Methods for modeling circular 3D epitopes for use in vaccine development

Abstract

The present invention relates to a method for modeling a cyclic 3D epitope from the surface of a target antigen, comprising the steps of: a) identifying in a 3D model representation of the target antigen one or more conserved region(s) likely to be / constitute a 3D epitope, said one or more conserved region(s) having one or more charged amino acid(s) selected in the group consisting of arginine (Arg), lysine (Lys), histidine (His), aspartic acid (Asp) and glutamic acid (Glu); b) performing molecular modeling on the one or more conserved region(s) identified in step a); c) providing a linear amino acid sequence likely to assemble one or more conserved region(s) within the 3D epitope; d) cyclizing the linear amino acid sequence in silico to mimic the 3D epitope as it exists in the target antigen; and e) obtaining a modeled cyclic 3D epitope.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the development of vaccines, in particular synthetic vaccines, by modelling circular 3D epitopes. [Background technology]

[0002] 2. Background of the Invention Interest in synthetic peptide immunogens as an approach to generate specific immune reagents such as antibodies, particularly neutralizing antibodies, has increased significantly over the past few decades.

[0003] In theory, many peptide sequences can be immunogenic, but in practice, not all are equally effective in eliciting antibodies that cross-react with intact cognate proteins. It is now recognized that many factors can affect the success of using peptide immunogens to raise specific antibodies. These include factors such as the number of peptides from one protein sequence used, the availability and accuracy of sequence data, the predicted secondary and tertiary structure of the intact protein, and finally the ease of synthesis of specific sequences. Although continuous improvements to synthesis methodology mean that recent aspects are less critical than past aspects, certain sequences may still be problematic (Hancock and O'Reilly; 2005. Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Chapter 2. Edited by: R. Burns c Humana Press Inc., Totowa, NJ).

[0004] Recently, scientists have developed the mimotope approach. Mimotopes are peptides that can mimic epitopes. Epitopes are specific surfaces of antigenic proteins that are recognized by antibodies. Epitopes can be linear or three-dimensional (3D), but 3D-epitopes are recognized by 80% of immunoglobulins (IgG) and are much more specific. The main approach to designing 3D-epitopes is to use molecular modeling to design peptides with 3D structures that can mimic epitopes.

[0005] So far, two main strategies have been developed in this field: the first approach is the in vivo production of the protein of interest by utilizing techniques that rely on the expression of recombinant cDNA in heterologous living organisms, specifically bacteria; the second approach is chemical-based in vitro peptide synthesis.

[0006] Synthetic peptides have been produced, but vaccination with synthetic peptides obtained by Fmoc chemistry often does not have a stable 3D structure and is only useful for recognizing linear epitopes. Summary of the Invention [Problem to be solved by the invention]

[0007] There is therefore a need to overcome these shortcomings and provide the state of the art with a reproducible method for modeling stable and robust 3D epitopes that can be used for the development of synthetic vaccines. [Means for solving the problem]

[0008] overview A first aspect of the present invention is a method for modeling a cyclic 3D epitope from the surface of a target antigen, comprising: a) identifying in a 3D model representation of the target antigen one or more conserved region(s) likely to be / constitute a 3D epitope, said one or more conserved region(s) having one or more charged amino acid(s) selected from the group consisting of arginine (Arg), lysine (Lys), histidine (His), aspartic acid (Asp) and glutamic acid (Glu); b) performing molecular modeling on one or more conserved region(s) identified in step a); c) providing a linear amino acid sequence that is amenable to assembling one or more conserved region(s) within the 3D epitope; d) in silico cyclization of the linear amino acid sequence to mimic the 3D epitope as it exists in the target antigen; e) obtaining a modeled circular 3D epitope; The present invention relates to a method comprising the steps of:

[0009] In some embodiments, the cyclic 3D epitope is a mimotope. In certain embodiments, the cyclic 3D epitope is synthetic. In some embodiments, the target antigen comprises or is selected in the group consisting of bacterial antigens, viral antigens, and cancer antigens. In certain embodiments, step a) is performed by aligning homologous and / or mutated sequences of one or more conserved region(s) to obtain atomic coordinates. In some embodiments, the one or more conserved region(s) are on the surface of the 3D model representation of the target antigen. In certain embodiments, step b) comprises performing one or more cycles of minimizing the energy of the force field induced by the atomic coordinates, in particular by one or more energy minimization algorithm(s). In some embodiments, the energy minimization algorithm(s) is associated with a molecular dynamics step. In certain embodiments, step d) is performed chemically, in particular by site-specific cross-linking. In some embodiments, step d) is carried out by substituting two amino acid residues in the linear sequence obtained in step c) with cysteine ​​residues, where the dihedral angles between each of the alpha carbons of each of the cysteine ​​residues and any one of the adjacent amino acid residues are compatible with the formation of a disulfide bridge.

[0010] A further aspect of the invention relates to cyclic 3D epitopes obtained by synthesis of cyclic 3D epitopes modelled by the method according to the invention.

[0011] In one aspect the invention relates to a pharmaceutical or vaccine composition comprising a cyclic 3D epitope according to the invention.

[0012] The present invention also relates to a cyclic 3D epitope or a pharmaceutical composition according to the invention for use in a method for vaccination of an individual.

[0013] In one aspect the invention relates to a cyclic 3D epitope or a pharmaceutical composition according to the invention for use in the generation of antibodies, in particular neutralising antibodies.

[0014] Another aspect of the invention relates to a cyclic 3D epitope or a pharmaceutical composition according to the invention for use for eliciting an immune response in an individual.

[0015] definition In the present invention, the following terms have the following meanings.

[0016] The term "about" preceding a number includes up to ±10% of the value of said number. It should be understood that the value to which the term "about" refers is the exact value that is specifically and preferably disclosed.

[0017] "Adjuvant" refers to a compound or combination of compounds that enhances the immune response in a vaccine composition. In one embodiment, an adjuvant is used with a vaccine composition, thus enhancing the immune response to infectious disease. For example, an adjuvant can increase the number of lymphocytes, increase the activation of lymphocytes; increase the compatibility of lymphocytes, and / or increase the survival rate of lymphocytes.

[0018] "Comprising" is intended to mean "containing," "including," and "comprehensive." In some embodiments, the term "comprising" also encompasses the term "consisting of."

[0019] "Cross-linker" refers to a series of chemical or amino acid sequences that physically link two domains or portions of a peptide or polypeptide by utilizing two reactive groups. In some embodiments, cross-linkers can be homobifunctional (two identical reactive groups) or heterobifunctional (two separate reactive groups) compounds.

[0020] "Cyclic 3D epitope" refers to an epitope in the form of a peptide, in which the side chains of two non-contiguous amino acid residues are covalently linked, resulting in a peptide with a 3D structure. In one embodiment, the covalent linkage is obtained chemically, in particular by using a cross-linking agent. In one embodiment, the covalent linkage is a disulfide bridge between two -SH groups belonging to two non-contiguous cysteine ​​residues.

[0021] "Molecular modeling" refers to a collection of techniques well known to those skilled in the art that provide realistic 3D structures of proteins or peptides with potential energies according to a force field. The potential energies of the force field can be modified by energy minimization and / or dynamics.

[0022] An "immunogenic composition" refers to a composition that is capable of eliciting an immune response in an individual upon contact with said individual.

[0023] An "immunogenic peptide" refers to a peptide that is capable of eliciting an immune response in an individual upon contact with said individual.

[0024] "Isolated peptide" refers to a peptide that has been removed from the environment in which it was synthesized. "Model" or "modeling" refers to the activity of generating in silico a representation or simulation of a target antigen of interest. In some embodiments, the target of interest is the surface of the target antigen.

[0025] "Linker" refers to a series of chemical or amino acid sequences that physically separate two domains or portions of a peptide or polypeptide. As used herein, "a peptide and a carrier protein are conjugated by a linker" means that the peptide and the carrier protein are indirectly covalently associated through the linker.

[0026] "Mimotope peptide" refers to a peptide, particularly a synthetic peptide, that mimics an epitope of an antigen found in nature. As used herein, a mimotope may be represented by a linear peptide, preferably a peptide with a 3D structure.

[0027] A "peptide" or "polypeptide" refers to a linear polymer of amino acids linked together by peptide bonds.

[0028] "Synthetic peptide" refers to a peptide that is not naturally occurring and / or that is synthesized chemically or through the use of recombinant technology.

[0029] "Vaccine composition" refers to a composition comprising one or more antigens and / or epitopes suitable for eliciting an immune response against those antigens and / or epitopes in an individual who is the recipient of said composition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Aiming to provide an easy, robust and reproducible method for designing stable 3D epitopes, we have shown that it is possible to block the 3D structure of a peptide that mimics the surface epitope of an immunogenic target in a conformation similar to the 3D epitope, specifically with disulfide bridges, which we have named the "mimotope approach".

[0031] In the mimotope approach, only one disulfide bridge is created.If there is a free cysteine ​​or a natural disulfide bridge with a cysteine ​​outside the epitope, it must be mutated with another amino acid residue, such as serine, to replace the free cysteine.It is advantageous to have only one disulfide bridge, because two or three disulfide bridges can generate different conformations with the same sequence.

[0032] To determine the optimal location for disulfide bridges, the method of the present invention uses a specific force field equation called MImotope MOdeling Force Field (MIMOFF) and a minimization algorithm, optionally combined with a dynamics step. This mathematical approach utilizes a force field corresponding to a set of equations that provide potential energy for peptide chains with respect to atomic 3D coordinates. The goal of energy minimization is to reduce the force field energy to an optimal potential energy with a negative value with respect to van der Waals energy by the energy minimization algorithm.

[0033] The present invention provides a method for modeling cyclic 3D epitopes from the surface of a target antigen, comprising the steps of: a) identifying in a 3D model representation of the target antigen one or more conserved region(s) likely to be / constitute a 3D epitope, the one or more conserved region(s) having one or more charged amino acid(s) selected from the group consisting of arginine (Arg), lysine (Lys), histidine (His), aspartic acid (Asp) and glutamic acid (Glu); b) performing molecular modeling on one or more conserved region(s) identified in step a); c) providing a linear amino acid sequence that is amenable to assembling one or more conserved region(s) within the 3D epitope; d) in silico cyclizing the linear amino acid sequence to mimic said 3D epitope as it exists in the target antigen; e) obtaining a modeled circular 3D epitope; The present invention relates to a method comprising the steps of:

[0034] In some embodiments, step b) is performed using the following equation E to calculate the energy of the force field:

number

[0035] The above equation E is thereby referred to as the MImotope Modeling Force Field (MIMOFF).

[0036] It should be understood that the equation E used in step b) can be divided into five distinct equations as follows: Equation E1:

number

number

number

number

number

[0037] In some embodiments, Equation E3 is applied to the orbital hybridization of carbon atoms. In some embodiments, Equation E3 reflects the energy associated with the dihedral angle for an SP2 carbon atom. In some embodiments, E3 reflects the energy associated with the dihedral angle for an SP3 carbon atom.

[0038] In some embodiments, equations E2, E3 and E5 are derived from the AMBER force field. In some embodiments, equation E4 is derived from the CHARMM force field. Both force fields are known to those of skill in the art.

[0039] In some embodiments, Equation E1 is specific to MIMOFF and uses its anharmonic functions to account for van der Waals energies as opposed to AMBER or CHARMM force fields, which use harmonic functions. In some embodiments, Equation E1 provides negative energies when the two bonded atoms respect the optimal van der Waals radii.

[0040] In some embodiments the cyclic 3D epitope is a mimotope.

[0041] In certain embodiments, the cyclic 3D epitope is synthetic.

[0042] In some embodiments, the epitope is a peptide. In practice, the peptide of the present invention can be synthesized by any suitable method known in the state of the art or derived therefrom. By way of example, methods for synthesizing peptides according to the present invention can be disclosed, for example, by Lloyd-Williams et al. (1997; Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press. 278), Merrifield (1963; Journal of the American Chemical Society. 85, 2149-54), Lewandowski et al. (2013; Science, Vol. 339(6116), 189-193).

[0043] In some embodiments, the target antigen is selected from the group including or consisting of a bacterial antigen, a viral antigen, and a cancer antigen.

[0044] As used herein, "bacterial antigen" and "viral antigen" refer to antigens of bacterial or viral origin, respectively, that are capable of eliciting an immune response in an individual, particularly a vertebrate individual, and more particularly a non-human mammal or a human.

[0045] In certain embodiments, the bacterial and / or viral antigens are directed against infectious diseases, in particular acute flaccid paralysis (AFM), anaplasmosis, anthrax, babesiosis, botulism, brucellosis, camprobacteriosis, kabapenem-resistant infections (CRE / CRPA), chancroid, chikungunya virus infection (chikungunya), chlamydiosis, ciguatera (harmful algal blooms (HAB)), Clostridium difficile infection, Clostridium perfringens (epsilon toxin), coccioidomycosis fungal infection (valley fever), COVID-19 (coronavirus disease 2019), Creutzfeldt-Jakob disease, transmissible sponge disease, CJD, Tryptosporidiosis (Crypto), Cyclosporiasis, Dengue (Dengue Fever), Diphtheria, Shiga Toxin Producing E. coli Infection (STEC), Eastern Equine Encephalitis (EEE), Ebola Hemorrhagic Fever (Ebola), Ehrlichiosis, Arbovirus or Parainfectious Encephalitis, Non-Porioenterovirus Infection (Non-Porioenterovirus), D68 Enterovirus Infection (EV-D68), Giardiasis (Giardia), Glanders, Gonococcal Infection (Gonorrhea), Inguinal Granulomatosis, Haemophilus influenzae type B (Hib or F-flu), Hantavirus Pulmonary Syndrome (HPS), Hemolytic Uremic Syndrome (HUS), Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Hepatitis F (Hep F), Hepatitis G (Hep G), Hepatitis H (Hep H), Hepatitis I (Hep I ... E), herpes, herpes zoster (shingles), histoplasmosis (histoplasmosis), human immunodeficiency virus / AIDS (HIV / AIDS), human papillomavirus (HPV), influenza (Flu), lead poisoning, legionnaires' disease (legionnaires' disease), leprosy (hansen's disease), leptospirosis, listeriosis (listeria), Lyme disease, lymphogranulomatosis gracilis (LGV), malaria, measles, melioidosis, viral meningitis, bacterial meningitis, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Multisystem Inflammatory Syndrome in Children (MIS-C), Mumps, Norovirus, Paralytic Shellfish Poisoning (Paralytic Shellfish Poisoning, Ciguatera), Pediculosis (Lice, Head and Body Lice), Pelvic Infection (PID), Pathophysiology (Pertussis), Bubonic Plague, Septicemic Plague, Pneumonic Plague (Black Plague), Pneumococcal Infection (Pneumonia), Poliomyelitis (Polio), Powassan, Psittacosis (Parrot Fever), Pediculosis (Pediculosis;Lice infestation), Pustular rash (Smallpox, Monkeypox, Cowpox), Q fever, Rabies, Ricin poisoning, Rickettsiosis (Rocky Mountain spotted fever), Rubella (Rubella), Salmonella gastroenteritis (Salmonella), Scabies infestation (Scabies), Mackerel, Septic shock (Sepsis), Severe acute respiratory syndrome (SARS), Shigella gastroenteritis (Shigella), Smallpox, Methicillin-resistant Staphylococcus aureus infection (MRSA), Staphylococcus aureus (Enterotoxin B) food poisoning (Staph food poisoning), Vancomycin-intermediate resistant Staphylococcus aureus infection (VISA), Vancomycin-resistant Staphylococcus aureus infection (VRSA), Group A (invasive) Streptococcus infection (Streptococcus aureus) The infection may be associated with an infectious disease selected from the group consisting of Group A (invasive), Group B Streptococcus infection (Strep-B), Streptococcal toxic shock syndrome (STSS, TSS), syphilis, tetanus infection (tetanus, trismus), trichomomatosis (trichomonas infection), Trichinella spiralis infection (trichinosis), tuberculosis (TB), tularemia (rabbit fever), Group D typhoid fever, typhoid, bacterial vaginosis (yeast infection), vaping-associated lung injury (e-cigarette-associated lung injury), chickenpox (varicella), Vibrio cholerae infection (cholera), Vibriosis (Vibrio), viral hemorrhagic fever (Ebola, Lassa, Marburg), West Nile virus, yellow fever, Yersinia infection, and Zika virus infection (Zika);

[0046] As used herein, the term "cancer antigen" is intended to refer to an antigen that can be found in high amounts in the blood of patients with a particular type of cancer.

[0047] Non-limiting examples of cancer include acute lymphoblastic leukemia, acute myeloblastic leukemia, adrenal gland cancer, bile duct cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumors, glioblastoma, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, kidney cancer, lung cancer, melanoma, Merkel cell skin cancer, mesothelioma, multiple myeloma, myeloproliferative disorders, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, salivary gland cancer, sarcoma, squamous cell carcinoma, testicular cancer, thyroid cancer, urothelial carcinoma, and uveal melanoma.

[0048] In practice, said cancer is a hematological cancer or a solid cancer.

[0049] As used herein, the term "blood cancer," also referred to as "hematological cancer," encompasses any cancer involving uncontrolled proliferation of blood cells, particularly white blood cells. Blood cancers include immunoblastic lymphadenopathy, leukemia, lymphoma (Hodgkin's and non-Hodgkin's lymphoma), and myeloma.

[0050] As used herein, the term "solid cancer" includes any cancer (also called malignant tumor) that forms a separate tumor mass, as opposed to a cancer that does not form a mass and diffusely infiltrates tissue.Solid cancer includes melanoma, breast cancer, colon cancer, kidney cancer, adrenocortical carcinoma, testicular teratoma, sarcoma of the skin, fibrosarcoma, lung cancer, adenocarcinoma, liver cancer, glioblastoma, prostate cancer, ovarian cancer and pancreatic cancer.

[0051] Non-limiting examples of cancer antigens include alpha-fetoprotein (AFP), cancer antigen 125 (CA125), cancer antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), human chorionic gonadotropin (hCG or beta-hCG), and prostate-specific antigen (PSA).

[0052] In some embodiments, the cancer antigen is a neoantigen.

[0053] As used herein, the term "neoantigens" refers to newly formed antigens not previously recognized by the immune system. Neoantigens, and therefore neoantigenic determinants (or neoepitopes), can form when proteins undergo further modification in biochemical pathways such as glycosylation, phosphorylation, or proteolysis.

[0054] Neoantigen mutations may be identified by comparing DNA isolated from tumors with that of normal sources.

[0055] Preferably, any suitable sequencing-by-synthesis platform can be used to identify mutations. Four major sequencing-by-synthesis platforms are currently utilized: Genome Sequencers from Roche / 454 Life Sciences, HiSeq Analyzer from Illumina / Solexa, SOLiD system from Applied BioSystems, and Heliscope system from Helicos Biosciences. Sequencing-by-synthesis platforms have also been described by Pacific Biosciences and VisiGen Biotechnologies. Each of these platforms can be used in the methods of the present invention.

[0056] In certain embodiments, the target antigen is a polypeptide or protein.

[0057] In certain embodiments, the 3D model representation of the target antigen may be obtained from a database. In some embodiments, the 3D model representation of the target antigen may be obtained by utilizing crystallography and / or NMR (nuclear magnetic resonance). In certain embodiments, the 3D model representation of the target antigen may be obtained by modeling from 3D model representation(s) of antigen(s) that are homologous to the target antigen. In some embodiments, the 3D model representation of the target antigen may be obtained by utilizing molecular modeling to predict 3D structure.

[0058] Non-limiting examples of algorithms for minimizing the energy of a force field derived from atomic coordinates include Steepest Descent, Conjugate Gradients, and the like.

[0059] In certain embodiments, step a) is performed by aligning homologous and / or mutated sequences of one or more conserved region(s) to obtain atomic coordinates.

[0060] In practice, step a) may be performed by aligning the sequences of homologous polypeptides or proteins. As used herein, the term "homologous polypeptides or proteins" refers to polypeptides or proteins that share substantial amino acid sequence identity. In practice, homologous polypeptides or proteins include orthologous polypeptides or proteins, paralogous polypeptides or proteins, and xenologous polypeptides or proteins.

[0061] The term "sequence identity" as used herein in the context of a relationship between the sequences of two or more polypeptides refers to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" refers to the percentage of perfect matches between the smaller of two or more sequences (i.e., "homology" according to InsightII), with gap alignment (if any) addressed by a particular mathematical model or computer program. The identity of related polypeptide or nucleic acid sequences can be readily calculated by known methods. Preferred methods for determining identity are designed to give the maximum match between the sequences tested. Methods for determining identity are described in published computer programs. Preferred computer program-based methods for determining identity between two sequences include GAP (Devereux et al., Nucl. Acid. Res. 2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, TBLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)) from the GCG program package. The BLASTX program is available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity. In one embodiment. "Identity" refers to a parameter measured over the entire length of the referenced sequence.

[0062] Indeed, amino acid sequence identity may be assessed by any suitable algorithm from the state of the art, non-limiting examples of which include CLUSTAL, CLUSTAL W, BLAST P, LALIGN, and the like.

[0063] By way of example, the percentage of amino acid identity may be determined using the CLUSTAL W software (version 1.83), with the parameters set as follows: - For slow / fine alignment: (1) gap opening penalty: 10.00; (2) gap extension penalty: 0.1; (3) protein weight matrix; BLOSUM; - For fast / approximate alignment: (4) gap penalty: 3; (5) K-tuple (language) size: 1; (6) optimal number of diagonals: 5; (7) window size: 5; (8) scoring method: PERCENT.

[0064] In some embodiments, the one or more conserved region(s) is on the surface of a 3D model representation of the target antigen.

[0065] As used herein, the term "surface" is intended to mean that when the target antigen is in aqueous solution, one or more conserved region(s) are exposed to the solvent.

[0066] In certain embodiments, step b) comprises performing one or more cycles of energy minimization of the derived force field according to the atomic coordinates, in particular according to one or more energy minimization algorithm(s), which are known in the art.

[0067] In some embodiments, the energy minimization is achieved by taking a derivative of the force field equation. In some embodiments, the energy minimization is achieved by taking a derivative of the equation used in step b) of the present invention, preferably the equation E(MIMOFF).

[0068] In some embodiments, the derivative of the force field equation provides the atomic coordinates that have the minimum potential energy of the force field.

[0069] The term "energy minimization" as used herein refers to an approach to find a set of coordinates that represents the minimum energy conformation for a given structure of a peptide, polypeptide or protein.It is commonly recognized in the state of the art that the energy of a peptide, polypeptide or protein can be characterized as a function of atomic coordinates.In practice, this energy function can depend on multiple parameters, such as bond energy and bond angle energy, which represent covalent bonds and bond angles, respectively; dihedral angle energy obtained from dihedral angles; van der Waals forces that determine the steric hindrance of atoms; and electrostatic forces that correspond to the long-range forces between charged atoms and partially charged atoms.

[0070] In practice, non-limiting examples of algorithms for energy minimization include Steepest Descent and Conjugate Gradient. In some embodiments, energy minimization may be performed by using the Discover module of InsightII® software with the equation used in step b) of the present invention, preferably the equation E(MIMOFF).

[0071] In some embodiments, the energy minimization algorithm(s) is associated with a molecular dynamics step.

[0072] "Molecular dynamics" as used herein is intended to refer to an approach for solving stalls in energy minimization in an energy wheel.

[0073] In practice, molecular dynamics is related to the Steepest Descent algorithm for energy minimization.

[0074] In some embodiments, the molecular dynamics step involves repeating one or more cycle(s) of energy minimization.

[0075] In some embodiments, a molecular dynamics step is performed when the minimization algorithm is shut down.

[0076] In some embodiments, the molecular dynamics step includes or consists of analyzing different potential energy trajectories of MIMOFF with different rounds of energy minimization. In some embodiments, the molecular dynamics step includes or consists of repeating one or more cycle(s) of energy minimization using different initial energies. In some embodiments, the molecular dynamics step includes or consists of repeating one or more cycle(s) of energy minimization using different potential energy values.

[0077] According to the invention, step c) consists of providing a linear amino acid sequence that is amenable to collecting one or more conserved regions(s) within the 3D epitope. As used herein, the term "providing a linear amino acid sequence" is intended to mean "determining a linear amino acid sequence". In one embodiment, the terms "providing" and "determining" are equivalent.

[0078] In some embodiments, two or more conserved regions within a 3D epitope may be aligned as one linear amino acid sequence. In certain embodiments, two or more conserved regions may be linked together by utilizing an amino acid linker, such as a glycine-rich amino acid linker, in particular comprising one or two glycine repeats.

[0079] The term "amino acid linker" as used herein refers to a peptide whose function is to provide a chain of peptide bonds to separate two or more peptides of interest in space.In practice, suitable amino acid linkers can be described in Chen et al. (Adv Drug Deliv Rev. 2013; 65(10): 1357-1369), Chichili et al. (Protein Sci. 2013; 22(2): 153-167), Crasto and Feng (Prot Engineer. 2000; 13(5), 309-312), Waldo et al. (Nat. Biotechnol. 1999; 17, 691-695).

[0080] In some embodiments, the amino acid linker has a length within the range of about 1 to about 100 amino acid residues, preferably about 5 to about 50 amino acid residues, and more preferably about 10 to about 25 amino acid residues.

[0081] As used herein, the expression "about 1 to about 100 amino acid residues" refers to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100 amino acid residues.

[0082] In effect, the structure of the epitope of interest is maintained and stabilized by cyclization.

[0083] In a particular embodiment, step d) is carried out chemically, in particular by site-specific cross-linking.

[0084] The term "site-specific cross-linking" as used herein is intended to refer to the step of chemically joining two or more selected amino acids by covalent bonds.

[0085] In some embodiments, site-specific cross-linking may be performed with a cross-linking agent.

[0086] Indeed, site-specific cross-linking can be mediated by cross-linking agents including homobifunctional cross-linkers and heterobifunctional cross-linkers.

[0087] As used herein, a "homobifunctional cross-linker" is intended to refer to a cross-linker that has identical reactive groups on either end of a spacer arm. Non-limiting examples of homobifunctional crosslinkers include adipic acid dihydrazide (CAS No. 1071-93-8), 1,4-bis[3-(2-pyridyldithio)propionamido]butane (CAS No. 141647-62-3), disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; CAS No. 207233-90-7), dimethyl 3,3'-dithio-bis(propionimidate) dihydrochloride (DTBP; CAS No. 38285-78-8), dimethyl pimeline diimidate dihydrochloride (DMP; CAS No. 58537-94-3), 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DTSP; CAS No. 57757-57-0), ethylene glycol-bis(succinic acid Examples of such crosslinkers include bis(N-hydroxysuccinimide ester) (CAS No. 70539-42-3), bis(N-succinimidyl) sebacic acid ester (DSSeb; CAS No. 23024-29-5), disuccinimidyl suberate (DSS; CAS No. 68528-80-3), and bis(sulfosuccinimidyl) suberate sodium salt (BS3; CAS No. 82436-77-9). In fact, homobifunctional crosslinkers may crosslink identical free reactive groups within the side chains of amino acid residues. For example, the free -OH group may be located on the side chains of amino acid residues such as aspartic acid (Asp), glutamic acid (Glu), serine (Ser), threonine (Thr), and tyrosine (Tyr), and the free -NH2 group may be located on the side chains of amino acid residues such as asparagine (Asn), glutamine (Gln), arginine (Arg), and lysine (Lys).

[0088] As used herein, "heterobifunctional crosslinker" refers to a crosslinker having different reactive groups at either end. Non-limiting examples of heterobifunctional crosslinkers include acetylene-PEG4-maleimide, maleimide-PEG-succinimidyl ester, azido-PEG4-phenyloxydiazole methylsulfone, LC-SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), PDPH (3-(2-pyridyldithio)propionylhydrazide) (CAS number: 115616-51-8), SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate) (CAS number: 72252-9), and the like. 6-1), SMPH (succinimidyl-6-((b-maleimidopropionamido)hexanoate), sulfo-SIAB (sulfo-succinimidyl(4-iodoacetyl)aminobenzoate), 3-(maleimido)propionic acid N-hydroxy-succinimide ester (BMPS; CAS number: 55750-62-4), 2-(2-((7-(tert-butoxy)-7-oxoheptyl)oxy)ethoxy)acetic acid (OtBu-PEG2 acid), and N-(β-maleimidopropionic acid) hydrazide, trifluoroacetate (BMPH).

[0089] In practice, the cross-linking agents may be commercially available, for example from Merck®, Themofisher Scientific®, Sigma Aldrich®, and may be used according to the manufacturer's instructions.

[0090] In some embodiments, step d) is carried out by substituting two amino acid residues in the linear sequence obtained in step c) with cysteine ​​residues, wherein the dihedral angles between each of the alpha carbons of the cysteine ​​residues and any one of the adjacent amino acid residues are compatible with the formation of a disulfide bridge.

[0091] The term "disulfide bridge" as used herein refers to a covalent bond between two -SH groups from the side chains of free cysteines. To create a disulfide bridge, the -SH side chain of a first free cysteine ​​must be located less than 0.1 nm away from another -SH side chain of a second free cysteine. The sp3 hybrid nature of the alpha carbon of each cysteine ​​allows for different orientations, but this flexibility is limited by the dihedral angle freedom of the peptide bond with the neighboring amino acid residue. The size of the side chain of the neighboring amino acid residue can directly affect the dihedral angle freedom.

[0092] In a particular embodiment, step d) may be preceded by a step of carrying out substitution(s) of one or more cysteine ​​residue(s) by one or more amino acid residue(s) selected in the group consisting of alanine (Ala), serine (Ser) and threonine (Thr), preferably alanine (Ala) and serine (Ser), more preferably serine (Ser) in the linear sequence obtained in step c). The linear peptide at the end of step d) may contain only two cysteine ​​residues, so that in practice the formation of only one disulfide bridge may be possible.

[0093] Another aspect of the invention also relates to cyclic 3D epitopes obtained by synthesis of cyclic 3D epitopes modelled by the method according to the invention.

[0094] A further aspect of the invention relates to a pharmaceutical or vaccine composition comprising a cyclic 3D epitope according to the invention.

[0095] In some embodiments, the pharmaceutical composition according to the present invention comprises a pharma- ceutically acceptable excipient. The term "pharma-ceutically acceptable excipient" as used herein refers to an excipient that does not cause adverse, allergic or other untoward reactions when administered to animals, preferably humans. It includes any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption retardants, and the like. For administration to humans, preparations must meet sterility, pyrogenicity, general safety and purity standards as required by regulatory authorities, such as the Food and Drug Administration (FDA in the United States) or the European Medicines Agency (EMA).

[0096] In some embodiments, the vaccine composition according to the invention comprises one or more adjuvants. As used herein, the term "adjuvant" refers to a component that enhances the immune response to an antigen and / or tailors it towards a desired immune response. Thus, the incorporation of an adjuvant into a vaccine formulation aims to enhance, promote and prolong the specific immune response towards a desired response to the vaccine antigen. The advantages of adjuvants include enhancing the immunogenicity of antigens, modifying the nature of the immune response, reducing the amount of antigen required for effective immunization, reducing the frequency of booster immunization required, and improving the immune response in the elderly and immunocompromised.

[0097] In some embodiments, the cyclic 3D epitopes according to the invention may be conjugated to a carrier protein, non-limiting examples of which include bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), multiple antigen peptides (MAP), or ovalbumin (OVA).

[0098] In one aspect the invention relates to a cyclic 3D epitope or a pharmaceutical composition according to the invention for use in a method for vaccination of an individual.

[0099] A further aspect of the present invention relates to a method for vaccinating an individual in need thereof comprising the step of administering a therapeutically effective amount of a cyclic 3D epitope or a pharmaceutical composition according to the present invention.

[0100] The present invention also relates to a cyclic 3D epitope or a pharmaceutical composition according to the invention for use in generating antibodies, in particular neutralizing antibodies.

[0101] In some embodiments, the present invention also relates to a method for the generation of antibodies, in particular neutralizing antibodies, in an individual in need thereof comprising administering a therapeutically effective amount of a cyclic 3D epitope or a pharmaceutical composition according to the present invention.

[0102] Another aspect of the invention relates to a cyclic 3D epitope or a pharmaceutical composition according to the invention for use in eliciting an immune response in an individual.

[0103] A further aspect of the present invention relates to a method for inducing an immune response in an individual in need thereof comprising the step of administering a therapeutically effective amount of a cyclic 3D epitope or a pharmaceutical composition according to the present invention.

[0104] In some embodiments, the individual in need is susceptible to an infectious disease or cancer. [Brief description of the drawings]

[0105] [Figure 1] FIG. 1 is a schematic diagram of the SARS-CoV-2 virus and SARS-CoV-2 virus particle showing the S (spike) protein. [Diagram 2] Scheme showing epitope sequences that correspond to only 5% of the S (spike) protein and are part of the receptor binding domain (RBD) that is highly conserved in all SARS-CoV-2 strains. "*" indicates the position of identical amino acid residues. ":" indicates the position of amino acid residues that share similar properties (polarity or charge). [Diagram 3]Scheme showing the structure of Mimocov2 peptide (SEQ ID NO: 3). The primary structure corresponds to highly conserved sequences in SARS-CoV-1 and SARS-CoV-2 RBD, which directly interacts with the ACE2 receptor. "*" indicates amino acid substitutions introduced to allow disulfide bridges and stabilize loop structures. The N-terminal type 1 beta turn and the C-terminal loop are the main secondary structures. The 3D structure was determined by the atomic coordinates of SARS-CoV-1, and disulfide bridges were generated only after energy minimization.

[0106] Working Example The present invention is further illustrated by the following examples.

[0107] Example 1: Design of 3D circular epitopes against the S (spike) protein of SARS-CoV-2 In late December 2019, multiple cases of pneumonia of unknown origin were reported from China and in early January 2020, it was announced to be caused by a novel coronavirus. The emerging SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2) is responsible for the ongoing outbreak of coronavirus disease 2019 (COVID19) (Walls et al., 2020). Even if an efficient drug treatment is found to cure COVID19, only a vaccine could rapidly eradicate the transmission of this virus. A vaccine is the only alternative to containment to limit deaths from this virus.

[0108] Despite extensive testing of the disease in China, the virus spread worldwide and COVID-19 was declared a pandemic by the World Health Organization (WHO) in March 2020. COVID-19 has induced more than 111 million cases and 2.46 million deaths worldwide in the last WHO report on COVID-19 (March 2020). A study in Lombardy (Italy) on a cohort of volunteers (n=1591) who were critically ill and admitted to public hospitals between February 20 and March 18, 2020, showed a global mortality rate of 26%, mostly male. Mortality was shown to be 35% in volunteers over 63 years old and 15% in those under 64 years old (Grasseli et al., 2020).

[0109] One of the main envelope proteins of the SARS-CoV-2 virus is called the S (spike) protein (Figure 1). Infected volunteers can generate antibodies against the nucleocapsid N protein and / or the S protein, but only antibodies eliciting the receptor binding domain (RBD) at the top of the S protein are neutralizing, i.e., these antibodies help cure from COVID-19.

[0110] The SARS-CoV-2 S (spike) protein interacts with a receptor called ACE2 for angiotensin-converting enzyme 2 to gain entry into human cells (Coutard et al., 2020 & Walls et al., 2020). Compared to other coronaviruses, the high affinity of the receptor-binding domain (RBD) of the SARS-CoV-2 S (spike) protein to the human ACE2 receptor may be responsible for the rapid viral spread of SARS-CoV-2 in humans (Ortega et al., 2020). A preventive vaccine must elicit an IgG response against the RBD sequence to neutralize human cell infection.

[0111] Synthetic peptides obtained with Fmoc chemistry are the safest vaccine approach and are currently used in more than 60 vaccines against SARS-CoV-2. Synthetic vaccines are sterile and can be stable for 3 years after lyophilization. Short peptides (less than 30 residues) are economically viable for mass vaccination. However, peptides have an unstable structure (or random coil) and are useful only for linear epitopes.

[0112] The first step for the design of a vaccine against SARS-CoV-2 (also called Mimocov2 vaccine) was to identify structural motifs in the SARS-CoV-2 RBD that bind to the ACE2 receptor (step a). Having made this design, but since the 3D structure of ACE2 with SARS-CoV-2 was still not published, we used the PDB file corresponding to the X-ray structure of the ACE2 receptor with the S protein of SARS-CoV-1 (Shang et al., 2020). After sequence alignment analysis between the SARS-CoV-1 and SARS-CoV-2 sequences (SEQ ID NO:1), it became possible to identify highly conserved regions in both SARS-CoV-1 and SARS-CoV-2 that interact with the ACE2 receptor. InsightII® Analysis of the Homology pulldown revealed that the conserved region of SARS-CoV-2 RBD matches the highly conserved region of SARS-CoV-1 and SARS-CoV-2 variants from the S (spike) protein, from amino acid residue 483 to amino acid residue 509 (peptide of SEQ ID NO:2: FIG. 2), as shown in Figure 2. This sequence exhibits low variability, likely because it corresponds to a sequence essential for these two viruses to enter human cells.

[0113] The sequence of the identified peptide has two structural motifs made up of a beta turn at the two ends (amino acid residue 483 to amino acid residue 491) and a beta turn at the other end (amino acid residue 506 to amino acid residue 509). The MERS-CoV virus has an insertion in the middle of this sequence between these two structural motifs. This insertion probably reduces the affinity to the ACE2 receptor. The first loop, strictly conserved among the three viruses, is of course a structural motif essential to fit into the ACE2 binding site.

[0114] Molecular modeling was carried out on the conserved region containing the two structural motifs forming the loop (step 3) using the MIMOFF equations to calculate the force field. In this loop, preserving the 3D structure, molecular modeling provides a model in which the structure is hardened by a disulfide bridge between cysteine ​​at position 497 and cysteine ​​at position 507. Molecular modeling teaches that it is not possible to create a disulfide bridge with cysteine ​​at position 488 to another part of the sequence. Therefore, to fix the loop structure, the only possibility was to carry out two amino acid substitutions, namely Phe497Cys and Pro507Cys, as shown in FIG. 3. To avoid different possible disulfide bridges, a third substitution was introduced, namely Cys488Ser. The side chain of serine at position 488 is very close to the cysteine ​​with an -OH group instead of -SH, providing the same constraint on the dihedral angles. The resulting peptide was named Mimocov2 peptide (SEQ ID NO: 3).

[0115] The atomic coordinates of SARS-CoV-1 (amino acid residue 483 to amino acid residue 509) were used as a template to determine the atomic coordinates of the Mimocov2 peptide using Homology from InsightII® software. From the X-ray structure of the ACE2 receptor and the S protein of SARS-CoV-1 (Shang et al., 2020), it was possible to model a peptide of less than 30 residues covering a loop of the SARS-CoV-2 S (spike) protein that interacts with the ACE2 receptor. This peptide belongs to the RBD. The peptide may overlap the backbone of the loop of the SARS-CoV-2 S protein. The disulfide bridge introduced within the peptide blocks the structure as it is in SARS-CoV-2. Only one box containing amino acid residue 483 to amino acid residue 509 was used to determine the atomic coordinates. Then, using Discover (InsightII®), energy minimization cycles were performed with free cysteines to determine potential structural changes due to the three substitutions. The Covalent Valence Force Field (CVFF) in the algorithm Steepest Descent® was used with the free cysteines so as not to induce constraints. After three minimization cycles and minimal variations with three different side chains, the two -SH side chains of cysteines were still in suitable positions to create disulfide bridges.

[0116] The resulting peptide, named Mimocov2 peptide (SEQ ID NO: 3), was thus confirmed to be a linear amino acid sequence capable of assembling the conserved regions of the SARS-CoV-2 RBD that bind to the ACE2 receptor (step c). Its cyclization in silico results in the modeled cyclic 3D epitope.

[0117] Example 2: Synthesis of Mimocov2 peptide (sequence SEQ ID NO:3) Mimocov2 peptide synthesis was carried out by Fmoc / tBu chemistry using the protecting groups Trt for Cys(c), Asn(N) and Gln(q) and tBu for Glu, Ser(s), Thr(t) and Tyr(Y). For chemical synthesis, Fmoc was deprotected with piperidine / DMF (25 / 75).

[0118] Amino acid residues were coupled with HCTU and NMM in the ratio A / HCTU / NMM (5eq / 5eq / 10eq). Peptide deprotection was carried out with TFA / water / DTT (90 / 5 / 5).

[0119] The synthesized peptides were analyzed by HPLC on a Symmetry C18 column, 100 Å, 5 μm, 4.6 mm×250 mm (Waters®) according to the manufacturer's instructions. The injection volume was 10 μL and the elution buffers A / B were: - A: 0.1% trifluoroacetic acid (TFA) in H2O; - B: 0.1% TFA in acetonitrile It was.

[0120] The results are presented in Table 1 below. [Table 1]

[0121] The Mimocov2 peptide was shown to be highly stable upon synthesis.

[0122] Example 3: Vaccination with Mimocov2 peptides Studies in animal models showed no toxicity.

[0123] Animal studies show that the Mimocov2 vaccine was able to induce an immune response against the RBD detectable by the Siemens test, and the IgG immune response was still detectable after six months and was high enough to provide protection against SARS-CoV-2 infection.

[0124] Sequences used herein SEQ ID NO:1 (YP_009724390.1 Surface glycoprotein of severe acute respiratory syndrome coronavirus 2)

[0125] SEQ ID NO:2 (domain of the S protein responsible for interaction with the ACE2 receptor) EGFNCYFPLQSYGFQPTNGVGYQPYR

[0126] SEQ ID NO:3 (Mimocov2 peptide according to the invention) EGFNSYFPLQSYGCQPTNGVGYQCYR

Claims

1. 1. A method for modeling a circular 3D epitope from the surface of a target antigen, comprising: a) identifying in a 3D model representation of the target antigen one or more conserved region(s) likely to be / constitute a 3D epitope, said one or more conserved region(s) having one or more charged amino acid(s) selected from the group consisting of arginine (Arg), lysine (Lys), histidine (His), aspartic acid (Asp) and glutamic acid (Glu); b) performing molecular modeling on said one or more conserved region(s) identified in step a) to calculate the force field energy according to the following equation (Equation E): [0010] where E is the energy of the force field, r is the radius between the two covalently bonded atoms, and r ０ is the ideal van der Waals radius at 298 K, θ is the valence angle between two covalent bonds, φ is the dihedral angle of the SP3 carbon, n is the multiplicity or periodicity of said dihedral angle, the parameters Hφ and Hθ are the respective force constants, the variables with subscript 0 are the respective equilibrium values, A is the distance between two atoms, B is the double distance between two non-bonded atoms, q is the atomic charge, and D is the Debie constant. providing a linear amino acid sequence that is amenable to assembling the one or more conserved region(s) within the 3D epitope using c) providing a linear amino acid sequence that is amenable to assembling said one or more conserved region(s) within said 3D epitope; d) in silico cyclizing said linear amino acid sequence to mimic said 3D epitope as it exists in said target antigen; e) obtaining a modeled circular 3D epitope; A method comprising:

2. The method of claim 1, wherein said cyclic 3D epitope is a mimotope.

3. The method of claim 1, wherein the cyclic 3D epitope is synthetic.

4. 2. The method of claim 1, wherein the target antigen comprises or is selected from the group consisting of a bacterial antigen, a viral antigen and a cancer antigen.

5. 2. The method of claim 1, wherein step a) is performed by aligning homologous and / or mutant sequences of said one or more conserved region(s) to obtain atomic coordinates.

6. 2. The method of claim 1, wherein the one or more conserved region(s) is / are on a surface of the 3D model representation of the target antigen.

7. 2. The method of claim 1, wherein step b) comprises performing one or more cycles of energy minimization of the force field guided by the atomic coordinates, in particular by one or more energy minimization algorithms, said energy minimization being achieved by utilizing derivatives of the equations used in step b).

8. The method of claim 7 , wherein the energy minimization algorithm(s) is associated with a molecular dynamics step.

9. The method according to claim 1, wherein step d) is carried out chemically, in particular by site-specific cross-linking.

10. 2. The method of claim 1, wherein step d) is carried out by substituting two amino acid residues in the linear sequence obtained in step c) with cysteine ​​residues, the dihedral angles between each of the alpha carbons of each of the cysteine ​​residues and any one of the adjacent amino acid residues being compatible for the formation of a disulfide bridge.

11. Circular 3D epitopes obtained by synthesis of cyclic 3D epitopes modelled by the method of claim 1.

12. A pharmaceutical or vaccine composition comprising the cyclic 3D epitope of claim 11.

13. 12. A pharmaceutical composition for use in a method of vaccination of an individual, comprising a cyclic 3D epitope as defined in claim 11.

14. A pharmaceutical composition for use in the generation of antibodies, particularly neutralizing antibodies, comprising a cyclic 3D epitope as defined in claim 11.

15. 12. A pharmaceutical composition for use in eliciting an immune response in an individual, the pharmaceutical composition comprising a cyclic 3D epitope according to claim 11.

Images