MXPA06015161A - Adjuvant compositions based on salmonella enterica serovar typhi porins. - Google Patents

Abstract

The present invention shows that Salmonella enterica serovar Typhi porins are capable of activating the innate immunological response upon the uptake thereof by macrophages, causing the cell signalling via the TLR-2 and TLR4 molecules, and inducing the cell activation upon stimulating the production of co-stimulation molecules. Said porins induce the production of pro- and anti-inflammatory cytokines and show an adjuvant activity regarding the response of antibodies against model antigens.

Description

ADJUVANT COMPOSITIONS BASED ON PORINES OF Salmonella enterica serovar Typhi FIELD OF THE INVENTION The invention relates to the use of the porins of Salmonella enterica serovar Typhi to prepare adjuvant compositions useful for the production of vaccines, as well as to adjuvant compositions containing porins of Salmonella enterica serovar Typhi.

BACKGROUND OF THE INVENTION Through evolution, the immune system has developed the ability to respond more quickly and efficiently to a second infection. This property has been defined as immunological memory (1). This memory response is responsible for conferring the immune status of organisms, that is, the ability to protect against reinfection. The generation of immunological memory is the basis for the development of vaccines (2,2-4) (5,6). The first prototypes of vaccines that were used were based on attenuated microorganisms such as the rabies and anthrax vaccine (7) developed by Pasteur, later Calmette and Guérin (8) and Theiler (9) resumed the practice in the development of the vaccines against tuberculosis and yellow fever respectively. With the advance in experimental techniques, it reached the attenuation of some other viruses, which allowed the development of vaccines against polio (Sabin), measles and chicken pox respectively, among these vaccines is also the yellow fever vaccine YF-17D which is the vaccine with the highest efficiency currently known. However, the great disadvantage of this type of vaccines is that they can trigger the disease in individuals with subclinical or transient immunodeficiencies, which represents a risk during vaccination campaigns. Almost simultaneously, vaccines were developed using inactivated microorganisms, such as the vaccine against typhoid, cholera and the inactivated vaccine against polio, which was used together with Sabin in the vaccination campaign that led to the eradication of ?? (10) · however, the presence of lipopolysaccharide and other pyrogenic bacterial components and produce strong adverse side effects, making these vaccines impractical for general vaccination programs. To avoid the side effects induced by vaccines based on inactivated microorganisms, the antigens of the microorganisms that have relevance in the induction of the protective effect were looked for and these were purified, unfortunately the purification of these subunits brought about the decrease of immunogenicity and the ability to confer long-term protection of the vaccine and therefore the need to apply a greater number of doses to seek the protective effect (11-13) In order to increase immunogenicity, protective capacity and the generation of immunological memory many substances have been used as adjuvants that when administered mixed with the vaccine induce a more powerful and long-lasting protective immune response. Adjuvants are substances capable of enhancing or increasing the immune or cellular immune response against an antigen (14). The best known adjuvants are microbial products such as bacterial extracts, although some aluminum salts, mineral oils and various synthetic products have also been described as adjuvants (15,16). One of the best known adjuvants is Freund's complete adjuvant (FCA). The FCA consists of an emulsion containing mineral oil, lanolin and dead tubercle bacilli. A subcutaneous or intramuscular injection of this mixture causes a strong local and granulomatous inflammatory reaction so its use is prohibited in humans and is only used in some animal models with prior authorization. The lipopolysaccharide (LPS) of Gram-negative bacteria can also act as an immunostimulant, however, it causes a large number of adverse effects (14, 17). Among many other activities, LPS activates B lymphocytes, macrophages and T lymphocytes, induces inflammatory reactions, activates alternate complement pathway and elevates glucocorticoid production. Lipid A is the component of LPS responsible for both its toxic activity and its immunostimulating activity. Aluminum salts with monovalent ions of NH4 +, Na + and K +, can form soluble compounds such as sulfates or insolubles such as oxides, hydroxides and aluminum phosphates. The former cause the aggregation of proteins, while the latter adsorb them and thus prevent their diffusion (14,17). Currently, numerous vaccines are prepared by adsorbing the antigens to aluminum compounds. However, because these immunogens can not be lyophilized, they require a cold network for transport and storage, which increases the price of vaccines enormously. Other studies have shown that the immunogenicity of antigens can be significantly increased when they are inoculated after they have been conjugated to synthetic polymers or adsorbed on the surface of inert plastic or bentonite particles. However, these procedures do not offer more stimulating power than aluminum salts and can generate adverse reactions. The role of liposomes as adjuvants was established in 1974 when a strong humoral immune response to diphtheria toxoid included in liposomes was observed after injection into mice. Unlike other adjuvants, it does not induce the formation of granulomas at the administration site, nor hypersensitivity reactions in mice. In subsequent years, it was demonstrated that the adjuvanticity of liposomes is applicable to a large variety of bacterial, viral, parasitic antigens and associated antigens a tumor; however, they have certain disadvantages such as high cost and low stability. Immunostimulatory complexes (ISCOMs, Iscotec AB, Uppsala, Sweden) are complexes consisting of lipids, saponins and antigens that form spontaneously when mixed at appropriate concentrations.

The composition of the ideal ISCOM depends on different factors such as immunogenicity of the incorporated antigen, degree and purification of the saponin Of all the above examples it is worth noting that the only adjuvant approved for use in humans is alumina, thus, it is very important to generate new adjuvants with greater immunopotentiating capacity, with a high margin of safety, high stability and low cost. Despite the extensive experience with the use of adjuvants, until a few years ago, the molecular mechanisms involved in the immunopotentiating effect of these preparations began to be clarified. The discovery of TLRs as one of the main receptors of LPS changed the concept of molecular recognition mediated by the innate immune response and allowed to delineate the mechanism of adjuvanticity. Thus, Janeway develops a theory in which he proposes that innate immunity selectively recognizes molecules mainly of microorganisms by receptors present on the surface of effector cells of the immune system, to such receptors he called them pattern recognition receptors (PRRs) and the molecules that are recognized by these are called pathogen-associated molecular patterns (PAMPs) (18) PAMPs are important molecules for the structure and function of microorganisms, a great variety of PAMPs have been described, among the which are: lipopolysaccharide (LPS) (19), Lipoteichoic acid (LT), peptidoglycan (PG) from Gram-positive bacteria, the lipophopeptide glycan from Entamoeba histolytica (20), Lipoarabinomanana (LAM) from Mycobacterium, flagellin , non-methylated CpGs, and double-stranded RNA. The outer membrane proteins termed porins of gram-negative bacteria such as Neisseria (21), Shigella (22), S. typhimurium (23) and Haemophilus (24) have also been described as PAMPs.

The immunopotentiating effect of adjuvants begins with the activation of dendritic cells and macrophages through PRRs (such as TLRs) that transduce the chemical signal to the cell nucleus activating genes involved in the synthesis of cytokines, endocytosis and phagocytosis. By favoring endocytosis of the antigen, it is prevented that it is metabolized and thus transported to the secondary lymphoid organs for presentation to the T lymphocytes and for the activation of B lymphocytes (25). The activation of the innate immune response leads to a better presentation of antigen by the antigen-presenting cells (APC), due to the increase of MHC molecules in addition to co-stimulatory molecules, which makes the activation of T lymphocyte better, producing cytokines that favor the production of antibodies and change of isotype. For B lymphocytes to be activated, it is required that their antigen receptor (BCR) recognize their antigen, recognition through BCRs activates B lymphocyte and induces cell differentiation and production of IgM class antibodies. The cytokines generated by the LT cooperators promote the change of isotype in B lymphocytes in this way induces the production of other classes of antibodies such as IgG and its subclasses, IgA, IgE (26). The classification of adjuvants can be done according to the mechanism of action involved in the adjuvanticity. For the present study, the most important ones are the immunostimulatory molecules. This type of adjuvant activates the innate immune system mainly through the TLRs (25,27). In this type of adjuvants are a large number of bacterial products and their analogs, such as: Freund's complete adjuvant containing Mycobacterium tuberculosis H37Rv killed, lipopolysaccharide, peptidoglycan, bacterial CpG-DNA, synthetic products such as MPL, some inactivated toxins as CTB or LTk63. Recent studies suggest that this ability to activate the innate immune system either through the binding of adjuvants through Toll-like receptors or other types of PRRs is of utmost importance not only for the increase and establishment of a protective response to short term but is largely related to long-term protection (28,29). As mentioned above, a large number of bacterial products have been tried to be used as adjuvants (25) due to their capacity to activate the innate immune response, which is of great importance to enhance the magnitude and duration of the protective immune response ( 11, 28). Thus, in the bacterial surface are a large number of molecules with adjuvant potential because they are powerful stimulators of the immune response. In particular, the proteins of the The outer membrane of Gram-negative bacteria have been identified as strong immunogens and one of the main and highly immunogenic components of this are the proteins called porins. Porins are pores or non-specific protein channels that can be crossed by small hydrophilic molecules with an exclusion limit of approximately 600Da. Transmembrane proteins are assembled as trimers of identical subunits of cylindrical shape similar to a barrel and each homotrimer is very stable to the action of detergents and proteases (30,31). In the Medical Research Unit in Immunochemistry we have identified that the porins of S. typhi, is one of the targets of the immune response against S. typhi in patients with typhoid fever (32), these proteins were purified and the protection studies in mice showed that they induce 90% protection against the challenge of up to 500 LD50 of S. typhi (33.34). It was also shown that these proteins are the target not only of the antibodies, but also of T lymphocytes (35). In turn, we have reported that Germanier's oral vaccine induces cellular immune response against porins and OMPs of S. typhi, probably due to a self-limited infection produced by the Ty21a strain capable of generating protection through cellular and humoral responses similar to those they generate in convalescent patients of typhoid fever. These results show that porins are good immunogens and that they are targets of the cellular immune response against S. typhi. Both the peripheral blood mononuclear cells (PBMCs) of patients with typhoid fever and the PBMCs of volunteers Humans vaccinated orally with Ty21a responded against porins and outer membrane proteins of S. typhi (36).

SUMMARY OF THE INVENTION The present invention is based, at least in part, on the discovery that Porcine Salmonella serovar Typhi porins can be used as an immunological adjuvant when co-administered with an antigen.

Thus, according to the present invention, adjuvant compositions based on porphyrins of Salmonella enterica serovar Typhi useful for the preparation of vaccines are provided. The invention finds application in any vaccine, but can be particularly in subunit vaccines, conjugate vaccines, DNA vaccines, recombinant vaccines. The vaccine can be therapeutic or prophylactic.

In another aspect of the invention, the use of Salmonella enterica serovar Typhi porins is provided to prepare adjuvant compositions useful for the preparation of vaccines.

In another aspect of the invention, there is provided a vaccine comprising the porcine adjuvant composition of Salmonella enterica serovar Typhi together with one or more antigens. Any antigen or combination of antigens can be used in the vaccines of the invention, including for example sequences of nucleic acids encoding one or more antigenic proteins or peptides; glycoproteins; polysaccharides and other carbohydrates; fusion proteins; lipids; glycolipids, peptides resembling polysaccharides; mixtures of carbohydrates and proteins; carbohydrate-protein conjugates; cells or extracts thereof; dead, attenuated cells or extracts thereof, tumor cells or extracts thereof; viral particles (eg, attenuated viral particles or viral components) and allergens. The antigen may comprise a bacterial, viral, fungal, protozoan or prion antigen. Other available antigens include neoantigens, tumor-associated antigens, and autoantigens.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "therapeutic vaccine" is intended to define a subclass of vaccines that in addition to their prophylactic properties have therapeutic properties. These vaccines have application in the treatment of diseases, infections or existing conditions and can have great importance as agents for the treatment of cancer, AIDS and malaria.

As used herein, the term co-administration attempts to define the sequential, concurrent or separate administration of the referred components. The concurrent administration covers the case where the referred compounds are physically mixed before administration. Sequential administration covers circumstances in which the compounds referred to are administered separately with some degree of temporary separation (typically from several minutes to several hours, although in some cases the administration of the co-administered compounds can be separated for a period of one or more days).

The term neoantigen is used here to define any antigenic determinant expressed again. Neoantigens can be produced from conformational changes in a protein, with newly expressed determinants (especially on the surface of infected or transformed cells), or as a result of a complex formation of one or more molecules or as a result of cutting a molecule with a presentation resulting from new antigenic determinants.

The term tumor-associated antigen is used herein to define an antigen present in transformed cells (malignant or tumor) which is absent (or present in low amounts or in a different cellular compartment) in normal cells of the type on which the tumor is located. origin Oncogenic viruses can also induce the expression of tumor antigens.

The vaccines of the invention can be used in the treatment or prophylaxis of a wide range of diseases and disorders. Viral targets include diseases and disorders in which any of the following viruses (or classes of viruses) are involved: Retroviridae, Picomaviridae, Calciviridae, Togaviridae, Flaviridae, Coronoviridae, Rhabdoviradae, Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bungaviridae, Arenaviridae, Birnaviridae, Hepadnaviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, Iridoviridae and unclassified viruses (for example, the etiological agents of spongiform encephalopathies), HCV virus (causing hepatitis A or B) ), Norwalk virus and related. Of the above mentioned, particularly preferred are HIV, Hepatitis A, Hepatitis B, Hepatitis C, rabies, poliovirus, influenza, meningitis, smallpox, rubella, encephalitis, papilloma, yellow fever, respiratory syncytial, parvovirus, chikungunya, hemorrhagic fever and Herpes. In such incorporations the antigen selected for use in vaccines derives from those antigens present in naturally occurring viruses.

Bacterial targets include Gram-positive and Gram-negative bacteria. Examples of bacteria which can be targeted by the vaccines of the invention include, but are not limited to: Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacterium spp (eg M. tuberculosis, M. leprae, M. avium, M. intracellulare, M. kansaii and M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactie (Group B Streptococcus), Streptococcus viridans, Streptococcus faecalis, Streptococcus bovis, any of the anaerobic species of the genus Streptococcus, Streptococcus pneumoniae, Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium spp. (including C. diphtheriae), Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella spp (including K. pneumoniae), Pasteurella multocida, Bacteroides spp. , Fusobacterium nucleatum, Streptobacillus monilijormis, Treponema pallidium, Treponema pertenue, Leptospira spp., Rickettsia spp. and Actinomyces spp. (including A. israelil). In such incorporations the antigen selected for use in vaccines derives from those antigens present in bacteria occurring naturally (or expressed / induced during infection).

Fungal targets include Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis and Candida albicans. In such incorporations the antigen selected for use in vaccines derives from those antigens present in fungi naturally occurring (or expressed / induced during infection).

Prozoan targets include Plasmodium spp. (including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax), Toxoplasma spp. (including T. gondii and T. crut), Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp.

Cancer and proliferative disorders include cancer of solid tissue and those of the lymphatic and blood systems (including Hodgkin's disease, leukemias, lymphomas, multiple myeloma, and Waldenstrom's disease), melanomas (including melanoma of the eye), adenomas, sarcomas, solid tissue carcinomas, melanoma, lung cancer, thyroid, salivary glands, leg, tongue, lips, bile ducts, pelvis, mediastinum, urethra, Kaposi's sarcoma (for example when associated with AIDS); skin cancer (including malignant melanoma), cancer of the digestive tract (including cancer of the head, neck, esophagus, stomach, pancreas, liver, colon, rectum, and anus), cancer of the genital and urinary system (including kidney cancer, bladder , testicle, and prostate), cancer in women (including breast, cervical, ovarian, and choriocarcinoma), as well as brain, bone, nasopharyngeal, retroperitoneal, and cancer of unknown primary site. In such embodiments, the antigen selected for use in vaccines are the neoantigens or tumor-associated antigens present in the malignant cells and / or tissues.

Allergic disorders include atopic allergy, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, hypereosinophilia, irritable bowel syndrome, allergen-induced migraine, bacterial allergy, bronchial allergy (asthma), contact allergy (dermatitis), pollen allergy, allergy to medications, allergy to pickets, food allergy, physical allergy (including cold urticaria or angioedema), heat allergy (cholinergic urticaria) and photosensitivity. In such additions the antigen selected for use in vaccines are derived from those antigens present in the allergen, including pollen, insect venom, fungal spores, drugs and specific proteins to the following genera: Canis, Dermatophagoides, Felis, Ambrosia, Lolium, Cryptomeria, Alder, Agnus, Betula, Quercus, Festuca and Bromus.

The compositions and vaccines of the invention contain Salmonella enterica serovar Typhi porins, optionally together with one or more auxiliary adjuvants and / or pharmaceutically acceptable excipients.

When Porcine Salmonella enteric serovar Typhi are formulated together with a pharmaceutically acceptable excipient, any excipient can be used, including for example inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.

The pharmaceutical compositions can take any available form, and include for example tablets, capsules, solutions, suspensions, powders, granules and aerosols.

Tablets for oral use may contain the Salmonella enterica serovar Typhi porins mixed with pharmaceutically acceptable excipients, such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Available inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are available as disintegrating agents. The binding agents may include starch and gelatin, while the lubricating agents, if present, will generally be magnesium stearate, stearic acid, or talc.

Capsules for oral use include hard gelatine capsules, in which the porins of Salmonella enterica serovar Typhi are mixed with a solid diluent, and the soft gelatin capsules, wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

Formulations for rectal administration may be presented as a suppository with an available base comprising for example cocoa butter or a salicylate.

Formulations for vaginal administration may be presented as presentations of buffers, creams, gels, pastes, foams or spray containing in addition to the active agent, ingredients such as appropriate carriers.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, the porins of Salmonella enterica serovar Typhi will generally be provided in sterile aqueous solutions or suspensions, buffered at an appropriate pH and isotonicity.

When used adjunct, the porins of Salmonella enterica serovar Typhi can be formulated with one or more medications. In particular, they can be used in combination with antitumor, antimicrobial, anti-inflammatory, antiproliferative, and / or immunostimulatory agents. For example, Porins can be used with antiproliferative agents such as atocines, including IL-2 and IL-12, ineriorones and inducers thereof, TNF, TGF, as well as myelosuppressive and / or chemotherapeutic agents (such as doxorubicin, 5-fluorouracil, cyclophosphamide and methotrexate), isoniazid (for example in the prevention or treatment of peripheral neuropathy) and with analgesic (for example NSAIDs) for the prevention and treatment of gastroduodenal ulcers.

The amount of the porins administered can vary extensively according to the dose unit used, the treatment period, age, weight, kind of treatment attached, and sex of the patient to be treated, the nature and extent of the disorder to be treated, the nature of the antigen administered.

The vaccines of the invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, aerial, rectal, vaginal and topical (including buccal and sublingual).

The invention will now be described with reference to specific examples, which are merely for illustrative purposes. These examples do not in any way attempt to limit in any way the purpose of the described invention. Said examples up to now constitute the best method currently contemplated for the practice of the invention.

EXAMPLES Example 1. Intake of porins by macrophages. Mice of the BALB / C strain were sacrificed by cervical dislocation and fixed to a mouse template. Both femur were removed and placed in a tube with PBS. In the biological safety hood, the femurs were washed with 70% alcohol for 10 seconds and then 3 times with PBS. The ends of the femur were cut leaving exposed the channel to extract the bone marrow using 2.5 ml of DMEM medium supplemented for each end. The extract was collected in sterile conical tobs of 15 ml. And it was centrifuged at 1100 rpm / 1 min / 4 ° C. The supernatant from each tube was transferred to another sterile conical tube and centrifuged at 1100 rpm / 10min / 4 ° C. The supernatant was discarded, the buttons of the tubes were collected in one and resuspended in a final volume of 6 ml. With bone marrow medium. It was placed 1 mi. Of the cell suspension in boxes for low adhesion cell culture and it was completed to a final volume of 20 ml. With bone marrow medium. The boxes were incubated at 37 ° C with 5% CO2 for 6 days. On the third day, 5 ml was added to each box. Of bone marrow medium (supplemented DMEM, SFB, penicillin / streptomycin, M-CSF). On the sixth day, the middle of the boxes was discarded and washed twice with 10 ml. PBS cold and left on ice with 10 ml. From PBS. The cells were peeled off and the suspensions were placed in sterile 15 ml conical tubes, which were centrifuged at 1100 rpm / 10min / 4 ° C. The supernatant was discarded, all the buttons were collected in a single tube and resuspended in DMEM supplemented to a known volume. The number of cells in the hematocytometer was obtained making sure that more than 80% of the crop will be viable. The necessary amount of cell suspension was placed in a 6-well plate so that each of them had 1 X 10 6 cells and the volume of each well was completed to 2 ml. With DMEM supplemented. It was incubated overnight before the experiment at 37 ° C under a 5% C02 atmosphere.

They were placed on circular coverslips around 200,000 bone marrow derived macrophages (MDMO) obtained in the previous step, later they were stimulated with ^ g / ml of porins during 45 min. To eliminate the non-internalized antigen, washings were carried out with PBS + SFB 2%. The preparation was fixed with 3% formaldehyde for 20 min. At 4 ° C. Once the fixing time has ended, a wash is carried out under the same conditions. The first rabbit anti-porin antibody was then placed, dilution 1: 100, and incubated at room temperature in a humid chamber for 30 min. Washes were performed and the second anti-rabbit antibody labeled with TRICT (50-600 nm) was placed. The preparations were mounted for further analysis by confocal microscopy. In the cells that were stimulated, a large amount of red fluorescence was observed corresponding to the porins that were captured by the cell, in comparison with the cells that were not treated with porins.

Example 1. Signaling through Toll receivers mediated by porins. HEK293 cells were cultured in DMEM medium supplemented with 10% FBS, at 37 ° C and 5% C02. The plasmids containing the TLR2 and TLR4 genes and the reporter gene of luciferase were propagated in the E. coli strain DH5a. The bacterium became competent by treatment with calcium chloride, was transformed with the corresponding plasmids by thermal shock (45 min at 4 ° C, 90 sec at 42 ° C, and 5 min at 4 ° C) and was cultured in Luria medium with 100 μg / ml of ampicillin for 18 hours. The plasmids were extracted from the bacteria with the Plasmad Midi Kit from Qiagen, according to the protocol provided by the manufacturer. To perform the transfection, 300,000 HEK293 cells were placed per well in 24-well plates and incubated for 24 h at 37 ° C and 5% C02. Subsequently, the culture medium was removed and 500 μ? per well of a solution containing 5 μg of plasmid DNA and 10 μ9 of LipofectaMINE (GIBCO BRL, 2 mg / ml). It was incubated at 27 ° C for 1 h, then the medium was removed and incubated for 24 h at 37 ° C to allow expression of the corresponding TLR. After the incubation time, the corresponding agonists were added at a concentration of 10 μg / ml, the cells were incubated for 6 to 18 h more to allow the expression of the luciferase. In this system luciferase is under the control of L-selectin, which has 5 NF-γ binding sites, so this enzyme is expressed when NF- ?? it is activated through the TLRs. The luciferase activity was evaluated with the Promega Luciferase Detection System, according to the protocol provided by the manufacturer; the readings were made in a Fluoroskan AFCENT luminometer (Labsystems).

The porins were able to signal for two different TLRs, TLR-2 and TLR-4 / MD2. This signaling was very similar to that induced by the LPS in 7LR-4 / MD2 (around 25 relative units of light), although it must be considered that the amount of LPS contained in the porins is 50 times less than the amount of LPS used in a control. In the case of TLR-2, it is observed that the porins induce a high response (around 10 relative units of light), although it is much lower than that obtained with peptiglicana (around 20 relative units of light), which is a potent TLR-2 agonist Example 3. Secretion of cytokines in dendritic cells stimulated by porins. From the dendritic cells obtained by Example 1 stimulated at various times (6, 12 and 24 h) with 1 μg / ml of porins (OmpC and OmpF), the concentration was determined by ELISA of the cytokines TNF-a, IL- 6 and IL-10. The porins were able to induce the production of cytokines. In the case of the production of IL-6, porcine OmpF induced a large amount (1800 pg / ml) of this cytokine, having a maximum peak of 6 h. For its part, the porcine OmpC induced a peak production at 12 h (1700 pg / ml). For TNF-a behavior similar to that obtained for IL-6 was observed (OmpF = 800 pg / ml at 6 h, and OmpC = 1000 pg / ml at 24 h). On the other hand, IL-10 levels at 6 h were relatively low; however, the expression of said cytokine was progressively increased up to 24 h when stimulated with OmpF. Stimulation with OmpC did not induce any production of IL-10.

Example 4. Increase in the expression of costimulatory molecules in dendritic and macrophage cells mediated by porins. Dendritic cells and macrophages obtained as in example 1 were stimulated for 12 h or 48 h respectively with porins to analyze the cellular expression of co-stimulatory molecules CD80 and CD86. The results show that the porins are capable of activating both macrophages and dendritic cells increasing the expression of said costimulatory molecules. In this sense, the porins induced an expression of the co-stimulatory molecules CD80, and CD86, comparable to that obtained with the stimulation with LPS.

Example 5. Porin-mediated adjuvant effect on the humoral immune response. Groups of mice immunized with model antigen, OVA or HEL, in the presence or absence of porins were used to determine the potential of the porins as adjuvant. The mice thus immunized were bled at 0, 4, 8, 12, 20, 30, 90, 120, and 400 days to analyze the production of anti-OVA or anti-HEL antibodies. When evaluating the adjuvant effect of the porins, OmpC and OmpF on OVA, we can observe that at day 8 OmpF was the only one able to induce an adjuvant effect. At day 120, it was observed that both OmpC and OmpF had an adjuvant effect compared to the antigen alone. In the case of immunization with HEL, it can be observed that the adjuvant effect begins on the eighth day post-immunization and that it is maintained until day 120, indicating that the induced effect is of long duration, which has a great relevance for its use in vaccines.

Claims (8)

1. The use of at least one porin selected from the OmpC and OmpF group of Salmonella enterica serovar Typhi to prepare an adjuvant composition useful for the preparation of vaccines.

2. An adjuvant composition useful for the preparation of vaccines characterized in that it contains at least one porin selected from the OmpC and OmpF group of Salmonella enterica serovar Typhi.

3. A vaccine composition comprising: a) an adjuvant composition containing at least one porin selected from the group of OmpC and OmpF of Salmonella enterica serovar Typhi and b) an antigen or combination of antigens.

4. The vaccine composition according to claim 3, wherein the vaccine can be a subunit vaccine, a vaccine conjugate, a DNA vaccine, or a recombinant vaccine.

5. The vaccine composition according to claims 3-4, wherein the vaccine can be therapeutic or prophylactic.

6. The vaccine composition according to claims 3-5, wherein the antigen is selected from the group of genes which encode one or more proteins directly involved with the biosynthesis of the antigen or peptide, as well as the combinations of the antigen or peptide with glycoproteins, polysaccharides, fusion proteins, lipids, glycolipids, polysaccharide-like peptides, carbohydrate-protein conjugates, cells or extracts of the same, dead or attenuated cells or extracts thereof, tumor cells or extracts thereof, attenuated viral particles or viral components, and allergens.

7. The vaccine composition according to claims 3-6, wherein the antigen is selected from the group: bacterial, viral, fungal, protozoan, prionic, neoantigen, tumor-associated antigen, and autoantigen antigens.

8. The vaccine composition according to claims 3-7, characterized in that it additionally contains an auxiliary adjuvant and pharmaceutically acceptable excipients. The vaccine composition according to claims 3-8, characterized in that they are pharmaceutically adapted in the form of tablets, capsules, solutions, suspensions, powders, granules or aerosols.