Platform for developing recombinant multiantigen vaccines

Abstract

The present invention relates to molecular genetics, structural biology, immunology, and microbiology. The present application is directed to compositions and methods for preparing immunogenic compositions. More specifically, one embodiment of the present invention provides an immunogenic macrocomplex comprising a heteromeric protein complex to be used as a carrier for obtaining recombinant multiantigen vaccines with self-adjuvant capacity (SAMAP - Self Adjuvanted Multi Antigen Platform).

Description

[0001] Platform for developing recombinant multiantigen vaccines.

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to molecular genetics, structural biology, immunology, and microbiology. This application is directed to compositions and methods for preparing immunogenic compositions. More specifically, one embodiment of the present invention provides an immunogenic macrocomplex comprising a heteromeric protein complex to be used as a carrier for obtaining recombinant multiantigen vaccines with self-adjuvant capability, called SAMAP (Self Adjuvanted Multi Antigen Platform).

[0004] BACKGROUND OF THE INVENTION

[0005] Vaccines represent the most effective human invention, saving millions of lives globally each year. The vast majority of vaccines used today are based on technologies that are over a hundred years old, using live, inactivated, or attenuated pathogens. The effectiveness of these vaccines is due to the fact that they stimulate an immune response against multiple antigens (immunological targets) at once, as they contain all the antigens of the pathogen. In addition to antigens, these pathogens possess molecules that stimulate the immune system, such as lipopolysaccharides, lipoteichoic acid, flagellin, cell wall fragments, DNA, RNA, among others, which act as powerful immunostimulating agents (adjuvants). Thus, traditional vaccines have high immunogenicity.

[0006] Not all species of pathogens can be cultivated in the laboratory or produced on an industrial scale, which is why it is not possible to develop vaccines for these organisms using traditional technologies. For this reason, there are still several diseases that could be prevented through vaccination, awaiting the development of an effective and safe vaccine.

[0007] Recombinant DNA technology overcomes the need to cultivate pathogens to obtain antigens, which can now be produced by genetically modified organisms. This has led to the creation of vaccines (subunit vaccines) that are safer than traditional vaccines.

[0008] However, after many studies, there are no more recombinant vaccines, apart from the virus-like particles (VLPs) of the Hepatitis B and Human Papillomavirus vaccines and the COVID-19 vaccines, based on the "spike" antigen. Subunit vaccines, based on highly purified proteins, are indeed safer, but induce low levels of protective immunity, despite being used with potent adjuvants. The exceptions are viruses, for which a single well-structured epitope can induce neutralizing antibodies, which does not occur with bacteria or parasites.

[0009] One strategy to enhance the immune response against recombinant antigens is to create nanoparticles that carry multiple copies of the antigen or several antigens. The presence of multiple copies of antigen subunits on the surface of a particle can reproduce the repetitiveness of the pathogen and simulate the natural interaction between pathogen and host.

[0010] The size of the antigen particle is crucial for its interaction with Antigen-Presenting Cells (APCs), such as Dendritic Cells (DCs). Particles between 20 and 200 nm can easily reach DCs in the lymph nodes, while larger particles (approximately 1-6 µm) remain at the injection site and are absorbed by peripheral macrophages. Proteins and peptides are easily absorbed and are not retained in the lymph nodes, where lymphocyte maturation occurs in the presence of the antigen; therefore, they need to be associated with carrier proteins or polymers to increase their immunogenicity.

[0011] Synthetic peptides containing epitopes can also be considered antigens. Among the patents for multiantigen vaccines that use peptides are WO2021 / 138721, for a vaccine against Streptococcus pyogenes, and BR112022023366, for Alzheimer's disease, among others. These peptides are produced separately and subsequently conjugated to a carrier molecule that will provide the size of the antigenic particle.

[0012] Document WO2021 / 138721 describes a peptide-based vaccine comprising a hydrophobic sequence of 10 leucine residues (Leu10) followed by an immunomodulatory PADRE sequence (Pan-DR epitopes) and the antigenic epitope (J8, PL1 and 88 / 30). To obtain the multiantigenic complex, these peptides are mixed in aqueous solution, expecting vesicles to form due to the hydrophobic region, which does not structurally represent the same platform as the present invention.

[0013] US5580563 describes a platform similar to WO2021 / 138721, which replaces the sequence of ten leucines with a lipid that can be inserted into liposomes. This platform does not contain immunomodulatory sequences, and to obtain particles with multiple antigens it is necessary to mix different lipopeptides (lipid-modified peptides) with lipids capable of forming liposomes. Thus, the US5580563 platform differs from the platform of the present invention in that it has a portion specifically targeted for anchoring to the liposome membrane, which is not present in the platform of the present invention.

[0014] W02022 / 060488 describes a multi-epitope vaccine for the treatment of Alzheimer's disease, based on peptides derived from beta-amyloid protein, chemically conjugated to a carrier protein (core) which can be KLH, serum albumin, ovalbumin, mutated diphtheria toxin, among others. However, the platform of W02022 / 060488 differs from the platform of the present invention in that this technology requires separate production of the antigens, peptides, and subsequent conjugation with the carrier (core). Thus, it does not structurally represent the same platform.

[0015] Recently, the use of self-assembled nanoparticles (SANPs), which can be modified through protein engineering, has been suggested for the development of new antigenic platforms. The fields of application for SANPs are not limited to preventive vaccines; they are also used for therapeutic vaccines, such as anti-tumor vaccines.

[0016] In this regard, US2018 / 0071400 describes a platform with a protein core linked to multiple antigens, wherein said core corresponds to the homotrimer of the modified archaea Glnkl protein (Methanocaldococcus jannaschii). However, the platform of US2018 / 0071400 differs from the platform of the present invention in that it does not include immunomodulatory sequences and has as its core a single protein that forms multimers, which does not correspond to the platform of the present invention.

[0017] US patent 2020 / 10766932 describes a technology called "Multiple Antigen Presenting System" (MAPS), in which different antigens are loaded onto a polysaccharide matrix. However, this technology does not have immunomodulatory sequences and requires producing the recombinant antigens separately and then combining them with a polymer, unlike the technology proposed here.

[0018] The use of nanoparticles containing multiple copies of a single antigen may enhance the immune response against the antigen, but this does not mean it is effective in protecting against the target pathogen. As mentioned earlier, the success of traditional vaccines is due to their ability to attack multiple targets simultaneously and to contain considerable amounts of adjuvants from the pathogen itself.

[0019] The inclusion of immunomodulatory molecules in nanoparticles, along with antigens, means "mimicking" the pathogen, which is why this strategy is called "Pathogen-Like Particles (PLPs)," and can improve vaccine performance.

[0020] The PLP systems currently used, such as synthetic particles, Virus-like particles (VLPs), and liposomes, have limitations in terms of modification to add more than one antigen and immunomodulatory sequences to their structure, as well as difficulties for large-scale production.

[0021] The present invention solves the aforementioned problems of the technique by means of a multiantigenic self-adjuvant protein platform (SAMAP platform) that has the capacity to carry various antigens and immunomodulatory sequences, forming Pathogen-Like Particles (PLPs) that are 100% recombinant.

[0022] BRIEF DESCRIPTION OF THE INVENTION

[0023] The present invention relates to molecular genetics, structural biology, immunology, and microbiology. This application is directed to compositions and methods for preparing immunogenic compositions. More specifically, one embodiment of the present invention provides an immunogenic macrocomplex comprising a heteromeric protein complex to be used as a carrier for obtaining recombinant multiantigen vaccines with self-adjuvant capability, called SAMAP (Self Adjuvanted Multi Antigen Platform).

[0024] The present invention describes a multiantigenic self-adjuvant protein platform comprising:

[0025] (a) a central protein core;

[0026] (b) one or more immunomodulatory sequences; and

[0027] (c) two or more protein or peptide antigens from the same or different pathogens.

[0028] The present invention also relates to the production of recombinantly expressed self-assembling nanoparticles (SAMAPs) in genetic fusion with immunomodulatory sequences and polypeptide and protein antigens, useful as vaccine candidates, which are obtained in a single purification process, eliminating the need for separate antigen production and subsequent chemical conjugation or affinity association with a carrier molecule.

[0029] The immunogenic compositions thus obtained can induce humoral and cellular responses against multiple antigens simultaneously. The immunomodulatory sequences carried in the immunogenic complex ensure that this response is effective and long-lasting.

[0030] The SAMAP platform can be used to develop vaccines that require the use of more than one antigen in their composition and that need a greater stimulation of humoral and cellular immune response. Among the diseases that can be prevented by multiantigen vaccines are malaria, schistosomiasis, tuberculosis, pneumonia caused by Streptococcus pneumoniae, leptospirosis, Lyme disease, among others, as well as diseases of veterinary importance such as bovine anaplasmosis, bovine and canine babesiosis, ehrlichiosis, dirafilariasis, haemonchosis, among others.

[0031] The present invention further describes a nucleic acid molecule encoding said platform, a composition comprising said platform, a process for manufacturing said platform, and the uses of said platform for manufacturing a medicament to improve or enhance the response of multiantigen vaccines against different pathogens, or for manufacturing a medicament to prevent bovine anaplasmosis.

[0032] BRIEF DESCRIPTION OF THE FIGURES

[0033] Figure 1A: Side view of the schematic diagram of the molecular structure of prefoldin from Pyrococcus horikoshii.

[0034] Figure 1B: Top view of the schematic diagram of the molecular structure of prefoldin from Pyrococcus horikoshii.

[0035] Figure 2A: Side view of the schematic of the Pyrococcus horikoshii prefoldin nucleus.

[0036] Figure 2B: Top view of the schematic diagram of the prefoldin nucleus of Pyrococcus horikoshii.

[0037] Figure 3: Restriction map of the pMR-Esa1-alpha plasmid for the expression of the alpha subunit of the SAMAP01 platform in Escherichia coli. The ampicillin resistance gene (AmpR) and the pSC101 plasmid partition sequence (par) are highlighted.

[0038] Figure 4: Restriction map of the pMRK-Esa1-beta plasmid for the expression of the beta subunit of the SAMAP01 platform in E. coli. The kanamycin resistance gene (KanR) and the pSC101 plasmid partition sequence (par) are highlighted.

[0039] Figure 5: Structural diagram of the hetero-hexameric SAMAP01 platform, where dark gray represents the alpha subunits; light gray represents the beta subunits, and the letters C and N indicate the antigen fusion sites on the platform.

[0040] Figure 6: Diagram of the molecular structure of flagellin.

[0041] Figure 7: Diagram of the molecular structure of the DO and D1 domains of flagellin. The N1 and C1 ends correspond to the N-terminal sequence, and N2 and C2 to the C-terminal of flagellin.

[0042] Figure 8: Diagram of the molecular structure of the alpha subunit region of prefoldin, chosen for insertion into flagellin.

[0043] Figure 9: Diagram of the chimeric structure formed by the DO and D1 domains of flagellin and the core region of the alpha subunit of prefoldin.

[0044] Figure 10: Restriction map of the pMR-Esa2-alpha plasmid for the expression of the alpha subunit of the SAMAP02 platform (Flagellin) in E. coli.

[0045] Figure 11: Structural scheme of the SAMAP02 platform. Heterohexameric structure of the modified Prefoldin core fused with the D0-D1 domains of the FliC flagellin. Dark gray - alpha subunits; light gray - beta subunits. Flagellin in red. The letters C and N indicate the antigen fusion sites to the platform.

[0046] Figure 12: Structural model of the Z-domain of the S. aureus alpha protein.

[0047] Figure 13: Restriction map of the pMR-Esa3-alpha plasmid for the expression of the alpha subunit of the SAMAP03 platform (Z-domain) in E. coli.

[0048] Figure 14: Schematic of the hetero-hexameric structure of the Prefoldin core, carrying copies of the Z domain of S. aureus protein A. Dark gray - alpha subunits; light gray - beta subunits. The letters C and N, as well as the arrow, indicate the antigen fusion sites on the platform.

[0049] Figure 15: Restriction map of the pMRK-Sa1-beta-AMA plasmid for the expression of the beta subunit of the SAMAP01 platform with the sequences of the Anaplasma marginale antigens OMP8, OMP7, MSP2, VirB'O and VirB9.

[0050] Figure 16: Restriction map of the pMR-Sa2-alpha-AMA plasmid for the expression of the alpha subunit of the SAMAP02 platform (flagellin) with the sequence of the Anaplasma marginale MSP1a antigen.

[0051] Figure 17: Restriction map of the pMR-Sa3-alpha-AMA plasmid for the expression of the alpha subunit of the SAMAP03 platform (Z domain) with the sequence of the Anaplasma marginale MSP1a antigen.

[0052] Figure 18A: Purification of the Sa3-ama platform. Chromatogram of the Gel-Filtration process on Superdex 200 resin.

[0053] Figure 18B: Purification of the Sa3-ama platform. Analysis of the protein present in the first peak of the chromatography.

[0054] Figure 19: Kinetics of antibody induction against peptides, stimulated by immunization with the experimental formulations. Serum dilution 1 / 800.

[0055] Figure 20: Recognition of epitopes present in the peptide vaccine by sera from animals immunized with the oily formulations of the peptides and the Sa3-ama platform (final time, 56 days). Serum dilution 1 / 500.

[0056] Figure 21: Recognition of antigens by the serum of mice immunized with the Sa3-ama antigen (SAMAP03-AMA). Serum dilution 1 / 12800 (final time, 56 days).

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Unlike other technologies for multi-antigen vaccines, such as MAPS, the SAMAP platform described in the present invention has the advantage of being 100% recombinant and having all components produced in a single step, resulting in better performance in terms of scalability and production cost.

[0059] In addition to antigens, the SAMAP platform carries immunomodulatory sequences that can target and enhance the immune response, as needed for the development of a particular vaccine.

[0060] The SAMAP platform is composed of proteins that can be obtained through mRNA vaccine technology, eliminating the need for antigen purification. This technology (mRNA) cannot be applied to platforms that require the conjugation of antigens with carrier molecules.

[0061] The following are definitions related to the components and functionality of the SAMAP platform. MULTIANTIGENIC PROTEIN PLATFORM

[0062] The present invention describes a multiantigenic self-adjuvant protein platform comprising:

[0063] (a) a central protein core;

[0064] (b) one or more immunomodulatory sequences; and

[0065] (c) two or more protein or peptide antigens from the same or different pathogens.

[0066] Specifically, the main components of SAMAP are:

[0067] (a) a central protein core comprising a thermostable heteromeric protein, which is a genetically modified multimeric protein complex / conjugate, used as a carrier;

[0068] (b) one or more immunomodulatory sequences such as universal T-cell epitopes, proteins that interact with components of the immune system, such as Flagellin, the Z domain of Staphylococcus aureus protein A, chemokines, among others, used to enhance the immune response against the antigens loaded onto the platform;

[0069] (c) two or more protein or peptide antigens from the same or different pathogens.

[0070] In a preferred embodiment, the core protein conjugate comprises a thermostable heteromeric protein.

[0071] In a preferred embodiment, the thermostable heteromeric protein is a prefoldin, preferably wherein the prefoldin is a prefoldin from Pyrococcus horikoshii.

[0072] Pyrococcus horikoshii is a hyperthermophilic archaea that lives in hot springs at temperatures of 98°C. To survive in these conditions, evolution has selected for extremely stable proteins to withstand high temperatures. Prefoldin is a molecular "chaperone" that maintains the structure of proteins under stress conditions, meaning that this protein is more stable than common P. horikoshii proteins.

[0073] The structure of the PhPFD prefoldin from P. horikoshii strain OT3 was determined by crystallography and shows a heterohexameric protein composed of two subunits (alpha and beta), which form a core with six alpha helix projections (tentacles) that form a large central cavity (Figure 1). The "tentacles" have high flexibility, allowing them to enlarge the central cavity and capture denatured or misfolded proteins, which then regain their structure in the prefoldin cavity. This complex was successfully produced in the Escherichia coli system, remaining stable after a heat treatment at 80°C for 30 minutes, used to remove host proteins by precipitation of denatured proteins.

[0074] In this application, the sequences of the alpha and beta subunits that form the core, without the "tentacles," were selected (Figure 2). Instead of the "tentacles," sequences of the selected antigens were added, as well as sequences that stimulate the immune response (immunomodulators).

[0075] Several immunomodulatory sequences, which are related to peptides and proteins, have been successfully used to increase the immunogenicity of recombinant antigens. In terms of vaccine antigen production, immunomodulators can help reduce the amount of antigen per dose and make a vaccine viable for production and commercialization.

[0076] In a preferred embodiment, said immunomodulatory sequence(s) is / are selected from (a) T-helper epitopes, (b) T-cell chemoattractant peptides (chemokines); (c) FliC flagellin from Salmonella enterica serovar Typhimurium; (d) S. aureus protein A Z-domain, (e) complement component C3d; and / or combinations thereof, wherein:

[0077] (i) T-helper epitopes are derived from antigens studied in detail, such as the HIV envelope protein (PCLUS3 epitope), the F protein of canine distemper virus, the hemagglutinin of influenza virus, sequences derived from tetanus and diphtheria toxins and their combination (TpD), or artificial sequences, such as the PADRE sequence (universal epitope Pan DR);

[0078] (ii) the chemokines are CCL2 (MCP-1), CCL3 (MIP-1 a), CCL4 (MIP-1[3], CCL5 (RANTES), CXCL8 (IL-8), CXCL10 (IP-10), CXCL12 (SDF-1), CXCL13 (BLC), CCL11 (eotaxin), CCL19 (MIP-3|3), CCL21 (SLC), CXCL1 (GROa), CXCL2 (GROP), CXCL9 (MIG), CCL17 (TARC), CCL20 and CCL22 (MDC);

[0079] (iii) flagellin FliC is from Salmonella enterica serovar Typhimurium-,

[0080] (iv) the Z-domain is a derivative of the immunoglobulin-binding domain of Staphylococcus aureus protein A.

[0081] In a preferred embodiment, the platform also includes other immunomodulatory sequences, such as M-cell ligand, li-Key motif, and Cel-1000 sequence, and combinations thereof.

[0082] In a preferred embodiment, the aforementioned OMP proteins are OMP7 and OMP8, and combinations thereof.

[0083] Below is a list of immunomodulatory sequences that can be included in the SAMAP platform.

[0084] A. T-HELPER EPITOPES

[0085] Antigens perform better in immunization tests when they contain sequences that can be presented by MHC class II molecules (T-cell epitope). The MHC II, with the attached peptide, can interact with the CD4 receptors of effector cells, triggering an immune response.

[0086] However, it is possible that important targets for vaccine development may not contain these sequences. The addition of T-cell epitopes derived from potent antigens (such as bacterial toxins and viral proteins) to non-immunogenic peptides and proteins has increased their immunogenicity, making the vaccines more effective.

[0087] Examples of universal T-helper epitopes that can be carried in the SAMAP complex are T-helper peptides derived from antigens studied in detail, such as the HIV envelope protein (PCLUS3 epitope), the F protein of canine distemper virus, influenza virus hemagglutinin, sequences derived from tetanus and diphtheria toxins and their combination (TpD). In addition to natural antigen sequences, there are artificial sequences, such as the PADRE sequence (universal Pan DR epitope) and sequences described in W02020 / 132275, among others. These sequences increase vaccine potency by activating CD4+ helper T cells and aiding in the generation of high titers of IgG antibodies and combinations.

[0088] B. T-CELL CHEMIOATTRACTIVE PEPTIDES

[0089] Chemokines have been explored for their potential use as vaccine adjuvants due to their ability to recruit immune cells, such as dendritic cells and T cells, to the vaccination site, which facilitates the uptake of antigens by antigen-presenting cells, generating a more robust immune response.

[0090] The chemosins that can be loaded onto the SAMAP platform are: CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP-1|3), CCL5 (RANTES), CXCL8 (IL-8), CXCL10 (IP-10), CXCL12 (SDF-1), CXCL13 (BLC), CCL11 (eotaxin), CCL19 (MIP-3(3)), CCL21 (SLC), CXCL1 (GROa), CXCL2 (GROP), CXCL9 (MIG), CCL17 (TARC), CCL20 and CCL22 (MDC).

[0091] The immune response can be directed depending on the chemokine carried on the SAMAP platform. For example, chemokines such as CXCL10 and CXCL11 are associated with Th1 responses, while CCL20 has been associated with Th2 responses.

[0092] C. DOMAIN Z

[0093] There are pathogen molecules that interact with immune system cells and induce an effective response during infection. For example, the A protein of Staphylococcus aureus. The Z domain, derived from the immunoglobulin-binding domain of the A protein, has the property of binding to receptors present on antigen-presenting cells (APCs), thus increasing the immune response against the target antigen.

[0094] D. FLAGELINA FLIC

[0095] Flagellin, one of the structural subunits of bacterial flagella, is recognized in the host organism as one of the "pathogen-associated molecular patterns" (PAMPs), which alerts the immune system to the presence of a pathogenic agent.

[0096] Flagellin is primarily identified through its interaction with Toll-Like Receptor 5 (TLR5), found on the surface of antigen-presenting cells (APCs), such as macrophages and dendritic cells. Activation of TLR5 by flagellin induces the release of cytokines and chemical mediators, essential for the development of the immune response, activating response mechanisms of both the innate and acquired immune systems.

[0097] Therefore, flagellin can be used as an antigen carrier or as an adjuvant, co-administered via mucosal or parenteral routes.

[0098] E. COMPONENT C3D OF THE COMPLEMENT

[0099] The fusion of antigens with the C3d component can help interact with B cells and follicular dendritic cells, where there are receptors for C3d. This interaction significantly increases the efficiency of the immune response compared to using the antigen alone.

[0100] F. OTHER IMMUNOMODULATING SEQUENCES

[0101] In addition to the sequences mentioned above, other immunomodulatory sequences can be used with the SAMAP platform, such as the M-cell ligand, the li-Key motif, and the Cel-1000 sequence, and combinations thereof. G. ANTIGENS

[0102] Regarding antigens, they are included in the platform in the form of polypeptides (proteins) or peptides (epitopes) from the same or different pathogens, depending on the vaccine being developed. The literature has reported the need to include more than one antigen in the vaccine composition, and to induce a greater humoral and cellular immune response, in order to develop vaccines that can prevent diseases such as malaria, schistosomiasis, tuberculosis, pneumonia caused by Streptococcus pneumoniae, leptospirosis, Lyme disease, among others, as well as diseases of veterinary importance such as bovine anaplasmosis, bovine and canine babesiosis, ehrlichiosis, dirafilariasis, haemonchosis, among others. The antigens identified and tested in the development of these vaccines can be loaded into the SAMAP complex in combination with immunomodulatory sequences that direct the immune response according to the needs of each application.

[0103] In a preferred embodiment, said two or more protein or peptide antigens from the same or different pathogens comprise 2 to 6 polypeptide (protein) antigens or 2 to 16 different peptide (epitope) antigens, wherein the peptide or polypeptide antigens are from a pathogenic organism or from a cancer or tumor.

[0104] In a preferred embodiment, said antigens are from Anaplasma marginale and are selected from the MSP antigen, the OMP proteins, the VirB9 and VirB10 antigens, and combinations thereof.

[0105] In a preferred embodiment, the aforementioned MSP antigen is MSP 1a, MSP2, and combinations thereof.

[0106] In a preferred embodiment, the aforementioned OMP proteins are OMP7 and OMP8, and combinations thereof.

[0107] In a preferred embodiment, the present invention claims a nucleic acid molecule consisting of the nucleotide sequence SEQ ID NO: 9, and degenerate sequences thereof, which encode the amino acid sequence SEQ ID NO: 8.

[0108] In a preferred embodiment, the present invention claims a nucleic acid molecule consisting of the nucleotide sequence SEQ ID NO: 12, and degenerate sequences thereof, which encode the amino acid sequence SEQ ID NO: 11. In a preferred embodiment, the present invention claims a nucleic acid molecule consisting of the nucleotide sequence SEQ ID NO: 18, and degenerate sequences thereof, which encode the amino acid sequence SEQ ID NO: 17.

[0109] In a preferred embodiment, the present invention claims a nucleic acid molecule consisting of the nucleotide sequence SEQ ID NO: 22, and degenerate sequences thereof, which encode the amino acid sequence SEQ ID NO: 21.

[0110] In a preferred embodiment, the present invention claims a nucleic acid molecule consisting of the nucleotide sequence SEQ ID NO: 25, and degenerate sequences thereof, which encode the amino acid sequence SEQ ID NO: 24.

[0111] In a preferred embodiment, the present invention claims a nucleic acid molecule consisting of the nucleotide sequence SEQ ID NO: 27, and degenerate sequences thereof, which encode the amino acid sequence SEQ ID NO: 26.

[0112] In a preferred embodiment, the present invention claims a nucleic acid molecule consisting of the nucleotide sequence SEQ ID NO: 29, and degenerate sequences thereof, which encode the amino acid sequence SEQ ID NO: 28.

[0113] Examples of the use of multi-antigen vaccines are the patents described above: WQ2021 / 138721, BR112022023366, WQ2021 / 138721, US5580563, WQ2022 / 060488, WQ2022 / 060488 and US2018 / 0071400.

[0114] In the application example to be described of the present invention using the SAMAP platform, the antigens described in patent US2020 / 0095289 were considered for the development of a vaccine for bovine anaplasmosis based on synthetic peptides. To obtain a recombinant vaccine, the epitopes of the antigens MSP1a, MSP2, OMP7, OMP8, VirB9 and VirBIO of Anaplasma marginale, described in this patent, were loaded onto the SAMAP platform.

[0115] Unlike other multi-antigen vaccine technologies, the SAMAP platform can carry both antigens and peptides, which can be produced together in a single manufacturing process. Therefore, the SAMAP platform can be used for the development of vaccines for both humans and veterinary use. [Image caption: Nucleic acid molecule encoding the multi-antigen protein platform]

[0116] The present invention further describes a nucleic acid molecule that encodes a multiantigenic protein platform, as defined in the present invention, and which comprises a combination of nucleotide sequences selected from SEQ ID: NO 9, SEQ ID: NO 12, SEQ ID: NO 18, SEQ ID: NO 22, SEQ ID: NO 25, SEQ ID: NO 27, SEQ ID: NO 29, SEQ ID: NO 30, SEQ ID: NO 32, and SEQ ID: NO 34.

[0117] COMPOSITION COMPRISING THE MULTIANTIGENIC PROTEIN PLATFORM

[0118] The present invention further describes an immunogenic composition comprising a multi-antigen immunogenic system obtained using a SAMAP platform, as defined in the present invention, or a nucleic acid molecule encoding the protein components of this system, as defined in the present invention, and one or more pharmaceutically acceptable excipients, vehicles or diluents.

[0119] In a preferred embodiment, the composition of the present invention further comprises one or more excipients, vehicles or diluents.

[0120] In a preferred embodiment, the excipients, vehicles, or diluents are pharmaceutically acceptable and known in the art, including, but not limited to, adjuvants, vehicles, excipients, glidants, sweetening agents, diluents, preservatives, colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, solvents, and emulsifiers acceptable for use in humans or animals. Preferably, one or more excipients, vehicles, or diluents comprise sugars, starches, cellulose and its derivatives, gelatin, talc, cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide, oils, glycols, polyols, esters, agar; buffering agents, alginic acid, water, saline solution, Ringer's solution, alcohols, phosphate buffer solutions, and any other compatible substances used in pharmaceutical compositions.

[0121] Professionals in this field are highly capable of determining the pharmaceutically acceptable excipients, vehicles, and diluents for each composition and purpose.

[0122] PROCESS FOR MANUFACTURING A MULTIANTIGENIC PROTEIN PLATFORM The present invention further describes a process for manufacturing a multiantigenic protein platform, as defined in the present invention, comprising the following steps:

[0123] (a) select the core protein, immunomodulators and antigens of interest and the nucleotide sequences that encode them;

[0124] (b) construct with these sequences the genes that code for each of the subunits that make up the SAMAP platform (alpha and beta), each containing the coding sequences for the T-cell epitopes, immunomodulatory sequences and the epitopes of interest;

[0125] (c) clone these sequences into two Escherichia coli T7 expression plasmids with different selection markers (resistance to different antibiotics, ampicillin and kanamycin). Single plasmids can also be used for co-expression of recombinant proteins in Escherichia coli or Pichia pastoris systems.

[0126] (d) cotransform an Escherichia coli host cell with plasmids encoding the alpha and beta subunits of the SAMAP platform and select transform cells in the presence of ampicillin and kanamycin;

[0127] (e) cultivate the selected transformants in selective media and induce the expression of recombinant proteins with ITPG (isopropyl [3- D-thiogalactopyranoside]) or, preferably, lactose, so that the alpha and beta subunits of the SAMAP platform, synthesized in the bacterial cytoplasm, self-assemble;

[0128] (f) After induction, the immunogenic complex is purified from the soluble fraction of the bacterial cytoplasm using the following procedures:

[0129] • Cell lysis;

[0130] • Clarification of the lysate;

[0131] • Tangential Filtration;

[0132] • Ion-exchange chromatography;

[0133] • Molecular exclusion chromatography.

[0134] In a preferred embodiment, the present invention further describes a process for manufacturing a multiantigenic protein platform, as defined in the present invention, comprising the following steps:

[0135] (a) select the core protein, immunomodulators and antigens of Anaplasma marginale and the nucleotide sequences that encode them; (b) construct with these sequences the genes that encode for each of the subunits that make up the SAMAP platform (alpha and beta), each containing the coding sequences for the T-cell epitopes, immunomodulatory sequences and Anaplasma marginale epitopes,

[0136] (c) clone these sequences into two Escherichia coli T7 expression plasmids with different selection markers (resistance to different antibiotics, ampicillin and kanamycin). Single plasmids can also be used for co-expression of recombinant proteins in Escherichia coli or Pichia pastoris systems.

[0137] (d) cotransform an Escherichia coli host cell with plasmids encoding the alpha and beta subunits of the SAMAP platform and select transform cells in the presence of ampicillin and kanamycin;

[0138] (e) cultivate the selected transformants in selective media and induce the expression of recombinant proteins with ITPG (isopropyl [3- D-thiogalactopyranoside]) or, preferably, lactose, so that the alpha and beta subunits of the SAMAP platform, synthesized in the bacterial cytoplasm, self-assemble;

[0139] (f) After induction, the immunogenic complex is purified from the soluble fraction of the bacterial cytoplasm using the following procedures:

[0140] • Cell lysis;

[0141] • Clarification of the lysate;

[0142] • Tangential Filtration;

[0143] • Ion-exchange chromatography;

[0144] • Molecular exclusion chromatography.

[0145] In the Escherichia coli system, plasmids with T7 promoters are used under lac operator control. In the case of Pichia pastoris, plasmids with inducible (AOX1) or constitutive (GAP) promoters can be used.

[0146] The purification of recombinant antigen from the SAMAP platform can be performed using tangential filtration through membranes with different pore sizes, chromatographic procedures using different resins, such as ion exchange, hydrophobic interaction, molecular exclusion, dassalting, mix-mode resins, among others.

[0147] The SAMAP complex can also be purified by immobilized metal affinity chromatography (IMAC) when at least one of its subunits contains the fusion with the 6xHis-tag.

[0148] According to the present invention, the process for manufacturing the multiantigen protein platform still comprises a single purification process.

[0149] In a preferred embodiment, the host cell is a bacterial, yeast, mammalian, or insect cell.

[0150] In a preferred embodiment, expression is preferentially induced by lactose, in the case of using Escherichia coli T7 plasmids.

[0151] Uses of the Multiantigen Protein Platform

[0152] The present invention further describes the use of a multiantigenic protein platform, as defined in the present invention, which is for the manufacture of a medicament to improve or enhance the immune response against Anaplasma marginale.

[0153] The present invention further describes the use of a multiantigenic protein platform, as defined in the present invention, which is for the manufacture of a medicament to improve or enhance the response of multiantigenic vaccines against different pathogens.

[0154] The present invention further describes the use of a multiantigenic protein platform, as defined in the present invention, for the manufacture of a medicament to prevent or treat anaplasmosis.

[0155] In a preferred embodiment, the medicine is in the form of a vaccine.

[0156] In a preferred embodiment, the drug is suitable for oral or nasal administration.

[0157] DESCRIPTION OF THE SEQUENCES

[0158] The following are descriptions of the sequences of the present invention as per Table 1:

[0159] EXAMPLES OF SAMAP PLATFORM DEVELOPMENT SCAFFOLD SELECTION - PREFOLDINA CORE

[0160] The core of P. horikoshii prefoldin was chosen to serve as the scaffold for the platform (as shown in Figure 1). After structural analysis, the sequences corresponding to the “core” of the alpha and beta subunits were separated, forming two beta barrels at the top of the prefoldin, to include all the amino acids that hold the core structure together (Figure 2).

[0161] Core of the alpha subunit: corresponds to amino acids 37-107 of the sequence deposited in GenBank WP_048053149.1 (alpha subunit of prefoldin from Pyrococcus horikoshii), with the K106S substitution (SED ID No. 01: alpha subunit of prefoldin in the core).

[0162] Core of the beta subunit: corresponds to amino acids 36-79 of the sequence deposited in GenBank WP_010884634.1 (beta subunit of prefoldin from Pyrococcus horikoshii), with the E77S substitution (SED ID No. 02: beta subunit of the prefoldin core). The amino and carboxyl terminal ends of the subunits are close to each other (Figure 2). A loop-helix sequence was chosen to be fused to the carboxyl-terminal end of the core proteins in order to alter the orientation and separate the amino and carboxyl terminal ends.

[0163] For this purpose, a small amino acid sequence 68-80 was selected from the WP_010884339.1 repository (Pyrococcus horikoshii potassium channel protein), with the substitutions A69V, R70G, I79K, which forms the turn-helix structure (SEQ ID No. 03: turn-helix sequence).

[0164] This sequence was added to the C-terminal ends of the core proteins, and the molecules were subjected to Molecular Dynamics under different conditions, including heating to 80°C, and the turn-helix structure remained stable.

[0165] T-CELL EPITOPE SELECTION

[0166] The addition of T-cell epitopes derived from potent antigens, such as bacterial toxins and viral proteins, to non-immunogenic peptides and proteins significantly increases immunogenicity. These sequences will also serve as linkers to better expose the antigens on the prefoldin core platform. For this purpose, we chose the following sequences:

[0167] Canine Distemper Virus F protein: amino acids 264 - 280 of AJO72810.1 (F protein, Canine morbillivirus). (SEQ ID No 04: F protein epitope);

[0168] Influenza virus hemagglutinin: amino acids 518 - 532 of AFD32428.2 (Influenza A (H3N1) virus hemagglutinin) (SEQ ID No 05: hemagglutinin epitope);

[0169] Tetanus Toxin: amino acids 827 - 843 of WP_011100836.1 (tetanus toxin, Clostridium tetani) (SEQ ID No 06: tetanus toxin epitope);

[0170] Diphtheria Toxin: amino acids 356 - 370 of WPJD72564851. 1 (toxin, Corynebacterium diphtheriae) (SEQ ID No 07: diphtheria toxin epitope).

[0171] Depending on the application of the SAMAP platform, the quantity and nature of these epitopes can be modified.

[0172] CONSTRUCTION OF THE SAMAP01 PLATFORM

[0173] The SAMAP01 platform corresponds to the first version of the SAMAP platform. It consists of prefoldin core subunits modified with their carboxyl-terminal ends to change their orientation and the fusion of T-cell epitopes at the amino and carboxyl-terminal ends. The following sequences were added to the alpha subunit:

[0174] SEQ ID No 04: F protein epitope;

[0175] SED ID No 01: alpha subunit of prefoldin in the nucleus;

[0176] SEQ ID No 03: helix-turn sequence;

[0177] SEQ ID No. 05: hemagglutinin epitope; which results in the amino acid sequence called ALFA01 (SEQ ID No. 08).

[0178] A nucleotide sequence encoding SEQ ID No. 08 was designed. At the 5' end of the designed gene, the sequence “CCATGGGCTCGAGGACGGTACC” was added, containing restriction sites for the enzymes Ncol (CCATGG), Xhol (CTCGAG), and Kpnl (GGTACC). At the 3' end of the designed gene, the sequence “AGATCTGAGGACACTAGTTAAGCTT” was added, containing sites for the enzymes Bglll (AGATCT) and Spel (ACTAGT) separated by the hexanucleotide GAGGAC, and at the end, the Hindi II recognition sequence (AAGCTT). The addition of restriction sites aims to facilitate antigen cloning in fusion with the components of the SAMAP01 platform (SEQ ID No. 09 = gene for ALFA01).

[0179] The following sequences were joined for the beta subunit:

[0180] SEQ ID No 06: epitope of tetanus toxin;

[0181] SED ID No. 02: beta subunit of the prefoldin nucleus;

[0182] SEQ ID No 03: helix-turn sequence;

[0183] SEQ ID No. 07: diphtheria toxin epitope; which results in the amino acid sequence called BETA01 (SEQ ID No. 11).

[0184] A nucleotide sequence encoding SEQ ID No. 11 was designed. At the 5' end of the designed gene, the sequence “CATATGCGGGGATCCGACCTGCAG” was added, containing the cleavage sites for the enzymes Ndel (CATATG), BamHI (GGATCC), and Pstl (CTGCAG). At the 3' end of the designed gene, the sequence “GTCGACGAGGAATTCGAAGCTT” was added, containing the sites for the enzymes Sall (GTCGAC), EcoRI (GAATTC), and Hindlll (AAGCTT) (SEQ ID No. 12 = gene for BETA01).

[0185] Cloning of synthetic genes in co-expression vectors

[0186] The genes synthesized for the ALFA01 and BETA01 subunits were cloned into the Escherichia coli pMRK and pMR expression vectors, via the Ndel-Hindll and Ncol-Hindl II restriction sites, respectively, resulting in the pMR-Esa1-alpha (Figure 3) and pMRK-Esa1-beta (Figure 4) plasmids.

[0187] After transformation of E. coli cells with pMR-Sa1-alpha and pMRK-Sa1-beta plasmids, selection with the antibiotics ampicillin and kanamycin, and induction with isopropyl-[3-D-thiogalactopyranoside (IPTG), the ALFA01 and BETA01 subunits are synthesized in the cytoplasm of the bacteria, which self-assemble to form the SAMAP01 platform.

[0188] The SAMAP01 platform, shown schematically in Figure 5, can be used to load antigens onto the carboxyl- and amino-terminal ends of the ALFA01 and BETA01 subunits that comprise it.

[0189] Thus, in the BETA01 subunit, antigens can be cloned by N-terminal fusion via the BamHI-Pstl restriction sites, and by C-terminal fusion via Sall-EcoRI. In the case of the ALFA01 subunit, fusion at the N-terminal ends occurs via Xhol-Kpnl and C-terminal fusion via Bglll-Spel.

[0190] Therefore, in the SAMAP01 system it is possible to:

[0191] Clone at least four different antigens. It is possible to use antigen sequences in the form of polyproteins, or hybrid peptides, to increase the number of antigens per complex.

[0192] The platform created allows for the purification of all antigens in a single process.

[0193] The presence of T-cell epitopes will increase the immunogenicity of the platform and the memory of the immune response.

[0194] CONSTRUCTION OF THE SAMAP02 PLATFORM (WITH FLAGELLIN)

[0195] The SAMAP02 platform is a version that includes bacterial flagellin as an immunomodulatory molecule. Flagellin is one of the most potent adjuvant molecules, but its use is limited due to its limited ability to fuse antigens to this molecule.

[0196] Figure 6 shows the structure of Flagellin with four domains designated D0, D1, D2, and D3. Domains D2 and D3 are not relevant to immunomodulatory activity; on the contrary, they can cause adverse reactions during vaccination. The amino and carboxyl terminal ends are close to each other, and it is only possible to fuse proteins at one end at a time, which limits the use of Flagellin as an antigen carrier.

[0197] For the addition of Flagellin to the SAMAP platform, the D2 and D3 regions were excluded, leaving the sequences that make up the DO and D1 domains (Figure 7).

[0198] Amino-terminal fragment of Flagellin: amino acids 3 - 178 of ABJ98818.1 (mid-domain variant of flagellin phase I C131 Salmonella enterica subsp. enterica serovar Typhimurium) (SEQ ID No 14: N-terminal sequence of flagellin).

[0199] Carboxyl-terminal fragment of Flagellin: amino acids 234 - 327 of ABJ98818.1 (mid-domain variant of flagellin phase I C131 Salmonella enterica subsp. enterica serovar Typhimurium) (SEQ ID No 15: C-terminal sequence of flagellin).

[0200] SUBUNIT ALFA02

[0201] The aim was to link the N- and C-terminal fragments of flagellin to the core of the alpha subunit of prefoldin. After structural analysis to plan the best way to accomplish this task, the following region of the alpha subunit was chosen:

[0202] Core of the alpha subunit for flagellin binding: corresponds to amino acids 32-111, L34S modification of the sequence deposited in GenBank WP_048053149.1 (alpha subunit of prefoldin from Pyrococcus horikoshii), (SED ID No. 16: alpha subunit of the prefoldin core for flagellin) (Figure 8).

[0203] The C1 end of the N-terminal fragment of flagellin shown in Figure 7 (SEQ ID No. 14) was fused to the N-terminal end of the alpha subunit (SEQ ID No. 16) in Figure 8, separated by the “GGG” linker.

[0204] The N2 end of the C-terminal fragment of flagellin (SEQ ID No. 13) shown in Figure 7 is fused to the C-terminal end of the alpha subunit (SEQ ID No. 16) in Figure 8, with the linker “EGG” between the two sequences.

[0205] As a result of these fusions, we have the following sequence (SEQ ID No. 17: ALFA02-Flagelin). The resulting structure of this fusion is shown in Figure 9.

[0206] A nucleotide sequence encoding SEQ ID No. 17 was designed. At the 5' end of the designed gene, the restriction site for the Xhol enzyme (CTCGAG) was added. At the 3' end of the designed gene, the sequence AGATCTGAGGACACTAGT was added, containing the sites for the Bglll (AGATCT) and Spel (ACTAGT) enzymes separated by the hexanucleotide GAGGAC, and the Hindi 11 recognition sequence (AAGCTT). The addition of restriction sites aims to facilitate antigen cloning in fusion with the components of the SAMAP02 platform. (SEQ ID No. 18 = gene for ALFA02-FLA). CLONING OF THE SYNTHETIC ESA2-ALFA GENE IN PMR-ESA1-ALFA PLASMIDS:

[0207] The gene synthesized for the ALFA02-FLA subunit (SEQ ID No. 18) was cloned into the pMR-ESa1-alpha plasmid, through the Xhol and Hindlll restriction sites, to replace the ALFA01 gene, resulting in the pMR-Esa2-alpha plasmid (Figure 10).

[0208] After co-transformation of E. coli cells with the pMR-Sa2-alpha and pMRK-Sa1-beta plasmids, and selection with the antibiotics ampicillin and kanimycin, and induction with IPTG, the subunits ALFA02 and BETA01 are synthesized in the cytoplasm of the bacteria, which self-assemble to form the SAMAP02 platform.

[0209] The SAMAP02 platform, shown schematically in Figure 11, can be used to load antigens onto the carboxyl and amino terminal ends of the BETA01 subunits, as well as onto the carboxyl-terminal end of the ALFA02-FLA subunit.

[0210] Thus, in the BETA01 subunit, antigens can be cloned via N-terminal fusion through the BamHI-Pstl restriction sites, and via C-terminal fusion through Sall-EcoRI. In the case of the ALFA02-FLA subunit, fusion at the C-terminal end occurs through the Bglll-Spel restriction sites.

[0211] Therefore, in the SAMAP02 system it is possible to:

[0212] Clone at least three different antigens. It is possible to use antigen sequences in the form of polyproteins to increase the number of antigens per complex.

[0213] The platform developed allows for the purification of all antigens together with Flagellin in a single production process.

[0214] The presence of T-cell epitopes of the BETA01 subunit (tetanus toxin and diphtheria toxin), along with Flagellin bound to the ALFA02-FLA subunit, will increase the immunogenicity of the platform and the memory of the immune response.

[0215] The Flagellin domains included in the platform will bind the SAMAP02 platform to TLR5 receptors, inducing an immune response. This variant is expected to outperform SAMAP01 and SAMPA03.

[0216] CONSTRUCTION OF THE SAMAP03 PLATFORM (WITH THE Z-DOMAIN)

[0217] The Z-domain of S. aureus protein A (Figure 12) is capable of interacting with the Fc regions of antibodies and similar structures present in B cells and antigen-presenting cells (APCs), which increases the immunogenicity of antigens fused to this domain. For this project, the following sequence was used:

[0218] Z-domain of S. aureus protein A: amino acids 61 - 120 of ALO52730.1 (synthetic construct of protein A) (SEQ ID No 20: Z-domain).

[0219] The structure formed by this sequence of amino acids:

[0220] To increase the exposure of this domain and facilitate its interaction with the receptor, SEQ ID No. 20 was linked to the ALFA01 subunit (SEQ ID No. 08) of the SAMAP01 platform using a linker sequence, SEGTATRSGGSA, designed to insert the restriction sites of the Kpnl and Mlul enzymes. The resulting amino acid sequence was named Z-ALFA03 (SEQ ID No. 21):

[0221] A nucleotide sequence encoding SEQ ID No. 21 was designed. At the 5' end of the designed gene, the restriction site for the Xhol enzyme (CTCGAG) was added. The Kpnl (GGTACC) and Mlul (ACGCGT) linker sequences were introduced. At the 3' end of the designed gene, the AGATCTGAGGACACTAGT sequence was added, containing the sites for the Bglll (AGATCT) and Spel (ACTAGT) enzymes separated by the hexanucleotide GAGGAC, and at the end, the Hindi 11 recognition sequence (AAGCTT). The addition of restriction sites aims to facilitate antigen cloning in fusion with the components of the SAMAP03 platform. (SEQ ID No. 22 = gene for Z-ALFA03).

[0222] Cloning of the synthetic gene in the PM R-SA1-alpha vector:

[0223] The gene synthesized for the ALFA03 subunit (Z-domain) (SEQ ID No. 22) was cloned into the pMR-ESa1-alpha plasmid, through the Xhol and Hindlll restriction sites, to replace the ALFA01 gene, resulting in the pMR-ESa3-alpha plasmid (Figure 13).

[0224] After co-transformation of E. coli cells with pMR-Sa3-alpha and pMRK-Sa1-beta plasmids, selection with the antibiotics ampicillin + kanamycin and induction with IPTG, the subunits ALFA03 and BETA01 are synthesized, which self-assemble to form the SAMAP03 platform.

[0225] The SAMAP03 platform, shown schematically in Figure 14, can be used to load antigens onto the carboxyl-terminal and amino-terminal ends of the BETA01 subunit, and onto the carboxyl-terminal end of the Z-ALFA03 subunit. It is also possible to introduce sequences between the ALFA subunit sequences and the Z-domain, as indicated by arrows in Figure 14.

[0226] Thus, in the BETA01 subunit, antigens can be cloned via N-terminal fusion through the BamHI-Pstl restriction sites, and via C-terminal fusion through Sall-EcoRI. In the case of the Z-ALFA03 subunit, fusion at the C-terminal end is achieved through the Bglll-Spel restriction sites. Cloning between the Z-domain and the Alpha subunit is possible using the Kpnl and Mlul enzyme sites. Therefore, in the SAMAP03 system it is possible to:

[0227] Clone at least four different antigens. It is possible to use antigen sequences in the form of polyproteins to increase the number of antigens per complex.

[0228] The platform developed allows for the purification of all antigens along with the Z-domain in a single process.

[0229] The presence of T-cell epitopes of the BETA01 subunit (tetanus toxin and diphtheria toxin), together with the Z-domain of S. aureus protein A, will increase the immunogenicity of the platform and the memory of the immune response.

[0230] The Z-domains included in the platform will link the SAMAP03 platform to receptors present on the surface of antigen-presenting cells and immune response-mediating T cells. This variant is expected to outperform SAMAP01.

[0231] EXAMPLE 2 (ANAPLASMA)

[0232] Anaplasma marginale is the most common livestock pathogen worldwide, with a major impact on animal health. The disease causes a high mortality and morbidity rate in cattle populations, significantly affecting meat exports.

[0233] This bacterium infects red blood cells, which are destroyed in the spleen, causing anemia in cattle. While mammalian erythrocytes appear to be the only site of infection, in ticks there is a complex developmental cycle with infection of different cell types.

[0234] Despite the importance of this disease to animal health, there is still no widely commercially available vaccine for bovine anaplasmosis.

[0235] Following immunoproteomics and immunobioinformatics analysis, epitopes of these targets were identified with the potential to be used as antigens in multiantigen vaccines. Subsequently, some sequences were joined using a glycine residue sequence (GGGG) as a spacer to give rise to the hybrid peptides of patent US2020 / 0095289.

[0236] TABLE 2: COMPOSITION OF HYBRID PEPTIDES WITH EPITOPES OF A. ANTIGENS.

[0237] MARGINAL (US2020 / 0095289)

[0238] The hybrid peptides were tested as antigens in experimental vaccines in mice, both individually and in pools. Recognition of the peptides by the serum of immunized animals was tested, as well as with the serum of an animal (calf) immunized with the traditional vaccine obtained from the blood of an infected animal. Recognition of the peptides with sera from diseased animals was also tested. A pool of five hybrid peptides shown in Table 2 showed positive results for protection against experimental and natural infection with A. marginale in calves.

[0239] SELECTION OF PEPTIDES TO BE USED IN THE RECOMBINANT MSP ANTIGEN VACCINE

[0240] The MSP1a antigen is the most abundant on the surface of Anaplasma. It is responsible for the interaction with the erythrocyte, therefore being a primary target in vaccine development. For this reason, it is one of the best-characterized Anaplasma antigens.

[0241] For this antigen, an epitope recognized by a monoclonal antibody capable of neutralizing Anaplasma (neutralizing epitope) was described, whose sequence is repeated several times in the MSP1 antigen a: Q / EASTSS. An immunodominant epitope was also identified, which is also repeated several times at the N-terminal end of MSP1 a: SSAGGQQQESS.

[0242] Considering that there is no size limitation in the synthesis of recombinant antigen, it was decided to use two repetitive sequences of MSP1α (F and beta), each containing a neutralizing epitope and an immunogenic epitope, as well as sequences flanking the N- and C-terminal of the repeats, present in the hybrid peptides of Table 2: “SEÇVSLQPTDSSSASGQQQE” present in P54, and “SKVASVEYILAAR” present in the hybrid peptides P28 and P53. (SEQ ID No 24: MSP1α repeats).

[0243] A nucleotide sequence encoding SEQ ID No. 20 was designed. At the 5' end of the designed gene, the cleavage site sequence for the Bglll enzyme (AGATCT) was added, and at the 3' end, the Spel cleavage site (ACTATG) (SEQ ID No. 25: gene for MSP1a).

[0244] OMP ANTIGENS

[0245] Outer Membrane Proteins (OMPs) are located in the outer membrane of Gram-negative microorganisms, such as Anaplasma marginale. Their structure contains eight antiparallel beta sheets that form a barrel (beta-barrel structure) embedded in the membrane and four loops that project into the external environment.

[0246] The hybrid peptides have the following sequences for OMP7 and OMP8:

[0247] OMP7: GASSVDALTATKLVAAALGH (P44 peptide);

[0248] OMP8: GASTDDAAAAAKIVAMAYGR (peptides P44 and P50).

[0249] Structural models of these proteins were obtained using the AlphaFold2 program to verify the location and exposure of these sequences. With this information, the QKGTLSYT sequence in the OMP8 peptide was determined.

[0250] A sequence was drawn containing the epitopes OMP8, OMP7, the linker sequence GGGS and MSP2 (QVDRLANALGK, present in the peptides P28 and P53). (SEQ ID No 26: OMPs Antigen).

[0251] A nucleotide sequence encoding SEQ ID No. 26 was designed. At the 5' end of the designed gene, the cleavage site sequence for the BamHI enzyme (GGATCC) was added, and at the 3' end, the cleavage site for Pstl (CTGCAG) (SEQ ID No. 27: gene for the OMPs antigen).

[0252] VIRB ANTIGENS

[0253] The VirB9 and VirBI O antigens are components of the Type IV Secretion System (T4SS), which the bacterium uses to secrete and inject its virulence factors into host cells.

[0254] Of all the components of the Type IV Secretion System base, only VirB9 has a portion exposed outside the cell. The following sequences were considered in the design of the recombinant antigen:

[0255] VirBI O (2) FSESMKALIKKYVDTSKPTIYVDQGTVMKV (P51 ) VirB (1 ) LQGHMIDAVLETAINSDIPGVLRAIVSRDV (P54)

[0256] VirB9 (1 ) AMGDSVHWKVKPVDNKLFIMP (P41 and P53)

[0257] A polypeptide containing these sequences was designed, with the spacers EAAAK and GGEG: (SEQ ID No. 28: VirBs Antigen).

[0258] A nucleotide sequence encoding SEQ ID No. 28 was designed. At the 5' end of the designed gene, the cleavage site sequence for the Sall enzyme (GTCGAC) was added, and at the 3' end, the cleavage site for the EcoRI enzyme (GAATTC) was added (SEQ ID No. 29: gene for the VirBs antigen).

[0259] Obtaining recombinant SA2-AMA and SA3-AMA antigens; Construction of expression plasmids.

[0260] To obtain recombinant antigens, expression vectors containing the sequences of the designed antigens were first constructed and genetically fused to the components of the SAMAP platform.

[0261] SEQ ID No. 27 (OMPs) and SEQ ID No. 29 (VirBs) were cloned into the pMRK-Sa1-beta plasmid (Figure 4) via the BamHI+Pstl and Sal+EcoRI sites, respectively, resulting in the pMRK-Sa1-beta-AMA plasmid (Figure 15). SEQ ID No. 30 shows the sequence of the pMRK-Sa1-beta-AMA expression cassette, which encodes the Sa1-beta-AMA protein (SEQ ID No. 31).

[0262] In turn, SEQ ID No. 25 (MSP1a) was cloned by the Bglll + Spel sites in the pMR-Sa2-alpha (Figure 10) and pMR-Sa3-alpha (Figure 13) plasmids, resulting in the pMR-Sa2-alpha-AMA (Figure 16) and pMR-Sa3-alpha-AMA (Figure 17) plasmids, respectively. SEQ ID No. 32 shows the sequence of the pMR-Sa2-alpha-AMA expression cassette, which encodes the Sa2-beta-AMA protein (SEQ ID No. 33). SEQ ID No. 34 shows the sequence of the pMR-Sa3-alpha-AMA expression cassette, which encodes the Sa3-alpha-AMA protein (SEQ ID No. 35).

[0263] Expression and Purification of Recombinant Antigens

[0264] The BL21 Star (DE3) strain (Thermo Fischer) was transformed with the following plasmid pairs: pMRK-Sa1-beta-AMA + pMR-Sa2-alpha-AMA = SAMAP02-AMA; pMRK-Sa1-beta-AMA + pMR-Sa3-alpha-AMA = SAMAP03-AMA. Transformants were selected in the presence of the antibiotics ampicillin and kanamycin to ensure co-transformation safety. Clones resistant to both antibiotics were grown in 5 ml of LB medium to verify the co-expression of the proteins Beta01-AMA and AlphaO2-AMA for transformants before (1); and Beta01-AMA and AlphaO3-AMA for transformants before (2). Clones with the highest band intensity were frozen in the presence of glycerol and stored at -80°C.

[0265] Using material from one cryotube of each producing clone, 25 ml of LB medium supplemented with 100 mg / ml ampicillin and 50 mg / ml kanamycin was inoculated, and cultured for 14-16 hours at 37°C, 250 rpm agitation.

[0266] Using O / N culture, Terrific Broth autoinduction medium (glucose + lactose) supplemented with 200 mg / ml ampicillin and 100 mg / ml kanamycin was inoculated. A 1% inoculum relative to the culture volume was used. The culture was infused at 37°C, 250 rpm agitation, for 20 hours. Cells were recovered by centrifugation and stored at -20°C until use for protein purification.

[0267] The cells were resuspended in 200 mL of lysis buffer A (50 mM Tris-HCl pH 9.5, 8% Sucrose, 0.1% Triton X-100, 0.1% Tween-80, 10 mM EDTA, 10 mM 2-mercaptoethanol) at a ratio of 15 mL of lysis buffer per gram of wet cell mass. The material was then cooled on ice to 4°C.

[0268] The cells were lysed in a Panda 200 High Pressure Homogenizer (HPG) by recirculation at 1200 bar for 3 minutes. The lysate was clarified by centrifugation (6000xg, 8°C, for one hour). Finally, the supernatant was filtered through a 0.22 mm membrane.

[0269] The clarified material was filtered through a 300 kDa MWCO membrane using TFF. The retained material was concentrated and washed with 5 volumes of processing buffer 1 (50 mM Tris-HCl pH 8.0; 8% Sucrose; 0.1% Triton X-100; 0.1% Tween-80).

[0270] The permeate material from the 300 kDa membrane was concentrated on the 30 kDa MWCO membrane by TFF, and washed with 3 volumes of processing buffer 1. The diafiltrate material was washed with 5 volumes of processing buffer 2 (25 mM Tris-HCl pH 8.0; 0.03% Tween-80).

[0271] The diafiltrated material was loaded onto the Q-sepharose XL column, and the target protein was eluted by washing the column in “Steps” of 0, 15, 25, and 40% of buffer B (2 + 2M NaCl processing buffer).

[0272] The antigen eluted from the ion-exchange chromatography was conditioned using a superdex-300 gel-filtration column previously equilibrated with conditioning buffer (25 mM Tris-HCl pH 8.0; 0.1% sucrose; 0.03% Tween-80; 0.1 mM EDTA). The peak fractions containing the antigen (SAMAP02-AMA, or SAMA03-AMA) were pooled and filtered under sterile conditions on a 0.2 μm membrane.

[0273] Figure 18 shows the purification result of the Sa3-ama platform (SAMAP03-AMA). It can be observed that the subunits co-purify in the form of complexes with a higher molecular mass than that calculated for the monomers. A similar result was obtained for the Sa2-ama platform (SAMAP02-AMA).

[0274] IMMUNOGENICITY STUDIES OF RECOMBINANT ANTIGENS

[0275] To determine if the purified complexes are immunogenic, an immunization experiment was conducted on Balb / c mice with the following formulations (CEUA IB N 3841110219):

[0276] Aqueous formulations (volume 100mL):

[0277] - Sa2-ama (10mg);

[0278] - Sa3-ama (10 mg);

[0279] - Sa2-ama (10 mg) + Saponin (25 mg);

[0280] - Five synthetic peptides (4 mg each, 20 mg total);

[0281] Water-in-oil emulsion formulations (100 ml volume):

[0282] - Sa3-ama (10 mg) + Saponin (25mg + 50ml_ Montanide ISA V50;

[0283] - Five synthetic peptides (4µg each, 20 mg total) + Saponin (25 mg) + 50 mL Montanide ISA V50.

[0284] Mice were immunized intramuscularly 4 times at 14-day intervals. Figure 19 shows the results of antibody detection against the peptide mixture used to sensitize the ELISA plates. The pooled sera from each group were used at a 1 / 800 dilution.

[0285] Peptides in aqueous solution fail to stimulate an immune response, even with 4 doses (black line in Figure 19). However, their formulation in an oil emulsion with saponin (formulation tested in the field), antibodies against the peptides are induced from the first dose (dark blue line in Figure 19).

[0286] The Sa2-ama platform (SAMAP02-AMA, with flagellin) in aqueous solution required three doses to induce antibody formation (blue line in Figure 19). The addition of saponin to the aqueous solution of Sa2-ama increases its immunogenicity, inducing antibody formation after the second dose (red line in Figure 19).

[0287] Finally, the Sa3-ama platform (Z domain of S. aureus protein A) stimulates the immune response from the first dose, but subsequent doses do not noticeably increase the immune response (green line in Figure 19). However, its formulation in oil emulsion with saponin induces a strong immune response from the first dose (brown line in Figure 19), similarly to hybrid peptides.

[0288] Analysis of the recognition of epitopes separately, by dot-blot, using sera from animals immunized with the oil formulations (1 / 500 dilution), shows a similarity in the response induced by the peptide and recombinant vaccines (Figure 20). The biggest difference corresponds to the epitope of the MSP3 antigen, whose sequence is absent in the recombinant complexes.

[0289] Finally, the proportion of antibodies specifically directed against peptides with A. marginale epitopes and SAMAP platform sequences was verified. To this end, the “core” platform, formed by the P. horikoshii sequences that make up the platform (with and without 6xHis-tag fusion), and the SAMAP01 platform, which contains the core sequences plus 4 T-cell epitopes incorporated into the platform, were purified. Antibody titration experiments were performed using ELISA with these antigens. Figure 21 shows the results obtained.

[0290] The results show that immunizing mice with the recombinant antigen Sa3-ama (SAMAP03-AMA) is able to stimulate an immune response directed primarily against the Anaplasma marginale epitopes included in the platform.

[0291] The example of using the SAMAP platform shows that it is possible to generate an immune response against a large number of epitopes carried in antigenic complexes, and that they can be produced in a single purification process, without the need for conjugation. Therefore, it is possible to apply this technology to the development of multi-antigen vaccines.

Claims

CLAIMS 1. Multiantigen Immunogenic Platform, characterized by comprising: (a) a central protein core; (b) one or more immunomodulatory sequences; and (c) two or more protein or peptide antigens from the same or different pathogens.

2. PLATFORM, according to claim 1, characterized in that the central protein conjugate comprises a thermostable heteromeric protein.

3. PLATFORM, according to claim 1, characterized in that the thermostable heteromeric protein is a prefoldin, preferably in that the prefoldin is a prefoldin from Pyrococcus horikoshii.

4. PLATFORM, according to claim 1, characterized in that said one or more immunomodulatory sequences are selected from (a) T-helper epitopes, (b) T-cell chemoattractant peptides (chemokines); (c) FliC flagellin from Salmonella enterica serovar Typhimurium; (d) Z-domain of S. aureus protein A, (e) C3d component of complement; and / or combinations thereof.

5. PLATFORM, according to claim 4, characterized by: (i) because T-helper epitopes are derived from antigens studied in detail, such as the HIV envelope protein (PCLUS3 epitope), the F protein of canine distemper virus, the hemagglutinin of influenza virus, sequences derived from tetanus and diphtheria toxins and their combination (TpD), or artificial sequences, such as the PADRE sequence (universal epitope Pan DR); (ii) the chemokines are CCL2 (MCP-1), CCL3 (MIP-1a), CCL4 (MIP-1 P), CCL5 (RANTES), CXCL8 (IL-8), CXCL10 (IP-10), CXCL12 (SDF-1), CXCL13 (BLC), CCL11 (eotaxin), CCL19 (MIP-3p), CCL21 (SLC), CXCL1 (GROa), CXCL2 (GROP), CXCL9 (MIG), CCL17 (TARC), CCL20 and CCL22 (MDC); (iii) because flagellin FliC is from Salmonella enterica serovar Typhimurium-, (iv) because the Z-domain is a derivative of the immunoglobulin-binding domain of Staphylococcus aureus protein A.

6. PLATFORM, according to any one of claims 1 to 5, characterized by further comprising other immunomodulatory sequences, as a ligand for M cells, li-Key motif and Cel-1000 sequence, and their combinations.

7. PLATFORM, according to claim 1, characterized by comprising 2 to 6 different polypeptide antigens (proteins) or 2 to 16 different peptide antigens (epitopes), wherein the peptide or polypeptide antigens are from a pathogenic organism or from a cancer or tumor.

8. PLATFORM, according to claim 1, characterized in that one or more antigens are from Anaplasma marginale and are selected from the MSP antigen, the OMP proteins, the VirB9 and VirB10 antigens, and combinations thereof.

9. PLATFORM, according to claim 8, characterized in that said MSP antigen is MSP1a, MSP2, and combinations thereof.

10. PLATFORM, according to claim 8, characterized in that said OMP proteins are OMP7 and OMP8, and combinations thereof.

11. NUCLEIC ACID MOLECULE, characterized by consisting of the nucleotide sequence of SEQ ID NO: 9, and degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO:

8.

12. NUCLEIC ACID MOLECULE, characterized by consisting of the nucleotide sequence of SEQ ID NO: 11, and degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO:

10.

13. NUCLEIC ACID MOLECULE, characterized by consisting of the nucleotide sequence of SEQ ID NO: 16, and degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO:

15.

14. NUCLEIC ACID MOLECULE, characterized by consisting of the nucleotide sequence of SEQ ID NO: 19, and degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO:

18.

15. NUCLEIC ACID MOLECULE, characterized by consisting of the nucleotide sequence of SEQ ID NO: 21, and degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO:

20.

16. NUCLEIC ACID MOLECULE, characterized by consisting of the nucleotide sequence SEQ ID NO: 23, and degenerate sequences thereof, which encode the amino acid sequence SEQ ID NO:

22.

17. NUCLEIC ACID MOLECULE, characterized by consisting of the nucleotide sequence of SEQ ID NO: 24, and degenerate sequences thereof, which encode the amino acid sequence of SEQ ID NO:

25.

18. IMMUNOGENIC COMPOSITION, characterized by comprising a multiantigenic immunogenic platform, as defined in any one of claims 1 to 10, or a nucleic acid molecule, as defined in any one of claims 11 to 17, and one or more pharmaceutically acceptable excipients, vehicles or diluents.

19. PROCESS FOR MANUFACTURING A MULTI-ANTIGENIC PROTEIN PLATFORM, as defined in any one of claims 1 to 10, characterized by comprising the following steps: (a) select the core protein, immunomodulators and antigens of interest and the nucleotide sequences that encode them; (b) construct with these sequences the genes that code for each of the subunits that make up the SAMAP platform (alpha and beta), each containing the coding sequences for the T-cell epitopes, immunomodulatory sequences and the epitopes of interest; (c) clone these sequences into two Escherichia coli T7 expression plasmids with different selection markers (resistance to different antibiotics, ampicillin and kanamycin); (d) cotransform an Escherichia coli host cell with plasmids encoding the alpha and beta subunits of the SAMAP platform and select transform cells in the presence of ampicillin and kanamycin; (e) cultivate the selected transformants in selective media and induce the expression of recombinant proteins with ITPG (isopropyl [3- D-thiogalactopyranoside]) or, preferably, lactose, so that the alpha and beta subunits of the SAMAP platform, synthesized in the bacterial cytoplasm, self-assemble; (f) After induction, the immunogenic complex is purified from the soluble fraction of the bacterial cytoplasm using the following procedures: • Cell lysis; • Clarification of the lysate; • Tangential Filtration; • Ion-exchange chromatography; and • Molecular exclusion chromatography.

20. PROCESS, according to claim 19, characterized by further comprising a single purification process.

21. PROCESS, according to claim 19, characterized in that the antigen and epitopes of interest are from Anaplasma marginale.

22. PROCESS, according to claim 19, characterized in that the host cell is a bacterial, yeast, mammalian, or insect cell.

23. PROCESS, according to claims 19 to 22, characterized in that the expression is preferentially induced by lactose, in the case of the use of Escherichia coli T7 plasmids.

24. PROCESS, according to claim 20, characterized in that the purification step is carried out using tangential filtration procedures on membranes with different pore sizes, chromatographic procedures using different resins, such as ion exchange, hydrophobic interaction, molecular exclusion, dassalting, mix-mode resins, among others.

25. PROCESS, according to claim 24, characterized by the possibility of purification by immobilized metal affinity chromatography (IMAC) for the purification of the SAMAP complex when at least one of its subunits contains the fusion with the 6xHis-tag.

26. USE OF A MULTIANTIGENIC PROTEIN PLATFORM, as defined in any one of claims 1 to 10, characterized by being used in the manufacture of a medicament to improve or enhance the response of multiantigenic vaccines against different pathogens.

27. USE OF A MULTIANTIGENIC PROTEIN PLATFORM, as defined in any one of claims 1 to 10, characterized in that it is used in the manufacture of a medicament to prevent bovine anaplasmosis.

28. USE, according to any of claims 26 to 27, characterized in the medicine being in the form of a vaccine or a medicine for oral or nasal administration.

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