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BR-102024017991-A2 - Platform for developing recombinant multiantigen vaccines.

BR102024017991A2BR 102024017991 A2BR102024017991 A2BR 102024017991A2BR-102024017991-A2

Abstract

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).

Inventors

  • CELSO RAUL ROMERO RAMOS
  • MIRYAM MARROQUIN QUELOPANA
  • ANA MARISA CHUDZINSKI-TAVASSI

Assignees

  • IMUNOTICK PESQUISA E DESENVOLVIMENTO TECNOLÓGICO S.A
  • INSTITUTO BUTANTAN

Dates

Publication Date
20260317
Application Date
20240830

Claims (20)

  1. 1. MULTIANTIGENIC 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. 2. PLATFORM, according to claim 1, characterized in that the central protein conjugate comprises a thermostable heteromeric protein.
  3. 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. 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. 5. PLATFORM, according to claim 4, characterized: (i) by the T-helper epitopes being 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) by the chemokines being CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CXCL8 (IL-8), CXCL10 (IP-10), CXCL12 (SDF-1), CXCL13 (BLC), CCL11 (eotaxin), CCL19 (MIP-3β), CCL21 (SLC), CXCL1 (GROα), CXCL2 (GROβ), CXCL9 (MIG), CCL17 (TARC), CCL20 and CCL22 (MDC);(iii) because the 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. 6. PLATFORM, according to any one of claims 1 to 5, characterized by further comprising other immunomodulatory sequences, such as M-cell ligand, Ii-Key motif and Cel-1000 sequence, and combinations thereof.
  7. 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. 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. 9. PLATFORM, according to claim 8, characterized in that said MSP antigen is MSP1a, MSP2, and combinations thereof.
  10. 10. PLATFORM, according to claim 8, characterized in that said OMP proteins are OMP7 and OMP8, and combinations thereof.
  11. 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. 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. 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. 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. 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. 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. 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. 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. 19. PROCESS FOR MANUFACTURING A MULTIANTIGENIC PROTEIN PLATFORM, as defined in any one of claims 1 to 10, characterized by comprising the following steps: (a) selecting the core protein, immunomodulators and antigens of interest and the nucleotide sequences that encode them; (b) constructing 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 epitopes of interest; (c) cloning these sequences into two Escherichia coli T7 expression plasmids, with different selection markers (resistance to different antibiotics, ampicillin and kanamycin); (d) co-transforming an Escherichia coli host cell with the plasmids encoding the alpha and beta subunits of the SAMAP platform and selecting the transformants in the presence ampicillin and kanamycin; (e) cultivate the selected transformants in selective medium and induce the expression of recombinant proteins with ITPG (isopropyl β-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 by the following procedures: (g) Cell lysis; (h) Lysate clarification; (i) Tangential filtration; (j) Ion-exchange chromatography; and (k) Size exclusion chromatography.
  20. 20. PROCESS, according to claim 19, characterized by further comprising a single purification process.

Description

FIELD OF THE INVENTION [001] 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 capability called SAMAP (Self Adjuvanted Multi Antigen Platform). BACKGROUND OF THE INVENTION [002] 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. [003] Not all species of pathogens can be cultivated in the laboratory or produced on an industrial scale, so for these organisms, it is not possible to develop vaccines 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. [004] Recombinant DNA technology overcomes the need to cultivate pathogens to obtain antigens, which can now be produced by genetically modified organisms. Thus, vaccines (subunit vaccines) have been created, which are safer than traditional vaccines. [005] 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, which are based on the “spike” antigen. [006] 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. [007] 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. [008] 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, so they need to be associated with carrier proteins or polymers to increase their immunogenicity. [009] 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. [010] 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. [011] 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. [012] WO2022/060488 describes a multi-epitope vaccine for the treatment of Alzheimer'