteins.
- the soluble antigenic extract is diluted to a concentration of 1 mg / ml.
- the dilution of the soluble antigenic extract is followed by a filtration step or any other method to maintain sterile conditions.
- the composition according to the invention is characterized in that the immunity adjuvant of nanoparticulate type is the adjuvant MONTANIDE IMS 3012 ®.
- the composition of the invention contains 50% of the soluble antigenic extract and 50% of the nanoparticulate adjuvant MONTANIDE IMS 3012 ®.
- the composition of the present invention is characterized in that the immunity adjuvant of oil-in-water emulsion adjuvant is Montanide ISA 35 ®.
- the composition of the invention contains 75% soluble and 25% antigenic extract of the oil-in-water emulsion adjuvant Montanide ISA 35 ®.
- the invention relates to the use of a soluble antigenic extract according to the present invention, for the preparation of a pharmaceutical composition for preventing or treating an infection with R. equi in a mammal.
- the mammal is an equine.
- R. equi infections in mammals are the cause of various diseases, including diseases of the respiratory system. Examples of R.
- equi-induced diseases include, but are not limited to, foal, pyo-granulomatous bronchopneumonia, multifocal ulcerative enterocolitis and typhlitis often associated with suppurative adenitis of the mesenteric and colonic lymph nodes, septic arthritis, osteomyelitis, and more rarely, lymphangitis, suppurated myositis and cellulitis, and subcutaneous abscesses.
- the disease In adult horses, the disease is sporadic and results in pulmonary or colonic adenopathies, infected wounds; the bacterium was also isolated on equine fetuses during abortions. Rhodococcus equi infections have been described in other mammalian species (cattle, pigs, sheep, goats, llamas, dogs, cats ...) for which, in general, the clinical expression of the disease remains rare. In most cases, the lesions observed are adenopathies, suppurated or not; pneumonia is exceptional but has been reported, among others, in sheep, goats and llamas.
- the use according to the present invention is characterized in that infection with R. equi induces a disease of the respiratory system, including pneumonia. More preferably, the use according to the present invention is characterized in that the pharmaceutical composition further comprises an adjuvant of the immunity.
- the immunity adjuvant is of the nanoparticulate type or of the oil-in-water emulsion type.
- the use according to the present invention is characterized in that the pharmaceutical composition further comprises an adjuvant of immunity nanoparticulate type which is the adjuvant MONTANIDE IMS 3012 ®.
- the use according to the present invention is characterized in that the pharmaceutical composition contains 50% of the soluble antigenic extract and 50% of the nanoparticulate adjuvant MONTANIDE IMS 3012 ®.
- the use according to the present invention is characterized in that the pharmaceutical composition further comprises an adjuvant of immunity of oil-in-water emulsion which is the adjuvant Montanide ISA 35 ®.
- the pharmaceutical composition contains 75% of soluble antigenic extract and 25% of adjuvant oil-in-water emulsion MONTANIDE ® ISA 35.
- the pharmaceutical composition for preventing or treating R. equi infection may be administered in any suitable manner in a mammal, preferably an equine.
- the use according to the present invention is characterized in that the pharmaceutical composition is administered to said mammal intramuscularly or subcutaneously.
- the use according to the present invention is characterized in that the mammal is selected from man and non-human mammals, such as cattle, pigs, sheep, goat, cat.
- the mammal is an equine, such as in particular the horse.
- said pharmaceutical composition is administered to the foal after birth, preferably in 1 to 4 doses depending on the degree of contamination of the environment.
- the use according to the invention is characterized in that the pharmaceutical composition is intended for the prevention or pre-natal treatment of a pregnant foal, said pharmaceutical composition being administered to the pregnant horse.
- said pharmaceutical composition is administered in 1 to 4 doses depending on the degree of contamination of the environment.
- Figure 1 Anti-i? IgG levels. equi in mares immunized with antigens containing VapA protein (Group 1), mares immunized with killed R. equi whole bacteria (Group 2) and control mares. IgG levels were determined by ELISA. The results are expressed in arbitrary units (AU) + 1 standard error. Statistical significance of the differences between Groups 1 (A), 2 (•) and control (B): * p ⁇ 0.05, ** p ⁇ 0.01, *** p0.001.
- Figure 2 Western-blot detection of anti-VapA serum antibodies. The technique illustrates the binding of the antibodies contained in the sera tested to the 15 KDa band corresponding to VapA.
- VapA rabbit anti-VapA polyclonal antibody (arrow)
- FIG. 3 Kinetics of arG-R.equi IgGs in foals from mares immunized with antigens containing the VapA protein (Group 1, "Gl"), mares immunized with killed R. equi whole bacteria (Group 2,
- Figure 4 Opsonisation of R. equi by neutrophils of mares and foals, controls ([]) group 2 (() and group 1 (
- EXAMPLE 1 PROTOCOL FOR PREPARING VACCINE ANTI-R. EQUI FOR GRAVID MARS (EXTRACTION OF ANTIGENS TO TRITON)
- the objective of this example is to evaluate the effect of immunization of a pregnant mare with a potential vaccine containing VapA associated with a nanoparticulate adjuvant, on specific IgG levels and serum opsonizing activity, transmitted to the foal via colostrum, and the occurrence of R. equi pneumonia.
- the purified solution of R. equi proteins containing Vap A was prepared from strain 85F and characterized by western-blot.
- An injection was composed of 1 mg of protein diluted in 0.5 ml of tris acetate buffer, pH 8.5, 2% Triton X-100 (Sigma, St Quentin Fallavier, France), combined with 0.5 ml of adjuvant aqueous nanoparticles (IMS 3012, SEPPIC Laboratories, Castres, France).
- the control animals received the same solution without the proteins.
- Group 2 mares were immunized with a saline solution containing 1.10 9 fixed bacteria, derived from a culture from a foal colt that died of rhodococcosis in one of the stud farms.
- the animals that received the autovaccine during the previous gestation were distributed 9/24 in group 1 and 8/8 in group 2.
- the animals in the control group (15 mares) were paired with those in group 1 according to the term dates.
- the numbers of foals included in the study were respectively 23, 7 and 13 in groups 1, 2 and control, because of a case of stillbirth related to dystocia in group 1 and an interruption of follow-up in the other 3 cases.
- IgGs were assayed by ELISA method.
- 96-well plates Nath Generation Nunc,
- the secondary peroxidase-coupled goat anti-horse IgG antibody (1/1600, Bethyl Laboratories, Montgomery, TX, USA) was incubated for one hour at 37 ° C.
- 100 ⁇ l of TMB (3,3 ', 5,5'-tetramethylbenzidine) ready for use (Uptima, Interchim, Montlucon, France) were deposited and incubated for 20 min at room temperature.
- the reaction was stopped by the addition of H 3 P ⁇ 4 IM.
- the optical density was read at 415 nm on an automatic spectrometer. The retained OD value was determined by subtracting the average OD of the control wells from the OD of the experimental sera.
- the results are expressed in arbitrary units (mean OD for 1/1000 dilution, and 1/10000 and 1/50000 OD extrapolated to 1/1000).
- the limit of detection was experimentally determined by Western blot with a serum free of antibodies against Rhodococcus equi.
- the VapA-containing protein solution migrated on a 12% SDS-PAGE gel.
- the proteins were transferred to a nitrocellulose membrane for 90 min at 90 volts.
- the membranes were blocked overnight at 40 ° C. in a solution of TBS-T containing 5% BSA and then washed three times with TBS-T ("Tris buffer saline" or salt buffer, known to man art, associated with Tween).
- TBS-T Tris buffer saline
- the presence of 15 Kda VapA bands was monitored with a polyclonal anti-VapA rabbit antibody (1/2000) prepared by immunization with VapA peptides and revealed (1/30000) by secondary goat anti-IgG antibodies. rabbit coupled with alkaline phosphatase (Sigma, St Quentin Fallavier, France).
- Table 1 Number of animals with antibodies to Vap A detected by western-blot in mares before / after immunization and foals.
- IgG dosed with ELISA were significantly more abundant in colostrum of mares in group 1 (mean 2.124 AU ⁇ 0.035) and group 2 (mean 1.928 ⁇ 0.160) than in control mares (mean 1 ⁇ 0.147)
- Bacterial strain Rhodococcus equi 85F deposited at the CNCM on July 2, 2004 under the number 1-3250.
- Adjuvant IMS 3012 (France SEPPIC 5)
- Antigen Sl antigen solution diluted to 1 mg / ml and filtered (0.22 .mu.m)
- the adjuvant and the antigen are mixed (vol: vol) and then the mixture is stirred gently for 10 min at 40 ° C.
Abstract
Description
Claims
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Application Number | Priority Date | Filing Date | Title |
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FR0408136A FR2873386B1 (en) | 2004-07-22 | 2004-07-22 | VACCINE COMPOSITION AGAINST RHODOCOCCUS EQUI |
PCT/FR2005/001793 WO2006021643A1 (en) | 2004-07-22 | 2005-07-12 | Vaccine composition against rhodococcus equi |
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EP1769065A1 true EP1769065A1 (en) | 2007-04-04 |
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EP05788683A Ceased EP1769065A1 (en) | 2004-07-22 | 2005-07-12 | Vaccine composition against rhodococcus equi |
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US (1) | US8052978B2 (en) |
EP (1) | EP1769065A1 (en) |
CA (1) | CA2574516A1 (en) |
FR (1) | FR2873386B1 (en) |
WO (1) | WO2006021643A1 (en) |
Families Citing this family (2)
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GB0919733D0 (en) * | 2009-11-11 | 2009-12-30 | Univ Edinburgh | Immune system modulating composition |
IT201900014121A1 (en) | 2019-08-06 | 2021-02-06 | Univ Degli Studi Di Camerino | MARINOMONAS EF1 AND RHODOCOCCUS EF1 FOR THE PRODUCTION OF SECONDARY METABOLITES |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4803070A (en) | 1986-04-15 | 1989-02-07 | Ribi Immunochem Research Inc. | Immunological emulsion adjuvants for polysaccharide vaccines |
HU212924B (en) | 1989-05-25 | 1996-12-30 | Chiron Corp | Adjuvant formulation comprising a submicron oil droplet emulsion |
DE69332031T2 (en) | 1993-01-08 | 2002-12-19 | Csl Ltd | VACCINE PREPARATIONS |
CA2125426A1 (en) * | 1994-06-08 | 1995-12-09 | University Of Guelph | Rhodococcus equi gene sequence |
GB9705588D0 (en) | 1997-03-18 | 1997-05-07 | Anglia Research Foundation | Stable particle in liquid formulations |
AU6253898A (en) | 1997-12-16 | 1999-07-05 | Chiron Corporation | Use of microparticles combined with submicron oil-in-water emulsions |
CN101219217A (en) | 1998-05-07 | 2008-07-16 | 科里克萨有限公司 | Adjuvant composition and methods for its use |
US6585973B1 (en) | 1998-10-29 | 2003-07-01 | Henry M. Jackson Foundation For The Advancement Of Military Medicine | Method for preparing solid phase conjugated vaccine |
HUP0200302A2 (en) * | 1999-02-18 | 2002-05-29 | Rmf Dictagene Sa | Malaria vaccine |
TR200103333T2 (en) | 1999-05-20 | 2002-04-22 | Pharmasol Gmbh | Auxiliary drug (SBA) that improves endurance and biological compatibility |
US20020058040A1 (en) * | 1999-12-23 | 2002-05-16 | Stephen Grimes | Stable immunogenic composition for frozen storage |
FR2825276B1 (en) | 2001-05-31 | 2004-11-26 | Seppic Sa | SOLID FORM IMMUNITY ADDICTIVE AND VACCINE CONTAINING THEM |
US20040038303A1 (en) | 2002-04-08 | 2004-02-26 | Unger Gretchen M. | Biologic modulations with nanoparticles |
-
2004
- 2004-07-22 FR FR0408136A patent/FR2873386B1/en active Active
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2005
- 2005-07-12 EP EP05788683A patent/EP1769065A1/en not_active Ceased
- 2005-07-12 WO PCT/FR2005/001793 patent/WO2006021643A1/en active Application Filing
- 2005-07-12 US US11/572,293 patent/US8052978B2/en active Active
- 2005-07-12 CA CA002574516A patent/CA2574516A1/en not_active Abandoned
Non-Patent Citations (3)
Title |
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EDELMAN R: "The development and use of vaccine adjuvant", MOLECULAR BIOTECHNOLOGY, HUMANA PRESS, INC, US LNKD- DOI:10.1385/MB:21:2:129, vol. 21, no. 2, 1 June 2002 (2002-06-01), pages 129 - 148, XP002976315, ISSN: 1073-6085 * |
PRESCOTT J F ET AL: "Use of a virulence-associated protein based enzyme-linked immunosorbent assay for Rhodococcus equi serology in horses", EQUINE VETERINARY JOURNAL, vol. 28, no. 5, 1996, pages 344 - 349, ISSN: 0425-1644 * |
See also references of WO2006021643A1 * |
Also Published As
Publication number | Publication date |
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WO2006021643A1 (en) | 2006-03-02 |
US8052978B2 (en) | 2011-11-08 |
FR2873386B1 (en) | 2011-01-14 |
FR2873386A1 (en) | 2006-01-27 |
US20080008726A1 (en) | 2008-01-10 |
CA2574516A1 (en) | 2006-03-02 |
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