AU2005279641A1 - Immunogenic compositions - Google Patents

Description

WO 2006/024122 1 PCT/BR2005/000179 "IMMUNOGENIC COMPOSITIONS" Field of the invention The present invention is related to a novel formulation of immunogenic composition. This composition 5 contains antigens in different forms and an adjuvant, both entrapped in biodegradable microspheres. Principles of the invention: The introduction of biodegradable polymers in the pharmaceutical industry, such as lactic acid polymers (PLA) 10 and co-polymers of lactic and glycolic acid (PLGA) with different characteristics of degradation allowed the development of new drug and vaccine delivery systems. The use of microspheres prepared with these co polymers in the development of vaccines has allowed 15 reaching different immunogenic compositions with optimization of antigen presentation to the immune system. These optimized presentation to antigen presenting cells (APC) associated to the chemical composition of microspheres permitted the development of single-shot 20 vaccines allowing that a single formulation to carry several doses of a vaccine increasing the releasing period and presentation of the antigen to APC. This technique has been applied to protect the antigen and to form a depot in the site of administration resulting 25 in the recruitment of immune competent cells. Particles lower than 10 micrometers are selectively taken up by antigen presenting cells, which can optimize the immune response. Particles with higher diameters can be desirable in order to slowly release the antigen in the WO 2006/024122 2 PCT/BR2005/000179 site of administration. These formulations have many applications in the field of vaccinology for Human and animal health, where the antigen may be a purified subunit, a protein or peptide, 5 and a gene vector like plasmid DNA. Despite of the enthusiastic results observed in experimental animal models, generally mice and guinea pigs, these vaccines have failed to be effective in large animals, probably due to the need of optimization of 10 formulation, schedule, dose and also the antigenic composition. The patent BR-PI010 3 8 87 describes the use of a vaccine obtained from microspheres formulation containing at least one antigen, for example a gene vector or a purified 15 protein, associated to an adjuvant. The protein model was a mycobacterial 65kDa heat shock protein, hsp-65, entrapped in its recombinant form or in the form of a gene vector that codify it. The gene vector or the purified protein is presented 20 entrapped into PLGA microspheres formulations associated to an adjuvant, for example the trehalose dymicolate (TDM). The formulation described in the patent BRPI0103 8 8 7 can be used in the prevention or treatment infectious diseases where the induction of protective immune response 25 is dependent of the production of pro-inflammatory cytokines such as gamma-interferon. However this vaccine, despite the induction of significant amounts of gamma-interferon in small and large WO 2006/024122 3 PCT/BR2005/000179 animals, using mice and bovines as models, does not allow a sustained levels of cytokines over long periods. The duration of protection resulted from vaccination is associated to the residence time of the antigen in the 5 body. In some vaccination schedules it is necessary the administration of boosters in order to sustain the protective immune response. Recently, the concept of prime-boost has been explored taking in account the heterologous booster. It means that 10 the same antigen is presented to immune system in different forms during the prime and booster doses. In this strategy, it is very important that the used formulation in the prime must be able to induce a protective immune response. The booster has the function of 15 expanding and maintain the immune response initially stimulated. The most common prime-boost protocols involve the use of DNA vaccines or purified subunits during the initial phase, using live carriers, such as recombinant virus or 20 bacteria expressing the desired antigen, in the boosters. The prime-boost strategy involves the use of different vaccines, each one codifying the same antigen, administered in days or weeks intervals. Such strategy has been able to increase the immune 25 response in different animal and infectious disease models (McShane, H. (2002) Prime-boost immunization strategies for infectious diseases. Curr. Opin. Mol. Ther., 4(l):23-27). As previous described the most part of these prime boost protocols are related to prime the immune responses WO 2006/024122 4 PCT/BR2005/000179 with plasmid DNA and booster with live carriers (Robinson, H.L. (2003) Prime boost vaccines power up in people. Nat. Med., 9(6):642-643; Cheevers, W.P., Snekvik, K.R., Trujillo, J.D., Kumpula-McWhirter, N.M., Pretty, On Top 5 K.J., Knowles, D.P. (2003) Prime-boost vaccination with plasmid DNA encoding caprine-arthritis encephalitis lentivirus env and viral SU suppresses challenge virus and development of arthritis. Virology., 30 6 (1) :116-125; Zanotto, C., Elli, V., Basavecchia, V., Brivio, A., 10 Paganini, M., Pinna, D., Vicenzi, E., De Giuli Morghen, C., Radaelli, A. (2003) Evaluation in rabbits of different anti-SHIV vaccine strategies based on DNA/fowlpox priming and virus-like particle boosting. FEMS Immunol. Med. Microbiol., 35(1):59-65; Pancholi, P., Perkus, M., 15 Tricoche, N., Liu, Q., Prince, A.M. (2003) DNA immunization with hepatitis C virus (HCV) polycistronic genes or immunization by HCV DNA priming-recombinant canarypox virus boosting induces immune responses and protection from recombinant HCV-vaccinia virus infection in HLA-A2.1 20 transgenic mice. J. Virol.; 77(l):382-390; McConkey, S.J., Reece, W.H., Moorthy, V.S., Webster, D., Dunachie, S., Butcher, G., Vuola, J.M., Blanchard, T.J., Gothard, P., Watkins, K., Hannan, C.M., Everaere, S., Brown, K., Kester, K.E., Cummings, J., Williams, J., Heppner, D.G., Pathan, 25 A., Flanagan, K., Arulanantham, N., Roberts, M.T., Roy, M., Smith, G.L., Schneider, J., Peto, T., Sinden, R.E., Gilbert, S.C., Hill, A.V. (2003) Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by WO 2006/024122 5 PCT/BR2005/000179 recombinant modified vaccinia virus Ankara in humans. Nat. Med. ; 9 (6) :729-735) . Despite the good experimental results in these protocols, the disadvantage of the use of live carriers 5 still exists. This strategy re-introduces the controversies related to the safety of live vaccines. In prevention of tuberculosis, the prime-boost strategy has been evaluated combining the priming with plasmid DNA and the maintenance of the immune response with 10 attenuated BCG or recombinant protein (McShane, H., Brookes, R., Gilbert, S.C., Hill, A.V. (2001) Enhanced immunogenicity of CD4(+) t-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime boost vaccination regimen for murine tuberculosis. Infect 15 Immun.; 69 (2) : 681-686; Skinner, M.A., Buddle, B.M., Wedlock, D.N., Keen, D., de Lisle, G.W., Tascon, R.E., Ferraz, J.C., Lowrie, D.B., Cockle, P.J., Vordermeier, H.M., Hewinson, R.G. (2003a) A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces 20 protection against bovine tuberculosis. Infect. Immun.; 71(9):4901-4907; Vordermeier, H.M., Lowrie, D.B., Hewinson, R.G. (2003) Improved immunogenicity of DNA vaccination with mycobacterial HSP65 against bovine tuberculosis by protein boosting. Vet. Microbiol.; 93(4):349-359; Skinner, M.A., 25 Ramsay, A.J., Buchan, G.S., Keen, D.L., Ranasinghe, C., Slobbe, L., Collins, D.M., de Lisle, G.W., Buddle, B.M. (2003b) A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated WO 2006/024122 6 PCT/BR2005/000179 strains of Mycobacterium bovis against bovine tuberculosis. Immunology;108( 4 ) :548-555). These protocols generally requires more than one dose of DNA for the induction of the immune response followed by 5 boosters, being necessary the administration of many doses of DNA. Aiming at to reach attractive applications of DNA vaccines, many efforts have been done in order to optimize the targeting of functional DNA in the cells. The gene vector may be able to reach the target cells in its active 10 form, which means that many barriers may be avoided. The entrapment of plasmid DNA in biodegradable polymeric microparticles is one interesting strategy which has been proposed with success in order to overcome these barriers without using live cells (Lunsford, L., McKeever, 15 U., Eckstein, V., Hedley, M.L. (2000) Tissue distribution and persistence in mice of plasmid DNA encapsulated in a PLGA-based microsphere delivery vehicle. J. Drug Target.; 8 (1) :39-50; Briones, M., Singh, M., Ugozzoli, M., Kazzaz, J., Klakamp, S., Ott, G., O'Hagan, D. (2001) The 20 preparation, characterization, and evaluation of cationic microparticles for DNA vaccine delivery. Pharm Res.; 18 (5) :709-712). The patent BR-PI0103 887 additionally describes that PLGA microspheres using lactide and glycolide co-polymers 25 co-encapsulating DNA-hsp65 and the adjuvant TDM are able to protect mice from the challenge with a virulent strain of Mycobacterium tuberculosis after a single dose. More details on this vaccine are presented by Lima et al. (Lima, K.M., Santos, S.A., Lima, V.M., Coelho-Castelo,

A.A.,

WO 2006/024122 7 PCT/BR2005/000179 Rodrigues, J.M. Jr., Silva, C.L. (2003) Single dose of a vaccine based on DNA encoding mycobacterial hsp65 protein plus TDM-loaded PLGA microspheres protects mice against a virulent strain of Mycobacterium tuberculosis. Gene Ther.; 5 10 (8) :678-685.) . This formulation allowed the reduction of the number of injections and also the dose of DNA, after verifying that the entrapment of DNA in microspheres protects DNA from degradation by nucleases in vivo. The evidence of the 10 functionality of plasmid DNA allows concluding that the DNA is released in the cytosol and it is able to reach the cell nucleus. The patent describes the use of DNA or recombinant protein entrapped into PLGA microspheres in association 15 with trehalose dymicolate. Otherwise, the patent does not describe the concomitant use of protein and DNA associated in the same formulation. Despite the efforts in the search for safer vaccines the state of art does not describes a controlled release 20 technology associating the prime-boost strategy to a single-shot vaccine using non-live carriers, which can be administered by different routes allowing the targeting to specific cell populations. SUMMARY OF INVENTION 25 In this present invention we describe a novel immunogenic formulation, made by different compositions of PLGA, with different rates of poly lactic and glycolic acid, presenting release kinetics profiles of the recombinant protein, hsp65, dependent of the polymer WO 2006/024122 8 PCT/BR2005/000179 composition. Such polymers should be preferentially composed by lactic and glycolic acid molecules. Increasing the number of lactic monomers in the polymer composition the period of degradation in the body 5 also increases and, consequently, the entrapped molecule is slowly released. Thus, the polymers composed by lactic and glycolic monomers present an essential condition for the development of single-shot vaccines exploring the prime boost concept, containing protein and DNA in the same 10 formulation. One important reason for the development of such novel formulation refers to the need of more effective vaccines to protect against infectious diseases, or other diseases, which can be controlled by a suitable and sustained immune 15 response. Other important application is related to tumor processes where the induction of an immune response is important to control the development of the disease. Brief Description of Figures Figure 1 illustrates the kinetic of recombinant 20 protein release from PLGA 50:50 (A) and PLGA 75:25 (B) microspheres. Figure 2 illustrates the cytokine profile in calves after vaccination with DNA-hsp65 in different formulations. Figures 3a and 3b illustrate the antibody profiles in !5 calves after vaccination with DNA-hsp65 in different formulations: (a) IgGl, (b) IgG2a. Detailed Description of the invention This present invention refers to the formulation of novel immunogenic compositions containing antigens in WO 2006/024122 9 PCT/BR2005/000179 different forms entrapped in biodegradable microspheres containing adjuvants. This present invention describes an immunogenic composition that includes in a single dose the association 5 of microspheres containing one antigen in the form of DNA vaccine and a second antigen in the form of subunit protein. This composition was able to induce a specific immune response of Th1 pattern which was sustained over the 10 evaluated period. In this present invention the DNA is entrapped in PLGA microspheres with higher hydrophilic degree allowing its release from microspheres in a period inferior to 30 days, before total protein release. 15 The protein was entrapped in higher hydrophobic degree PLGA microspheres presenting kinetic release in a period superior to 30 days. The mixture of both formulations permits, after a single dose administration, the faster release of DNA from 20 microspheres stimulating the immune system to trigger a response characterized by the production of gamma interferon and IgG2a subtype against the antigen. The posterior release of the antigen in the protein form, entrapped in slow release polymers, has the ability 25 to induce the expansion of the subpopulations primed by the DNA acting as a booster. The presence of an adjuvant, preferentially the trehalose dymicolate is very important for the success of the induction of the immune response.

WO 2006/024122 10 PCT/BR2005/000179 Thus, this formulation allows jointing the prime and booster doses in the same formulation allowing the reduction of costs, more successful vaccination programs and increased coverage. 5 This ability to induce an immune response can be attributed to the stimulation of CD4+ cells that produce gamma-interferon and cytotoxic CD8+ cells. The process of entrapment of hsp65 and/or DNA-hsp65 into microspheres does not compromise its ability to induce the immune response. 10 This invention presents an immunogenic composition, which is composed by, at least one gene vector and one protein, associated or not to an adjuvant, entrapped into biodegradable microspheres with an average diameter inferior to 20 micrometers, preferentially with an average 15 diameter comprised between 0.5 and 10 micrometers. The composition can be alternatively composed by one protein and a gene vector and an adjuvant. The antigens are preferentially antigens from parasites and other pathogenic agents including viral antigens, intra and extra cellular 20 bacterial antigens. The adjuvants are preferentially those that stimulate pro-inflammatory cytokines. More preferentially, the antigens of the present invention may be entrapped in PLGA microspheres with rate 25 of PLA and PGA monomers varying from 00 to 100 and 100 to 00. Particularly, can be used the plasmid that codify the Mycobacterium protein, such as the gene that codify the hsp65 and also the protein itself may be entrapped in the WO 2006/024122 11 PCT/BR2005/000179 purified or recombinant form, or peptides derived of the same protein or yet antigens that can stimulate the immune response taking profit of the prime boost technology. The formulation can also contain trehalose dymicolate 5 entrapped in the same formulation of PLGA microspheres. Theses systems containing trehalose dymicolate and the plasmid DNA containing the gene that codify the hsp65 protein and also the hsp65 protein, can be used as vaccine against tuberculosis. 10 Additionally this formulation can be also applied in the therapy of tuberculosis when the disease is already installed or in diseases that requires the induction of a Thl pattern to be controlled. The microencapsulation of a gene vector, antigen and 15 adjuvant, according to the procedures of the present invention can be obtained as the following protocols: Protocol A: (i) dissolution of polymeric material composed by lactic and glycolic acids derivatives (relation in % of weight ranging from 100:00 to 00:100) into an 20 immiscible or partially immiscible organic solvent in water. The concentration of polymer in this solution varies from 0.2 a 10%. Methylene chloride, ethyl acetate and Chloroform among others, are examples of organic solvents used in this process; (ii) solubilization of lipophilic 25 antigen and adjuvant in the polymer solution; (iii) addition of the solution obtained at (ii) into an aqueous phase constituted by a polyol, eg. Polyvinyl alcohol, to obtain an oil-in-water emulsion; (iv) agitation of the emulsion in an appropriate velocity (200 a 1000 rpm) WO 2006/024122 1 2 PCT/BR2005/000179 allowing the formation of small microspheres with diameter smaller than 10 micrometer, preferentially ranging from 0.5 to 5 micrometer and e; (v) solvent evaporation to form a microspheres suspension and microspheres recovery 5 optionally by centrifugation or filtration; (vi) Addition of a' second antigen (soluble or dispersed into the aqueous phase) and water elimination by liophilization or other drying method. Protocolo B: (i) dissolution of polymeric material 10 composed by lactic and glycolic acids derivatives (relation in % of weight ranging from 100:00 to 00:100) into an immiscible or partially immiscible organic solvent in water. The concentration of polymer in this solution varies from 0.2 a 10%. Methylene chloride, ethyl acetate and 15 Chloroform among others, are examples of organic solvents used in this process. Lipophilic substances as antigens and adjuvants should be added in this solution; (ii) solubilization of genetic vector, antigen and/or adjuvant with hydrophilic nature into an aqueous phase which volume 20 ranges from 0.1 to lmL; (iii) addition of the solution obtained at (ii) into the polymeric solution obtained at (i) and agitation with.rotation ranging from 500 to 15,000 rpm, to form an water-in-oil emulsion or dispersion; (iv) addition of the water-in-oil emulsion or dispersion to an 25 emulsifing aqueous phase, constituted by a polyol, eg. Polyvinyl alcohol, to obtain an water-in-oil-in-water double emulsion; (iv) agitation of the emulsion in an appropriate velocity allowing the formation of small microspheres with diameter smaller than 10 micrometer, WO 2006/024122 13 PCT/BR2005/000179 preferentially ranging from 0.5 to 5 micrometer and e; (v) solvent evaporation to form a microspheres suspension and microspheres recovery optionally by centrifugation or filtration; (vi) and water elimination by liophilization or 5 other drying method. It is important to mention that other methods described in the state of art can be employed to obtain the microspheres entrapping antigens and/or adjuvants described in this invention. 10 The Table I summarize the different compositions formulated and used in the characterization of the present invention. The Table II summarizes the entrapment rate and average diameter of microspheres containing protein, 15 plasmid DNA and trehalose dimicolate. Table I Formulation Composition Control Me Suspension of pCDNA3 TDM-loaded PLGA 50:50 microspheres DNA Me Suspension of pCDNA3-hsp65 plus TDM-loaded PLGA 50:50 microspheres Prime-boost Me Suspension of a mixture composed by 50% of pCDNA3-hsp65 plus TDM-loaded PLGA 50:50 microspheres and 50% of recombinant hsp65 plus TDM-loaded PLGA 75:25 microspheres WO 2006/024122 14 PCT/BR2005/000179 Table II Ratio of Formulation entrapment Medium (micrograms/mg of Diameter microspheres) (micrometer) DNA Protein PLGA 50:50 Microspheres 4,11 - 7,3 containing TDM plus pCDNA3 PLGA 50:50 Microspheres 4,97 - 7,6 containing TDM plus pCDNA3 hsp65 PLGA 50:50 Microspheres 6,40 - 5,8 containing pCDNA3-hsp65 PLGA 50:50 Microspheres - - 9,3 containing TDM PLGA 50:50 Microspheres - 1,70 1,4 containing TDM plus r-hsp65 PLGA 75:25 Microspheres - 1,70 4,8 containing TDM plus r-hsp65 I The present invention is described in details by means of the examples below. It is necessary to point out that the invention is not limited to these examples but includes variations and modifications inside the limits in which it 5 works. EXAMPLE 1 - Plasmid Obtaining The pCDNA3-hsp65 construct was derived from the pCDNA3 vector (Invitrogen@, Carlsbad, CA, USA), previously digested with Bam HI and Not I (Gibco BRL, Gaithersburg, 10 MD, USA); a 3.3 kb fragment, corresponding to the M. leprae hsp65 gene and the CMV intron A, was then inserted in the vector. Plasmid pCDNA3 without the hsp65 gene was used as control. DH5a E. coli transformed with plasmid pCDNA3 or the plasmid containing the hsp65 gene (pCDNA3-hsp65) was 15 cultured in LB liquid medium (Gibco BRL, Gaithersburg, MD, WO 2006/024122 15 PCT/BR2005/000179 USA) containing ampicillin (100ptg/mL) . The plasmids were purified using the Qiagen Endofree Plasmid Purification kit (Qiagen Inc, Valencia, CA, USA) . Plasmid concentration was determined by spectrophotometry at X= 260 and 280 nm using 5 the Gene Quant II apparatus (Pharmacia Biotech, Buckinghamshire, UK). EXAMPLE 2 - Recombinant Protein Obtaining E. coli BL21 transformed with the plasmid containing the mycobacterial hsp65 gene was cultured in LB medium 10 containing ampicillin (100 ptg/pL) . The bacterial growing was monitored by spectrophotometry at X 600nm in a Shimadzu UV-1650 spectrophotometer. When the OD reached the value of 0.6, the culture was induced with 0.lM of IPTG (Gibco, BRL, Gaithersburg, MD, USA) and incubated at 30'C under 15 agitation for 4 hours. The cells were harvested centrifugation, suspended in citrate buffer and disrupted by sonication. Afetr centrifugation, the supernatant was discharged and the pellet dissolved with urea 8 M.Protein purification was done by affinity cromatography. 20 EXAMPLE 3 - Preparation of Microspheres containing plasmid and recombinant protein. Microspheres were obtained by the double emulsion/solvent evaporation technique. Briefly, 30 ml dichloromethane solution containing 400 mg of polymer PLGA 25 50:50 or PLGA 75:25 (Resomerfrom Boehringer Ingelheim, Ingelheim, Germany) was emulsified with 0.3 ml of an inner aqueous phase containing 5 mg of DNA (pCDNA3 or pCDNA3 hsp65) or 1 mg of recombinant hsp65 protein using a T25 Ultraturrax homogenizer (IKA@ - Labortechnik, Germany) to WO 2006/024122 16 PCT/BR2005/000179 produce a primary water-in-oil emulsion. This emulsion was then mixed with 100 ml of an external aqueous phase containing 1% to 3% poly-(vinyl alcohol) (Mowiol@ 40-88, Aldrich Chemicals, Wankee, WI, USA) as surfactant, to form 5 a stable water-in-oil-in-water emulsion. The mixture was stirred for 6 hours with a RW20 IKA homogeniser for solvent evaporation. Microspheres were collected and washed 3 times with sterile water, freeze-dried and stored at 4*C. EXAMPLE 4 - Preparation of microspheres containing 10 trehalose dimicolate "and" plasmid "and" protein Microspheres were obtained by the double emulsion/solvent evaporation technique. Briefly, 30 ml dichloromethane solution containing 400 mg of polymer PLGA 50:50 or PLGA 75:25 (Resomerfrom Boehringer Ingelheim, Ingelheim, 15 Germany) was emulsified with 0.3 ml of an inner aqueous phase containing 5 mg of DNA (pCDNA3 or pCDNA3-hsp65) or 1 mg of recombinant hsp65 protein using a T25 Ultraturrax homogenizer (IKA@ - Labortechnik, Germany) to produce a primary water-in-oil emulsion. This emulsion was then mixed 20 with 100 ml of an external aqueous phase containing 1% to 3% poly-(vinyl alcohol) (Mowiol@ 40-88, Aldrich Chemicals, Wankee, WI, USA) as surfactant, to form a stable water-in oil-in-water emulsion. The mixture was stirred for 6 hours with a RW20 IKA homogeniser for solvent evaporation. 25 Microspheres were collected and washed 3 times with sterile water, freeze-dried and stored at 4 0 C. EXAMPLE 5: Kinetic of protein release The recombinant hsp65 protein was initially entrapped into PLGA 50:50 or PLGA 75:25 microspheres. The kinetics of WO 2006/024122 17 PCT/BR2005/000179 protein release from microspheres was evaluated by ressuspending 30 mg of protein-loaded microspheres in 3 mL of PBS containing sodium azide (0.05% w/v) . The suspension was maintained at 37'C under constant agitation at 200 rpm. 5 In pre-established time intervals, samples of the supernatant (0.1 mL) were collected and replaced with fresh buffer. The protein concentration in the supernatant was determined by using the Comassie Reagent. The evaluation of the in vitro protein release kinetic from these particles 10 allowed the selection of the more suitable formulation able to sustain the immune response. A new vaccine composed by PLGA 75:25 microspheres containing recombinant protein plus PLGA 50:50 microspheres containing the plasmid (DNA-hsp65) and an adjuvant was able to elicit a sustained immune 15 response. This effect is better observed when the protein is associated to plymers which present molar ratio greater that 50% in lactic acid, preferentialy PLGA 75:25 or PLGA 85:15. As shown on Figure 1, Polymer with 50% of lactic acid (PLGA 50:50) presented a fast protein release profile, 20 releasing 80% of entrapped protein over five days. However, PLGA 75:25 microspheres were able to sustain the protein release over a longer time interval. This effect could be attributed to the lower hydrophilic characteristic of PLGA 75:25 which are associated to less glycolic acid in 25 polymeric chain. EXAMPLE 6: Evaluation of immune response in vaccinated cattle. Twelve female calves Nelore - Red Angus- Senepol crossbred calves, aged 7 to 10 months, were divided in four groups of WO 2006/024122 18 PCT/BR2005/000179 3 animals. Background levels of in vitro IFN-y response against mammalian and avian tuberculin PPD were. determined prior to they purchase and non-responding animals were selected. The animals were immunized by intramuscular 5 injection of the following formulations: 3 doses of naked DNA-hsp65 in saline administered in a 2 weeks time interval; a single dose of microspheres-based formulations. The 2 microspheres formulation evaluated were DNA-hsp65 plus TDM-loaded microspheres and the prime-boost 10 microspheres as described on Table I. Tirty days after vaccination, the cellular immune response was evaluated by cell proliferation and interferon gamma secretion using ELISPOT. The humoral immune response was evaluated by the measurement of anti-hsp65 specific antibody in serum. 15 Since the encapsulation of antigen into PLGA microspheres allows the development of controlled release delivery systems, where we can design the release profile of the encapsulated material, we evaluated the use of a vaccine formulation based on a mixture of two different 20 PLGA microspheres, composed by faster and slower release profiles, containing DNA encoding hsp65 and the recombinant hsp65 protein respectively, aiming at DNA priming and protein boost after a single dose vaccination. In mice, Prime-Boost formulation was able to induce high levels of 25 anti-hsp65 specific antibodies. The serum levels of these specific antibodies remaining high 90 days after vaccination while the DNA-Me formulation was not able to sustain the antibodies levels in the same fashion. Production of IFN-y was significant in mice vaccinated with WO 2006/024122 1 9 PCT/BR2005/000179 DNA-Me and Prime-Boost formulations being that Prime-Boost vaccinated mice sustained the high levels of this cytokine during all period evaluated. Moreover, when the same Prime Boost formulation was applied to cattle, we showed for the 5 first time, that a DNA vaccine was able to elicit humoral and cellular specific immune response after a single-dose administration in large animals (Figures 2 and 3). The invention here described, as well as the aspects presented, must be considered as one among the all possible 10 concretizations. It should be clarified that the invention is not limited to these concretizations and one with know how in the technique will realize that any particular characteristic introduced must be understood as something described to facilitate the comprehension and can not be 15 done without ignoring the inventive concept described here.

Claims (4)

1. A pharmaceutical composition able to induce a sustainable immune response, where this formulation is composed by polymeric biodegradable microspheres containing 5 one antigen and one gene vector, associated or not to an adjuvant, where this formulation is capable of release the gene vector in a period inferior to the antigen release, and the release of entrapped material is sustained over a period not inferior to 30 days, and it is able to induce a 10 specific immune response, cellular and humoral, after administration of one or several doses.

2. A pharmaceutical composition as described in claim 1, characterized by containing an antigen and a functional gene vector, and an adjuvant, which can be administered by 15 upper air way (intratracheal, pulmonar and nasal), oral and parenteral routes.

3. A pharmaceutical composition as described in claim 1 which is composed by one antigen and functional gene vector, and an adjuvant, characterized by microspheres 20 constituted acid lactic polyesters and co-polymers of lactide and glycolide in the proportions of 00:100 or

100-:00. 4. A pharmaceutical composition as described in claim 1, which is composed by an antigen and a functional gene 25 vector, associated or not to an adjuvant, characterized by the fact that the microspheres containing the first antigen, that could be the gene vector, are preferentially constituted by PLGA co-polymers which degradates over the first 30 days after administration in hot blood animals, WO 2006/024122 2 PCT/BR2005/000179 allowing the release of most gene vector entrapped into the first 30 days. 5. A pharmaceutical composition as described in claim 1, which is composed by an antigen and a functional gene 5 vector, associated or not to an adjuvant, characterized by the fact that the microspheres containing the second antigen, are preferentially constituted by PLGA co-polymers which degradate totally in a period greater than 30 days after administration in hot blood animals, allowing the 10 sustained release of the antigen. 6. A pharmaceutical composition as described in claim 1, which comprehends one antigen and a functional gene vector, associated or not to an adjuvant, characterized by the fact that the first antigen may be at the external 15 phase and not entrapped, in association to microspheres charged with a second antigen, preferentially constituted by PLGA co-polymers which degradate totally in a period greater than 30 days after administration in hot blood animals, allowing the sustained release of the antigen. 20 7. A pharmaceutical composition as described in claim 1, which is composed by an antigen and a functional gene vector, associated or not to an adjuvant, characterized by the fact that the microspheres containing the antigen, are preferentially constituted by PLGA co-polymers which 25 composition allows the sustained release of the antigen over a time period greater than the period of the total or partial gene vector release. 8. A pharamaceutical composition as described in claim 1 which comprehends one antigen and a functional gene WO 2006/024122 . 3 PCT/BR2005/000179 vector, associated or not to an adjuvant, characterized by the fact that antigen-loaded microspheres are preferentially constituted of PLGA co-polymers in lactic/glycolic rates of 75:25 or 85:15, and that the gene 5 vector is not entrapped or is entrapped into microspheres constituted of PLGA 50:50 co-polymers. 9. A pharmaceutical composition as described in claim 1 characterized by microspheres with an average diameter inferior to 20 micrometers, preferentially comprised 10 between 0.5 to 10 micrometers. 10. A pharmaceutical composition as described in claim 1 characterized by the fact that a single dose is able to elicit both cellular and humoral immune responses after administration in hot blood animals. 15 11. A pharmaceutical composition as described in claim 1 where the antigen is a protein or a peptide. 12. A pharmaceutical composition as described in claim 1 where the antigen is a 65kDa heat shock protein, hsp65, of Mycobacterium. 20 13. A pharmaceutical composition as described in claim 1 where the functional gene vector is a plasmid DNA that codify the 65kDa heat shock protein, hsp65, of Mycobacterium. 14. A pharmaceutical composition as described in claim 25 1 where the adjuvant is the trehalose dymicolate. 15. Method of treatment using a pharmaceutical composition as described in claim 1 that elicit, after administration by pulmonary, nasal, oral or parenteral route, an immune response characterized by: WO 2006/024122 4 PCT/BR2005/000179 - Stimulation of cytokines production, such as IL-6, IL-10, IL-12, gamma-IFN, TNF-alfa; - Increase of nitric oxide production by alveolar macrophages; 5 - Stimulation of local and/or systemic immune response. 16. Method of treatment using a pharmaceutical composition as described in claim 1, characterized by the ability to induce an immune response able prevent the immune system against infectious diseases in hot blood 10 animals. 17. Method of treatment using a pharmaceutical composition as described in claim 1, characterized by the ability to induce an immune response able treat infectious diseases in hot blood animals. 15 18. Method of treatment using a pharmaceutical composition as described in claim 1, characterized by employment in the control and treatment of endo and ectoparasites infestations in hot blood animals. 19. Method of treatment using a pharmaceutical 20 composition as described in claim 1, characterized by employment in the treatment of infectious diseases, endo and ectoparasites, tumor process, asthma and autoimmune processes, inducing an immune response able to paralyze, reduce or eliminate the multiplication process of tumor 25 cells. 20. Method of treatment using a pharmaceutical composition as described in claim 1, to treat tumor process characterized by inducing an immune response able to WO 2006/024122 5 PCT/BR2005/000179 paralyze, reduce or eliminate the multiplication process of tumor cells. 21. Use of the pharmaceutical composition as described in claim 1 to induce immune response able to prevent the 5 immune system against infectious diseases in hot blood animals. 22. Use of the pharmaceutical composition as described in claim 1 to induce immune response against endo and ectoparasites in hot blood animals.