WO2003059383A1

WO2003059383A1 - Brucellosis vaccine composition comprising poly($g(e)-caprolactone) microparticles as an adjuvant

Info

Publication number: WO2003059383A1
Application number: PCT/ES2002/000367
Authority: WO
Inventors: Carlos Gamazo De La Rasilla; Juan Manuel Irache Garreta; José BLASCO MARTINEZ; María del Mar GOÑI LEZA; María Isabel MURILLO MARTIN; María Jesús GRILLO DOLSET; Clara Marin Alcala; Monserrat Barberan Pelegrin; Judith REÑE GONZALEZ
Original assignee: Instituto Cientifico Y Tecnologico De Navarra, S.A.
Priority date: 2001-07-31
Filing date: 2002-07-23
Publication date: 2003-07-24
Also published as: ES2190350B1; AR034950A1; AU2002365205A1; ES2190350A1

Abstract

The invention relates to a subcellular vaccine composition comprising a substance which is immunogenically-active against Brucella spp. and an adjuvant comprising poly(ε-caprolactone) microparticles encapsulating, in a mixture, the aforementioned substance which is immunogenically-active against Brucella spp. and different complexing and solubilising agents.

Description

COMPOSITION OF VACCINE AGAINST BRUCELOSIS THAT INCLUDES POLY MICROPARTICLES (ε-CAPROLACTONE) AS ADJUVANT

FIELD OF THE INVENTION

This invention relates to a brucellosis vaccine composition comprising an immunogenic substance active against Brucella spp. and an adjuvant comprising poly (ε-caprolactone) microparticles.

BACKGROUND OF THE INVENTION

1. Brucellosis and vaccines

Human brucellosis is usually characterized by sweating and acute or intermittent fever with several clinical manifestations. The most frequent signs are lymphadenopathy, hepato-splenomegaly and ostearticular, genital and cardiac complications (endocarditis being the main cause of the few deaths that it causes today). However, the reservoir of the disease is animals, in which the disease usually goes without external signs and the most frequent clinical manifestations are abortions, the birth of unviable animals and alterations in the genital apparatus of males (Blasco et al., Ovis, treaty of pathology and sheep production, 1990). In infected animals, bacteria are excreted during abortion or childbirth; they are found in large quantities (up to ten billion bruises per gram) in colostrum and milk, in vaginal exudate and in the organs of the aborted fetus.

Bacteria belonging to the genus Brucella have a fairly wide range of hosts. Six species are known and, although they are characterized by a certain exclusivity in host selection, they also produce cross infections. These species are: B. abortus, which infect cattle; B. meli tensis, sheep and goats; B. suis, the pig; B. neotomae, the desert rat; B. ovis the sheep, and B. canis, the dogs. With the exception of B. ovis and B. neotomae, all are pathogenic for man (Young and Corbel, In: Brucellosis: Clinical and laboratory aspects of human infection. CRC Press inc., Boca mouse, Florida. 1989).

Brucellosis in humans and animals is increasing in certain parts of the world, especially in developing countries in the Mediterranean area, the Middle East, West Asia and some regions of Africa and Latin America. In the Mediterranean and Middle East basin the annual incidence in humans varies from less than 1 to 78 cases per 100,000 inhabitants; however, more than 550 cases have been described in some endemic areas where control measures are not being applied in animals. Up to 77 cases per 100,000 inhabitants have been described in certain communities in southern European countries where control measures in animals are mandatory. In addition, despite being a mandatory declaration, since it is a frequently asymptomatic infection, the number of real cases could be twenty times that of those registered. In a recent study of a country in the Arabian peninsula, 20% of the human population presented antibodies against Brucella, with more than 2% of active disease (data from WHO-1999).

In Spain, although the incidence of the disease has been decreasing progressively, it remains an endemic disease, with an incidence rate of 7.6 per 100,000 inhabitants, higher than that of the countries around us. With regard to economic losses, these are estimated at around 14,000 million pesetas per year, not counting those associated with the disease in man. As an indicator of the importance of this disease, keep in mind that the program to eradicate ovine, caprine and bovine brucellosis of the European Union, received more than half of funds for the control of infectious diseases in animals in 1997.

Obviously, the control of human brucellosis necessarily involves the control and eradication of the disease in animals. It has also been seen that in Spain and other countries with extensive production systems the only effective alternative for the control of B. Meli tensis in sheep and goats is the use of vaccination programs.

The most universally used prophylaxis system against ovine and caprine brucellosis consists in the subcutaneous inoculation of complete doses of the vaccine strain Rev 1 (Blasco et al., Ovis, a pathology and sheep production treaty. 1990), an attenuated live strain developed by the selection of reversing strains of streptomycin-dependent B mutants. meli tensis. This vaccination system allows the vaccine strain to pass into the general circulation and be distributed throughout the organism of the vaccinated animal, persisting several months in it. The method confers a strong immunity but induces the formation of a high rate of circulating antibodies (against smooth lipopolysaccharide, LPS-S) that persist until several years after vaccination and make it impossible to differentiate infected animals from vaccinated animals by not there are differences in the antigenic structure of Rev 1 and that of the smooth field strains (Gamazo et al., Infect. Immun. 57 (1989) 1419-1426). Consequently, the use of vaccines that induce a high level of antibodies against LPS-S would be technically advised against. In addition, said Rev 1 vaccine, although effective in lambs, is less effective in the vaccination of adult animals, and is virulent for humans, so its use is prohibited in those countries where B infection has been eradicated. meli tensis.

In a previous study, the inventors have shown that Brucella species have a pattern of Very similar outer membrane proteins, and identical in different strains of smooth and rough species from different geographical origins. This fact, together with the existence of cross-reactions between them, and the intense immunogenicity that the outer membrane proteins possessed, suggests that these antigens could serve as effective vaccines for the prophylaxis of brucellosis (Gamazo et al., Infect. Immun. 57 (1989) 1419-1426). In fact, the outer membrane proteins and the complexes that they form with LPS are excellent diagnostic antigens (in particular, in the case of B. ovis infection). Thus, most of the moruecos infected by B. ovis have high levels of circulating antibodies against outer membrane proteins (Riezu-Boj et al., Infec. Immun. 58 (1990) 489-494). 2. Adjuvants in general

The exploitation of the new generation vaccines, which include subcellular vaccines (membrane complexes, synthetic peptides, recombinant proteins, DNA, etc.), is being limited mainly due to their deficient immunogenicity and the lack of appropriate adjuvants.

An adjuvant is any substance that increases the immune response to an antigen with which it is mixed. The adjuvants act essentially by means of three mechanisms: (i) forming an antigen deposit at the place of application of the vaccine, from which the antigen is released for a variable period of time; (ii) supplying the antigen to the antigen presenting cells; and (iii) inducing the secretion of interleukins. Depending on the type of adjuvant, one type or another of immunity will preferably be stimulated. Thus, for example, the most frequently used alumina, preferably stimulates the production of antibodies (preferential induction of Th2 lymphocytes). Other adjuvants are also capable of stimulating cellular immunity (via Thl) _: lipid A (lipopolysaccharide component of the gram negative bacteria envelope); muramyl dipeptide [MDP] (from the cell wall of Mycobacterium); liposomes (composed mainly of phospholipids), microparticles of biodegradable polymers (polylactic, polyglycolic), plant polymers (ISCOM, from Quilaja saponaria), etc.

(Bowersock & Martin, Adv. Drug Del. Rev., 38 (1999) 167-194).

Although in veterinary practice the restrictions are minor, currently, aluminum hydroxide and phosphate are the only adjuvants approved for use in man. However, these adjuvants cannot be lyophilized and are not effective for many vaccines, even causing allergic problems after a re-immunization. Therefore, the development of new galenic systems as adjuvants, such as those mentioned above, is being promoted: liposomes and microparticles.

Liposomes and microparticles are new drug delivery systems, called vectors, that have had great development in recent years. These pharmaceutical forms allow designing a more rational and better adapted therapy, by increasing the efficacy and specificity of the biologically active drug or molecule that they incorporate. Among the advantages that these systems bring to therapeutics, the following are worth mentioning (Couvreur & Puisieux. Adv. Drug Del. Rev., 10 (1993) 141-162):

(i) increase the stability of the material (drug, antigen, etc.) that they incorporate during the manufacture, transport and storage of the medicine;

(ii) protect the encapsulated material or associated with these pharmaceutical systems against their chemical, enzymatic or immunological inactivation in the environmental conditions of the organism;

(iii) improve the transport of the biologically active molecule to hard-to-reach places and its penetration into the cell; Y (iv) prolong the residence time of the drug in the organism (interesting for those molecules with high clearance and low biological half-life) and, control its release. The microparticles are solid particle type systems of size equal to or greater than 1 micrometer (generally accepted between 1 and 250 μm) that can have porous or vesicular matrix structure (Orecchioni and Irache, Formes pharmaceutiques pour application lócale. Lavoisier Tech & Doc, Paris , 1996, 441-457). According to the manufacturing techniques used and depending on the morphology of the particles, it is possible to distinguish three categories of microparticles (Benoit et al., Microencapsulation methods and industrial applications, Marcel Dekker, New York, 1996, 35-71):

(i) microcapsules or spherical particles constituted by a solid coating containing in its interior a solid, liquid or pasty substance [each microcapsule constitutes a reservoir system that gives rise to a state of maximum heterogeneity];

(ii) microparticles: spherical particles constituted by a continuous network of support or polymeric material in which the substance to be encapsulated is dispersed to the molecular state (solid solution) or to the particular state (solid dispersion) [this structure, in a state of maximum homogeneity , constitutes a matrix system]; Y

(iii) homogeneous microcapsules (multinuclear forms) or heterogeneous microparticles (particular dispersions): they are intermediate systems between the two possible states of heterogeneity (microcapsules) and homogeneity

(microparticles), and are identified by the presence of rich and poor areas in active principle and by having an internal structure of crystalline dispersion type.

In general, the main advantages of microparticles can be summarized in that they offer a protection of the material and / or encapsulated drug from its eventual degradation in storage and / or biological conditions and allow sustained release profiles over time, without the need for repeated administrations. The microparticles can be obtained from natural or synthetic materials. The former include proteins (albumin, collagen, gelatin) and polysaccharides

(hyaluronic acid, alginic acid, chitosan). Synthetic materials include hydroxy acid poly-esters, poly-orthoesters, polycarbonates, poly-amino acids, poly-anhydrides, poly acrylamides and poly-alkyl-α-cyanoacrylates. In any case, the most commonly used materials in the manufacture of microparticles are biodegradable polymers of the poly-ester type. Among them, the following polymers may be mentioned: polylactic acid (PLA), polylactic acid and glycolic acid (PLAGA) copolymers, poly (hydroxybutyric acid) and poly (ε-caprolactone) (PEC).

Microparticle manufacturing procedures can be classified into three main groups: physicochemical, chemical and mechanical. The selection of a microparticle manufacturing process depends on the physical and chemical properties of the active principle / polymer couple and the final characteristics of the microparticles to be prepared (granulometry, internal structure, active principle load, release profile, wettability, etc.) depending mainly on the route of administration chosen. On the other hand, the use of microparticles may be interesting to attempt a vaccination through the intramuscular, subcutaneous or oral routes. The purpose of intramuscular or subcutaneous administration of microparticles is to form a reservoir or reservoir of medication at the place of administration from which the encapsulated active ingredient is released in a controlled manner. In that area the polymeric support slowly degrades and releasing the encapsulated drug by diffusion and erosion. The degradation rate of the system depends on its shape, size and the material used.

Oral administration of microparticles can be used for the vectorization of Peyer's plaques or other specific areas of the mucosa. In fact, oral vaccination has been proposed as an alternative to parenteral because of the latter's disadvantages. It should be borne in mind that most bacteria and viruses access the host through mucous membranes so it has been suggested that to confer the highest level of protection, the place of immunization should be parallel to the place of infection (Childers et al., Reg. Immunol., 3 (1990) 8-16; Rubas et al., ^" . Microencaps., 7 (1990) 385-395). Peyer plates (PP) along with other aggregates of lymphoid tissue that they are located in the gastrointestinal tract they are specialized in the uptake and absorption of macromolecules, bacteria, viruses and other pathogenic organisms present in the intestinal lumen and, therefore, are responsible for the initiation of the intestinal immune response (Wolf & Bye, Ann Rev. Med., 35 (1984) 95-112; Kreuter, Adv. Drug Deliv. Rev., 1 (1991) 71-86; Gilligan & Po, Int. J. Pharm., 75 (1991) 1-24 Two solid arguments demonstrate this statement: (i) the cytoplasm of these cells is poor in lysosomes, with which the material absorbed is practically not degraded, and (ii) these cells pour the captured material into a medium rich in immunocompetent cells (O'Hagan, Clin. Pharmacokinet , 22 (1992) 1-10; Jepson et al., Cell Tissue Res. , 271 (1993) 399-405). Once the particles have passed through the PP (through the M cells), they migrate to the mesenteric vessels through the lymphatic capillaries (Jani et al., Int. J. Pharm., 84 (1992) 245-252 ; Jani et al., Int. J. Pharm., 86 (1992) 239-246) and an event cascade is established that produces IgA secretion in the different mucous membranes of the organism (Bergmann et al., Int. Arch. Allergy Appl. Im unol., 87 (1998) 334-335; Eldridge et al., J ^" . Control. Reí., 11 (1990) 205-214).

The factors that influence the degree of capture of the particles by the PP are (i) the hydrophilic / lipophilic balance of the system, (ii) the surface charge and (iii) the size. The uptake of particles by PP seems restricted to particles smaller than 10 μm (Eldridge et al., Adv. Exp. Med. Biol. 251 (1989) 191-202). The greater than 5 μm are fixed to the PP, while those of a smaller diameter are transported to the efferent lymphatic vessels (Eldridge et al., J. Control. Reí., 11 (1990) 205-214). Also, it has been seen that the arrival in the general circulation of the administered particles increases with decreasing size (Nefzger et al., J. Pharm. Sci., 73 (1984) 1309-1311). On the other hand, as hydrophilicity and / or the surface charge of the particles increases, the capture and / or absorption by the PP decreases (Eldridge et al., J ^" . Control. Reí., 11 (1990) 205-214).

SUMMARY OF THE INVENTION

The invention generally faces the problem of developing effective vaccines for the prevention of infection caused by Brucella spp. The solution provided by this invention is based on the use of microparticles, of the heterogeneous microsphere type or homogeneous microcapsule, based on biodegradable synthetic polymers, of the polyester type, capable of encapsulating protective antigens against Brucella spp. The inventors have found that the use of poly (ε-caprolactone) microparticles as an adjuvant, together with an immunogenically active substance against Brucella spp., Provides effective vaccines against brucellosis, as illustrated in the accompanying Example 2 to this description. Accordingly, an object of this invention is a brucellosis vaccine composition comprising an immunogenic substance active against Brucella spp. and an adjuvant comprising poly (ε-caprolactone) microparticles.

A further object of this invention is a process for obtaining said vaccine composition.

Another additional object of this invention is the use of microparticles of poly (ε-caprolactone) as an adjuvant, in the preparation of a vaccine against brucellosis.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a photograph showing the morphology of the multiple emulsion (Aι / 0) A ₂ , obtained by optical microscopy, during the manufacturing process of microparticles with HS HS antigen. ovis [see Example 1.2].

Figure 2 is an SEM photograph of the poly (ε-caprolactone) microparticles loaded with the HS-CD antigen complex [see Example 1.2].

Figure 3 is a graph showing the release kinetics of the HS antigen from the poly (ε-caprolactone) microparticles [see Example 1.3]. Figure 4 is a graph showing the release profile of the HS antigen from the poly (ε-caprolactone) microparticles. The release is expressed in ng of HS per mg of microparticles [see Example 1.3].

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a vaccine composition comprising an immunogenically active substance against Brucella spp. and an adjuvant, characterized in that said adjuvant comprises poly (ε-caprolactone) microparticles [PEC] encapsulating said immunogenically active substance against Brucella spp.

In a particular embodiment, said vaccine composition is a subcellular or acellular (avirulent) vaccine composition.

The immunogenically active substance against Brucella spp. it can be any substance that elicits an immune response in an animal in contact with a Brucella spp. antigen, and includes antigens or membrane extracts, synthetic peptides, recombinant proteins, DNA, etc., preferably, membrane antigens (lipopolysaccharide complexes with outer membrane proteins).

In a particular embodiment, the immunogenically active substance against Brucella spp. It is an extract of the outer membrane of B. ovis [see Example 1] which, after a heat treatment in saline medium, releases the HS antigen complex (Gamazo et al., Infect. Immun., 57 (1989) 1419-1426), which contains phospholipids, outer membrane proteins and lipopolysaccharide. Its characterization can be performed based on the percentage of proteins, for example, by the method of Lowry (Lowry et al., J. Biol. Chem 193 (1951) 265-275) and the amount of lipopolysaccharide, for example, by KDO trial (Osborn et al., Proc. Nati. Acad. Sci. USA 50 (1963) 4449-506). The B. ovis HS antigen is immunogenic as the results obtained previously show (Blasco et al., Vet. Immunol. Immunopathol. 37 (1993) 257-270).

In another particular embodiment, the immunogenically active substance comprises external membrane antigens of B. ovis (lacking LPS-S) capable of stimulating a correct protective immune response against smooth and rough strains of Brucella spp. without interfering with diagnostic tests against smooth strains.

The immunogenically active substance against Brucella spp. can be mixed with a stabilizing compound capable of stabilize it In a particular embodiment, said stabilizing compound is a cyclodextrin, preferably, a β-cyclodextrin that acts as a solubilizer and stabilizer of said immunogenically active substance against Brucella spp. Cyclodextrins are excipients for pharmaceutical use with multiple practical applications for the solubilization and formation of complexes with drugs (Patent WO9961062).

As an adjuvant, microparticles of a biodegradable synthetic polymer are used, specifically poly (ε-caprolactone) [PEC] capable of encapsulating immunogenically active substances against Brucella sp. The use of PEC has the advantage that in its degradation it will not confer an acidic medium to the medium such as occurs with the use of polylactic agents with the subsequent loss of their structural integrity and antigenicity (Gander et al., Intern. Symp. Control. Reí. Bioact. Mater. 20 (1993) 65-66; Schwendeman et al., Dev. Biol. Stand. 87 (1996) 293-306; Benoit et al., Int. J. Pharm., 184 (1999) 73-84). Other advantages of PEC include its hydrophobicity, its in vitro stability and its low cost, which is more viable in formulations intended for veterinary use. PEC is a biocompatible polymer and slowly hydrolyzes in the body to give water-soluble degradation products that will be excreted or metabolized (Pitt et al., In: Chasin M., Langer. R., (Eds.) Biodegradable polymers as Drug Delivery Systems, Marcel dekker, New York (1990) 71-120).

PEC microparticles containing the immunogenically active substance against Brucella spp. encapsulated therein may also contain in addition to the stabilizing compound of said immunogenically active substance, for example, a cyclodextrin, surfactants used in the preparation of the multiple emulsion from which the PEC microparticles containing said substance are obtained inside immunogenically active against Brucella spp., according to the procedure used in this invention for the manufacture of PEC microparticles comprising solvent removal after having formed a multiple emulsion [these aspects will be described in more detail when describing the manufacturing process of said PEC microparticles containing the immunogenically active substance against Brucella encapsulated inside]. In a particular embodiment, said surfactants are (i) a copolymer of ethylene oxide and propylene oxide and (ii) polyvinyl alcohol. Accordingly, the invention provides PEC microparticles, useful as a subcellular vaccine against brucellosis, which have the following composition, in dry weight:

Poly (ε-caprolactone): 74.6%, Cyclodextrin complex / immunogenically active substance against Brucella spp .: 3.0%,

Copolymer of ethylene oxide and propylene oxide: 22.4%, and

Polyvinyl alcohol: sufficient quantity. The vaccine composition provided by this invention contains an effective amount of immunogenically active substance against Brucella spp. in the form of PEC microparticles that encapsulate said immunogenically active substance against Brucella spp. In a particular embodiment, said PEC microparticles contain the concrete composition previously mentioned.

The vaccine composition provided by this invention can be administered by any appropriate route of administration, for example parenterally, preferably subcutaneously, or also through mucous membranes, preferably orally or conjunctivally.

In a particular embodiment, the vaccine composition is a sustained release composition over time, so that a part of the immunogenically active substance against Brucella spp. encapsulated is released quickly, while another part does it continuously over time. In one embodiment of the invention [see example 1], the vaccine composition is a sustained release formulation that has a pulse of release of the immunogenically active substance during the first day of incubation and another two pulses 2 days later and the day 14. From this point on, the immunogenically active substance is released continuously in very low doses.

The vaccine composition provided by this invention is suitable for the prophylaxis of brucellosis. Several trials have shown its effectiveness in experimental animals, achieving a level of protection equivalent to that achieved with the live attenuated reference vaccine Rev 1. However, the incorporation of the antigen complex in the biodegradable microparticles of PEC presents, among others, the following advantages over the current reference vaccine Rev 1: (i) vaccine safety; (ii) possibility of administration in a single dose; and (iii) no interference with diagnostic tests against smooth strains.

In a particular embodiment, the vaccine composition of the invention protected against B. ovis both subcutaneously (SC) and orally, similarly to the reference vaccine Rev 1 [see Example 2], while in another particular embodiment, the vaccine composition of the invention protected against B. SC abortion, presenting statistically similar levels of protection to those conferred by the commercial vaccine Rev 1 [see Example 2]. The microencapsulation of a Brucella external membrane antigenic complex (HS) in PEC biodegradable microparticles induces adequate immunity and facilitates vaccination campaigns, since it allows reducing the number of administrations needed, as it is a controlled release formulation, and, In addition, it is safe for man and could be used in B-free countries. meli tensis.

Among the reasons that justify the industrial interest of the vaccine composition provided by this invention are the following:

1) use of biodegradable pharmaceutical forms under normal organism conditions and manufactured from polymers and materials accepted in pharmaceutical and medical practice, - 2) administration of a vaccine preparation, based on microparticles, which protects the encapsulated antigen against its premature inactivation;

3) preparation and manufacture of a vaccine preparation, based on the use of microparticles, which has a sustained release over time;

4) administration of a vaccine preparation, based on microparticles containing an antigen extract from a rough strain, which protects against brucella infections in the smooth or rough phase; 5) administration of a vaccine preparation that does not induce the formation of circulating antibodies against LPS-S, which would allow its sero-diagnostic differentiation with respect to animals infected by the smooth strains; Y

6) administration of a vaccine preparation that given its characteristics (safety, avirulence) could be susceptible to being used in man.

PEC microparticles can be obtained by conventional methods, such as those mentioned in the Background of the Invention. In a particular embodiment, the manufacturing process of the PEC microparticles was carried out by the solvent evaporation method after the formation of a multiple emulsion (Cohen et al., Pharm. Res. 8 (1991) 713-720; Couvreur et al., Adv. Drug. Del. Rev. 28 (1997) 85-96; 0 = Donnell et al., Adv. Drug. Del. Rev. 28 (1997) 25-42). The invention provides a method for obtaining the vaccine composition provided by this invention comprising (i) the preparation of a complex comprising the immunogenically active substance against Brucella spp., For example, a complex formed by a cyclodextrin, such as β-cyclodextrin, and said immunogenically active substance against Brucella spp., and (ii) the incorporation of said complex into poly (ε-caprolactone) microparticles. The preparation of said cyclodextrin-immunogenically active substance complex against Brucella spp. it comprises the mixing (kneading) of said immunogenically active substance against Brucella spp. with an aqueous solution of cyclodextrin, followed by removal of water, for example, by stirring in a mortar until complete evaporation of water. Alternatively, the cyclodextrin-immunogenically active substance complex against Brucella spp. it can be obtained by dry mixing between cyclodextrin and the immunogenically active substance against Brucella spp.

The incorporation of the cyclodextrin-immunogenically active substance complex against Brucella spp. in the microparticles of poly (ε-caprolactone) it comprises the steps of: a) resuspending the cyclodextrin-immunogenically active substance complex against Brucella spp. in water, in the presence of a surfactant; b) preparing a solution of poly (ε-caprolactone) in an organic solvent; c) mixing the solutions obtained in steps a) and b) to form an aqueous-oil type emulsion in which the internal phase comprises the dissolution of the cyclodextrin-immunogenically active substance complex against Brucella spp. and the external phase is the dissolution of poly (ε- caprolactone); d) prepare an aqueous solution of polyvinyl alcohol; e) mixing the emulsion obtained in step c) with said aqueous solution of polyvinyl alcohol to form a multiple emulsion of type (water in oil) in water, in which the internal phase comprises the primary emulsion obtained in step c) and the external phase is the aqueous solution of polyvinyl alcohol; f) remove the organic solvent, thereby forming the microparticles of poly (ε-caprolactone) containing the cyclodextrin-immunogenically active substance complex against Brucella spp .; g) purifying said poly (ε-caprolactone) microparticles containing the cyclodextrin-immunogenically active substance complex against Brucella spp .; and h) optionally, lyophilize said poly (ε-caprolactone) microparticles containing the cyclodextrin-immunogenically active substance complex against Brucella spp.

More specifically, the cyclodextrin-immunogenically active substance against Brucella spp. it is collected with an aqueous solution of a surfactant, such as a copolymer of ethylene oxide and polypropylene oxide

(Pluronic F-68), a surfactant that helps the formation of the emulsion, and then is added onto a solution of PEC in an organic solvent, for example, dichloromethane, and an A / 0 emulsion is formed, for example , with an ultrasound probe for 1 minute; The emulsion formed has an oily external phase that contains in its interior an aqueous internal phase in which the antigenic complex is found. The emulsion obtained is added on a second aqueous phase containing polyvinyl alcohol as a surfactant and is emulsified, for example, with an Ultraturrax ^® , to form a multiple emulsion (Aχ / θ) A which is checked by optical microscopy. For the microparticles to form, the solvent Organic must diffuse in the aqueous phase and evaporate at the water-air interface. The final emulsion is vigorously stirred at room temperature for 3-4 hours until complete evaporation of the organic solvent at which time the microparticles are formed.

Next, the microparticles formed by conventional methods are collected, for example, by centrifugation or vacuum filtration and washed with distilled water to remove traces of the surfactant used in the formation of the second emulsion. Subsequently, the size of the microparticles is measured by any conventional method, for example, by laser diffractometry. Finally, the microparticles are lyophilized, being in the form of a white lyophilized powder, an optimal form for their conservation, storage and easy reconstitution.

In a particular embodiment, approximately 90% of the PEC microparticles containing the encapsulated antigen therein have a particle size of less than 2.5 μm, their final lyophilized form is spherical and without pores.

The following examples illustrate the invention and should not be considered in a limiting sense of the scope thereof. In the examples, biodegradable microparticles of PEC (Aldrich-Chemie) are used, containing an antigenic complex extracted from the rough strain of the genus Brucella, B. ovis REO 198, as a subcellular vaccine against infections caused by B. ovis and B. abortus Together with the antigen, β-cyclodextrin (Aldrich-Chemie) and Pluronic F-68 (Sigma) are added for its protection and stabilization. In its formation the multiple emulsion is checked by optical microscopy (Mod. BH-2, Olympus). Once formed, the size of the microparticles was determined by laser diffractometry (Mastersizer S-Malvern Instru ents / Optilas, Spain). The antigen was analyzed by the bicinconic acid method (BCA- Sigma, Spain), specific for proteins (Smith et al., Anal. Biochem. 150 (1985) 76-85). The particles formed are lyophilized (Virtis Genesis 12EL. USA) and the final shape of the microparticles was observed by scanning electron microscopy (Mod. DSM 940 A Zeiss Digital Scanning Electron Microscopy).

EXAMPLE 1 Preparation and characterization of biodegradable microparticles with HS antigen of B. ovis

1.1 Extraction, characterization and treatment of the HS antigenic complex of B. ovis REO 198 The antigen used is an extract of the outer membrane of B. ovis called HS (Hot Saline) since, in saline medium and with heat it releases the antigenic complex. This antigen contains phospholipids, outer membrane proteins and rough lipopolysaccharide (LPS-R), therefore, does not contain O chain.

HS extract of B. ovis was obtained by a previously described method (Gamazo et al., Infect. Immun. 57 (1989) 1419-1426). After the culture of the bacteria, B. ovis REO 198, in flasks containing tripticase-soy (TSB), the bacteria are centrifuged at 7,000 xg for 20 minutes and washed twice with saline, live cells are resuspended in saline (10 g of wet cells in 100 mL) and heated at ^" 100 ° C in flowing steam for 15 minutes. After centrifugation (12,000 xg, 15 min), the supernatant is dialyzed for 5 days at 4 ° C against several changes with deionized water. The dialyzed material centrifuge for 5 hours at 100,000 xg (23,000 rpm), and the sediment (HS) is resuspended in deionized water, lyophilized and stored at room temperature The yield obtained after the process was 1.24% ± 0.26 (n = 5).

The characterization includes the determination of the percentage of proteins and lipopolysaccharide (LPS). The Determination of the amount of protein was carried out by the Lowry method. The antigenic extract HS of B. ovis contains approximately 45% protein.

The amount of LPS is determined indirectly by measuring one of its exclusive markers, the KDO, by the thiobarbituric acid method. By this method, 1.4% of KDO was obtained, representing 45% of LPS-R.

Prior to incorporation into the microparticles, the HS antigen must be treated with pharmaceutical excipients in order to form a complex mixture that allows its encapsulation in the microparticles. A wet procedure or a dry procedure can be used for this. By the wet process, 4 mg of the HS antigen are kneaded together with 4 mg of β-cyclodextrin for 30 minutes, after the addition of 1 mL of double-distilled water. Subsequently, this mixture is dried in an oven at 60 ° C. By the dry procedure, the HS antigen (4 mg) is mixed, for 30 minutes, in a mortar with the 4 mg of β-cyclodextrin.

In both cases, the mixture of HS antigen and cyclodextrin (from now on HS-CD complex), in the form of solid powder, is the material that must be encapsulated in the microparticles.

1.2 Manufacture and physicochemical characterization of the microparticles with HS antigen of B. ovis

The HS-CD complex is resuspended in 1 mL of a solution of a copolymer of ethylene oxide and propylene oxide (Pluronic F-68) at 6% w / v in water. On the other hand, a lipophilic organic phase is prepared by dissolving 200 mg of poly (ε-caprolactone) in 5 mL of dichloromethane (4% w / v). Subsequently, the hydrophilic aqueous phase (containing the antigenic complex) is added over 5 mL of the lipophilic organic phase (containing the microparticle-forming polymer) and the mixture is emulsified with an ultrasound probe (1 minute, 12- 13 Volts) This first emulsion of an aqueous or hydrophilic phase in an oily or lipophilic (Ai / O) phase is emulsified with a second aqueous phase formed by a 0.5% w / v polyvinyl alcohol (PVA) solution in water (30 mL ), by using an Ultraturrax at 13500 rpm for two minutes. The result is to obtain a multiple emulsion (Aχ / 0) A ₂ [see Figure 1], characterized by small droplets of aqueous phase (where the HS-CD complex is) surrounded by other drops of lipophilic phase (where it is the polymer forming the microparticles) and these, in turn, dispersed in an outer aqueous phase.

The next step is the evaporation of the organic solvent for the formation and obtaining of the microparticles. For this, the multiple emulsion is vigorously stirred for 3-4 hours at room temperature until complete evaporation of the organic solvent.

Subsequently, the microparticles are collected by some method of separation (centrifugation, filtration, etc.) and washed with distilled water twice. When centrifugation is used (10,000 rpm for 10 minutes), the supernatants are removed and the microparticles are resuspended in 5 mL of ultrapure water. Finally, the suspended microparticles are lyophilized either directly (i) directly or (ii) after the addition of a cryoprotectant or adjuvant at a concentration of 5% w / v (sucrose, mannitol, Pluronic F-68).

The formula resulting from the suspension of the microparticles before lyophilizing is as follows:

Poly (ε-caprolactone) 4% w / v

HS-CD 0.4% w / v antigenic complex

- 0.2% w / v HS antigen

- β-cyclodextrin 0.2% w / v Pluronic F-68 1.5% w / v Purified water csp 5 mL Prior to lyophilization, the size of the microparticles is determined by laser diffractometry (Mastersizer S). Table 1 shows the sizes corresponding to the different batches of microparticles expressed in volume, with D (v, 0.1) being the size below which 10% of the microparticles are found.

Table 1 shows the main physicochemical characteristics of the microparticles obtained. The yields of the manufacturing process of the microparticles were obtained by determining their weight at the end of the lyophilization process. Starting from 200 mg of polymer, the amount transformed into a microparticle was determined at the end of the process, and expressed as a percentage with respect to the initial mass of the polymer.

The external appearance of the particles was studied by scanning electron microscopy (SEM) with a gold coating. It was found that the poly (ε-caprolactone) microparticles loaded with the HS-CD antigen complex were spherical and had smooth, pore-free surfaces [see Figure 2].

The quantification and detection of the antigen was carried out by the bicinconic acid method. For this, 20 mg of microparticles are dispersed in 0.1 N NaOH and kept under magnetic stirring for 18 hours. Subsequently, the dispersion is centrifuged at 15,000 rpm for 15 minutes and the supernatant is analyzed by the colorimetric method of bicinconic acid for proteins after validation of the method. The validation of this method has been performed with the antigen dissolved in 0.1 N NaOH previously kneaded with the same amount of β-cyclodextrins. The solution of bicinconic acid together with 5% cupric sulfate in 100: 2 ratio is added to the sample (2 mL in each), the mixture is maintained at 37 ° C for half an hour and then measured spectrophotometrically at 562 nm. The antigen load is expressed as the amount of antigen in μg per mg of microparticles and the encapsulation efficiency was determined by relating the total amount of antigen encapsulated in the microparticles to the initial amount of antigen. Table 1 shows the antigen load in the microparticles expressed in μg of antigen per mg of microparticles and the encapsulation efficiency (%).

Table 1 Physico-chemical characteristics of poly (ε-caprolactone) microparticles loaded with the HS-CD antigen complex

Size (μm) Rdto ¹ Antigen Encapsulated efficacy ² encapsulation

Mean D (0.1) D (0.5) D (0.9) (μg HS / mg) (%)

1.34 0.38 1.09 2.54 71% 5.18 ± 1.52 30.73 ± 5.89

manufacturing performance expressed in percentage by weight with respect to the initial polymer.

² Encapsulated HS antigen expressed in μg of HS antigen per mg of microparticle.

1.3 In vitro release of the antigen from the microparticles

For the release study, 30 mg of lyophilized microparticles were dispersed using a vortex in 1 mL of phosphate buffer (0.01 M, pH 7.4), 0.02% sodium azide as a bacteriostatic agent. The samples were kept under constant stirring in a bath at 37 ± 1 ° C and at certain time intervals (1, 3, 6 hours, 1 day, 2, 4, 7, 14, 21 and 28 days) the suspension was centrifuged (17000 rpm for 5 min) collecting the supernatant. This was centrifuged again in eppendorf tubes (22,500 rpm for 15 min) and the amount of antigen released was determined using the bicinconic acid method.

Figure 3 shows the antigen release profile from the microparticles, expressed as a percentage accumulated over time. The percentage released after the first hour of incubation corresponds to the antigen found on the surface of the microparticles, this portion would be the first to be recognized by the immune system.

The amount of HS antigen released per mg of microparticles per day is shown in Figure 4. It is interesting to observe how there is a release pulse during the first day of incubation and another two pulses 2 days later and day 14. From At this point, the HS antigen is released continuously in very low doses.

EXAMPLE 2 In vivo study of the protection conferred by orally administered microparticles to experimentally infected mice Lots of female BALB / c mice of 8-10 weeks of age were used, each consisting of 5 animals. Vaccination was carried out with the indicated preparations at a rate of 20 μg of antigen per animal (oral and SC), also including as controls: (i) the same amount of the corresponding empty particles; (ii) 20 μg / animal of free, non-encapsulated antigens; (iii) 5 x 10 ⁴ CFU (colony forming units) / animal of the vaccine strain B. meli tensis Rev 1; and (iv) PBS: 0.1 ml (orally or SC).

Eight weeks after vaccination, they were inoculated intraperitoneally with 5 x 10 ⁴ CFU of the virulent rough strain B. ovis PA, or with 5 x 10 ⁴ CFU of the virulent smooth strain of reference B. abortus 2308. Animals infected with B. abortus 2308 were sacrificed at 2 weeks post-infection, while those infected with B. PA ovis was at 3 weeks post-infection. After sacrifice, aseptic extraction of the spleens was performed and subsequent individual homogenization in 10 mL of PBS. Splenic counts of the number of CFUs of the virulent strain were performed by decimal dilutions, plating on TSA plates and incubation for 3-5 days at 37 ° C in an atmosphere with 10% C0 ₂ . The mean and standard deviation of the splenic counts corresponding to each lot were calculated, previous logarithmic transformation. Statistical comparisons of the means obtained in each lot with respect to their corresponding control lot were made using the Dunnett multiple comparison test. The results are presented in Tables 2 and 3.

Table 2 IP infection with 5 x 10 ⁴ of B. ovis PA and sacrifice 3 weeks post-infection

VACCINE ORAL ROUTE SUBCUTANEOUS ROUTE

Log media (SD), DUNNETT test *

BO2101 4.77 (2.61) * 3.12 (1.83) ** MP empty 6.92 (0.16) 5.24 (1.74) HS free 6.51 (0.51) 7, 20 (0.30) Rev 1 4.88 (2.03) * 3.72 (1.61) ** PBS 7.24 (0.30) 7.24 (0.35)

* S = significant (P <0.05); ** (P <0.01), vs PBS control Table 3 IP infection with 5 x 10 ⁴ of B. abortus 2308 and sacrifice 2 weeks post-infection ORAL VACCINE SUBCUTANEOUS ROUTE

Log average (SD), DUNNETT test * BO2101 6.16 (0.28) 3.90 (0.67) **

Empty MP 5.91 (0.43) 4.79 (1.38)

Ag HS free 5.74 (0.39) 5.42 (0.66)

REVI 2.86 (1.13) **

PBS 5.95 (0.44) 5.95 (0.44)

* S = significant (P <0.05); ** (P <0.01), vs PBS control

Vaccine preparation based on microparticles protected against B. ovis both by SC and Oral route, in a similar way to the reference vaccine Rev 1; also protected by SC route B. abortus (reduction of splenic infection by 2 logs compared to the unvaccinated control), presenting statistically similar levels of protection as those conferred by the commercial vaccine Rev 1.

Claims

1. A subcellular vaccine composition against brucellosis comprising: a) an immunogenically active substance against Brucella spp. ; b) an adjuvant, characterized in that it comprises microparticles of poly (ε-caprolactone) that encapsulate said immunogenically active substance against Brucella spp .; and c) a stabilizing compound of said immunogenically active substance against Brucella spp. with which it forms a complex.

2. Vaccine composition according to claim 1, characterized in that said stabilizing compound of said immunogenically active substance against Brucella spp. It is a cyclodextrin.

3. Vaccine composition according to claim 2, characterized in that said stabilizing compound of said immunogenically active substance against Brucella spp. is β-cyclodextrin.

4. Vaccine composition according to any one of claims 1 to 3, characterized in that it further comprises a copolymer of ethylene oxide and propylene oxide.

5. Vaccine composition according to any of claims 1 to 4, characterized in that said immunogenically active substance against Brucella spp. It comprises an outer membrane complex of Brucella spp.

6. Vaccine composition according to claim 5, characterized in that said immunogenically active substance against Brucella spp. it is an extract of the outer membrane of B. ovis called HS, which comprises phospholipids, outer membrane proteins and lipopolysaccharide.

7. Vaccine composition according to any of claims 3 to 6, characterized in that the β-cyclodextrin-immunogenically active substance complex against Brucella spp. encapsulated in said poly (ε-caprolactone) microparticles is present in an amount of 3.0% by dry weight per poly (ε-caprolactone) microparticle.

8. Vaccine composition according to any one of claims 1 to 7, characterized in that it is administered parenterally or through mucous membranes.

9. Vaccine composition according to claim 8, characterized in that it is administered orally.

10. Vaccine composition according to claim 8, characterized in that it is administered conjunctivally.

11. Vaccine composition according to claim 8, characterized in that it is administered subcutaneously.

12. Vaccine composition according to any of claims 1 to 11, characterized in that it is formulated in a sustained release formulation.

13. A method for obtaining a vaccine composition according to any of claims 1 to 12, comprising: a) the preparation of a complex comprising the immunogenically active substance against Brucella spp. and a cyclodextrin; and b) incorporation of said complex into microparticles of poly (ε-caprolactone).

14. A method according to claim 13, wherein the preparation of said cyclodextrin-immunogenically active substance complex against Brucella spp. It comprises the kneading steps of an aqueous solution of cyclodextrin with said immunogenically active substance against Brucella spp., and water removal.

15. The method according to claim 13, wherein the preparation of said cyclodextrin-immunogenically active substance complex against Brucella spp. It comprises the dry physical mixture between cyclodextrin and the immunogenically active substance against Brucella spp.

16. The method according to claim 13, wherein the incorporation of the cyclodextrin-immunogenically active substance complex against Brucella spp. in the microparticles of poly (ε-caprolactone) it comprises the steps of: a) resuspending the cyclodextrin-immunogenically active substance complex against Brucella spp. in an aqueous solution of a surfactant (surfactant I); b) preparing a solution of poly (ε-caprolactone) in an organic solvent; c) mixing the solutions obtained in steps a) and b) to form an aqueous-oil type emulsion in which the internal phase comprises the dissolution of the cyclodextrin-immunogenically active substance complex against Brucella spp. and the external phase is the organic solution of poly (ε-caprolactone); d) preparing an aqueous solution of an excipient with surface properties (surfactant II); e) mixing the emulsion obtained in step c) with said aqueous solution of polyvinyl alcohol to form a multiple emulsion of type (water in oil) in water, in which the internal phase comprises the primary emulsion obtained in step c) and the external phase is the aqueous solution of surfactant II; f) remove the organic solvent, thereby forming the microparticles of poly (ε-caprolactone) containing the cyclodextrin-immunogenically active substance complex against Brucella spp .; g) purifying said poly (ε-caprolactone) microparticles containing the cyclodextrin-immunogenically active substance complex against Brucella spp .; and h) optionally, lyophilize said poly (ε-caprolactone) microparticles containing the cyclodextrin-immunogenically active substance complex against Brucella spp.

17. The method of claim 16, wherein said surfactant I is a copolymer of ethylene oxide and propylene oxide.

18. The method of claim 16, wherein said surfactant II is polyvinyl alcohol.

19. A method according to any of claims 13 to 18, wherein the microparticles of poly (ε-caprolactone) containing the immunogenically active substance against Brucella spp. They have the following dry weight composition:

Poly (ε-caprolactone): 74.6%,

Cyclodextrin / immunogenically active substance complex against Brucella spp .: 3.0%, and

Copolymer of ethylene oxide and propylene oxide:

22.4%

20. Use of microparticles of poly (ε-caprolactone) containing β-cyclodextrin and a copolymer of ethylene oxide and propylene oxide, as an adjuvant in the preparation of a brucellosis vaccine composition.