MXPA00005513A - An over-expressing homologous antigen vaccine and a method of making the same - Google Patents

Abstract

This invention relates to an over-expressing homologous antigen vaccine, a method of producing the same, and use of the vaccine for prophylaxis or treatment of vertebrates at risk of or suffering form disease caused by a pathogenic micro-organism. The vaccine is an attenuated or avirulent pathogenic micro-organism that over-expresses at least one homologous antigen encoded by at least one gene derived form the pathogenic micro-organism, and may also express a heterologous antigen.

Description

A VACCINE OF SQBREEXPRESSION OF ANTIGEN HOMOLOGOUS AND METHOD TO PREPARE The invention described here was made with a contribution from the United States Department of Agriculture. America. Therefore, the American government has certain rights over this invention. The invention relates to a vaccine for overexpression of homologous antigens, a method for producing the vaccine and a method for using the vaccine for the prophylaxis or treatment of a vertebrate that suffers from a pathogen or is at risk of suffering from a pathogen The vaccine is derived from an attenuated or avirulent version of the pathogen, and over expresses one or several genes of the pathogen, thus providing greater immunity than the immunity induced by a vaccine of the same pathogen without overexpression of a gene. BACKGROUND OF THE INVENTION Vaccines are used to protect against diseases that are caused by pathogens. These pathogens are microbial organisms, such as bacteria and viruses that affect animals, including humans. Vaccines are developed primarily from a pathogen by producing and administering either a) an attenuated or avirulent version of the pathogen; b) the dead pathogen; c) antigens or mixtures of protective antigens extracted from the pathogen (homologous antigens); or d) a microorganism expressing one or more protective antigens encoded by cloned genes originating from a microbial pathogen different from the vaccine strain (heterologous antigens). The vaccines for both bacteria and viruses are prepared from microorganisms that express one or more protective antigens, in accordance with that described by K. Jones and M. Sheppard in Designer Vaccines, CRC Press (1997). The vaccines are made to produce an immune system response in the recipient which consists of at least one of the following: antibody-mediated immune response or T cell-mediated immune response, thereby preventing future infections by a pathogen, or It is well fought against a current pathogenic infection. Particularly, vaccines against facultative intracellular pathogens, those that grow inside the cells of the infected host, must induce a strong and appropriate cell-mediated immune response. In contrast, vaccines against ligated extracellular pathogens must induce an immune response mediated by appropriate antibodies. Frequently, independently of the pathogen, an appropriate combined immunological response mediated by antibodies and cells causes sufficient protection or alleviation of the infection. In order to achieve this protection or relief against infection, vaccines can express one or more homologous antigens, heterologous antigens, or a combination of both. Vaccines can be administered to vertebrates both to prevent and treat infections caused by pathogens. Thus, vaccines are administered frequently to prevent the spread of a disease caused by a pathogen. Particularly, herd animals, such as cows, goats, sheep and pigs are vaccinated to prevent the spread of a disease among members of the herd.

In addition, since certain diseases can spread among vertebrates, including can pass between various animals and between animals and humans, vaccines are used to prevent the spread of disease among several species, usually by its administration to the infected animal and other infected animals. in the immediate vicinity. However, other animals in the area that are less likely to acquire the disease may also be vaccinated as a prophylactic measure. For example, an infected cow and her herd not yet infected can be vaccinated to treat the disease and prevent its further spread. As a prophylactic measure, other animals that have a propensity to contract the disease from the infected cow, such as neighboring cows, sheep or humans, can also be vaccinated.

It has been found that vaccines derived from an attenuated or avirulent version of a pathogen are highly effective in preventing or fighting an illness caused by this pathogen. Particularly, it is known that said attenuated or avirulent pathogens can be modified to express heterologous antigens (antigens derived from a pathogen of a different species). In order to express heterologous antigens in a desired attenuated or avirulent pathogen, a gene encoding an antigen capable of providing protection against the pathogen is identified from the deoxyribonucleic acid of a heterologous species. The desired gene is isolated and then inserted into a plasmid capable of replication and expression in the attenuated or avirulent pathogen. The plasmid is then introduced into the attenuated or avirulent pathogen and causes expression of the heterologous antigen when administered to a vertebrate. An example of such an expression of a heterologous antigen is the bacterial Salmonella vaccine, which expresses a protein of Streptococcus spaA. See North American Patent 4,888,170. This vaccine comprises an avirulent derivative of a pathogenic microbe of the genus Salmonella, which in turn expresses a recombinant gene derived from a pathogen of the species Streptococcus mutans, thus producing an antigen capable of inducing an immune response in a vertebrate against the pathogen.

A further example of heterologous expression is Vibrio cholera vaccines. Several live attenuated strains of Vibrio cholera have been used to vaccinate humans against cholera. See Kaper, J.B., et al., New and improved vaccines against cholera in New Generation Vaccines (new and improved vaccines against cholera in new generation vaccines) (MM Levin et al editions). Marcel Deker, Inc., NY. 1997. Some of these strains overexpress heterologous antigens. See Butterton, J.R. and S.B. Calderwood, Attenuated Vibrio cholera as a live vector for expression of foreign antigens in New Generation Vaccines (Vibrio cholera attenuated as a live vector for the expression of fox antigens in new generation vaccines) (MM Levin et al.) Marcel Deker, Inc. , NY, 1997. The immunity induced by attenuated vaccine strains is the result of the induction of antibodies that have either antibacterial and / or antitoxic activities. Some strains have been attenuated by the removal of several genes encoding toxigenic components, including the A subunit of the cholera toxin encoded by the ctxA gene. However, for a strain of cholera vaccine to be fully protective, it is necessary that the ctxB gene encoding subunit B (to which subunit A binds) is expressed to allow the production of antibodies that neutralize cholera toxin. . The ctxB gene has been overexpressed in Vibrio cholera for the purpose of producing large amounts of cholera toxin B subunit (CTB) antigen. The overexpressed antigen CTB is collected, purified and used as a subunit vaccine which is the extracted CTB antigen. See Lebens M., et al., 1993, Biotechnology (NT) Dec; 11: 1574-1578. However, even when an overexpressed antigen has been used as a vaccine, an attenuated or avirulent pathogen of Vibrio cholera overexpressing the ctxB gene, or any other homologous gene, has not been used as a live vaccine. Another example of heterologous expression is in Mycobacterium spp vaccines, which are used to prevent tuberculosis in humans. The GroEL protein of Mycobacterium tuberculosis induces a protective immunity. when expressed by the transfected macrophage groEL gene (Silva, CL and Lowrie, DB, 1994, Immunology 84: 244-248), which indicates that the GroEL protein is a protective antigen if it is presented to T cells by this type of antigen presenting cell (APC). Vaccines with naked DNA that employ Mycobacterium genes that encode several antigens (hsp 70, 85kDa, 65kDa, 36kDa, 6kDa), can also induce a protective immunity. See Lowrie, D.B. et al., 1997, Vaccine 15: 834-838; Tascon, E. et al., 1996, Nat. Med. 2: 888-892; and Lozes, E. et al., 1997, Vaccine 15: 880-333. It is believed that naked DNA vaccines function because they transfect APCs (Chattergon, M. et al., 1997, FASEB J. 11: 753-763) which in turn present the antigen appropriately to T cells, thus inducing a mediated immunity to T cells. protective cells. M. bovis BCG, a live attenuated strain of Mycobacterium is used to induce a protective immunity against M. tuberculosis infection in humans. Fine, PM. 1988, Br. Med. Bull. 44:91. Antigen vaccines developed against Brusellosis offer examples of expression of homologous antigens where the antigen is derived from the same species as the attenuated pathogen. The bruselosis is an infectious bacterial disease that can be transmitted to humans by animals. It is caused by several species of pathogenic aerobic bacteria of the genus Brucella. In animals, brusellosis can result in miscarriage and infertility. In humans, it causes fever, malaise and headache. This disease has been studied extensively resulting in the development of numerous vaccines. It is known that existing strains of Brucella vaccine, such as for example strains 19 and RB51 of B.abortus and the strain REVI of B. melitensis, can protect both against the Brucella species from which they were derived and protect in a crossed against an infection caused by other species, such as B. abortus, B. melitensis, B. ovis, B. suis, B. canis and B. neotomae. See Winter, A.J. et al., 1996, Am. J. Vet. Res., 57: 677; P. Nicolettis in Animal Brucellosis, CRC Press (1990), pages 284-296; J.M. Blasco in Animal Brucellosis, CRC Press (1990), pages 368-370; and G.C. Alton in Animal Brucellosis, CRC Press (1990), pages 395-400. New strains of B. melitensis VTRM1 and B. suis VTRS1 also provide cross protection against several Brucella species. See Winter, A.J. et al., Am. J. Vet. Res., 57: 677. In the past, one of the vaccines most frequently used to prevent bovine Bruselosis was strain 19, from B. abortus, in accordance with that described by P. Nicoletti in Animal Brucellosis, CRC Press (1990), pages__ 284-296. This particular strain of B. abortus provided immunity in cattle with a protection range of 65 to 75% according to numerous variables such as the age of the cattle at the moment of the application of the vaccine, the dose administered, the route of administration and the prevalence of bruselosis in the vaccinated herd. Strain RB51 of B. Abortus, a new live attenuated Brucella vaccine (marketed as RB-51®), is a stable vaccine approved for use in the United States of America. Véasne Schurig, G.G. et al., 1991, Vet. Microbiol. 26: 359; and Colby, L., 1997, M. Sc. Thesis, Virginia Tech, Blacksburg, Va. The attenuation of strain RB51 is indicated by studies in mice, goats and cattle. See, Schurig, G.G., 1991, Vet. Microbiol. 28: 171; Palmer R.M. et al., 1997, Am. J. Vet Res. 58: 472; Roop, R.M. et al., 1995, Res. Vet. Science, 51: 359; and Zambrano, A.J. et al., 1995, Archives of Veterinary Medicine XXVIII, Extraordinary No.: 119-121. Compared to the protection offered by strain 19, it has been shown that strain RB51 in single vaccine application protocols offers similar protection in cattle. See Cheville, N.F. et al., 1993, Amer. J. Vet Research 53: 1881; and Cheville, N.F. et al., 1995, Amer. J. Vet Research, 57: 1153. In addition, oral administration of strain RB51 in mice and cattle provided protective immunity. See Stevens, M.G. et al., 1996, Infect. Immun. 64: 534. Particularly, the mouse model indicates that the protective immunity to Bruselosis induced by the RB51 strain is only mediated by the T cells since a passive transfer of antibodies induced by RB51 does not protect against the disease, while the transfer of T cells adoptive if it protects against the disease. See Bagchi, T., 1990, M.Sc. Thesis, Virginia Tech. Blacksburg, Va; Jiménez deBagues, M.P. et al., 1994, Infect. Immun. 62: 4990. It is believed that the vaccine with RB-51® provides protection by inducing the production of interferon-gamma capable of activating macrophages and specific cytotoxic T cells in the patient that can kill macrophages infected with Brucella. Even though RB-51®, derived from strain 2308 of B. abortus is the best vaccine currently against Brusellosis in animals, it is not 100% effective. None of the current vaccines against Bruselosis is totally effective. Accordingly, research continues on promising strains, such as the RB51 strain of B. abortus. For example, the expression of heterologous antigens by the RB51 strain of B. abortus has been described by S. Cravero, et al., 1995, Review of the 4th. Symposium on International Veterinary Immunology, Julio, Davis, Ca., abstract No. 276; and S. Cravero et al., 1996, Researchers' Conference on Animal Diseases, November, Chicago, abstract No. 150. Overexpression of a homologous antigen by Brucella has been described as a research tool for the purpose of complementing specific removal mutants. for the study of the HtrA protein in B. abortus (PH Elzer, Inf. Immun., 1994, 62: 4131), and for the study of physiological functions in accordance with what was commented by R. Wright in a Verbal Presentation of the Conference of Research on Brucella on November 9, 1997 in Chicago, Illinois. However, overexpression of Brucella homologous antigens and other pathogens, with or without concomitant expression of a heterologous antigen, has not been studied for use in vaccines. The overexpression of homologous antigens has previously been used primarily as a research tool, in accordance with what has been described above. An attenuated or avirulent pathogen modified to overexpress a homologous antigen has not been used as a live vaccine. However, we have found that a vaccine that is an attenuated or avirulent pathogen that overexpresses one or more homologous antigens, as described herein, will offer greater protection against a pathogenic disease than attenuated pathogen vaccines that express wild type levels of the same homologous antigens. Accordingly, the invention focuses on a vaccine, a means of producing the vaccine, and its use for the prophylaxis and treatment of a pathogenic disease wherein the vaccine is an attenuated or avirulent pathogen overexpressing at least one homologous antigen, thus providing greater protection against disease and better treatment of the disease caused by the non-attenuated pathogen in the vertebrate. SUMMARY OF THE INVENTION The invention is directed to a live vaccine that is an attenuated or avirulent pathogen that overexpresses one or more homologous antigens of a pathogen, a method to produce it, and a method to treat animals, including humans, with the vaccine. This vaccine increases the level of protection against the non-attenuated pathogen compared to attenuated pathogen vaccines that express wild-type levels of homologous antigens of the pathogen. In this matter, the overexpression of homologous antigen vaccines will induce a strong immune response mediated by cells and / or a strong humoral antibody response against the non-attenuated pathogen in the vaccinated patient. Particularly, it is the purpose of this invention to provide a method for producing a vaccine that is an attenuated or avirulent pathogen overexpressing a homologous antigen, and immunizing an animal, including humans, with the vaccine in such a manner that the vaccine induces a strong immune response mediated by cells or mediated by antibodies against a virulent pathogen, thereby providing complete protection, such as for example sterile immunity, against a challenge by the virulent pathogen. It is a further object of the present invention to provide a method for producing a vaccine that is an attenuated or avirulent pathogen overexpressing a homologous antigen, and immunizing an animal with the vaccine in such a manner that the vaccine causes the overexpression of a homologous antigen and the expression of a heterologous antigen, both providing protection against the virulent pathogen in the vaccinated patient. It is a further object of the present invention to provide a homologous overexpression vaccine, a device for preparing said vaccine and a method for using the vaccine for prophylaxis and treatment of Brusellosis in animals, especially bovines. BRIEF DESCRIPTION OF THE DRAWINGS The appended figures are intended to help explain and underline more particularly the invention described herein. Particularly: Figure 1 is a diagram showing the derivation of a homologous antigen from a Brucella species, and the insertion of the antigen in a vaccine strain of Brucella species; Figure 2 shows a construction of recombinant plasmids for overexpression of SOD (A) copper / zinc and GroES and GroEL (B) in strain RB51 of B. abortus; Figure 3 demonstrates the clearance of B. abortus strain 2308B from the spleens of mice vaccinated with the RB51 strain of B. abortus overexpressing copper / zinc SOD or GroES / EL, and Figure 4 demonstrates cytotoxic activity by lymphocytes relative to cells infected with Brucella from mice vaccinated with the RB51 strain of B. abortus overexpressing copper / zinc SOD or GroES / EL. DETAILED DESCRIPTION OF THE INVENTION The invention is directed to a vaccine for the immunization of vertebrates against disease caused by a pathogen, wherein the vaccine comprises an attenuated or avirulent pathogen overexpressing one or more homologous antigens encoded by at least one gene of the pathogen, where each antigen is able to induce a protective immune response against the pathogen. This homologous overexpression antigen vaccine is produced by the genetic manipulation of live, attenuated microbes, through a process that presents the steps of: a) selecting a gene that encodes an analogous antigen capable of directly or indirectly stimulating protective immunity against a pathogenic microorganism (pathogen), and b) inserting said gene in an attenuated or avirulent version of the pathogen in such a way that the homologous antigen is overexpressed. The resulting overexpression homologous antigen (OVA) vaccine is prepared more specifically through the following steps: a) extraction of deoxyribonucleic acid from a pathogenic microorganism; b) identifying a deoxyribonucleic acid gene, wherein said gene encodes at least one antigen capable of stimulating a protective immunity against the pathogenic microorganism; c) insertion of said gene into a plasmid capable of replication and expression in the pathogenic microorganism; and d) introducing said plasmid in an attenuated or avirulent version of the pathogenic microorganism. The resulting vaccine synthesizes the antigen as a result of the transcription and translation of the gene located in at least two sites, i.e., the genome and the plasmid. Particularly, it is preferred that the plasmid be of the multiple copy type, such that it can produce a greater number of the protective antigen than the single genomic copy generated in another way. The above method can be used to create homologous overexpression antigen vaccines for many different diseases. Overexpression of the antigen usually increases both the immune response of T cells and antibodies, thus significantly increasing the level of protection in the patient. Since both types of immune response are improved, both intracellular and extracellular pathogens are affected, thus offering greater protection against the pathogen. For example, a vaccine against the pathogenic microorganism Brucella can be produced. Particularly, the pathogen can be selected from any Brucella species, including B. abortus, B. melitensis, B. ovis, B. suis, B. canis and B. neotomae. The pathogen used to produce the vaccine is preferably selected from a specific strain of Brucella, such as, for example, strain 19 of B. abortus, strain RB51 of B. abortus, strain VTRM1 of B. melitensis, strain VTRS1 of B suis and the REVI strain of B. melitensis. It is particularly helpful if the vaccine is prepared with one or more of the following: a SOD Cu / Zn gene, a GroES gene, or a GroEL gene from the RB51 strain of B. abortus.

Particularly, it is preferred that the above genes be obtained from a genomic library of pUC19 of strain 2308 of B. abortus. A vaccine produced in accordance with the above specifications is especially effective for the prophylaxis or treatment of diseases such as Bruselosis. For example, an effective vaccine for the prophylaxis or treatment of a bovine against Brusellosis according to the present invention is an attenuated or avirulent derivative of the RB51 strain of B. abortus capable of overexpressing at least one homologous antigen. Particularly, it is preferred that the antigen be encoded by one or more of a SOD Cu / Zn gene, a GroES gene or a GroEL gene, preferably selected from a genomic library of pUC19 from a strain 2308 of B. abortus. It is further preferred that the attenuated or avirulent derivative also express a heterologous antigen capable of inducing a protective immunity against B. abortus. The method of prophylaxis or treatment of a vertebrate that suffers from a pathogenic microorganism is the following: a) extract deoxyribonucleic acid from the pathogenic microorganism; b) identifying at least one gene encoding at least one deoxyribonucleic acid antigen, where the antigen is capable of stimulating a protective immunity against the pathogenic microorganism; c) inserting the at least one gene into a plasmid capable of replication and expression in the pathogenic microorganism; d) transforming an attenuated or avirulent version of the pathogenic microorganism with the plasmid to form a vaccine; and e) administering an effective amount of a vaccine to the vertebrate. The vaccine employed for the method for prophylaxis and treatment may be an original vaccine strain or a modified existing vaccine strain. For example, the RB51 strain of B. abortus can be modified to overexpress a homologous antigen, thus producing a new strain capable of being used in a vaccine for the prophylaxis or treatment of brusellosis, especially in bovines. In particular, a new vaccine against Brucella can be prepared by: 1) selecting a gene encoding a protective antigen from a strain of Brucella; 2) inserting the pathogen gene into a multiple copy plasmid, capable of replication and expression in Brucella; and 3) introducing the plasmid into Brucella through a transformation of this type. In this way, one or more homologous antigens can be overexpressed. Additionally, one or more heterologous antigens can be expressed in the vaccine by methods known in the art.

By overexpression of one or more homologous antigens of a given pathogen, an immunological response of T cells and / or major antibodies against the pathogen is stimulated in the vertebrate treated with the vaccine produced from the attenuated or avirulent pathogen, providing greater protection against the attenuated pathogen. In addition, additional protection can be offered by additional expression of one or more heterologous antigens by the attenuated or avirulent pathogen by means known to a person with certain knowledge in the art. The resulting overexpression homologous vaccine may be administered in an effective dose to promote the prophylaxis or treatment of a disease caused by the pathogen in the desired vertebrate. As a person with certain knowledge in the field knows, dosages should be adjusted for each patient based on factors such as weight, age, and environmental factors. The effective dose can be administered in any effective way based on the type of animal being treated, its age and its condition. EXAMPLES Example 1: Two OHAVs were constructed by overexpression of either the SOD Cu / Zn gene or the GroES and GroEL genes in the RB51 strain of B. abortus. The genes for SOD Cu / Zn, GroES and GroEL were initially obtained from a genomic library of pUC19 from strain 2308 of B. abortus. As shown in figure 2, inserts containing these genes together with their own promoters were removed from the pBA113 (SOD) and pBA2131 (GroES and GroEL) regions and subcloned into pBBRIMCS, a broad host range plasmid that has been used routinely in research on Brucella. The resulting recombinant plasmids were called pBBSOD and pBBGroES / EL (Figure 2). Strain RB51 of B. abortus was transformed with these plasmids by electroporation. Brucella containing the plasmids were selected by plating the bacteria transformed into trypticase soy agar plates containing 30 μg / mL chloramphenicol. To determine the overexpression of. Cloned genes, antibiotic-resistant colonies were cultured individually in trypticase soy broth and bacterial extracts were employed as antigens in an immunotinsion analysis. Strain RB51 containing pBBSOD (RB51SOD) and pBBGroES /? L (RB51GroESL) overexpressed SOD Cu / Zn and GroEL, respectively, compared to strain RB51 containing pBBRIMCS alone (RB51pBB). Protection studies in mice: Groups of 8 mice were vaccinated by inoculation, intraperitoneally, of 4 x 108 colony-forming units (cfu) either of strain RB51SOD, RB51GroESL, RB51pBB or -RB51 in 0.5 mL of saline. A group of mice was inoculated with 0.5 mL of saline as control. After 6 weeks, 5 mice from each group were challenged intraperitoneally with 2.5 x 104 cfu of virulent strain 2308. The three remaining mice in each group were used to characterize the immunological responses. Two weeks after challenge with the virulent strain 2308, the mice were euthanized and the cfu of strain 2308 was determined by spleen. Mice immunized with strain RB51SOD had a significantly lower number of bacteria compared to mice immunized with strain RB51. In mice immunized with strain RB51GroESL, the number of observable bacteria was found at the lower limit of the detection method (<20 cfu / spleen). Characterization of immunological responses ~~~ After 6 weeks of vaccination, the serum of 3 mice in each group was collected for analysis of the humoral antibody response. These mice were euthanized and the lymphocytes harvested from their spleens were used to study the cell-mediated immune response. As shown in FIG. 3, mice vaccinated with strain RB51 developed antibodies against GroEL but did not develop antibodies against SOD Cu / Zn. In contrast, mice vaccinated with strain RB51SOD developed a strong antibody response against Cu / Zn SOD, and mice vaccinated with strain RB51GroESL developed a stronger antibody response against GroEL protein (figure 3) than the response presented by mice vaccinated with strain RB51. These results indicate an increased response of antibodies by OVA. The cell-mediated immune response was characterized by determining the cytotoxic activity of lymphocytes to cells infected by Brucella. The specific activity of splenic lymphocytes was increased in vitro by co-culture with Brucella-infected macrophages treated with mitomycin C as stimulation cells. A cytotoxicity assay was performed using increased lymphocytes as effector cells (E) and macrophages infected with Brucella as target (T) cells. In the assay, E and T cells were mixed in two different ratios, 10: 1 and 5: 1. The percentage of specific lysis of target cells was calculated for each E: T ratio using standard methods (figure 4). Lymphocytes from mice vaccinated with RB51SOD and RB51GroESL exhibited increased cytotoxic activity compared to mice that received a saline solution or were vaccinated with the RB51 strain. This increased cytotoxic lymphocyte activity (indicated by the percentage increase in specific lysis) correlates directly with the improved protection observed in mice against challenge with strain 2308 of B. abortus virulenta; the higher the level of protection, the higher the specific cytotoxic activity. Example 2: An OVA was constructed by overexpression of the ctxB gene in Vibrio cholera. The gene was obtained from the deoxyribonucleic acid of the pathogen and inserted into a plasmid capable of replication and expression in the pathogen. The resulting recombinant plasmid is used to transform Vibrio cholera through electroporation. The plasmids were placed in dishes and selected by means known in the art. The resulting overexpression homologous vaccine strain promotes the overproduction of antibodies that neutralize the cholera toxin, thus providing greater protection for the prophylaxis and treatment of cholera in humans. Example 3: An OHAV is constructed by overexpressing the groEL gene of Mycobacterium tuberculosis in a Mycobacterium species. The gene is obtained from the deoxyribonucleic acid of the pathogen and inserted into a plasmid capable of replication and expression in the pathogen. The resulting recombinant plasmid is used to transform a Mycobacterium species through electroporation. The plasmids are placed in dishes and selected by means known in the art. The resulting overexpression homologous vaccine strain promotes the overproduction of GroEL proteins, thereby providing greater protection for the prophylaxis and treatment of tuberculosis in humans. Particularly, the overexpression of the groEL gene encoding the GroEL protein in BCG of M. bovis offers greater protective immunity against tuberculosis because it is known that BCG vaccines are focused towards antigen protection cells such as macrophages, thus offering a way to introduce the antigens in the T cells, inducing an immunity mediated by protective cells. The above examples are presented for illustrative purposes only. The scope of the present invention is not limited to the examples, but is described in the specification and the appended claims. Those with certain knowledge in the art will recognize methods and materials that could be substituted instead of those described above, and these methods and materials are encompassed by the above disclosure and the following claims.

Claims (19)

1. A vaccine for the immunization of vertebrates against a disease caused by a pathogen, wherein said vaccine comprises an attenuated or avirulent derivative of said pathogen overexpressing at least one homologous antigen encoded by at least one gene of said pathogen and wherein said at least one antigen is able to induce a protective immune response against the pathogen.
2. The vaccine according to claim 1, wherein said attenuated or avirulent derivative of said pathogen further expresses one or more heterologous antigens.
3. The vaccine according to claim 1, wherein the pathogen is selected from the group consisting of Brucella, Mycobacterium, and Vibrio.
4. A vaccine for the prophylaxis or treatment of a vertebrate against Bruselosis, wherein said vaccine comprises an attenuated or avirulent pathogen of Brucella, where the attenuated or avirulent pathogen overexpresses at least one homologous antigen encoded by at least one gene of said pathogen, and where the at least one antigen is able to induce a protective immune response in the vertebrate against brusellosis.
5. The vaccine according to claim 4, wherein the pathogen is selected from the group consisting of B. abortus, B. melitensis, B. suis, and B. canis.
6. 6. The vaccine according to claim 4, wherein Brucella is the RB51 strain of B. abortus.
7. The vaccine according to claim 6, wherein the at least one gene is a SOD Cu / Zn gene.
8. 8. The vaccine according to claim 7, wherein the Cu / Zn gene is obtained from a genomic library of pUC19 of strain 2308 of B. abortus.
9. 9. The vaccine according to claim 6, wherein the at least one gene is one or both of the following: GroES gene and GroEL gene.
10. 10. The vaccine according to claim 9, wherein the GroES gene and the GroEL gene are obtained from a genomic pUC19 library of strain 2308 of B. abortus.
11. 11. The vaccine according to claim 4, wherein the vertebrate is a bovine.
12. 12. An attenuated or avirulent version of the RB51 strain of B. abortus overexpressing at least one homologous antigen capable of stimulating a protective immunity against brusellosis.
13. 13. The attenuated or avirulent version of the B. abortus strain RB51 of claim 12, wherein the at least one homologous antigen is encoded by at least one gene selected from the group consisting of a SOD Cu / Zn gene, a gene GroES and a GroEL gene.
14. 14. A method for the prophylaxis or treatment of a vertebrate at risk of being affected by a pathogenic microorganism or suffering from a pathogenic microorganism, comprising the administration of an effective amount of an over-expression homologous antigen vaccine, wherein said vaccine is an attenuated or avirulent version of said pathogenic microorganism overexpressing at least one homologous antigen of the pathogenic microorganism.
15. 15. The method according to claim 14, wherein the vaccine further expresses a heterologous antigen.
16. 16. The method according to claim 14, wherein the pathogenic microorganism is Mycobacterium k Vibrio.
17. 17. The method according to claim 16, wherein the vertebrate is a human being.
18. 18. A method for the prophylaxis or treatment of a vertebrate at risk of suffering Brusellosis or suffering from Brusellosis, which comprises the administration of an effective amount of a vaccine, wherein said vaccine is an attenuated or avirulent pathogen overexpressing at least a homologous antigen encoded by at least one gene of said attenuated or avirulent pathogen.
19. 19. The method according to claim 18, wherein said attenuated or avirulent pathogen further expresses a heterologous antigen. The method according to claim 18, wherein the at least one gene is a SOD Cu / Zn gene in the RB51 strain of B. abortus. . The method according to claim 20, wherein the Cu / Zn gene is obtained from a genomic library pUC19 of strain 2308 of B. abortus. . The method according to claim 18, wherein the at least one gene is one or both of the following: GroES gene and GroEL gene in the RB51 strain of B. abortus. . The method according to claim 22, wherein the GroES gene and the GroEL gene are obtained from a pUC19 genomic library of the B. abortus strain 2308. . A method for the prophylaxis or treatment of a vertebrate at risk of suffering from a pathogenic microorganism or suffering from a pathogenic microorganism, comprising the steps of: a) extracting deoxyribonucleic acid from the pathogenic microorganism; b) identifying at least one gene encoding at least one deoxyribonucleic acid antigen wherein said at least one antigen is capable of stimulating a protective immunity against the pathogenic microorganism; c) inserting the at least one gene into a multiple copy plasmid capable of replication and expression in the pathogenic microorganism; d) transforming an attenuated or avirulent version of the pathogenic microorganism with the plasmid to form a vaccine; and e) administering an effective amount of said vaccine to the vertebrate. . The method according to claim 24, wherein said attenuated or avirulent version of the pathogenic microorganism further expresses one or more heterologous antigens. . The method according to claim 24, wherein the pathogenic microorganism is selected from the group consisting of Brucella, Mycobacterium, and Vibrio. The method according to claim 26, wherein the pathogenic microorganism is selected from the group consisting of B. abortus, B. melitensis, B. suis, B. ovis, B. neoto ae and B. canis. The method according to claim 27, wherein the pathogenic microorganism is the RB51 strain of B. abortus. The method according to claim 28, wherein the at least one gene is a SOD Cu / Zn gene. The method according to claim 28, wherein the at least one gene is one or both of the following: GroES gene and GroEL gene.