MX2009013222A - Adjuvant compositions based on salmonella enterica serovar typhi omps2 porins. - Google Patents

Abstract

The present invention relates to the use of the Salmonella enterica serovar Typhi OmpS2 porin for preparing an adjuvant composition, and a vaccine composition comprising the OmpS2 porin and one or more microbial and/or cancerous antigens.

Description

ADJUVANT COMPOSITION BASED ON PORINA OMPS2 DE Salmonella enterica serovar Typhi FIELD OF THE INVENTION The invention relates to the use of the porcine OmpS2 of Salmonella enterica serovar Typhi to prepare an adjuvant composition useful for the production of vaccines, as well as an adjuvant composition containing said porin, and a vaccine composition comprising the porcine OmpS2. plus an antigen of bacterial or tumoral origin.

BACKGROUND OF THE INVENTION Vaccines are formulations capable of inducing the host mount an immune response for therapeutic or prophylactic purposes, the mechanism by which they are able to generate this type of response is through the activation of the immune system of the host through the administration of antigens of the agents that cause the disease. Most of the successful vaccines used are constituted by antigenic components of a pathogen that are recognized by the cells of the adaptive immune response, in addition to molecules called Pathogen-associated molecular patterns (PAMP) capable of being counted by the pattern recognition receptors (PRR, for its acronym in English) in the cells of the innate immune response generating their activation. Such vaccines are based primarily on attenuated or dead forms of microbes, and can be extremely potent prophylactic therapeutic agents.

However, the presence of other components present in the structure of the microorganism not involved with the establishment of the immune response in attenuated or killed vaccines can cause undesirable side effects, decreasing the safety of the vaccine preparation which has limited the use of vaccines to It is based on complete microorganisms and has stimulated considerable efforts to refine the components of the vaccine, promoting the development of subunit vaccines, conjugate vaccines and DNA vaccines.

Subunit vaccines are based on antigens isolated from different microorganisms or synthetic antigens created using chemical and / or recombinant techniques. Conjugated vaccines involve the coupling of relatively non-immunogenic antigens to harder immunogenic carrier proteins. The DNA vaccines release the antigen in the form of coding nucleic acid and thus allow, after administration, the endogenous expression of the coding DNA. Such vaccines normally require an appropriate vector to deliver the encoding nucleic acid to an appropriate compartment of the host.

Although subunit, conjugate or DNA vaccines are generally well tolerated with few undesirable side effects, they generally lack the PAMPs that are required for the activation of the innate response. In this way, they typically exhibit less power when compared to the antigenic mixture present in many of the vaccines that are composed of complete microorganisms. Another important point is that the nature of synthetic low molecular weight antigens does not allow the establishment of a good immune response and by themselves they are not highly immunogenic, which even when they lack potentially dangerous contaminants does not make them a good option for the generation of new vaccines.

Therefore, a challenge of modern vaccine design is to develop strategies to efficiently induce the innate and adaptive response to mimic a natural infection and to achieve powerful responses against defined antigens, with limited toxicity. The great effort is currently focused on the adjuvants of the immune response, which are essential to make new types of viable vaccines, more effective, and safer and with cheaper manufacturing processes.

An adjuvant is any compound or composition that increases the strength and / or duration of an immune response against antigen. The key functional characteristics of an adjuvant include its ability to increase an appropriate immune response against the target antigen, long-term safety in extensive application, and flexibility in use with different antigens / diseases. Despite its wide use in vaccine preparations, the mechanisms of action of most adjuvants are only partially known.

The only adjuvant currently licensed for human use with any antigen is alumina (aluminum salts). This can precipitate soluble antigens in the area near the injection site, where macrophages and dendritic cells can process it. In this way, it acts as an antigen-releasing vehicle, mediating a more effective presentation of the antigen to the innate system.

However, a recent study has shown that alumina also has an enhancing effect via cytokine-mediated control of the Th1 / Th2 response (Brewer et al., 1999. The Journal of Immunology, 163: 6448-6454). The immunological stimulus exhibited by alumina is strongly polarized towards a Th2 type response. This is an important limitation, since it is now recognized that the Th1-type immune response is essential to generate protective immunity against many infectious agents (viral, bacterial, and intracellular protozoa).

Another potent immunological adjuvant is bacterial lipopolysaccharide endotoxin (LPS), and specifically its active portion, lipid A. Native lipid A is reactogenic and pyrogenic; however, chemical modifications in its structure have given rise to non-toxic preparations that preserve their adjuvant activity. Clinical trials have been conducted with the purified lipid A of Salmonella minnesota R595 and detoxified by removing the phosphate group from the reducing end of the disaccharide skeleton by acid hydrolysis, to produce monophosphoryl lipid A (MPL). This MPL can also be detoxified by 3-O-acetylation with a slight alkaline hydrolysis and can be encapsulated in liposomes composed of phospholipids and cholesterol.

On the other hand, the US patent application US 2003091599 describes adjuvant oligonucleotides having at least one non-methylated CpG dinucleotide and discloses that the CpG DNA induces a Th1 pattern of production of cytokines dominated by IL-12 and IFN-gamma, with a small secretion of Th2-type cytokines, thus, CpG directly activates monocytes, macrophages, and dendritic cells to secrete a variety of cytokines, including high levels of IL-12. These cytokines stimulate NK cells to secrete IFN-gamma and also increase their lytic activity.

Another known adjuvant is that described in the US patent US 5976539; said patent describes the potential use of IL-12 as an adjuvant and highlights its potential as an activator of Th1 type responses. Its potential is accentuated in vaccines against infections that require an increased immune response mediated by cells for effective protection against pathogens. However, its use has been limited due to the high cost of its production and formulation and its use in humans has high reactogenicity and pleiotropic effects (which can lead to problems of dosage and unexpected side effects).

On the other hand, the international patent application W08809336 describes several fractions of immunologically active saponins having adjuvant activity. Fractions have been used successfully in veterinary practice and they are derived from the bark of the South American Quillajasaponana tree. The substantially pure saponins have adjuvant activity at a much lower concentration than the heterogeneous preparations of saponins, and do not exhibit their toxic effects. For example QS-21, also known as QA21, is a fraction purified by HPLC disclosed (as QA21) in US Patent 5057540. Saponin adjuvants QS-21 stimulate the immune response type 1 and 2 and have been used with great success in large-scale clinical trials of prophylactic and therapeutic vaccines.

Additionally, the international patent application W09014837 describes several submicron oil / water emulsions as well as their use as adjuvants. In particular, this document describes adjuvant compositions comprising a metabolizable oil and an emulsifying agent, wherein the oil and the emulsifying agent are present in the form of an oil / water emulsion having oil droplets substantially less than 1 micrometer in diameter. One of the emulsions described, a microfluidized emulsion system consisting of polysorbate 80 and sorbitan trioleate MF59, has recently been approved in Italy for use in a flu vaccine. However, MF59, like alumina, stimulates a strongly polarized type 2 response.

Thus, there is a need for adjuvants and low toxicity adjuvant systems, which stimulate both branches of the immune system, which improve the safety and efficacy of existing vaccines and are suitable for use with synthetic and subunit vaccines.

In this sense, the present invention relates to a new adjuvant, the porcine OmpS2 of S. typhi. This porin is not expressed in laboratory conditions, such as changes in osmolarity or under stress conditions, such as changes in pH and temperature or in anaerobiosis or in the presence of bile salts and cationic peptides (Oropeza et al., 1999). , Mol.Microbio !. 32: 243-252).

SUMMARY OF THE INVENTION In one aspect, the present invention provides an adjuvant composition based on the porcine OmpS2 of Salmonella enterica serovar Typhi.

In another aspect of the invention, the use of OmpS2 porcine Salmonella enterica serovar Typhi is provided to prepare adjuvant compositions useful for the preparation of vaccines.

In yet another aspect of the invention, there is provided a vaccine comprising an OmpS2 porcine adjuvant composition of Salmonella enterica serovar Typhi and one or more antigens.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term therapeutic vaccine attempts to define a subclass of vaccines which have therapeutic properties (and not only prophylactic). Said vaccines may have a prophylactic activity in addition to their therapeutic potential. They find application in the treatment of diseases, infections or existing conditions. They come to be of great importance as agents for the treatment of cancer, AIDS and malaria.

Any antigen or combination of antigens can be used in vaccines of the invention, including for example nucleic acids which encode one or more antigenic proteins or peptides; glycoproteins, polysaccharides and other carbohydrates; fusion proteins; lipids; glycolipids, glycopeptides; carbohydrates and proteins in mixture; carbohydrate-protein conjugates; cells or extracts thereof; dead or 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.

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 (eg agents etiological findings of spongiform encephalopathies), HCV virus (causing hepatitis A and B), Norwalk virus and related viruses. 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, dengue, 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 may 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. Y Actinomyces spp. (including A. israelil). In such incorporations the antigen selected for use in vaccines derives from those antigens naturally occurring in bacteria (or expressed / induced during infection, toxoids and toxins).

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 naturally occurring fungi (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. cruf), 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 tract digestive (including cancer of the head, neck, esophagus, stomach, pancreas, liver, colon, rectum, and anus), cancer of the genital and urinary system (including cancer of the kidney, 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 incorporations the antigen selected for use in vaccines are the neoantigens to 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, and 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 the OmpS2 of Salmonella enterica serovar Typhi, optionally together with one or more auxiliary adjuvants and / or pharmaceutically acceptable excipients.

When the porcine OmpS2 of enteric Salmonella serovar Typhi is 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 adjuvant or vaccine 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 OmpS2 porcine of Salmonella enterica serovar Typhi 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 gelatin hard capsules, in which the porcine OmpS2 of enteric Salmonella serovar is mixed with a solid diluent, and the soft gelatine capsules, wherein the ingredient Active 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 well known in the art.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, OmpS2 porcine Salmonella enterica serovar Typhi will generally be provided in sterile aqueous solutions or suspensions, buffered at appropriate pH and isotonicity.

When used adjunct, the porcine OmpS2 of Salmonella enteric 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, the porins can be used with antiproliferative agents such as atocines, including IL-2 and IL-12, interferons and inducers thereof, TNF, TGF, as well as myelosuppressive and / or chemotherapeutic agents (such as doxorubicin). -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 porin 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, transdermal, mucosal, air, rectal, vaginal and topical (including buccal and sublingual).

The invention will now be described with reference to specific examples.

They are for illustrative purposes only and do not attempt in any way to limit the purpose of the disclosed invention in any way. Said examples up to now constitute the best method currently contemplated for the practice of the invention.

EXAMPLES Example 1. Purification of OmpS2 porin.

Obtaining the OmpS2 porcine from S. typhi is done through the modified Nikaido method. S. typhi pTrc99A-S2 is grown in 9 liters of minimal medium A (70g K2HP04, 30g KH2P0, 10g (NH4) 2S04, 5g citrate sodium, 10g yeast extract, 50g of glucose, 0.1% MgSO4) until obtaining an optical density of 1.0 to 540 nm corresponding to the late logarithmic phase of the growth of the bacteria. It is harvested by centrifugation at 7,000 rpm for 15 minutes at 4 ° C. The bacterial biomass is weighed and resuspended in 0.05M Tris-CI solution (pH 7.7) until 50 mL of bacterial suspension is obtained. It is broken by sonication (Sonicator Ultra Sonio) for one hour thirty minutes and the broken bacterium suspension is centrifuged at 7,000 rpm 30 minutes at 4 ° C.

The supernatant is removed and treated with 25μ? of DNase 10 000 U / mL and 25μ? of RNAse 10 000 U / mL and 2.77 mL of 1 M gCI2 solution for each 10 g of wet biomass obtained from the harvest, for 30 minutes at 37 ° C 120 rpm to eliminate DNA and RNA. The free RNA and DNA biomass was ultracentrifuged at 45,000 rpm for 45 minutes at 4 ° C. The button is resuspended in 100 mL of Tris HCI-SDS 2% solution, incubated 30 minutes at 32 ° C, with orbital shaking at 120 rpm and ultracentrifuged at 40,000 rpm, 30 minutes at 20 ° C. The resulting button is resuspended in 25 mL of Tris HCI-SDS 2% solution, incubated again for 30 minutes at 32 ° C, with orbital shaking at 120 rpm and ultracentrifuged at 40,000 rpm 30 minutes at 20 ° C. The button is resuspended in 20 mL of Nikaido SDS 1% buffer (Tris 0.05M, NaCl 0.4M, EDTA 0.005M) pH 7.7. Incubate 2 hours at 37 ° C, with orbital shaking at 120 rpm and ultracentrifuge at 40,000 rpm 45 minutes at 20 ° C. The supernatant is recovered.

The supernatant is purified on a Sephacryl S-200 column (XK100 Pharmacia) at a flow rate of 5 mL / min with Nikaido SDS buffer 0.5% pH 7. 7. Collect the peak obtained in the chromatogram and dialyze with PBS solution for 4 days to eliminate the SDS.

OmpS2 porin is obtained in fractions 75 to 100 of the chromatogram. Afterwards, an electrophoretic polyacrylamide gel run with SDS (SDS-PAGE) was carried out, for staining with coomasie blue and another for silver staining in order to corroborate the presence of the porin, as well as the integrity of it. Porcine OmpS2 is observed as a band of 40 kDa, which corresponds to the weight reported in the literature for this protein.

Example 2. Effect of OmpS2 porin on mouse monocytes.

Mice of the BALB / c strain are sacrificed by dislocation and both femurs are removed, under sterile conditions. The ends of the femur are cut leaving a channel to elute the bone marrow using 2.5 mL of supplemented DMEM medium for each end. The eluate is collected and resuspended in 6 mL with bone marrow medium. 1 mL of the cell suspension is placed in boxes for low adhesion cell culture and is completed to a final volume of 15 mL with bone marrow medium. The boxes are incubated at 37 ° C with 5% C02 for 6 days.

On the sixth day, the middle of the boxes is discarded. The cells are detached and the necessary amount of cell suspension is placed in a 6-well plate so that in each of them there are 1 x 106 cells and the volume in each well at 2mL with supplemented DMEM. It is incubated overnight before the experiment at 37 ° C in a 5% CO2 atmosphere.

Plates of 6 wells with 1 x 106 macrophages derived from bone marrow in 1 mL of DMEM medium are placed, they are stimulated with 1pg / ml of the porcine OmpS2 of S. typhi, 2 ng / mL of LPS of E. coli, as positive control 100 ng / mL of E. coli LPS and, as a negative control, DMEM medium, are incubated at 37 ° C in a 5% C02 atmosphere. At 6, 12 and 24 hours after stimulation, all the supernatant is collected and they discard the cells. The supernatant is stored at -70 ° C until its analysis of the different cytokines.

Porcine OmpS2 is able to induce IL-6, TNF and MCP-1 in MDMO. The positive control (100 ng of LPS) induced all the cytokines evaluated, the 2 ng of LPS only induced a very small amount of MCP1. So it can be said that the effect is due to the porin and not to the possible contamination of LPS. MCP-1 was induced at 24 hours, and TNF-a was induced at 12 hours. The presence of IL-6 was observed only at 12 hours.

Example 3. Adjuvant effect of OmpS2 porin on immunization with ovalbumin (OVA) Groups of BALB / c mice were immunized intraperitoneally with 100 g of OVA in the absence or presence of adjuvant (10 μg OmpS2, 10 μg porins). Control mice received isotonic saline. Samples of Blood was collected from the facial vein at different times. Individual serum samples were frozen at -20"C until analysis.

The 96-well ELISA plates were fixed by adding 100 pL of binding solution per well (15pg of OVA / 1 OOpL and 5pg of HELJIOOpL). The plates were incubated 1 hour at 37 ° C and overnight at 4 ° C. They were then washed four times with wash solution (PBS-Tween20 0.1%). Subsequently, the plates were blocked by adding each well 100μ? of blocking solution (PBS-Milk 0.5%). They were incubated for one hour at 37 ° C and subsequently washed four times with washing solution.

The sera to be analyzed were diluted 1: 40 in blocking solution with a final volume of 100 pL. 100 pL of the diluted sera were added to the first wells of the dilution plates, serial dilutions were made 1: 2 with blocking solution. The sera were transferred quantitatively and in the order corresponding to the previously sensitized ELISA plate. They were incubated 1 hour at 37 ° C and at the end of that time they were washed four times with washing solution. Later, 100 pL of the corresponding mouse anti-immunoglobulin antibody labeled with horseradish peroxidase diluted 1: 1000 in blocking solution was added. Incubation of the plates was carried out at 37 ° C for 1 hour, afterwards they were washed four times with washing solution. The development of the plates was done by adding 100 pL of development solution to each well. It was incubated in the dark at room temperature for 15 minutes and the reaction was stopped with 10 pL of 2.5N H2SO4. water well. The optical density was determined at a wavelength of 490 nm in an ELISA reader.

It was found that by co-administering OmpS2 porin with OVA, it is able to act as an adjuvant when finding high titers of OVA antibodies, of all subclasses of IgG, from the first days, a phenomenon that is not observed when only OVA is immunized without adjuvant. The adjuvant response was compared with that generated with the major porins (OmpC and OmpF) of S. typhi, which also has an adjuvant effect and the adjuvant effect of the porcine OmpS2 is lower than that generated with the majority of S. typhi porins.

In addition, the adjuvant effect was evaluated using another antigen, in this case HEL (hen egg lysozyme) and it was observed that the co-administration of OmpS2 with HEL is able to induce high anti-HEL antibody titers of subclass IgG1 and IgG2b from the first days, response that is not able to induce HEL immunization without adjuvant. When comparing the adjuvant effect of the porcine OmpS2 with the major porins of S. typhi, the porcine OmpS2 showed to be a better adjuvant when inducing higher levels of anti HEL antibodies.

Example 4. Recognition of porcine OmpS2 by TLRs HEK-293 cells were used, which present a permanent transfection with a plasmid that has the TLR-2 gene, TLR-2 / CD14, TLR-2 / TLR-6, TLR-4, or human TLR-5 under the control of the promoter of L-selectin, which is activated by NF-?? The selection antibiotic for the plasmid was blasticidin S (Blasticidin InVivoGen). These cells were used to determine the recognition of OmpS2 porin through the TLR encoded in transfected plasmid. HEK cells were plated for 12-well cell culture plates (Corning incorporated Cat. 3526). 300,000 cells per well were placed and stimulated with 1 pg of the OmpS2 porins. The cells were incubated for 24 hours at 37C. As a control, E. coli LPS (10 pg / mL or 2ng / ml), Zimosan (10 pg / mL), flagellin (1 pg / mL) and OmpC and OmpS2 porins degraded with proteinase K (0.5 pg / mL) were used. of proteinase for each 10 pg of porin). After incubation, the supernatants were taken and the concentration of IL-8 was determined as a measure of the recognition of the porins through the transfected TLR in the cells.

OmpS2 porin is recognized by both TLR-2, TLR-2 / CD14, TLR-2 / TLR-6 and TLR-4 and this recognition is from porin in native form, because after treatment with proteinase K no longer observe recognition for none of the TLRs evaluated. According to the prediction of probable sites of proteinase K cut on OmpS2, using the ExPASy PeptideCutter tool program, I predict 160 cut sites, which would generate peptides smaller than 13 amino acids.

OmpS2 porin was not recognized by TLR-5, which allows us to rule out flagellin contamination. OmpS2 porin, being an outer membrane protein, may contain traces of LPS, however, when evaluating the amount of LPS that it could contain, by means of the umulus amoebocyte binding test, the result of the test was negative, the limit of detection of this system is 0.2 ng of LPS, so if LPS contamination existed this would be less than 0.2 ng per pg of porin, therefore, the transfected HEK cells were stimulated with ten times more the possible amount of contaminating LPS (2 ng of LPS) and only recognition was observed with TLR-4, which is the LPS receptor, however, the effect observed with the 2 ng of LPS is not similar to that observed with the porcine OmpS2.

It can be speculated that the mixture of traces of LPS with a protein might be able to signal through TLR-2, TLR2 / CD14 and TLR2 / TLR-6, and for this reason porcine OmpS2 is recognized by those TLRs, however , the mixture of a protein such as OVA with 2 ng of LPS are not able to signal for these receptors.

Example 5. Antigenic effect of OmpS2 porin Groups of mice were immunized intraperitoneally, without adjuvant, in a total volume of 500μ ?, using isotonic saline solution as a vehicle with different doses of OmpS2 porin (1, 10, and 50 pg). Blood samples were obtained by indentation of the facial vein on days 0 to 360.

The specific antibody titer was evaluated by the technique of ELISA To this end, 96-well plates were sensitized for immunoassay (Corning®) with 100 pL per well of carbonate buffer (pH 8.6) containing 1 pg of S. typhi porins, incubated at 37 ° C for one hour (Shelter incubator). lab, 2100) and overnight at 4 ° C.

For the determination of the immunoglobulin titer, the plates were washed four times with washing solution (0.1% PBS solution of Tween 20) and blocked with 150 μl of 5.0% skim milk (Svelty, Nestle) dissolved in PBS incubating the plates at 37 ° C for one hour. Subsequently, they were washed four times with the washing solution, the plates were refrigerated until they were used.

The sera were diluted 1: 40 (5μ? _ In 195 μ? _) In PBS-5% milk, from this concentration serial dilutions were made in factor of two transferring 100 μ? _ Of the dilution to another well that contained 100 μ? of PBS-milk to complete the 12 wells of the conical bottom micro dilution plate (Corning). The dilutions were transferred to the immunoassay plate sensitized with porins, incubated at 37 ° C for one hour, washed 4 times with washing solution and 100 μl were added per well of respective anti-Ig antibody (goat anti-IgM antibody, total IgG, IgG1, IgG2a, IgG2b, IgG3 mouse conjugated to horseradish peroxidase, Z ymed) diluted 1: 1000 in PBS-milk, incubated for one hour at 37 ° C, washed four times with washing solution and revealed with the developer solution (0.5mg / mL o-phenylenediamine in citrate buffer pH 5.6 and H2O2) incubating in the absence of light for ten minutes, the reaction was stopped by adding 10 μl of 2.5 N sulfuric acid. The plates were read in a microplate reader (Dynex Technologies, MRX II) at 410 nm. The specific antibody titers were determined as that dilution in which the absorbance was equivalent to 3 times the background (it was generally equal to 0.3) and the titres were plotted logarithmically against the corresponding day. the serum. Positive mouse sera previously titrated for the respective antigen were used as positive controls.

It was observed that a single dose of 10 g of OmpS2 is capable of inducing a long-lasting antibody response both Ig and IgG.

Example 6. Induction of protection against S. typhi bacteria using OmpS2 porin.

As it has been observed, the porcine OmpS2 of S. typhi is an immunogenic antigen capable of inducing a long-lasting antibody response, which could mediate protection against infection. To corroborate this, it was decided to immunize groups of 6 mice with 10 pg of the porcine OmpS2 or 10 pg of OmpC as a control group via i. p. and they were re-immunized at day 15 with the same corresponding dose. The twenty-fifth day was challenged with 20 and 100 LD50 of S. typhi in a lot of 5% (Porcine stomach mucus type II, Sigma) dissolved in Tris-Base EDTA (TE) solution. They were kept under observation for 10 days, analyzing the percentage of survival.

It was observed that porcine OmpS2 is capable of conferring 80% protection against 20 LD50 of S. typhi. No protection was observed against 100 DI_5o, it is known that the majority porins are able to confer 90-100% protection against 100 LD50, so it was used as a control of our system. These data demonstrate that S. typhi OmpS2 porin is a immunogenic antigen capable of inducing protection against infection with the bacteria.

Claims (1)

1. The use of the porcine OmpS2 of Salmonella enterica serovar Typhi to prepare an adjuvant composition useful for the preparation of vaccines. An adjuvant composition useful for the preparation of vaccines characterized in that it contains the porcine OmpS2 of Salmonella enterica serovar Typhi. A vaccine composition comprising: a) an adjuvant composition containing the porcine OmpS2 of Salmonella enterica serovar Typhi, and b) an antigen or combination of antigens. The vaccine composition according to claim 3, wherein the antigen is selected from the group of: nucleic acids encoding peptides, glycoproteins, polysaccharides, fusion proteins, lipids, glycolipids, polysaccharide-like peptides, carbohydrate-protein conjugates, cells or extracts thereof, dead or attenuated cells or extracts thereof, tumor cells or extracts thereof, attenuated viral particles or viral components, and allergens. The vaccine composition according to claims 3-4, wherein the antigen is selected from the group: bacterial antigens, viral, fungal, protozoan, prionic, neoantigen, antigens associated with tumor, and autoantigens. The vaccine composition according to claims 3-5, characterized in that it additionally contains an auxiliary adjuvant and pharmaceutically acceptable excipients. The vaccine composition according to claims 3-6, characterized in that they are pharmaceutically adapted in the form of tablets, capsules, solutions, suspensions, powders, granules or aerosols.