WO2023111728A1 - Immunogenic peptides against canine distemper virus (cdv) - Google Patents

Immunogenic peptides against canine distemper virus (cdv) Download PDF

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WO2023111728A1
WO2023111728A1 PCT/IB2022/061236 IB2022061236W WO2023111728A1 WO 2023111728 A1 WO2023111728 A1 WO 2023111728A1 IB 2022061236 W IB2022061236 W IB 2022061236W WO 2023111728 A1 WO2023111728 A1 WO 2023111728A1
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amino acid
seq
peptides
cdv
positively charged
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French (fr)
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Santiago RENDON MARÍN
Julian RUIZ SAENZ
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Universidad Cooperativa De Colombia
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A61K39/175—Canine distemper virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present development is in the field of immunology and veterinary medicine, in particular it is related to the design of immunogenic peptides against the Canine Distemper Virus and compositions that comprise them, useful for the generation of immunity against said virus in wild fauna and domestic.
  • CDV Canine Distemper Virus
  • Carré's disease or distemper a highly contagious disease known as Carré's disease or distemper, which affects domestic dogs, as well as a wide variety of wild species.
  • Distemper is a disease with worldwide distribution that produces respiratory, digestive, skin and neurological symptoms, among others. There is no specific treatment for the disease and most efforts are focused on prevention by administering two or more doses of a vaccine between the sixth or seventh week of age to three or four months, followed by re-vaccination. annually throughout the life of the animal.
  • CDV has a high genomic substitution rate, which means that the circulating variants in the field differ by more than 10% compared to the strains used in vaccines. This implies consequences in the development of immunity and the appearance of the disease even in previously vaccinated animals, in addition to the worldwide re-emergence of infection in wildlife, for which the safety of commercially available vaccines has not been proven.
  • CDV belongs to the Morbillivirus genus of the paramyxovirus family and contains a single-stranded RNA genome with negative polarity that encodes 8 proteins, including the Hemagglutinin (H) and the Fusion (F) protein.
  • H glycoprotein has great genetic variation, where it is estimated that the circulating variants in the field differ by 10% with those used in conventional vaccines.
  • These variations nucleotides can lead to changes in the amino acid sequence of the H protein and, therefore, involve modifications in its structure, which can have consequences on its antigenicity, since it is considered one of the main antigenic determinants of CDV.
  • Vaccines against CDV are described in the state of the art.
  • US Pat. No. 8,647,637 describes immunogenic compositions and broad-spectrum vaccines containing newly identified CDV isolates collected from a given geographic area. These isolates exhibit attributes of both the European wild lineage and one or both of the Arctic and American-2 lineages of CDV. Additionally, this patent relates to vaccines based on nucleic acids encoding antigenic peptides or proteins, or antigenic epitopes or regions of peptides or proteins.
  • patent US 10076566 describes vectors that contain and express CDV polypeptides or antigens in vivo or in vitro, which generate an immune response in an animal against CDV. Also illustrated are compositions comprising such vectors and/or polypeptides and methods of vaccination against CDV, as well as methods for inducing an immunogenic or protective response against CDV and other canine viruses.
  • the present development refers to immunogenic peptides against CDV, designed by means of computational tools and immunogenic compositions that comprise said peptides, useful as a universal vaccine alternative to prevent CDV contagion in wild and domestic fauna.
  • FIG. 1 Schematic representation of the procedure for the design of immunogenic peptides against CDV.
  • FIG. 2 A Epitopes obtained from consensus CDV H protein
  • B Epitopes obtained from the consensus CDV F protein.
  • FIG. 3 Peptide sizes predicted from the consensus sequence of the CDV proteins.
  • the present development is based on the evaluation of the immunogenic potential of peptides designed from the genetic information of circulating CDV strains in the field, using computational tools.
  • consensus sequences were initially generated for CDV H and F proteins, which are considered the main antigenic determinants of the virus, added to their location in the viral joint, which are targets of humoral immunity, mediated by antibodies, based on the sequence of all lineages reported for this virus worldwide.
  • Xi is an amino acid selected from a positively charged amino acid, an uncharged amino acid, a special amino acid, and a hydrophobic amino acid;
  • X2 is three the same or different amino acids, selected from a positively charged amino acid, a negatively charged amino acid, an uncharged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid;
  • X3 is an amino acid, selected from a positively charged amino acid, an uncharged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid;
  • X4 is two the same or different amino acids, selected from a positively charged amino acid, a negatively charged amino acid, an uncharged amino acid, a hydrophobic amino acid, and an aromatic amino acid;
  • X5 is an amino acid selected from a positively charged amino acid, a negatively charged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid;
  • X7 is absent or is an amino acid selected from a positively charged amino acid, an uncharged amino acid, a hydrophobic amino acid, and an aromatic amino acid;
  • X7 is Xi or absent
  • Xg is absent or is one or two of the same or different amino acids selected from a positively charged amino acid and a hydrophobic amino acid, wherein the positively charged amino acid is selected from the group consisting of R, H, and K; the negatively charged amino acid is selected from the group consisting of D and E; the uncharged amino acid is selected from the group consisting of S, T, N, and Q; the special amino acid is selected from the group consisting of C, G, and P; the hydrophobic amino acid is selected from the group consisting of A, I, L, M, and V, and the aromatic amino acid is selected from the group consisting of W, Y, and F.
  • the positively charged amino acid is selected from the group consisting of R, H, and K
  • the negatively charged amino acid is selected from the group consisting of D and E
  • the uncharged amino acid is selected from the group consisting of S, T, N, and Q
  • the special amino acid is selected from the group consisting of C, G, and P
  • the hydrophobic amino acid is selected
  • the peptides of the present disclosure include amino terminal modifications such as biotinylation, acetylation, fatty acids for lipopeptide production, and PEGylation.
  • the peptides include carboxyl terminal modifications such as amidation.
  • the present disclosure refers to the immunogenic peptides against CDV that are shown in Table 1: Table 1. Amino acid sequences of designed immunogenic peptides.
  • the present disclosure also relates to the nucleic acid molecules encoding the CDV immunogenic peptides shown in Table 2.
  • the present disclosure relates to an immunogenic composition
  • an immunogenic composition comprising one or more immunogenic peptides against CDV and pharmaceutically acceptable excipients.
  • the immunogenic composition is a universal vaccine that can be applied to CDV-susceptible wildlife species in an effective and safe manner.
  • the excipients of the immunogenic composition are selected, not limited to the group consisting of preservatives, buffers, stabilizers, salts and surfactants.
  • the preservative in a particular modality, as a preservative it is used, without being limited to benzyl alcohol. In another particular modality, the preservative is in a concentration between 20 and 30 pg/ml.
  • the buffer in a particular modality, as a buffer it is used, without being limited to phosphate buffer.
  • the buffer is at a concentration between 5 and 100 nM.
  • glycerol is used as stabilizer, without being limited to.
  • the stabilizer is in a concentration between 0 and 10%.
  • aluminum hydroxide is used as the salt, without being limited to.
  • the salt is at a concentration between 0 and 300 nM.
  • polysorbate 20-80 is used as a surfactant, without being limited to.
  • the surfactant is in a concentration between 0.01 and 0.1% w/v.
  • CDV H and F protein sequences belonging to the lineages reported worldwide, were converted into a single multiphase file. Additionally, a multicast file of the sequences circulating in Colombia was made. On the other hand, sequences of canine MHC class I and II molecules and human template structures were downloaded from UniProt and Protein Data Bank (PDB), to be modeled based on the crystallographic structure of a human protein.
  • PDB UniProt and Protein Data Bank
  • CDV H and F protein sequences were performed using the Clustal Omega online tool (https://www.ebi.ac.uk/Tools/msa/clustalo/), in order to establish differences. between each of the sequences of this protein and the lineages that circulate worldwide. In addition, the generation of the generation of the p matrix to be able to quantify the differences by means of Mega 10. On the other hand, an alignment was generated for the CDV H and F protein of the variants of the lineages that co-circulate in Colombia. Finally, 4 consensus sequences were generated using the online tool https://www.ebi. ac.uk/Tools/msa/emboss cons/. with which the prediction analyzes were performed.
  • Example 3 Construction of a library of potentially immunogenic peptides of consensus sequences.
  • CTL cytotoxic T lymphocytes
  • the CTL tool integrates different prediction tools, with databases that report epitopes for different alleles of human leukocyte antigens (HLA for its acronym in English - human leukocyte antigens) class I.
  • This tool generates peptides based on different approximations, which take into account different mathematical models, Support Vector Machine (SVM) with a cut off of 1.2 (maximum 1.5), Artificial Nueral Network (ANN), with a cut off of 0.95 (value between 0 and 1) and a combination of these models, which allows obtaining immunogenic potentials.
  • SVM Support Vector Machine
  • ANN Artificial Nueral Network
  • This tool allows to determine linear peptides for B lymphocytes, which allow the direct detection of antigens.
  • This tool has a limit in the search, since it has a window of specific peptides. In this case those of 10 amino acids were predicted.
  • This tool can predict peptides of 12, 14, 16, 18 and 20 amino acids.
  • Con-1 has 4 sequences of 10 amino acids, which have the potential to be epitopes for B lymphocytes.
  • Con-3 has 3 sequences of 10 amino acids, which have the potential to be epitopes for B lymphocytes.
  • This tool makes it possible to determine epitopes for the various human alleles of MHC-II molecules. For each allele the top 10 was marked, however, the threshold value greater than 1.5 was considered for potential peptides, with the intention of being more stringent.
  • the alpha chain of the sequence of a canine MHC-I (DLA-I-88), (model_l), was modeled, since this is the only one that interacts with the peptides with immunogenic potential.
  • the template used is a canine HSC-I deposited in the PDB, 5F1N, which comes from a crystallization of canine molecules.
  • DLA-DR canine MHC-II of the DR allele
  • Model_2 and Model_3 respectively.
  • the 3D structure reported in the PDB, 4FQX was used as a template for both the alpha and beta chains.
  • This structure is from humans, from the same allele of the sequence that is found for canines (HLA-DR).
  • HLA-DR canines
  • the calculation of the identity of the templates was performed with respect to each of the modeled molecules based on their sequence.
  • Homology modeling was performed using the Modeller 9.24 software.
  • Model_l Model 57, which obtained the best score.
  • Model_2 Model 76, which obtained the best score.
  • Model_3 Model 7, which obtained the best score.
  • Model_2 and Model o_3 were put into a file as a complex based on the template structure, in order to evaluate the molecular docking.
  • This strategy allows determine the ability of two molecules to interact, through different non-covalent bonds such as hydrogen bonds and Van de Waals forces.
  • the prediction is made without delimiting an interaction site; instead, the tools present the most probable interaction region, based on algorithms that can predict that molecular pose.
  • Model_l The best models for each of the three proteins (Model_l, Model_2 and Model_3) were validated using three different computational tools, ProsaWeb, Swissmodel and TM-Align, in order to know if the models could be used in subsequent analyzes of molecular docking. with the predicted peptides.
  • a synthesis of the validation data for the three proteins is shown below. This table also includes the data of the molds to be able to establish a comparison with the data obtained for the models.
  • Figures 3 A and B show the size of the peptides predicted from the consensus sequence of CDV H and F proteins, respectively.
  • Example 7 Modeling of some peptides in their three-dimensional structure using PEPFOLD3.
  • the peptides that obtained the best scores were modeled using the PEPFOLD3 tool in order to perform molecular coupling of these peptides with the canine MHC molecules modeled in Example 5.
  • Peptides with immunogenic potential for B cells, or to be presented in the class 1 or class 2 context of MHC molecules were determined using the IEDB tool. After submitting the sequences, more than a thousand peptides were obtained. This tool is supplied with the consensus sequence of all the lineages of both the H and F proteins.
  • peptides from both the H protein and the F protein were selected, which had appropriate physicochemical properties of stability and half-life, as well as the best prediction values of each of the tools, in order to validate in silico, a smaller group of peptides and to be able to take these to the next steps of development.
  • the initially selected peptides are shown in Table 1.
  • Example 9 Molecular coupling of the selected peptides with the canine MHC corresponding to the molecule that presents it (class I, II) by means of CABSDOCK.
  • Each of the 12 peptides was subjected to molecular docking analysis using CABSDOCK, with the respective target molecule (either class I or class II, depending on its prediction), on the online platform. Both files are uploaded separately and after the analysis time, the tool delivers the most probable pose in which both molecules interact, taking into account a clustering analysis.
  • Each of the 12 peptides was subjected to molecular docking analysis using HPEPDOCK, with the respective target molecule (either class I or class II, depending on its prediction), on the online platform. Both files are uploaded separately and after the analysis time, the tool delivers the most probable pose in which both molecules interact, taking into account an analysis of the most probable pose.
  • Each of the 12 peptides was subjected to molecular docking analysis using CABSDOCK, with the respective target molecule (either class I or class II, depending on its prediction), on the online platform. Both files are uploaded separately and after the analysis time, the tool delivers the most probable pose in which both molecules interact, taking into account an analysis of the most probable pose.
  • Each of the 12 peptides was subjected to molecular docking analysis using HPEPDOCK, with the target molecule (TLR-2 or TLR-4), on the online platform. Both files are uploaded separately and after analysis time, the tool returns the most probable pose in which both molecules interact, taking into account an analysis of the most probable pose.
  • Example 10 In silico safety tests a- Blastp analysis of selected peptides to determine self homologous peptides.
  • Whether the peptides were potential viral antigens was determined by a machine learning scoring analysis, compared to other peptides for which there is experimental evidence of their antigenicity. c- ToxinPred analysis of the selected peptides to determine if the peptides are toxic compounds.

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Abstract

The present invention relates to immunogenic peptides against canine distemper virus (CDV) having the sequence X1-X2-X3-X4-X3-X5-X6-X7-X8, designed using computational tools, and immunogenic compositions comprising the peptides, which are useful as a universal vaccine alternative for preventing CDV infection in wild and domestic animals.

Description

PÉPTIDOS INMUNOGÉNICOS CONTRA EL VIRUS DISTEMPER CANINO (CDV) IMMUNOGENIC PEPTIDES AGAINST CANINE DISTEMPER VIRUS (CDV)
CAMPO TÉCNICO TECHNICAL FIELD
El presente desarrollo se encuentra en el campo de la inmunología y medicina veterinaria, en particular se relaciona con el diseño de péptidos inmunogénicos contra el Virus Distemper Canino y composiciones que los comprenden, útiles para la generación de inmunidad frente a dicho virus en fauna silvestre y doméstica. The present development is in the field of immunology and veterinary medicine, in particular it is related to the design of immunogenic peptides against the Canine Distemper Virus and compositions that comprise them, useful for the generation of immunity against said virus in wild fauna and domestic.
DESCRIPCIÓN DEL ESTADO DE LA TÉCNICA DESCRIPTION OF THE STATE OF THE ART
El Virus del Distemper Canino (CDV por su sigla en inglés Canine Distemper Virus), es el agente etiológico de una enfermedad altamente contagiosa conocida como enfermedad de Carré o moquillo, que afecta perros domésticos, así como una gran variedad de especies silvestres. El distemper es una enfermedad de distribución mundial que produce síntomas respiratorios, digestivos, cutáneos y neurológicos entre otros. No existe un tratamiento específico para la enfermedad y la mayoría de los esfuerzos se concentran en la prevención mediante la administración de dos o más dosis de una vacuna entre la sexta o séptima semana de edad hasta los tres o cuatro meses, seguida de re-vacunación anual durante toda la vida del animal . The Canine Distemper Virus (CDV) is the etiological agent of a highly contagious disease known as Carré's disease or distemper, which affects domestic dogs, as well as a wide variety of wild species. Distemper is a disease with worldwide distribution that produces respiratory, digestive, skin and neurological symptoms, among others. There is no specific treatment for the disease and most efforts are focused on prevention by administering two or more doses of a vaccine between the sixth or seventh week of age to three or four months, followed by re-vaccination. annually throughout the life of the animal.
El CDV tiene una alta tasa de sustitución genómica, lo que hace que las variantes circulantes en campo difieran en más del 10% frente a las cepas utilizadas en vacunas. Esto implica consecuencias en el desarrollo de la inmunidad y la aparición de la enfermedad incluso en animales previamente vacunados, además de la re-emergencia mundial de la infección en fauna silvestre, para la cual no se ha comprobado la seguridad de las vacunas disponibles comercialmente. CDV has a high genomic substitution rate, which means that the circulating variants in the field differ by more than 10% compared to the strains used in vaccines. This implies consequences in the development of immunity and the appearance of the disease even in previously vaccinated animals, in addition to the worldwide re-emergence of infection in wildlife, for which the safety of commercially available vaccines has not been proven.
El CDV pertenece al género Morbillivirus de la familia paramyxovirus y contiene un genoma RNA de cadena sencilla con polaridad negativa que codifica 8 proteínas, incluidas la Hemaglutinina (H) y la proteína de Fusión (F). La glicoproteína H posee gran variación genética, donde se estima que las variantes circulantes en campo difieren en un 10% con las utilizadas en las vacunas convencionales. Estas variaciones nucleotídicas pueden llevar a cambios en la secuencia de aminoácidos de la proteína H y, por ende, implicar modificaciones en su estructura, los cuales pueden traer consecuencias en la antigenicidad de esta, ya que es considerada uno de los principales determinantes antigénicos de CDV. El efecto de la variabilidad de esta proteína entre las diferentes variantes de CDV distribuidas a nivel mundial, trae consigo la necesidad de desarrollar estrategias vacunales que permitan una cobertura universal. Por tal razón, es preponderante la determinación de secuencias consenso de los epitopes inmunodominantes, con el fin de obtener dichas coberturas. CDV belongs to the Morbillivirus genus of the paramyxovirus family and contains a single-stranded RNA genome with negative polarity that encodes 8 proteins, including the Hemagglutinin (H) and the Fusion (F) protein. The H glycoprotein has great genetic variation, where it is estimated that the circulating variants in the field differ by 10% with those used in conventional vaccines. These variations nucleotides can lead to changes in the amino acid sequence of the H protein and, therefore, involve modifications in its structure, which can have consequences on its antigenicity, since it is considered one of the main antigenic determinants of CDV. The effect of the variability of this protein among the different CDV variants distributed worldwide, brings with it the need to develop vaccination strategies that allow universal coverage. For this reason, the determination of consensus sequences of immunodominant epitopes is paramount, in order to obtain such coverage.
Por lo anterior, se considera que el diseño de péptidos mediante herramientas de predicción de epitopes inmunogénicos con base en secuencias consenso, que tome como suministro la información genética y antigénica de las variantes circulantes alrededor del mundo, puede dar como resultado una alternativa inmunogénica efectiva y segura para el desarrollo de una vacuna universal que genere inmunidad contra los diferentes linajes del virus, que pueda ser aplicada a las especies susceptibles tanto de fauna doméstica como silvestre. Therefore, it is considered that the design of peptides using immunogenic epitope prediction tools based on consensus sequences, which take as a supply the genetic and antigenic information of the circulating variants around the world, can result in an effective and immunogenic alternative. safe for the development of a universal vaccine that generates immunity against the different lineages of the virus, which can be applied to susceptible species of both domestic and wild fauna.
En el estado de la técnica se han descrito composiciones inmunogénicas contra el CDV que incluyen aislamientos atenuados del CDV, ácidos nucleicos que codifican proteínas antigénicas y vectores que expresan antígenos del CDV, entre otras estrategias. A la fecha, se cuenta con algunas alternativas vacunales. Se conocen vacunas de tipo de virus vivo modificado (MLV, del inglés, modified live virus), la cual fue propuesta en la década de 1960, con base en una cepa ancestral, denominada Onderstepoort. Por tanto, considerando que esta variante incluso ya no circula en campo, la inmunidad generada por esta vacuna pueda disminuir para los demás linajes distribuidos a nivel mundial, además que no pueden ser utilizadas en ninguna especie para la cual no ha sido probada la seguridad, por tener la atenuación como plataforma de obtención. Existe una vacuna recombinante, que utiliza un esqueleto de virus de la viruela del canario (Canary pox), que expresa las proteínas H y F de CDV, la cual protege contra el desarrollo de distemper sintomático. Además, se cuenta con una plataforma de nueva generación, considerada una vacuna bivalente recombinante, que emplea el virus de la rabia, que expresa las proteínas H y F, la cual ha sido probada en caninos domésticos y hurones. Uno de los problemas que pueden enfrentar todas las plataformas vacunales disponibles, es la emergencia de variantes distribuidas geográficamente, las cuales poseen diferencias de aminoácidosen los principales determinantes antigénicos, lo cual pude llevar a la generación de una respuesta inmunológica insuficiente frente a las variantes de campo. In the state of the art, immunogenic compositions against CDV have been described that include attenuated CDV isolates, nucleic acids encoding antigenic proteins and vectors that express CDV antigens, among other strategies. To date, there are some vaccine alternatives. Modified Live Virus (MLV) type vaccines are known, which were proposed in the 1960s, based on an ancestral strain, called Onderstepoort. Therefore, considering that this variant is no longer circulating in the field, the immunity generated by this vaccine may decrease for the other lineages distributed worldwide, in addition to the fact that they cannot be used in any species for which safety has not been proven. for having attenuation as a platform for obtaining. There is a recombinant vaccine, which uses a skeleton of the canarypox virus (Canary pox), which expresses the H and F proteins of CDV, which protects against the development of symptomatic distemper. In addition, there is a new generation platform, considered a recombinant bivalent vaccine, which uses the rabies virus, which expresses the H and F proteins, which has been tested in domestic dogs and ferrets. One of the Problems that all available vaccine platforms can face is the emergence of geographically distributed variants, which have amino acid differences in the main antigenic determinants, which could lead to the generation of an insufficient immune response against field variants.
En el estado de la técnica se describen vacunas contra el CDV. Por ejemplo, la patente US 8647637 describe composiciones inmunogénicas y vacunas de amplio espectro que contienen aislamientos recientemente identificados del CDV, recolectados en un área geográfica determinada. Estos aislamientos exhiben atributos tanto del linaje silvestre europeo como de uno o ambos de los linajes ártico y americano-2 del CDV. Adicionalmente, esta patente se relaciona con vacunas basadas en ácidos nucleicos que codifican péptidos antigénicos o proteínas, o epitopes antigénicos o regiones de péptidos o proteínas. Vaccines against CDV are described in the state of the art. For example, US Pat. No. 8,647,637 describes immunogenic compositions and broad-spectrum vaccines containing newly identified CDV isolates collected from a given geographic area. These isolates exhibit attributes of both the European wild lineage and one or both of the Arctic and American-2 lineages of CDV. Additionally, this patent relates to vaccines based on nucleic acids encoding antigenic peptides or proteins, or antigenic epitopes or regions of peptides or proteins.
Por otra parte, la patente US 10076566 describe vectores que contienen y expresan polipéptidos o antígenos CDV in vivo o in vitro, los cuales generan una respuesta inmune en un animal contra el CDV. También se ilustran composiciones que comprenden dichos vectores y /o polipéptidos y métodos de vacunación contra el CDV, así como métodos para inducir una respuesta inmunogénica o protectora contra el CDV y otros virus caninos. On the other hand, patent US 10076566 describes vectors that contain and express CDV polypeptides or antigens in vivo or in vitro, which generate an immune response in an animal against CDV. Also illustrated are compositions comprising such vectors and/or polypeptides and methods of vaccination against CDV, as well as methods for inducing an immunogenic or protective response against CDV and other canine viruses.
Sin embargo, persiste la necesidad de una alternativa para generar inmunidad contra la mayor cantidad de linajes del virus en una vacuna universal que pueda ser aplicada tanto en fauna silvestre como doméstica y sea eficaz y segura, pues los péptidos sintéticos de tamaño inferior a 20 aminoácidos han demostrado ser seguros en diferentes modelos in vitro e in vivo. Además, una vacuna que tenga como inmunógeno una subunidad (péptidos inmunogénicos), no posee la capacidad replicativa que, si tienen las vacunas de virus vivo modificado o recombinante, para las cuales es necesario probarles su seguridad en cada una de las especies. BREVE DESCRIPCIÓN However, there is still a need for an alternative to generate immunity against the largest number of virus lineages in a universal vaccine that can be applied to both wild and domestic fauna and is effective and safe, since synthetic peptides of less than 20 amino acids have been shown to be safe in different in vitro and in vivo models. In addition, a vaccine that has a subunit (immunogenic peptides) as immunogen does not have the replicative capacity of modified or recombinant live virus vaccines, for which it is necessary to prove their safety in each of the species. SHORT DESCRIPTION
El presente desarrollo se refiere a péptidos inmunogénicos contra el CDV, diseñados mediante herramientas computacionales y composiciones inmunogénicas que comprenden dichos péptidos, útiles como una alternativa de vacuna universal para prevenir el contagio por CDV en fauna silvestre y doméstica. The present development refers to immunogenic peptides against CDV, designed by means of computational tools and immunogenic compositions that comprise said peptides, useful as a universal vaccine alternative to prevent CDV contagion in wild and domestic fauna.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 Representación esquemática del procedimiento para el diseño de péptidos inmunogénicos contra el CDV. FIG. 1 Schematic representation of the procedure for the design of immunogenic peptides against CDV.
FIG. 2 A. Epitopes obtenidos de la proteína H de CDV consenso; B. Epitopes obtenidos de la proteína F de CDV consenso. FIG. 2 A. Epitopes obtained from consensus CDV H protein; B. Epitopes obtained from the consensus CDV F protein.
FIG. 3 Tamaño de péptidos predichos de la secuencia consenso de las proteínas de CDV. A. Proteína H; B. Proteína F FIG. 3 Peptide sizes predicted from the consensus sequence of the CDV proteins. A. H protein; B. F protein
DESCRIPCIÓN DETALLADA DETAILED DESCRIPTION
Para propósitos de interpretar los términos usados a lo largo del presente documento se debe tener en cuenta su significado usual en el campo técnico, a menos que se incorpore una definición particular o el contexto indique claramente lo contrario. Adicionalmente, los términos utilizados en forma singular también incluirán la forma plural. For purposes of interpreting the terms used throughout this document, their usual meaning in the technical field should be taken into account, unless a particular definition is incorporated or the context clearly indicates otherwise. Additionally, terms used in the singular form will also include the plural form.
El uso de herramientas computacionales se ha convertido en una pieza clave para la investigación y el desarrollo de vacunas para diversos modelos de infección, entre los cuales se incluyen las terapias basadas en péptidos. The use of computational tools has become a key element in the research and development of vaccines for various infection models, including peptide-based therapies.
El presente desarrollo se basa en la evaluación del potencial inmunogénico de péptidos diseñados a partir de la información genética de cepas de CDV circulantes en campo, mediante herramientas computacionales. Para el diseño de los péptidos inmunogénicos contra el CDV de la presente divulgación, inicialmente se generaron secuencias consenso para las proteínas H y F del CDV, las cuales son consideradas, los principales determinantes antigénicos del virus, sumado a su localización en la articula viral, las cuales son blanco de la inmunidad humoral, mediada por los anticuerpos, con base en la secuencia de todos los linajes reportados para este virus a nivel mundial. The present development is based on the evaluation of the immunogenic potential of peptides designed from the genetic information of circulating CDV strains in the field, using computational tools. For the design of the immunogenic peptides against CDV of the present disclosure, consensus sequences were initially generated for CDV H and F proteins, which are considered the main antigenic determinants of the virus, added to their location in the viral joint, which are targets of humoral immunity, mediated by antibodies, based on the sequence of all lineages reported for this virus worldwide.
Posteriormente, estas secuencias se sometieron a varias herramientas de predicción de epitopes inmunogénicos. De un total de 1402 péptidos, predichos para moléculas del complejo mayor de histocompatibilidad (CMH) clase 1, clase 2 y células B, con diferentes herramientas computacionales como MHC2Pred, SVMTriP, CTLPred e IEDB de la Jolla se eligieron los que mostraron mejor valor de predicción positivo como potenciales antígenos y sus características fisicoquímicas favorables. Subsequently, these sequences were subjected to various immunogenic epitope prediction tools. From a total of 1402 peptides, predicted for class 1, class 2 major histocompatibility complex (MHC) molecules, and B cells, with different computational tools such as MHC2Pred, SVMTriP, CTLPred, and La Jolla IEDB, the ones that showed the best value of positive prediction as potential antigens and their favorable physicochemical characteristics.
Luego, se evaluó la capacidad que estos tienen para interactuar con moléculas del CMH de caninos, así como con receptores tipo toll, mediante acoplamiento molecular y finalmente, se evaluó la seguridad in silico, mediante diferentes herramientas para determinar si estos eran buenos antígenos, podían ser tóxicos o alérgenos, además, si existían péptidos propios de los caninos que fueran homólogos a estos. Then, the ability of these to interact with canine MHC molecules, as well as toll-type receptors, was evaluated through molecular docking and finally, in silico safety was evaluated, through different tools to determine if these were good antigens, could be toxic or allergenic, in addition, if there were peptides of the canines that were homologous to them.
De acuerdo con el procedimiento de diseño descrito anteriormente, se seleccionaron péptidos inmunogénicos contra el CDV que tienen la secuencia According to the design procedure described above, immunogenic peptides against CDV having the sequence
X1-X2-X3-X4-X3-X5-X6-X7-X8 en donde, X1-X2-X3-X 4 -X3-X 5 -X 6 -X 7 -X 8 where,
Xi es un aminoácido seleccionado entre un aminoácido cargado positivamente, un aminoácido no cargado, un aminoácido especial y un aminoácido hidrofóbico; Xi is an amino acid selected from a positively charged amino acid, an uncharged amino acid, a special amino acid, and a hydrophobic amino acid;
X2 es tres aminoácidos iguales o diferentes, seleccionados entre un aminoácido cargado positivamente, un aminoácido cargado negativamente, un aminoácido no cargado, un aminoácido especial, un aminoácido hidrofóbico y un aminoácido aromático; X2 is three the same or different amino acids, selected from a positively charged amino acid, a negatively charged amino acid, an uncharged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X3 es un aminoácido, seleccionado entre un aminoácido cargado positivamente, un aminoácido no cargado, un aminoácido especial, un aminoácido hidrofóbico y un aminoácido aromático; X4 es dos aminoácidos iguales o diferentes, seleccionados entre un aminoácido cargado positivamente, un aminoácido cargado negativamente, un aminoácido no cargado, un aminoácido hidrofóbico y un aminoácido aromático; X3 is an amino acid, selected from a positively charged amino acid, an uncharged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid; X4 is two the same or different amino acids, selected from a positively charged amino acid, a negatively charged amino acid, an uncharged amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X5 es un aminoácido seleccionado entre un aminoácido cargado positivamente, un aminoácido cargado negativamente, un aminoácido especial, un aminoácido hidrofóbico y un aminoácido aromático; X5 is an amino acid selected from a positively charged amino acid, a negatively charged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X7, está ausente o es un aminoácido seleccionado entre un aminoácido cargado positivamente, un aminoácido no cargado, un aminoácido hidrofóbico y un aminoácido aromático; X7 is absent or is an amino acid selected from a positively charged amino acid, an uncharged amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X7 es Xi o está ausente; y X7 is Xi or absent; and
Xg está ausente o es uno o dos aminoácidos iguales o diferentes seleccionados entre un aminoácido cargado positivamente y un aminoácido hidrofóbico, en donde el aminoácido cargado positivamente se selecciona del grupo que consiste en R, H y K; el aminoácido cargado negativamente se selecciona del grupo que consiste en D y E; el aminoácido no cargado se selecciona del grupo que consiste en S, T, N y Q; el aminoácido especial se selecciona del grupo que consiste en C, G y P; el aminoácido hidrofóbico se selecciona del grupo que consiste en A, I, L, M y V, y el aminoácido aromático se selecciona del grupo que consiste en W, Y y F. Xg is absent or is one or two of the same or different amino acids selected from a positively charged amino acid and a hydrophobic amino acid, wherein the positively charged amino acid is selected from the group consisting of R, H, and K; the negatively charged amino acid is selected from the group consisting of D and E; the uncharged amino acid is selected from the group consisting of S, T, N, and Q; the special amino acid is selected from the group consisting of C, G, and P; the hydrophobic amino acid is selected from the group consisting of A, I, L, M, and V, and the aromatic amino acid is selected from the group consisting of W, Y, and F.
En una modalidad, los péptidos de la presente divulgación incluyen modificaciones amino terminal tales como biotinilación, acetilación, ácidos grasos para la obtención de lipopéptidos y PEGilación. In one embodiment, the peptides of the present disclosure include amino terminal modifications such as biotinylation, acetylation, fatty acids for lipopeptide production, and PEGylation.
En otra modalidad, los péptidos incluyen modificaciones carboxilo terminal tales como la amidación. In another embodiment, the peptides include carboxyl terminal modifications such as amidation.
En un aspecto particular, la presente divulgación se refiere a los péptidos inmunogénicos contra el CDV que se enseñan en la Tabla 1 : Tabla 1. Secuencias de aminoácidos de péptidos inmunogénicos diseñados
Figure imgf000008_0001
In a particular aspect, the present disclosure refers to the immunogenic peptides against CDV that are shown in Table 1: Table 1. Amino acid sequences of designed immunogenic peptides.
Figure imgf000008_0001
La presente divulgación también se refiere a las moléculas de ácidos nucleicos que codifican los péptidos inmunogénicos contra el CDV que se enseñan en la Tabla 2. The present disclosure also relates to the nucleic acid molecules encoding the CDV immunogenic peptides shown in Table 2.
Tabla 2. Secuencias de nucleótidos que codifican los péptidos inmunogénicos diseñados
Figure imgf000008_0002
Figure imgf000009_0001
Table 2. Nucleotide sequences encoding the designed immunogenic peptides.
Figure imgf000008_0002
Figure imgf000009_0001
En un aspecto adicional, la presente divulgación se refiere a una composición inmunogénica que comprende uno o más péptidos inmunogénicos contra el CDV y excipientes farmacéuticamente aceptables. En una modalidad particular, la composición inmunogénica es una vacuna universal que puede ser aplicada a especies de fauna doméstica y silvestre susceptibles al CDV de manera eficaz y segura. In a further aspect, the present disclosure relates to an immunogenic composition comprising one or more immunogenic peptides against CDV and pharmaceutically acceptable excipients. In a particular embodiment, the immunogenic composition is a universal vaccine that can be applied to CDV-susceptible wildlife species in an effective and safe manner.
Los excipientes de la composición inmunogénica se seleccionan, sin limitarse al grupo que consiste en preservantes, amortiguadores, estabilizantes, sales y surfactantes. The excipients of the immunogenic composition are selected, not limited to the group consisting of preservatives, buffers, stabilizers, salts and surfactants.
En una modalidad particular, como preservante se emplea, sin limitarse a alcohol bencílico. En otra modalidad particular, el preservante está en una concentración entre 20 y 30 pg/ml. In a particular modality, as a preservative it is used, without being limited to benzyl alcohol. In another particular modality, the preservative is in a concentration between 20 and 30 pg/ml.
En una modalidad particular, como amortiguador se emplea, sin limitarse a buffer de fosfatos. En otra modalidad particular el amortiguador está en una concentración entre 5 y 100 nM. En una modalidad particular, como estabilizante se emplea, sin limitarse a glicerol. En otra modalidad particular el estabilizante está en una concentración entre 0 y 10%. In a particular modality, as a buffer it is used, without being limited to phosphate buffer. In another particular embodiment, the buffer is at a concentration between 5 and 100 nM. In a particular modality, glycerol is used as stabilizer, without being limited to. In another particular modality, the stabilizer is in a concentration between 0 and 10%.
En una modalidad particular, como sal se emplea, sin limitarse a hidróxido de aluminio. En otra modalidad particular la sal está en una concentración entre 0 y 300 nM. In a particular embodiment, aluminum hydroxide is used as the salt, without being limited to. In another particular embodiment, the salt is at a concentration between 0 and 300 nM.
En una modalidad particular, como surfactante se emplea, sin limitarse a polisorbato 20- 80. En otra modalidad particular el surfactante está en una concentración entre 0,01 y 0, 1 % p/v. In a particular modality, polysorbate 20-80 is used as a surfactant, without being limited to. In another particular modality, the surfactant is in a concentration between 0.01 and 0.1% w/v.
La presente invención será presentada en detalle a través de los siguientes ejemplos, los cuales son suministrados solamente con propósitos ilustrativos y no con el objetivo de limitar su alcance. The present invention will be presented in detail through the following examples, which are provided for illustrative purposes only and not for the purpose of limiting its scope.
EJEMPLOS EXAMPLES
Ejemplo 1. Secuencias para las proteínas H y F del CDV Example 1. Sequences for CDV H and F proteins
Las secuencias de las proteínas H y F de CDV de todos los linajes, reportados por Duque- Valencia y colaboradores (Duque- Valencia, J., Forero-Muñoz, N.R., Díaz, F.J. et al. Phylogenetic evidence of the intercontinental circulation of a Canine distemper virus lineage in the Americas. Sci Rep 9, 15747 (2019). https://doi.org/10.1038/s41598-019- 52345-9), se descargaron en formato fasta traducido a los aminoácidos, con el fin de construir archivos multifasta, alineamientos múltiples y secuencias consenso, con el fin de determinar el potencial inmunogénico de las proteínas H y F del CDV. Posteriormente todas las secuencias de las proteínas H y F del CDV descargadas, pertenecientes a los linajes reportados a nivel mundial, se convirtieron en único archivo multifasta. Adicionalmente, se realizó un archivo multifasta de las secuencias que circulan en Colombia. Por otra parte, se descargaron secuencias de moléculas del CMH, clase I y II de canino y estructuras molde de humano de UniProt y Protein Data Bank (PDB), para ser modeladas con base en la estructura cristalográfica de una proteína de humano. The sequences of CDV H and F proteins from all lineages, reported by Duque-Valencia et al. (Duque-Valencia, J., Forero-Muñoz, NR, Díaz, FJ et al. Phylogenetic evidence of the intercontinental circulation of a Canine distemper virus lineage in the Americas. Sci Rep 9, 15747 (2019). https://doi.org/10.1038/s41598-019-52345-9), were downloaded in amino acid translated fasta format, in order to construct multiphase files, multiple alignments and consensus sequences, in order to determine the immunogenic potential of CDV H and F proteins. Subsequently, all downloaded CDV H and F protein sequences, belonging to the lineages reported worldwide, were converted into a single multiphase file. Additionally, a multicast file of the sequences circulating in Colombia was made. On the other hand, sequences of canine MHC class I and II molecules and human template structures were downloaded from UniProt and Protein Data Bank (PDB), to be modeled based on the crystallographic structure of a human protein.
Ejemplo 2. Generación de secuencias consenso Example 2. Generation of consensus sequences
Se realizaron alineamientos múltiples de las secuencias de la proteína H y F de CDV mediante la herramienta en línea Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/), con el propósito de establecer las diferencias entre cada una de las secuencias de esta proteína y los linajes que circulan a nivel mundial. Además, la generación de la generación de la matriz p para poder cuantificar las diferencias mediante Mega 10. Por otra parte, se generó un alineamiento para la proteína H y F de CDV de las variantes de los linajes que co-circulan en Colombia. Finalmente, se generaron 4 secuencias consenso mediante la herramienta en línea https://www.ebi. ac.uk/Tools/msa/emboss cons/. con las cuales se realizaron los análisis de predicción. Multiple alignments of CDV H and F protein sequences were performed using the Clustal Omega online tool (https://www.ebi.ac.uk/Tools/msa/clustalo/), in order to establish differences. between each of the sequences of this protein and the lineages that circulate worldwide. In addition, the generation of the generation of the p matrix to be able to quantify the differences by means of Mega 10. On the other hand, an alignment was generated for the CDV H and F protein of the variants of the lineages that co-circulate in Colombia. Finally, 4 consensus sequences were generated using the online tool https://www.ebi. ac.uk/Tools/msa/emboss cons/. with which the prediction analyzes were performed.
Las secuencias obtenidas son: The sequences obtained are:
Con-1. Consenso de la secuencia de la proteína H tomando como base todas las secuencias de los linajes circulantes a nivel mundial (SEC ID NO: 25): >CDV H protein consensus all linages MLSYQDKVGAFYKDNARANSSKLSLVTEEQGGRRPPYLLFVLLILLVGIMALLAITGVRFHQVST SNMEFSRLLKEDMEKSEAVHHQVIDVLTPLFKIIGDEIGLRLPQKLNEIKQFILQKTNFFNPNREFD FRDLHWCINPPSKIKVNFTNYCDTIGIRKSIASAANPILLSALSGGRGDIFPPYRCSGATTSVGRVFP LSVSLSMSLISRTSEIINMLTAISDGVYGKTYLLVPDYIEGEFDTQKIRVFEIGFIKRWLNDMPLLQ TTNYMVLPENSKAKVCTIAVGELTLASLCVDESTVLLYHDSNGSQDGILVVTLGIFGATPMDQV EEVIPVAHPSVEKIHITNHRGFIKDSIATWMVPALVSEKQEEQKNCLESACQRKSYPMCNQTSWE PFGGGQLPSYGRLTLPLDPSIDLQLNISFTYGPVILNGDGMDYYESPLLDSGWLTIPPKNGTVLGLI NKASRGDQFTVIPHVLTFAPRESSGNCYLPIQTSQIMDKDVLTESNLWLPTQNFRYVIATYDISR DDHAIVYYVYDPIRTISYTYPFRLTTKGRPDFLRIECFVWDDDLWCHQFYRFEADITNSTTSVENL VRIRFSCNRSKP With-1. Consensus of the H protein sequence based on all the sequences of the circulating lineages worldwide (SEQ ID NO: 25): >CDV H protein consensus all lineages MLSYQDKVGAFYKDNARANSSKLSLVTEEQGGRRPPYLLFVLLILLVGIMALLAITGVRFHQVST SNMEFSRLLKEDMEKSEAVHHQVIDVLTPLFKIIGDEIG LRLPQKLNEIKQFILQKTNFFNPNREFD FRDLHWCINPPSKIKVNFTNYCDTIGIRKSIASAANPILLSALSGGRGDIFPPYRCSGATTSVGRVFP LSVSLSMSLISRTSEIINMLTAISDGVYGKTYLLVPDYIEGEFDTQKIRVFEIGFIKRWLNDMPLLQ TTNYMVLPENSKAKVCTIAVGELTLASLC VDESTVLLYHDSNGSQDGILVVTLGIFGATPMDQV EEVIPVAHPSVEKIHITNHRGFIKDSIATWMVPALVSEKQEEQKNCLESACQRKSYPMCNQTSWE PFGGGQLPSYGRLTPLDPSIDLQLNISFTYGPVILNGDGMDYYESPLLDSGWLTIPPKNGTVLGLI NKASRGDQFTVIPHVLTFAPRES SGNCYLPIQTSQIMDKDVLTESNLWLPTQNFRYVIATYDISR DDHAIVYYVYDPIRTISYTYPFRLTTKGRPDFLRIECFVWDDDLWCHQFYRFEADITNSTTSVENL VRIRFSCNRSKP
Con-3. Consenso de la secuencia de la proteína F tomando como base todas las secuencias de los linajes circulantes a nivel mundial (SEC ID NO: 26): >CDV F protein consensus all linagesWith 3. Consensus of the F protein sequence based on all the sequences of the circulating lineages worldwide (SEQ ID NO: 26): >CDV F protein consensus all lineages
MHNKIPKRSKTQTHTQQDLPQQHSTKSAETKTSQARHSTTSAQRSTHHGPRTSDRPVHYIMNRT RSCKQASHRSDNIPAHGDHEGIIHHTPESVSQGARSRPKRRQSNATNSGSQCTWLVLWCIGIASLF LCSKAQIHWNNLSTIGIIGTOSVHYKIMTRPSHQYLVIKLMPNVSLIDNCTKAELGEYEKLLNSVL EPINQALTLMTKNVKPLQSVGSGRRQRRFAGVVLAGAALGVATAAQITAGIALHQSNLNAQAIQ SLRTSLEQSNKAIEEIREATQETVIAVQGVQDYVNNELVPAMQHMSCELVGQRLGLKLLRYYTE LLSIFGPSLRDPISAEISIQALSYALGGEIHKILEKLGYSGNDMIAILESRGIKTKITHVDLPGKLIILSI SYPTLSEVKGVIVHRLEAVSYNIGSQEWYTTVPRYVATNGYLISNFDESSCVFVSESAICSQNSLY PMSPLLQQCIRGDTSSCARTLVSGTMGNKFILSKGNIVANCASILCKCYSTSTIINQSPDKLLTFIAS DTCPLVEIDGVTIQVGGRQYPDMVYESKVALGPAISLERLDVGTNLGNALKKLDDAKVLIDSSN QILETVRRSSFNFGSLLSVPILICTALALLLLIYCCKRRYQQTLKQNTKVDPTFKPDLTGTSKSYVR SL MHNKIPKRSKTQTHTQQDLPQQHSTKSAETKTSQARHSTTSAQRSTHHGPRTSDRPVHYIMNRT RSCKQASHRSDNIPAHGDHEGIIHHTPESVSQGARSRPKRRQSNATNSGSQCTWLVLWCIGIASLF LCSKAQIHWNNLSTIGIIGTOSVHYKIMTRPSHQYLVIKLMPNVSLIDNCTKAELGE YEKLLNSVL EPINQALTLMTKNVKPLQSVGSGRRQRRFAGVVLAGAALGVATAAQITAGIALHQSNLNAQAIQ SLRTSLEQSNKAIEEIREATQETVIAVQGVQDYVNNELVPAMQHMSCELVGQRLGLKLLRYYTE LLSIFGPSLRDPISAEISIQALSYALGGEIHKILEKLGYSGND MIAILESRGIKTKITHVDLPGKLIILSI SYPTLSEVKGVIVHRLEAVSYNIGSQEWYTTVPRYVATNGYLISNFDESSCVFVSESAICSQNSLY PMSPLLQQCIRGDTSSCARTLVSGTMGNKFILSKGNIVANCASILCKCYSTSTIINQSPDKLLTFIAS DTCPLVEIDGVTIQVGGRQYPDMVYES KVALGPAISLERLDVGTNLGNALKKLDDAKVLIDSSN QILETVRRRSSFNFGSLLSVPILICTALALLLLIYCCKRRYQQTLKQNTKVDPTFKPDLTGTSKSYVR SL
Se seleccionaron 168 péptidos para ambas secuencias con las secuencias consenso de todos los linajes para análisis posterior. 168 peptides were selected for both sequences with the consensus sequences of all lineages for further analysis.
Ejemplo 3. Construcción de librería de péptidos potencialmente inmunogénicos de secuencias consenso. a- Predicción para linfocitos T citotóxicos (CTL) con la herramienta CTL prcd: hl-p / c;xk;
Figure imgf000012_0001
< blnu Example 3. Construction of a library of potentially immunogenic peptides of consensus sequences. a- Prediction for cytotoxic T lymphocytes (CTL) with the CTL prcd tool: hl-p / c;xk;
Figure imgf000012_0001
< blnu
La herramienta para CTL integra diferentes herramientas de predicción, con bases de datos que reportan epitopes para diferentes alelos de antígenos de leucocitos humanos (HLA por su sigla en inglés - human leukocyte antigens) clase I. Esta herramienta, genera los péptidos con base en diferentes aproximaciones, que toman en consideración diferentes modelos matemáticos, Support Vector Machine (SVM) con un cut off de 1.2 (máximo 1.5), Artificial Nueral Network (ANN), con cut off de 0.95 (valor entre 0 y 1) y una combinación de estos modelos, lo cual permite la obtención de los potenciales inmunógenos. The CTL tool integrates different prediction tools, with databases that report epitopes for different alleles of human leukocyte antigens (HLA for its acronym in English - human leukocyte antigens) class I. This tool generates peptides based on different approximations, which take into account different mathematical models, Support Vector Machine (SVM) with a cut off of 1.2 (maximum 1.5), Artificial Nueral Network (ANN), with a cut off of 0.95 (value between 0 and 1) and a combination of these models, which allows obtaining immunogenic potentials.
Se obtuvieron 22 péptidos ANN, 6 péptidos SVM y en combinación, 28 péptidos para Con-1 y 43 péptidos ANN, 8 péptidos SVM y en combinación, 50 péptidos para Con-3. b- Determinación de péptidos lineales para linfocitos B con la herramienta http://sysbio.unl ,edu//prediction php 22 ANN peptides, 6 SVM peptides and in combination 28 peptides for Con-1 and 43 ANN peptides, 8 SVM peptides and in combination 50 peptides for Con-3 were obtained. b- Determination of linear peptides for B lymphocytes with the tool http://sysbio.unl ,edu//prediction php
Esta herramienta permite determinar péptidos lineales para linfocitos B, los cuales permiten la detección directa de antígenos. Esta herramienta tiene un límite en la búsqueda, puesto que tiene una ventana de péptidos específica. En este caso se predijeron los de 10 aminoácidos. Esta herramienta puede predecir péptidos de 12, 14, 16, 18 y 20 aminoácidos. This tool allows to determine linear peptides for B lymphocytes, which allow the direct detection of antigens. This tool has a limit in the search, since it has a window of specific peptides. In this case those of 10 amino acids were predicted. This tool can predict peptides of 12, 14, 16, 18 and 20 amino acids.
Con esta herramienta, se encontró un set de péptidos para linfocitos B de ambas proteínas, H y F de CDV, los cuales pueden hacer parte de la librería de péptidos. Con-1 tiene 4 secuencias de 10 aminoácidos, que tienen el potencial de ser epitopes para linfocitos B. Con-3 tiene 3 secuencias de 10 aminoácidos, que tienen el potencial de ser epitopes para linfocitos B. c- Epitopes para los diversos alelos humanos de las moléculas del CMH clase II, con la herramienta http://crdd.osdd.net/raghava/mhc2pred/index.html With this tool, a set of peptides for B lymphocytes of both CDV H and F proteins was found, which can be part of the peptide library. Con-1 has 4 sequences of 10 amino acids, which have the potential to be epitopes for B lymphocytes. Con-3 has 3 sequences of 10 amino acids, which have the potential to be epitopes for B lymphocytes. c- Epitopes for the various human alleles of MHC class II molecules, with the tool http://crdd.osdd.net/raghava/mhc2pred/index.html
Esta herramienta permite determinar epitopes para los diversos alelos humanos de las moléculas del CMH-II. Para cada alelo se marcó el top 10, sin embargo, el valor umbral mayor a 1.5 se consideró para péptidos potenciales, con la intención de ser más astringentes. This tool makes it possible to determine epitopes for the various human alleles of MHC-II molecules. For each allele the top 10 was marked, however, the threshold value greater than 1.5 was considered for potential peptides, with the intention of being more stringent.
Para algunos de los alelos ninguno de los péptidos superó el valor umbral, por lo cual se hizo una depuración y extracción de los péptidos potenciales. d- Epitopes para linfocitos B, con la herramienta
Figure imgf000013_0001
For some of the alleles, none of the peptides exceeded the threshold value, for which a purification and extraction of the potential peptides was carried out. d- Epitopes for B lymphocytes, with the tool
Figure imgf000013_0001
Esta herramienta permite predecir epitopes para linfocitos B mediante una escala de colores en donde se establece la probabilidad de los péptidos y los resultados obtenidos. Los epitopes de cada una de las secuencias consenso se basan en este código de colores. Los epitopes obtenidos de las proteínas H y F de CDV consenso se ilustran en la Figura 2. e- Recuperación de péptidos de DLA clase I This tool makes it possible to predict epitopes for B lymphocytes using a color scale where the probability of the peptides and the results obtained are established. The epitopes of each of the consensus sequences are based on this color code. The epitopes obtained from the consensus CDV H and F proteins are illustrated in Figure 2. e- Recovery of DLA class I peptides
Se presentaron 39 diferentes péptidos recuperados de la molécula DLA-88*508:01, 2 de la proteína H y 4 para la proteína F. Los péptidos para H y F de CDV se muestran en la Tabla 3 : There were 39 different peptides recovered from the DLA-88*508:01 molecule, 2 from the H protein and 4 from the F protein. The peptides for CDV H and F are shown in Table 3:
Tabla 3. Péptidos para las proteínas H y F
Figure imgf000014_0001
Table 3. Peptides for proteins H and F.
Figure imgf000014_0001
Todos estos estos péptidos fueron recuperados de células infectadas con la variante Ondertepoort. All of these peptides were recovered from cells infected with the Ondertepoort variant.
Ejemplo 4. Depuración y análisis de péptidos para cálculo de propiedades fisicoquímicas Example 4. Purification and analysis of peptides for calculation of physicochemical properties
Con el fin de establecer el potencial de cada uno de los péptidos obtenidos y continuar con el estudio in silico, se realizó una base de datos con toda la información de los péptidos obtenidos mediante las diferentes herramientas antes mencionadas y así poder hacer una depuración sistemática de la librería para seleccionar los mejores candidatos. Allí se consigna: 1. Código de péptido. 2. Secuencia. 3. Tamaño. 4. Región del genoma. 5. Tipo de epitope. 6. Herramienta de obtención. 7. En adelante, propiedades fisicoquímicas como: carga, peso molecular, punto isoeléctrico, índice de estabilidad, GRAVY, índice alifático y vida media estimada, calculadas con Protparam (btq>s://web, expasy.org/protparam/) . In order to establish the potential of each of the peptides obtained and continue with the in silico study, a database was created with all the information on the peptides obtained through the different tools mentioned above and thus be able to carry out a systematic purification of the peptides. the bookstore to select the best candidates. There it is recorded: 1. Peptide code. 2. Sequence. 3. Size. 4. Region of the genome. 5. Type of epitope. 6. Obtain Tool. 7. Hereinafter, physicochemical properties such as: charge, molecular weight, isoelectric point, stability index, GRAVY, aliphatic index and estimated half-life, calculated with Protparam (btq>s://web, expasy.org/protparam/).
Ejemplo 5. Modelación de algunas moléculas inmunes Example 5. Modeling of some immune molecules
Se modeló la cadena alpha de la secuencia de una CMH-I de canino (DLA-I-88), (modelo_l), puesto que esta es la única que interactúa con los péptidos con potencial inmunogénico. El molde utilizado es una CMH-I de canino depositado en el PDB, 5F1N, la cual proviene de una cristalización de moléculas de canino. Por otra parte, se tiene la secuencia de la una CMH-II de canino del alelo DR (DLA-DR), tanto las cadenas alpha y beta (Modelo_2 y Modelo_3, respectivamente). Para esta estructura, se utilizó como molde, la estructura 3D reportada en el PDB, 4FQX, tanto para la cadena alpha como la beta. Esta estructura, es de humanos, del mismo alelo de la secuencia que se tiene para caninos (HLA-DR). Además, se realizó el cálculo de la identidad de los moldes con respecto a cada una de las moléculas modeladas con base en su secuencia. Se realizaron modelaciones por homología mediante el software Modeller 9.24. The alpha chain of the sequence of a canine MHC-I (DLA-I-88), (model_l), was modeled, since this is the only one that interacts with the peptides with immunogenic potential. The template used is a canine HSC-I deposited in the PDB, 5F1N, which comes from a crystallization of canine molecules. On the other hand, we have the sequence of a canine MHC-II of the DR allele (DLA-DR), both the alpha and beta chains (Model_2 and Model_3, respectively). For this structure, the 3D structure reported in the PDB, 4FQX, was used as a template for both the alpha and beta chains. This structure is from humans, from the same allele of the sequence that is found for canines (HLA-DR). In addition, the calculation of the identity of the templates was performed with respect to each of the modeled molecules based on their sequence. Homology modeling was performed using the Modeller 9.24 software.
Luego de la corrida de cada una de las tres moléculas diferentes, se generaron 100 modelos de cada una, donde se selecciona el mejor modelo con base en tres puntajes que entrega el software: molpdf, DOPE y GA341. After running each of the three different molecules, 100 models of each one were generated, where the best model is selected based on three scores provided by the software: molpdf, DOPE and GA341.
Mejores modelos: Best models:
Modelo_l: Modelo 57, el cual obtuvo el mejor puntaje. Model_l: Model 57, which obtained the best score.
Modelo_2: Modelo 76, el cual obtuvo el mejor puntaje. Model_2: Model 76, which obtained the best score.
Modelo_3: Modelo 7, el cual obtuvo el mejor puntaje. Model_3: Model 7, which obtained the best score.
El Modelo_2 y Model o_3 se pusieron en un archivo como complejo con base en la estructura molde, con el fin de evaluar el docking molecular. Esta estrategia permite determinar la capacidad de dos moléculas para interactuar, mediante diferentes enlaces no covalentes como los puentes de hidrogeno y las fuerzas de Van de Waals. Para el caso de las herramientas en línea utilizadas, la predicción se hace sin delimitar un sitio de interacción, en cambio, las herramientas presentan la región de interacción más probable, con base en algoritmos que pueden predecir esa pose molecular. Model_2 and Model o_3 were put into a file as a complex based on the template structure, in order to evaluate the molecular docking. This strategy allows determine the ability of two molecules to interact, through different non-covalent bonds such as hydrogen bonds and Van de Waals forces. In the case of the online tools used, the prediction is made without delimiting an interaction site; instead, the tools present the most probable interaction region, based on algorithms that can predict that molecular pose.
Tabla 4. Identidades Table 4. Identities
Secuencia modelo y molde Identidad (%) Model Sequence and Template Identity (%)
Modelo l y 5F1N 95.62 Model l and 5F1N 95.62
Modelo_2 y 4FQXA 88.7 Model_2 and 4FQXA 88.7
Modelo_3 y 4FQXB 79.7 Model_3 and 4FQXB 79.7
Los resultados de las corridas para cada una de las proteínas, así como los mejores modelos para cada una de las proteínas fueron validadas mediante diferentes herramientas computacionales. The results of the runs for each of the proteins, as well as the best models for each of the proteins, were validated using different computational tools.
Los mejores modelos para cada una las tres proteínas (Modelo_l, Modelo_2 y Modelo_3), se validaron mediante tres herramientas computacionales diferentes, ProsaWeb, Swissmodel y TM-Align, con el fin de saber si los modelos podían ser utilizados en análisis posteriores de acoplamiento molecular con los péptidos predichos. A continuación, se muestra una síntesis de los datos de la validación de las tres proteínas. En esta tabla también se incluyen los datos de los moldes para poder establecer una comparación con los datos obtenidos para los modelos. The best models for each of the three proteins (Model_l, Model_2 and Model_3) were validated using three different computational tools, ProsaWeb, Swissmodel and TM-Align, in order to know if the models could be used in subsequent analyzes of molecular docking. with the predicted peptides. A synthesis of the validation data for the three proteins is shown below. This table also includes the data of the molds to be able to establish a comparison with the data obtained for the models.
Tabla 5. Resumen de validación Table 5. Validation summary
Protein Z Value Favorable region (%) TM Value Align AAProtein Z Value Favorable region (%) TM Value Align AA
Modelo l -9.4 98.16 0.99066 274Model l -9.4 98.16 0.99066 274
Modelo_2 -5.34 96.00 0.88193 177Model_2 -5.34 96.00 0.88193 177
Modelo_3 -5.27 94.33 0.89979 194Model_3 -5.27 94.33 0.89979 194
5F1N -9.19 95.60
Figure imgf000016_0001
4FQXB -5.33 96.17 5F1N -9.19 95.60
Figure imgf000016_0001
4FQXB -5.33 96.17
Ejemplo 6. Consolidación de datos obtenidos para las propiedades de los péptidos Example 6. Consolidation of data obtained for the properties of the peptides
Posterior al cálculo de cada una de las propiedades fisicoquímicas y la consolidación de todos los datos de los péptidos predichos mediante las diferentes herramientas, se realizó un consolidado de algunas variables importantes para la elección de los mejores candidatos. En la Figura 3 A y B, se observa el tamaño de los péptidos predichos de la secuencia consenso de las proteínas H y F de CDV, respectivamente. After the calculation of each one of the physicochemical properties and the consolidation of all the data of the peptides predicted by means of the different tools, a consolidation of some important variables was carried out for the election of the best candidates. Figures 3 A and B show the size of the peptides predicted from the consensus sequence of CDV H and F proteins, respectively.
Ejemplo 7. Modelación de algunos péptidos en su estructura tridimensional mediante PEPFOLD3. Example 7. Modeling of some peptides in their three-dimensional structure using PEPFOLD3.
Los péptidos que obtuvieron los mejores puntajes fueron modelados mediante la herramienta PEPFOLD3 con el fin de realizar acoples moleculares de estos péptidos con las moléculas del CMH de canino modeladas en el Ejemplo 5. The peptides that obtained the best scores were modeled using the PEPFOLD3 tool in order to perform molecular coupling of these peptides with the canine MHC molecules modeled in Example 5.
Se determinaron péptidos con potencial inmunogénico para células B, o para ser presentados en el contexto clase 1 o clase 2 de las moléculas del CMH mediante la herramienta del IEDB. Luego de someter las secuencias, se obtuvieron más de mil péptidos. A esta herramienta, se le suministra la secuencia consenso de todos los linajes tanto de la proteína H como de F. Peptides with immunogenic potential for B cells, or to be presented in the class 1 or class 2 context of MHC molecules were determined using the IEDB tool. After submitting the sequences, more than a thousand peptides were obtained. This tool is supplied with the consensus sequence of all the lineages of both the H and F proteins.
Ejemplo 8. Selección de péptidos con base en score y propiedades Example 8. Selection of peptides based on score and properties
Luego de tener todos los datos de predicción mediante las diferentes herramientas, se seleccionaron péptidos tanto de la proteína H como de la proteína F, que tuvieran propiedades fisicoquímicas de estabilidad y vida media apropiadas, así como los mejores valores de predicción de cada una de las herramientas, con el fin de validar in silico, un grupo más pequeño de péptidos y poder llevar estos a los siguientes pasos del desarrollo. Los péptidos seleccionados inicialmente se muestran en la Tabla 1. Ejemplo 9. Acoplamiento molecular de los péptidos seleccionados con la MHC de canino correspondiente a la molécula que lo presenta (clase I, II) mediante CABSDOCK. After having all the prediction data using the different tools, peptides from both the H protein and the F protein were selected, which had appropriate physicochemical properties of stability and half-life, as well as the best prediction values of each of the tools, in order to validate in silico, a smaller group of peptides and to be able to take these to the next steps of development. The initially selected peptides are shown in Table 1. Example 9. Molecular coupling of the selected peptides with the canine MHC corresponding to the molecule that presents it (class I, II) by means of CABSDOCK.
Cada uno de los 12 péptidos, fue sometido a análisis de acoplamiento molecular mediante CABSDOCK, con la molécula blanco respectiva (sea clase I o clase II, según su predicción), en la plataforma en línea. Se suben ambos archivos por separado y posterior al tiempo de análisis, la herramienta entrega la pose más probable en la que interactúan ambas moléculas, teniendo en cuanta un análisis de clustering o agrupamiento. Each of the 12 peptides was subjected to molecular docking analysis using CABSDOCK, with the respective target molecule (either class I or class II, depending on its prediction), on the online platform. Both files are uploaded separately and after the analysis time, the tool delivers the most probable pose in which both molecules interact, taking into account a clustering analysis.
Acoplamiento molecular de los péptidos seleccionados con la MHC de canino correspondiente a la molécula que lo presenta (clase I, II) mediante MDOCKPEP. Molecular coupling of the selected peptides with the canine MHC corresponding to the molecule that presents it (class I, II) by means of MDOCKPEP.
Cada uno de los 12 péptidos, fue sometido a análisis de acoplamiento molecular mediante HPEPDOCK, con la molécula blanco respectiva (sea clase I o clase II, según su predicción), en la plataforma en línea. Se suben ambos archivos por separado y posterior al tiempo de análisis, la herramienta entrega la pose más probable en la que interactúan ambas moléculas, teniendo en cuanta un análisis de la pose más probable. Each of the 12 peptides was subjected to molecular docking analysis using HPEPDOCK, with the respective target molecule (either class I or class II, depending on its prediction), on the online platform. Both files are uploaded separately and after the analysis time, the tool delivers the most probable pose in which both molecules interact, taking into account an analysis of the most probable pose.
Acoplamiento molecular de los péptidos seleccionados con la MHC de canino correspondiente a la molécula que lo presenta (clase I, II) mediante HPEPDOCK Molecular coupling of the selected peptides with the canine MHC corresponding to the molecule that presents it (class I, II) using HPEPDOCK
Cada uno de los 12 péptidos, fue sometido a análisis de acoplamiento molecular mediante CABSDOCK, con la molécula blanco respectiva (sea clase I o clase II, según su predicción), en la plataforma en línea. Se suben ambos archivos por separado y posterior al tiempo de análisis, la herramienta entrega la pose más probable en la que interactúan ambas moléculas, teniendo en cuanta un análisis de la pose más probable. Each of the 12 peptides was subjected to molecular docking analysis using CABSDOCK, with the respective target molecule (either class I or class II, depending on its prediction), on the online platform. Both files are uploaded separately and after the analysis time, the tool delivers the most probable pose in which both molecules interact, taking into account an analysis of the most probable pose.
Acoplamiento molecular de los péptidos seleccionados con la TLR-2 y TLR-4 mediante HPEPDOCK. Molecular coupling of the selected peptides with TLR-2 and TLR-4 using HPEPDOCK.
Cada uno de los 12 péptidos, fue sometido a análisis de acoplamiento molecular mediante HPEPDOCK, con la molécula blanco (TLR-2 o TLR-4), en la plataforma en línea. Se suben ambos archivos por separado y posterior al tiempo de análisis, la herramienta entrega la pose más probable en la que interactúan ambas moléculas, teniendo en cuanta un análisis de la pose más probable. Each of the 12 peptides was subjected to molecular docking analysis using HPEPDOCK, with the target molecule (TLR-2 or TLR-4), on the online platform. Both files are uploaded separately and after analysis time, the tool returns the most probable pose in which both molecules interact, taking into account an analysis of the most probable pose.
Ejemplo 10. Pruebas de seguridad in silico a- Análisis de Blastp de los péptidos seleccionados para determinar péptidos homólogos propios. Example 10. In silico safety tests a- Blastp analysis of selected peptides to determine self homologous peptides.
Con este análisis se busca determinar si los péptidos diseñados, tenían péptidos homólogos en proteínas propias de los caninos, lo cual sería una situación que se debe evitar, puesto que únicamente se desea generar una respuesta inmunológica frente al CDV, pero no frente a las proteínas propias del canino. Para ningún péptido, se tuvo un valor del 100 % tanto en la identidad como en la cobertura, lo que indica que no hay péptidos idénticos en las proteínas de los caninos. b- Análisis de VaxiJenv2.0 de los péptidos seleccionados para determinar si los péptidos son potenciales antígenos This analysis seeks to determine if the designed peptides had homologous peptides in canine proteins, which would be a situation that should be avoided, since it is only desired to generate an immune response against CDV, but not against the proteins characteristic of the canine. For no peptide, we had a value of 100% for both identity and coverage, indicating that there are no identical peptides in the canine proteins. b- VaxiJenv2.0 analysis of the selected peptides to determine if the peptides are potential antigens
Se determinó si los péptidos eran potenciales antígenos virales, mediante un análisis de scoring de máquinas de aprendizaje, comparado con otros péptidos de los cuales hay evidencia experimental de su antigenicidad. c- Análisis con ToxinPred de los péptidos seleccionados para determinar si los péptidos son compuestos tóxicos. Whether the peptides were potential viral antigens was determined by a machine learning scoring analysis, compared to other peptides for which there is experimental evidence of their antigenicity. c- ToxinPred analysis of the selected peptides to determine if the peptides are toxic compounds.
Se determinó si los péptidos eran potenciales compuestos tóxicos, mediante un análisis de scoring de máquinas de aprendizaje, comparado con otros péptidos de los cuales hay evidencia experimental de su toxicidad. d- Análisis con AllergenFPvl.O de los péptidos seleccionados para determinar si los péptidos son potenciales alérgenos Se determinó si los péptidos eran potenciales compuestos alérgenos, mediante un análisis de scoring de máquinas de aprendizaje, comparado con otros péptidos de los cuales hay evidencia experimental de su potencial alérgenico. Ejemplo 11. Cálculo de las propiedades fisicoquímicas de los péptidos seleccionados inicialmente Whether the peptides were potential toxic compounds was determined by a machine learning scoring analysis, compared to other peptides for which there is experimental evidence of their toxicity. d- Analysis with AllergenFPvl.O of the selected peptides to determine if the peptides are potential allergens Whether the peptides were potential allergenic compounds was determined by a machine learning scoring analysis, compared with other peptides for which there is experimental evidence of their allergenic potential. Example 11. Calculation of the physicochemical properties of the initially selected peptides
Se determinaron los datos de la naturaleza de los péptidos mediante la herramienta Protparam. Los resultados se encuentran en la Tabla 6. Tabla 6. Datos de la naturaleza de los péptidos
Figure imgf000020_0001
Data on the nature of the peptides were determined using the Protparam tool. The results are found in Table 6. Table 6. Data on the nature of the peptides
Figure imgf000020_0001

Claims

REIVINDICACIONES
1. Un péptido inmunogénico contra el Virus Distemper Canino (CDV) que tiene la secuencia 1. An immunogenic peptide against Canine Distemper Virus (CDV) having the sequence
X1-X2-X3-X4-X3-X5-X6-X7-X8 en donde, X1-X2-X3-X 4 -X3-X5-X 6 -X 7 -X 8 where,
Xi es un aminoácido seleccionado entre un aminoácido cargado positivamente, un aminoácido no cargado, un aminoácido especial y un aminoácido hidrofóbico; Xi is an amino acid selected from a positively charged amino acid, an uncharged amino acid, a special amino acid, and a hydrophobic amino acid;
X2 es tres aminoácidos iguales o diferentes, seleccionados entre un aminoácido cargado positivamente, un aminoácido cargado negativamente, un aminoácido no cargado, un aminoácido especial, un aminoácido hidrofóbico y un aminoácido aromático; X2 is three the same or different amino acids, selected from a positively charged amino acid, a negatively charged amino acid, an uncharged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X3 es un aminoácido, seleccionado entre un aminoácido cargado positivamente, un aminoácido no cargado, un aminoácido especial, un aminoácido hidrofóbico y un aminoácido aromático; X3 is an amino acid, selected from a positively charged amino acid, an uncharged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X4 es dos aminoácidos iguales o diferentes, seleccionados entre un aminoácido cargado positivamente, un aminoácido cargado negativamente, un aminoácido no cargado, un aminoácido hidrofóbico y un aminoácido aromático; X 4 is two the same or different amino acids, selected from a positively charged amino acid, a negatively charged amino acid, an uncharged amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X5 es un aminoácido seleccionado entre un aminoácido cargado positivamente, un aminoácido cargado negativamente, un aminoácido especial, un aminoácido hidrofóbico y un aminoácido aromático; X5 is an amino acid selected from a positively charged amino acid, a negatively charged amino acid, a special amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X7, está ausente o es un aminoácido seleccionado entre un aminoácido cargado positivamente, un aminoácido no cargado, un aminoácido hidrofóbico y un aminoácido aromático; X7 is absent or is an amino acid selected from a positively charged amino acid, an uncharged amino acid, a hydrophobic amino acid, and an aromatic amino acid;
X7 es Xi o está ausente; y X7 is Xi or absent; and
Xx está ausente o es uno o dos aminoácidos iguales o diferentes seleccionados entre un aminoácido cargado positivamente y un aminoácido hidrofóbico, en donde el aminoácido cargado positivamente se selecciona del grupo que consiste en R, H y K; el aminoácido cargado negativamente se selecciona del grupo que consiste en D y E; el aminoácido no cargado se selecciona del grupo que consiste en S, T, N y Q; el aminoácido especial se selecciona del grupo que consiste en C, G y P; el aminoácido hidrofóbico se selecciona del grupo que consiste en A, I, L, M y V, y el aminoácido aromático se selecciona del grupo que consiste en W, Y y F. Xx is absent or is one or two of the same or different amino acids selected from a positively charged amino acid and a hydrophobic amino acid, wherein the positively charged amino acid is selected from the group consisting of R, H, and K; the negatively charged amino acid is selected from the group consisting of D and E; the uncharged amino acid is selected from the group consisting of S, T, N, and Q; the special amino acid is selected from the group consisting of C, G, and P; the hydrophobic amino acid is selected from the group consisting of A, I, L, M, and V, and the aromatic amino acid is selected from the group consisting of W, Y, and F.
2. El péptido de acuerdo con la Reivindicación 1 que tiene la secuencia que se selecciona del grupo que consiste en: SEC ID NO: 1, SEC ID NO: 2, SEC ID NO: 3, SEC ID NO: 4, SEC ID NO: 5, SEC ID NO: 6, SEC ID NO: 7, SEC ID NO: 8, SEC ID NO: 9, SEC ID NO: 10, SEC ID NO: 11 y SEC ID NO: 12. 2. The peptide according to Claim 1 having the sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
3. El péptido de acuerdo con cualquiera de las Reivindicaciones 1 o 2, en donde el amino terminal está biotinilado, acetilado, PEGilado o enlazado a un ácido graso. 3. The peptide according to either of Claims 1 or 2, wherein the amino terminus is biotinylated, acetylated, PEGylated or linked to a fatty acid.
4. El péptido de acuerdo con cualquiera de las Reivindicaciones 1 a 3, en donde el carboxilo terminal está amidado. 4. The peptide according to any of Claims 1 to 3, wherein the carboxyl terminus is amidated.
5. Un ácido nucleico que codifica el péptido de acuerdo con cualquiera de las Reivindicaciones 1 o 2. 5. A nucleic acid encoding the peptide according to either of Claims 1 or 2.
6. El ácido nucleico de acuerdo con la Reivindicación 4, que tiene la secuencia que se selecciona del grupo que consiste en: SEC ID NO: 13, SEC ID NO: 14, SEC ID NO: 15, SEC ID NO: 16, SEC ID NO: 17, SEC ID NO: 18, SEC ID NO: 19, SEC ID NO: 20, SEC ID NO: 21, SEC ID NO: 22, SEC ID NO: 23 y SEC ID NO: 24. 6. The nucleic acid according to Claim 4, having the sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.
7. Una composición inmunogénica que comprende uno o más péptidos de acuerdo con la Reivindicación 1 y excipientes farmacéuticamente aceptables. 7. An immunogenic composition comprising one or more peptides according to Claim 1 and pharmaceutically acceptable excipients.
8. La composición de acuerdo con la Reivindicación 7, en donde la composición es una composición de vacuna universal. 8. The composition according to Claim 7, wherein the composition is a universal vaccine composition.
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