WO2022055375A1 - Recombinant live vaccine for sars-cov-2 based on recombinant salmonella enteritidis - Google Patents

Recombinant live vaccine for sars-cov-2 based on recombinant salmonella enteritidis Download PDF

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WO2022055375A1
WO2022055375A1 PCT/PE2021/000006 PE2021000006W WO2022055375A1 WO 2022055375 A1 WO2022055375 A1 WO 2022055375A1 PE 2021000006 W PE2021000006 W PE 2021000006W WO 2022055375 A1 WO2022055375 A1 WO 2022055375A1
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recombinant
sars
cov
vaccine
salmonella enteritidis
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PCT/PE2021/000006
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Spanish (es)
French (fr)
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Manolo Clemente FERNANDEZ DÍAZ
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Farmacológicos Veterinarios Sac
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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
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella

Definitions

  • the present invention is framed in the health sector, mainly in human health. Especially in the development of alternative live vaccines to counteract the pandemic caused by the SARS CoV-2 virus.
  • the present invention refers to a live recombinant vaccine based on a strain of SE, Salmonella enteritidis 3934 XXII XrpoS waaL (3934VacR) attenuated and rough which has been modified for the expression of a protein of the Sars-Cov-2 virus caused by the COVID-19 pandemic.
  • This strain of Salmonella enterica (3934VacR) is capable of expressing the heterologous protein xenoantigen RBD (receptor binding domain) external to protein S of the Sars-Cov-2 COV-19 virus.
  • the present invention also includes the use of this strain as a vaccine vector against the Sars-Cov-2 virus, via a process of insertion and integration of the genes that encode the RBD surface protein antigen of the Sars-Cov-2 virus.
  • Covid-19 The SE vector used has been genetically modified to function as a vehicle for the expression of the immunodominant genes of the RBD protein antigen of the Sars-Cov-2 virus, with the ultimate goal of stimulating an effective and lasting immune response against the virus.
  • Sars-Cov-2 virus The generated strain is totally safe because it is free of the twelve genes that encode the proteins of the GMP-di-cyclic secondary messenger signaling pathway, the RpoS sigma factor and the WaaL protein.
  • this strain is proposed as a new effective and safe recombinant live vaccine against the SArs-Cov-2 virus, suitable for vaccinating populations of mammals, especially humans, where all the components of this vaccine have been specifically designed. and developed with the three successive priority factors: efficacy, safety and cost.
  • Coronaviruses are enveloped RNA viruses distributed widely in humans, some mammals and birds causing respiratory, enteric, hepatic and neurological diseases (Weiss SR, Leibowitz JL.) (Masters PS, Perlman S.).
  • Weiss SR Leibowitz JL.
  • Six species of coronavirus are now known to cause human illness, and four of them — 229E, OC43, NL63 and HKU1 — are the most prevalent and typical cause of these viral infections.
  • Sa S, Wong G, Shi W, et al. The most outstanding clinical characteristics of the patients are shown in the article entitled "Clinical Characteristics of Coronavirus Disease 2019 in China" (Guan, Wei-jie et.aL).
  • document CN101838627 refers to a recombinant structure of salmonella choleraesuis, a bivalent genetic engineering vaccine and a method of preparation and application of the recombination of salmonella choleraesuis, where the Salmonella choleraesuis recombination does not comprise a resistance marker and expresses the major Cap proteins of porcine circovirus II antigen.
  • Salmonella choleraesuis recombination conservation number C501 pYA-delta 410RF2 which does not comprise the resistance marker, expresses the major antigenic sites of porcine circovirus II and is prepared by the invention of CCTCC M209314.
  • Salmonella choleraesuis recombination deletes asd genes that are necessary for salmonella choleraesuis growth and comprises a plasmid that can express asd genes and porcine circovirus II Cap protein antigenic site genes in salmonella choleraesuis recombination .
  • patent WO205/035556 refers to systems such as recombinant plasmids, viruses and prokaryotes that express the M, E and S proteins associated with the SARS-CoV membrane in cells, such as human cells, both in in vitro and in vivo where SARS-CoV M, E and S proteins spontaneously form SARS-CoV virus-like particles (SARS-CoV-VLP).
  • SARS-CoV-VLP SARS-CoV virus-like particles
  • the intracellular expression of the SARS-CoV M, E and S proteins and their association to form virus-like particles that present the viral proteins in their "natural" context causes the induction of an immune system response.
  • It also relates to methods for eliciting an immune response in animals, such as humans and other mammals, by identifying a subject at risk of developing SARS and administering to the subject one or more genetic constructs capable of expressing SARS-CoV M, E and Polypeptides. /o S.
  • the invention teaches that live attenuated prokaryotic strains must maintain a balance between attenuation and immunogenicity and that do not cause any disease or alter the normal physiology of the host.
  • WO201 1/126976 is also known which provides a modified reovirus S1 protein comprising a first antigenic epitope of a non-reovirus antigen located in the helical region of the protein.
  • the first epitope may insert into the alpha-helical region of the protein, or may replace one or more heptad repeats in the alpha-helical region of the protein.
  • the first epitope can be a viral epitope, among which the SARS-associated coronavirus is found, and a bacterial epitope of a bacterium, among which Salmonella spp.
  • US2018/296663 teaches a combination of a first component and a second component, wherein the first component is typically an immunogenic component and wherein the second component is typically an adjuvant component, wherein the first component (immunogenic ) comprises at least one nucleic acid molecule encoding at least one antigen, or a fragment or variant thereof.
  • the second (adjuvant) component of the combination comprises at least one adjuvant compound, wherein at least one adjuvant compound is preferably an immunopotentiating compound.
  • the second component (adjuvant) of the combination comprises at least one immune-enhancing compound, and/or at least one compound of the delivery system.
  • Pathogenic antigens are peptide or protein antigens preferably derived from a pathogen associated with an infectious disease, among which the SARS coronavirus is considered.
  • RNA vaccines that are based on the knowledge that RNA (for example, messenger RNA (mRNA)) can safely direct the cellular machinery of the body to produce almost any protein of interest, from native proteins to antibodies and other entirely new proteins.
  • mRNA messenger RNA
  • the constructs may have therapeutic activity inside and outside cells.
  • RNA (eg mRNA) vaccines can be used to induce a balanced immune response against hMPV, PrV, RSV, MeV and/or BetaCoV (eg MERS-CoV, SARS-CoV, HCoV-OC43 , HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1), or any combination of two or more of the above viruses, comprising cellular and humoral immunity, without risk of insertional mutagenesis, eg hMPV, PIV, RSV, MeV, BetaCoV (eg MERS-CoV, SARS-CoV, HCoV -OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKUl) and combinations thereof.
  • BetaCoV eg MERS-CoV, SARS-CoV, HCoV-OC43,
  • flagellin is a monomeric protein of approximately 500 amino acids that polymerizes to form the flagella associated with bacterial movement. Flagellin is expressed by a variety of flagellated bacteria (Salmonella typhimurium for example) as well as by non-flagellated bacteria (such as Escherichia coli) for detection of flagellin by cells of the innate immune system.
  • US2016/339097 teaches the preparation and isolation of coronavirus proteins and antigens, particularly porcine coronaviruses, and further provides viral proteins and antigens obtained from coronavirus-infected cells and compositions comprising the proteins and antigens. Describes a method for preparing coronavirus proteins and/or antigens, such as PEDV or PDCoV proteins and/or antigens, from cells infected with a coronavirus, such as PEDV or PDCoV. The proteins and/or antigens are harvested at an early time point after infection when most, or all, of the viral proteins and/or antigens remain associated with the infected cells.
  • coronavirus proteins and/or antigens such as PEDV or PDCoV proteins and/or antigens
  • the vaccine composition may comprise proteins and/or antigens from at least one additional pathogen which may be any pathogen that causes disease and/or infection in a porcine subject.
  • the recombinant vector was introduced into Salmonella Typhimurium SL3261 vaccine strain, and stable soluble expression of the chimeric protein was assessed and confirmed by Western blotting with polyclonal antisera. from C. parvum.
  • the present invention provides the development of a live and recombinant vaccine against Sars-Cov-2 that has a very high probability of success based on the stability of the recombinant protein, simplicity and ease of application that is would offer national and international public health using an attenuated SE strain as a vector.
  • (3) can be administered by intradermal, oral, and intranasal inoculation routes;
  • Figure 1 illustrates SDS-PAGE electrophoresis with coomassie staining performed with anti-HIS of the total extracts obtained.
  • Figure 2 illustrates SDS-PAGE electrophoresis with coomassie staining and anti-SARS-CoV-2 immunodetection of total protein extracts (Western Blot).
  • Figure 3 shows the results of the agglutination test that detects antibodies against the particulate antigen.
  • Figure 4 shows photographs of Peth dishes with stool samples from individuals immunized with the vaccine according to the present invention.
  • Figure 5 shows the results of the response of individuals immunized with the Salmonella enteritidis antigen expressing RBD of SARs_CoV-2 (Covid-19).
  • Figure 6 shows the results of immune cells from the spleen of mice immunized with the oral vaccine, according to the present invention, where it shows a great immunological activity of T cells that produce functional cytokines such as IFN-y (interferon gamma ) among other.
  • the present invention relates to a live recombinant vaccine based on a strain of Salmonella enteritidis that expresses the S protein of the Sars-2019-Cov-2 virus where the best expression has been achieved when the insert is in the plasmid instead of the chromosome. Likewise, it refers to a vaccine that requires said strain.
  • the present invention refers to the use of a strain of Salmonella enteritidis 3934 (deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT9332) for the treatment of SARS-Cov-2 and to the method to control SARS-CoV-2 infection by administering a live recombinant vaccine to mammals
  • the present invention relates to the development of a vaccine against SARS-Cov-2-2019 causing the COVID-19 pandemic, for which the ⁇ PR-LppOmpA-RBD-6XHis-TTterminator> cassette was constructed by PCR overlapping of two fragments of 670 and 671 base pairs. A linker (GGGSGGGS) was included between the LppOmpA fragment and the RBD fragment. Finally, the 1348 bp fragment obtained was subcloned into an expression plasmid specifically designed for the insertion of sequences in the attTn7 locus, which was used to transform the rough strain of Salmonella enteritidis 3934 AXII ArpoS Awaal (3934VacR) by electroporation. Transformant colonies were selected in the presence of ampicillin.
  • an expression "cassette” has been designed and built, which is characterized in that it includes:
  • the plasmid contains, apart from the recombination framework regions, an expression cassette that is integrated into the chromosome.
  • the resulting strain to which this invention refers is a modified mutant strain of Salmonella enteritidis that carries a deletion of the waaL gene, and therefore presents a rough phenotype, and expresses a gene that encodes the RBD protein domain of the S protein of Sars-Cov-2-2019, causing the Covid-19 pandemic.
  • Salmonella enteritidis is an abbreviation for Salmonella enterica serovar Enteritidis and the strain SE3934-Cap has been deposited under the name SALVAC CIRCO in the international depositary in accordance with the guidelines of the BUDAPEST treaty. Therefore, throughout the technical document, these names may be interchangeable.
  • a live vaccine or vaccine vector must be safe and effective, with fully controlled genotype and phenotype, avoiding the risk of reversion to virulence.
  • the strain must maintain a balance between the degree of attenuation and immunogenicity, remaining in the host organism long enough to give rise to a protective immune response against homologous and/or heterologous antigens.
  • the live recombinant vaccine comprises the strain of Salmonella enteritidis 3934 generated, which has as its main characteristics a drastic attenuation of the disease, where it is a registered and accepted strain for the present invention.
  • the Salmonella enteritidis 3934 strain is unable to form biofilms and has a very low survival rate in the environment, avoiding any risk associated with the period in which mammals, such as vaccinated animals, can excrete this strain. So it is also estimated that it may have a potential use in humans.
  • its genotype and phenotype are fully controlled and it does not have antibiotic resistance genes. The safety of live and attenuated bacteria has been verified in other models such as the 9R vaccine, whose reports scientifically rule out a potential reversion to the original virulent form (Okamoto., et al 2010 Brazilian Journal of Poultry Science).
  • the present invention also involves the use of a strain of Salmonella enteritidis 3934 (deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT9332) to which, by genetic engineering techniques, The twelve genes encoding diguanylate cyclase enzymes, the rpoS gene and the waaL gene have been deleted. This last mutation was made with the aim of obtaining a vaccine strain with a rough phenotype (Salmonella enteritidis 3934vac DwaaL) that confers protection to mammalian animals, including humans.
  • CECT Collection of Type Cultures
  • another aspect of the present invention comprises a vaccine strain with a rough phenotype that carries an expression cassette for the heterologous antigen RBD of the protein S of the Sars-Cov2-2019 virus that causes the 2019 coronavirus pandemic, which it confers immunity against viral infection caused by viruses in certain animals. That is, the CECT-9932 strain has been modified to express a protein of the heterologous RBD antigen of the protein S of the SARS-Cov-2-2019 virus.
  • the methodology used to generate the rough mutants generally uses the following steps: a) Construction of the integrative vector pKO:waaL b) Integration of the suicide vector pKO:waaL (first recombination) c) Exclusion of the integrative vector pKO :waaL (second recombination) d) Verification of the mutants by PCR Transformation of the expression cassette ⁇ PR-LppOmoA-RBD-6XHis-TT> in strain 3934:
  • the immunodetection experiment showed the presence of a specific band of approximately 35-38 KDa (which corresponds to the expected protein size) both in the strains in which the expression came from the plasmid (lines 2, 3 and 4) and in those strains with the expression cassette integrated in the chromosome (lanes 5, 6, 7 and 8).
  • Protein extracts from the wild type 3934Vac strain (WT) were used as negative control, in which no expression of the antigen was detected (line 9). Extracts from a clone of E. coi! containing the expression vector (line 1). The band is recognized by both the Anti-His and the Anti-SARS-CoV-2 antibodies (Fig. 2).
  • Vaccine compositions according to the present invention may be administered by any conventional route, including injection, oral, inhalation intranasal aerosol, eg, a nasal spray or drops, or by gradual infusion over time. Administration can be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous, or transdermal. In a preferred embodiment of the invention, the live recombinant vaccine is administered orally.
  • the vaccine compositions of the present invention may be administered in effective amounts.
  • An "effective amount" is that amount of a vaccine composition which, alone or in conjunction with additional doses, produces the desired immune response.
  • the presentation of the doses and/or packaging for the recombinant live vaccine for SARS-CoV 2 based on recombinant salmonella enteritidis can be any known form, for example, that is, the primary packaging, that is, the packaging that is in direct contact with the vaccine, it can be, but is not limited to, sterile glass or plastic ampoules, shachets (envelopes), either in single-dose or multi-dose vials.
  • the vaccine may be in one vial and the adjuvant or diluent in a separate vial, wherein the volume of vaccine and diluent in each vial is determined in accordance with normal practice.
  • the manufacturer and may be in secondary packaging, that is, a box containing several primary containers of the vaccine and/or the adjuvant or diluent.
  • These packages can be at a temperature between -5°C and 10°C, preferably between 5°C and 8°C; Therefore, it is an additional advantage of the recombinant live vaccine of the present invention, since it does not require special storage and/or transport devices to maintain extreme low temperatures.
  • the vaccine compositions comprise an adjuvant and/or excipient, wherein pharmaceutically acceptable adjuvants are defined as substances that increase antigen-specific immune responses by modulating immune cell activity.
  • adjuvants that can be used in the present invention for the recombinant live vaccine include, but are not limited to saponins, agonist antibodies to co-stimulatory molecules, Freund's adjuvant, muramyl dipeptide (MPD), bacterial DNA (oligo CpG), lipo- polysaccharides (LPS), MPL (Mozilla Public license) and synthetic derivatives, ipopeptides and liposomes, among others.
  • the adjuvant is an immunomodulator.
  • other preferred adjuvants may be squalene, Quillaja saponaria, and surfactants.
  • the vaccine according to the present invention comprises a strain of Salmonella Enteritidis modified according to the present invention which expresses proteins of the RBD region of the S1 domain of SARS CoV-2, sterile water and optionally, adjuvants such as squalene, quillaja saponaria and surfactants, [040]
  • the vaccine doses or concentrations thereof according to the present invention are formulated and administered in doses between a range of 10 5 CFU and 10 15 CFU; preferably, in a range between 10 8 CFU and 10 12 CFU according to any standard procedure in the art.
  • the administration can be carried out in one, two, three or more doses in recommended periods.
  • a subject is a mammal, preferably a human, and includes primate, bovine, equine, porcine, ovine, feline, and rodent.
  • Vaccine compositions according to the present invention may also optionally contain suitable preservatives, such as: benzalkonium chloride; chlorobutanol, parabens and thiomerosal, among others; inactivating agents such as formaldehyde, glutaraldehyde, propiolactone and beta-propiolactone are used in a trace level amount.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol, parabens and thiomerosal, among others; inactivating agents such as formaldehyde, glutaraldehyde, propiolactone and beta-propiolactone are used in a trace level amount.
  • Vaccine compositions suitable for parenteral administration conveniently comprise a sterile aqueous or nonaqueous vaccine preparation, which is preferably isotonic with the blood of the recipient.
  • a sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanedioL
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils are conventionally employed as a solvent or suspending medium.
  • fatty acids such as oleic acid can be used in the preparation of injectables.
  • a suitable carrier formulation can be found for subcutaneous, intravenous, intramuscular, oral administrations.
  • sterile water, yeasts, starches, gelatin, albumin, sucrose, lactose, sodium glutamate and glycine in pharmaceutically acceptable amounts can be used as adjuvants, vehicles and/or diluents.
  • Balb/c mice were divided into 7 groups of 6 mice each. A pre-immune blood sample was collected through a cut in the tail vein. Subsequently, each group was administered orally, 60 mL of culture medium with ampicillin, medium with Salmonella "wild type", medium with Salmonella modified with the PL-1 insert, as well as a treatment with the modification of PL-1 1, PL-12, PL-13 and one additional with a combination of all inserts. All groups received a concentration of 10 8 CFU/mL. Then 2 additional reinforcements were administered every 15 days, after taking a blood sample.
  • Figure 5 shows results where the response of individuals immunized with the Salmonella enteritidis antigen expressing RBD is observed. Note that the response increases with days and the chromosome 30 antigenic sample stands out at 35 days, while the response of the chromosome 23 microorganism is lower.
  • Slide or plate agglutination is a serological test used to detect antibodies against a particulate antigen. It is a quick and simple test, where the particulate antigen is deposited on a slide, in this case the modified Salmonella enteritidis, on whose surface the recombinant viral molecule RBD is expressed. An amount of immune serum from the antigen is deposited on the antigen. antigen-stimulated individual. For 2-3 minutes, the slide is rotated and/or shaken to promote binding between the antigen and the antibody to form antigen-antibody complexes. In a positive sample, the immunoglobulin molecules bound with the bacterial particles will be visible to the naked eye as they have a clumpy appearance as shown in Figure 3(B).
  • Salmonella Cellular immune response Interferon Gamma 5 week old female mice received the oral vaccine according to the present invention at 0 and 15 days with 10 8 CFU/ml. After vaccinating them at 15 and 30 DPV with Salmonella, they were euthanized and their spleens were aseptically removed. The organs were disaggregated, the cell suspension filtered and placed in a centrifuge tube containing 2 mL of Histopaque 1077 (Sigma), obtaining mononuclear cells.
  • the present invention is not limited to the scope of the microorganisms deposited in the patent since these represent a specific illustration of an aspect of the invention. Any microorganism or plasmid that is functionally equivalent to those described in the invention are included within the invention.

Abstract

The present invention relates to a recombinant live vaccine based on a Salmonella enteritidis strain expressing the S protein of the Sars-2019-Cov-2 virus, wherein the best expression has been achieved when the insertion is in the plasmid rather than the chromosome. It also relates to a vaccine that requires such strain. Reference is also made to the use of a Salmonella enteritidis 3934 strain (deposited in the Spanish Type Culture Collection (CECT) under accession number CECT9332) for the treatment of SARS-Cov-2 and to the method for controlling the SARS-CoV-2 infection by administering a live recombinant vaccine to mammals.

Description

VACUNA VIVA RECOMBINANTE PARA SARS-COV-2 BASADA EN SALMONELLA ENTERITIDIS RECOMBINANTE RECOMBINANT LIVE VACCINE FOR SARS-COV-2 BASED ON RECOMBINANT SALMONELLA ENTERITIDIS
SECTOR TECNOLÓGICO TECHNOLOGICAL SECTOR
[001] La presente invención se enmarca en sector de la salud, principalmente en la salud humana. Especialmente en el desarrollo de vacunas vivas alternativas para contrarrestar la pandemia causada por el virus del SARS CoV-2. [001] The present invention is framed in the health sector, mainly in human health. Especially in the development of alternative live vaccines to counteract the pandemic caused by the SARS CoV-2 virus.
RESUMEN DE LA INVENCIÓN SUMMARY OF THE INVENTION
[002] La presente invención hace referencia a una vacuna viva recombinante basada en una cepa de SE, Salmonella enterítidis 3934 XXII XrpoS waaL (3934VacR) atenuada y rugosa la cual ha sido modificada para la expresión de una proteína del virus Sars-Cov-2 causante de la pandemia COVID-19. Esta cepa de Salmonella entérica (3934VacR) es capaz de expresar el xenoantígeno proteico heterólogo RBD (receptor binding domain) externo de la proteína S del virus Sars-Cov-2 COV-19. La presente invención incluye además la utilización de esta cepa como vector vacunal frente al virus Sars-Cov-2, vía un proceso de inserción e integración de los genes que codifican el antígeno de la proteína de la superficie RBD del virus Sars-Cov-2 Covid-19. El vector de SE que se emplea, ha sido genéticamente modificado para funcionar como vehículo de expresión de los genes inmunodominantes del antígeno de la proteína RBD del virus Sars-Cov-2, con el fin último de estimular una respuesta inmune eficaz y duradera contra el virus Sars-Cov-2. La cepa generada es totalmente segura debido a que está exenta de los doce genes que codifican las proteínas de la ruta de señalización del mensajero secundario GMP-di-cíclico, el factor sigma RpoS y la proteína WaaL. Por lo anteriormente expuesto, esta cepa se propone como una nueva vacuna viva recombinante eficaz e inocua, contra el virus SArs-Cov-2, idónea para vacunar poblaciones de mamíferos, especialmente humanos, en donde todos los componentes de esta vacuna han sido específicamente diseñados y desarrollados con los tres factores prioritarios sucesivos de: eficacia, seguridad y costo. [002] The present invention refers to a live recombinant vaccine based on a strain of SE, Salmonella enteritidis 3934 XXII XrpoS waaL (3934VacR) attenuated and rough which has been modified for the expression of a protein of the Sars-Cov-2 virus caused by the COVID-19 pandemic. This strain of Salmonella enterica (3934VacR) is capable of expressing the heterologous protein xenoantigen RBD (receptor binding domain) external to protein S of the Sars-Cov-2 COV-19 virus. The present invention also includes the use of this strain as a vaccine vector against the Sars-Cov-2 virus, via a process of insertion and integration of the genes that encode the RBD surface protein antigen of the Sars-Cov-2 virus. Covid-19. The SE vector used has been genetically modified to function as a vehicle for the expression of the immunodominant genes of the RBD protein antigen of the Sars-Cov-2 virus, with the ultimate goal of stimulating an effective and lasting immune response against the virus. Sars-Cov-2 virus. The generated strain is totally safe because it is free of the twelve genes that encode the proteins of the GMP-di-cyclic secondary messenger signaling pathway, the RpoS sigma factor and the WaaL protein. Due to the above, this strain is proposed as a new effective and safe recombinant live vaccine against the SArs-Cov-2 virus, suitable for vaccinating populations of mammals, especially humans, where all the components of this vaccine have been specifically designed. and developed with the three successive priority factors: efficacy, safety and cost.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
[003] La emergencia y reemergencia de patógenos es un nuevo desafío para la salud pública. (Gao, GF.). Los coronavirus son virus ARN con cubierta, distribuidos ampliamente en humanos, algunos mamíferos y aves causando enfermedades respiratorias, entéricas, hepáticas y neurológicas (Weiss SR, Leibowitz JL.) (Masters PS, Perlman S.). Ahora se conocen seis especies de coronavirus que causan enfermedades humanas y cuatro de ellas — 229E, OC43, NL63 y HKU1 — son los más prevalentes y causantes típicos de estas infecciones virales. (Su S, Wong G, Shi W, et al.). Las características clínicas más destacadas de los pacientes se muestran en el artículo titulado “Clinical Characteristics of Coronavirus Disease 2019 in China” (Guan, Wei-jie et.aL). [003] The emergence and re-emergence of pathogens is a new challenge for public health. (Gao, GF.). Coronaviruses are enveloped RNA viruses distributed widely in humans, some mammals and birds causing respiratory, enteric, hepatic and neurological diseases (Weiss SR, Leibowitz JL.) (Masters PS, Perlman S.). Six species of coronavirus are now known to cause human illness, and four of them — 229E, OC43, NL63 and HKU1 — are the most prevalent and typical cause of these viral infections. (Su S, Wong G, Shi W, et al.). The most outstanding clinical characteristics of the patients are shown in the article entitled "Clinical Characteristics of Coronavirus Disease 2019 in China" (Guan, Wei-jie et.aL).
[004] Desde el punto de vista de patentes, se conoce el documento CN101838627 el cual se refiere a una estructura recombinante de salmonella choleraesuis, una vacuna de ingeniería genética bivalente y un método de preparación y aplicación de la recombinación de salmonella choleraesuis, donde la recombinación de salmonella choleraesuis no comprende un marcador de resistencia y expresa las principales proteínas Cap del antígeno del circovirus II porcino. El número de conservación de la recombinación Salmonella choleraesuis C501 (pYA-delta 410RF2) que no comprende el marcador de resistencia, expresa los principales sitios antigénicos del circovirus II porcino y se prepara mediante la invención de CCTCC M209314. La recombinación de salmonella choleraesuis elimina los genes asd que son necesarios para el crecimiento de la salmonella choleraesuis y comprende un plásmido que puede expresar los genes asd y los genes de los sitios antigénicos de las proteínas Cap del circovirus porcino II en la recombinación de salmonella choleraesuis. [004] From the point of view of patents, document CN101838627 is known which refers to a recombinant structure of salmonella choleraesuis, a bivalent genetic engineering vaccine and a method of preparation and application of the recombination of salmonella choleraesuis, where the Salmonella choleraesuis recombination does not comprise a resistance marker and expresses the major Cap proteins of porcine circovirus II antigen. Salmonella choleraesuis recombination conservation number C501 (pYA-delta 410RF2) which does not comprise the resistance marker, expresses the major antigenic sites of porcine circovirus II and is prepared by the invention of CCTCC M209314. Salmonella choleraesuis recombination deletes asd genes that are necessary for salmonella choleraesuis growth and comprises a plasmid that can express asd genes and porcine circovirus II Cap protein antigenic site genes in salmonella choleraesuis recombination .
[005] De otra parte, la patente WO205/035556se refiere a sistemas tales como plásmidos recombinantes, virus y procariotas que expresan las proteínas M, E y S asociadas a la membrana del SARS-CoV en células, tales como células humanas, tanto in vitro como in vivo en donde las proteínas SARS-CoV M, E y S forman espontáneamente partículas similares al virus del SARS-CoV (SARS-CoV-VLP). La expresión intracelular de las proteínas SARS-CoV M, E y S y su asociación para formar partículas tipo virus que presentan las proteínas virales en su contexto "natural", provoca la inducción de una respuesta del sistema inmunológico. También se refiere a métodos para producir una respuesta inmune en animales, tales como humanos y otros mamíferos, identificando a un sujeto en riesgo de desarrollar SARS y administrando al sujeto una o más construcciones genéticas capaces de expresar la SRAS- Polipéptidos CoV M, E y/o S. La invención enseña que las cepas procariotas vivas atenuadas deben mantener un equilibrio entre la atenuación y la inmunogenicidad y que no causan ninguna enfermedad ni alteren la fisiología normal del huésped. [005] On the other hand, patent WO205/035556 refers to systems such as recombinant plasmids, viruses and prokaryotes that express the M, E and S proteins associated with the SARS-CoV membrane in cells, such as human cells, both in in vitro and in vivo where SARS-CoV M, E and S proteins spontaneously form SARS-CoV virus-like particles (SARS-CoV-VLP). The intracellular expression of the SARS-CoV M, E and S proteins and their association to form virus-like particles that present the viral proteins in their "natural" context, causes the induction of an immune system response. It also relates to methods for eliciting an immune response in animals, such as humans and other mammals, by identifying a subject at risk of developing SARS and administering to the subject one or more genetic constructs capable of expressing SARS-CoV M, E and Polypeptides. /o S. The invention teaches that live attenuated prokaryotic strains must maintain a balance between attenuation and immunogenicity and that do not cause any disease or alter the normal physiology of the host.
[006] También se conoce el documento WO201 1/126976 el cual proporciona una proteína de reovirus S1 modificada que comprende un primer epítopo antigénico de un antígeno de no reovirus localizado en la región helicoidal de la proteína. El primer epítopo puede insertarse en la región alfa-helicoidal de la proteína, o puede reemplazar una o más repeticiones de heptada en la región alfa-helicoidal de la proteína. El primer epítopo puede ser un epítopo viral dentro de los cuales se encuentra el coronavirus asociado al SARS y un epítopo bacteriano de una bacteria dentro de las cuales se puede escoger la Salmonella spp. [006] WO201 1/126976 is also known which provides a modified reovirus S1 protein comprising a first antigenic epitope of a non-reovirus antigen located in the helical region of the protein. The first epitope may insert into the alpha-helical region of the protein, or may replace one or more heptad repeats in the alpha-helical region of the protein. The first epitope can be a viral epitope, among which the SARS-associated coronavirus is found, and a bacterial epitope of a bacterium, among which Salmonella spp.
[007] El documento US2018/296663 enseña una combinación de un primer componente y un segundo componente, en donde el primer componente es típicamente un componente inmunogénico y en donde el segundo componente es típicamente un componente adyuvante, en donde, el primer componente (inmunogénico) comprende al menos una molécula de ácido nucleico que codifica al menos un antígeno, o un fragmento o vahante del mismo. El segundo componente (adyuvante) de la combinación comprende al menos un compuesto adyuvante, en el que al menos un compuesto adyuvante es preferiblemente un compuesto inmunopotenciador. El segundo componente (adyuvante) de la combinación comprende al menos un compuesto potenciador inmunitaho, y/o al menos un compuesto del sistema de administración. Los antígenos patógenos son antígenos de péptidos o proteínas derivados preferiblemente de un patógeno asociado con una enfermedad infecciosa dentro de las cuales, se considera el coronavirus SARS. [007] US2018/296663 teaches a combination of a first component and a second component, wherein the first component is typically an immunogenic component and wherein the second component is typically an adjuvant component, wherein the first component (immunogenic ) comprises at least one nucleic acid molecule encoding at least one antigen, or a fragment or variant thereof. The second (adjuvant) component of the combination comprises at least one adjuvant compound, wherein at least one adjuvant compound is preferably an immunopotentiating compound. The second component (adjuvant) of the combination comprises at least one immune-enhancing compound, and/or at least one compound of the delivery system. Pathogenic antigens are peptide or protein antigens preferably derived from a pathogen associated with an infectious disease, among which the SARS coronavirus is considered.
[008] De otra parte, la patente WO2017/070626 enseña vacunas de ácido ribonucleico (ARN) que se basan en el conocimiento de que el ARN (por ejemplo, ARN mensajero (ARNm)) puede dirigir de manera segura la maquinaria celular del cuerpo para producir casi cualquier proteína de interés, desde proteínas nativas hasta anticuerpos y otras proteínas completamente nuevas. Los constructos pueden tener actividad terapéutica dentro y fuera de las células. Las vacunas de ARN (por ejemplo, ARNm) de acuerdo con dicha invención pueden usarse para inducir una respuesta inmune equilibrada contra hMPV, PrV, RSV, MeV y/o BetaCoV (por ejemplo, MERS-CoV, SARS-CoV, HCoV- OC43, HCoV -229E, HCoV-NL63, HCoV-NL, HCoV-NH y/o HCoV- HKU1), o cualquier combinación de dos o más de los virus anteriores, que comprendan inmunidad celular y humoral, sin riesgo de mutagénesis insercional, por ejemplo, hMPV, PIV, RSV, MeV, BetaCoV (p. ej., MERS-CoV, SARS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH y HCoV-HKUl) y combinaciones de los mismos. También enseña que la f lagelina es una proteína monomérica de aproximadamente 500 aminoácidos que se polimeñza para formar los flagelos asociados con el movimiento bacteriano. La flagelina es expresada por una variedad de bacterias flageladas (Salmonella typhimurium por ejemplo) así como por bacterias no flageladas (tales como Escherichia coi!) para la detección de flagelina por células del sistema inmunológico innato. [008] On the other hand, patent WO2017/070626 teaches ribonucleic acid (RNA) vaccines that are based on the knowledge that RNA (for example, messenger RNA (mRNA)) can safely direct the cellular machinery of the body to produce almost any protein of interest, from native proteins to antibodies and other entirely new proteins. The constructs may have therapeutic activity inside and outside cells. RNA (eg mRNA) vaccines according to said invention can be used to induce a balanced immune response against hMPV, PrV, RSV, MeV and/or BetaCoV (eg MERS-CoV, SARS-CoV, HCoV-OC43 , HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and/or HCoV-HKU1), or any combination of two or more of the above viruses, comprising cellular and humoral immunity, without risk of insertional mutagenesis, eg hMPV, PIV, RSV, MeV, BetaCoV (eg MERS-CoV, SARS-CoV, HCoV -OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH and HCoV-HKUl) and combinations thereof. It also teaches that flagellin is a monomeric protein of approximately 500 amino acids that polymerizes to form the flagella associated with bacterial movement. Flagellin is expressed by a variety of flagellated bacteria (Salmonella typhimurium for example) as well as by non-flagellated bacteria (such as Escherichia coli) for detection of flagellin by cells of the innate immune system.
[009] El documento US2016/339097 enseña la preparación y aislamiento de proteínas y antígenos de coronavirus, particularmente de coronavirus porcinos y proporciona además proteínas y antígenos virales obtenidos de células infectadas por coronavirus y composiciones que comprenden las proteínas y antígenos. Describe un método para preparar proteínas y/o antígenos de coronavirus, tales como proteínas y/o antígenos PEDV o PDCoV, a partir de células infectadas con un coronavirus, tales como PEDV o PDCoV. Las proteínas y/o antígenos se recolectan en un punto de tiempo temprano después de la infección cuando la mayoría, o la totalidad, de las proteínas y/o antígenos virales permanecen asociados con las células infectadas. La mayoría o la totalidad de las proteínas codificadas por coronavirus están dentro de las células infectadas o asociadas con la membrana celular de las células infectadas. En tales condiciones, relativamente pocas partículas de coronavirus, si las hay, están presentes en el entorno extracelular fuera de las células. La composición de vacuna puede comprender proteínas y/o antígenos de al menos un patógeno adicional que puede ser cualquier patógeno que cause una enfermedad y/o una infección en un sujeto porcino. [009] US2016/339097 teaches the preparation and isolation of coronavirus proteins and antigens, particularly porcine coronaviruses, and further provides viral proteins and antigens obtained from coronavirus-infected cells and compositions comprising the proteins and antigens. Describes a method for preparing coronavirus proteins and/or antigens, such as PEDV or PDCoV proteins and/or antigens, from cells infected with a coronavirus, such as PEDV or PDCoV. The proteins and/or antigens are harvested at an early time point after infection when most, or all, of the viral proteins and/or antigens remain associated with the infected cells. Most or all of the coronavirus-encoded proteins are within infected cells or associated with the cell membrane of infected cells. Under such conditions, relatively few, if any, coronavirus particles are present in the extracellular environment outside of cells. The vaccine composition may comprise proteins and/or antigens from at least one additional pathogen which may be any pathogen that causes disease and/or infection in a porcine subject.
[010] En la parte de artículos científicos, se conoce la publicación “Ora! delivery of SARS-CoV-2 DNA vaccines using attenuated salmonella thyphimurium as a carrier in rat" Este artículo muestra una investigación y exploración del uso de Salmonella typhimurium como vehículos para administrar plásmidos de expresión por vía oral. Se administró por vía oral Salmonella phoP atenuada que albergaba plásmidos de expresión eucañotas que codificaban la proteína de espícula de SARS-CoV-2 a ratas Wistar. Las ratas se inmunizaron por vía oral con Salmonella que portaba un plásmido de expresión eucañota una vez a la semana durante tres semanas consecutivas. La eficacia del procedimiento de vacunación se debió a la transferencia del plásmido de expresión del portador bacteriano al hospedador mamífero. La evidencia de tal evento podría obtenerse ¡n vivo e in vitro. Los resultados mostraron que todos los animales inmunizados generaron inmunidad humoral contra la proteína de espícula del SARS- CoV-2, lo que indica que una vacuna basada en Salmonella que lleva el gen Spike puede provocar respuestas inmunes humorales específicas del SARS-CoV-2 en ratas, y puede ser útil para el desarrollo de una vacuna protectora contra la infección por SARS-CoV-2. [010] In the part of scientific articles, the publication “Pray! delivery of SARS-CoV-2 DNA vaccines using attenuated salmonella typhimurium as a carrier in rat" This article shows an investigation and exploration of the use of Salmonella typhimurium as vehicles to deliver orally expressed plasmids. Attenuated Salmonella phoP was orally administered harboring eucalyptus expression plasmids encoding the spike protein of SARS-CoV-2 to Wistar rats.The rats were immunized orally with Salmonella harboring a eucalyptus expression plasmid once a week for three consecutive weeks. of the vaccination procedure was due to transfer of the plasmid from expression of the bacterial carrier to the mammalian host. Evidence for such an event could be obtained in vivo and in vitro. The results showed that all immunized animals generated humoral immunity against the spike protein of SARS-CoV-2, indicating that a Salmonella-based vaccine carrying the Spike gene can elicit SARS-CoV-2-specific humoral immune responses in rats, and may be useful for the development of a protective vaccine against SARS-CoV-2 infection.
[011] De otra parte, el artículo titulado “Oral vaccination with attenuated salmonella enterica serovar typhimurium expressing cap protein of pcv2 and its immunogenicity in mouse and swine models" el cual menciona que se utilizó la cepa SL3261 de vacuna atenuada de Salmonella entérica serovar Typhimurium como un sistema de administración de antígeno para la inmunización oral de ratones contra dos antígenos de Cryptosporidium parvum, Cp23 y Cp40. Cada antígeno se subclonó en el sistema de vector pTECHI , lo que permite que se expresen como proteínas de fusión con el fragmento C altamente inmunogénico de la toxina del tétanos bajo el control del promotor n i r B anaeróbicamente inducible. El vector recombinante se introdujo en la cepa de vacuna de Salmonella Typhimurium SL3261 , y la expresión soluble estable de la proteína quimérica se evaluó y confirmó mediante transferencia Western con antisueros policlonales de C. parvum. [011] On the other hand, the article entitled "Oral vaccination with attenuated salmonella enterica serovar typhimurium expressing cap protein of pcv2 and its immunogenicity in mouse and swine models" which mentions that the SL3261 strain of Salmonella enterica serovar Typhimurium attenuated vaccine was used as an antigen delivery system for oral immunization of mice against two Cryptosporidium parvum antigens, Cp23 and Cp40 Each antigen was subcloned into the pTECHI vector system, allowing them to be expressed as highly potent C-fragment fusion proteins. immunogenicity of tetanus toxin under the control of the anaerobically inducible nir B promoter.The recombinant vector was introduced into Salmonella Typhimurium SL3261 vaccine strain, and stable soluble expression of the chimeric protein was assessed and confirmed by Western blotting with polyclonal antisera. from C. parvum.
[012] Teniendo en cuenta el estado del arte, es evidente que si bien, actualmente existen diferentes métodos para detección del virus causante del Covid-19, a la fecha no existen vacunas comerciales contra el Sars-Cov-2, sin embargo, existen otras que están procesos muy avanzados y que próximamente podrían estar en el mercado. Estas vacunas varían en el tipo de adyuvante y naturaleza del antígeno. Por lo tanto, el problema que resuelve la presente invención es proporcionar una vacuna viva recombinante para el SARS-CoV-2 (Covid 19) que pueda generar una respuesta inmune efectiva y prolongada. [012] Taking into account the state of the art, it is clear that although there are currently different methods for detecting the virus that causes Covid-19, to date there are no commercial vaccines against Sars-Cov-2, however, there are others that are very advanced processes and that could soon be on the market. These vaccines vary in the type of adjuvant and nature of the antigen. Therefore, the problem that the present invention solves is to provide a live recombinant vaccine for SARS-CoV-2 (Covid 19) that can generate an effective and prolonged immune response.
[013] En este sentido, la presente invención proporciona el desarrollo de una vacuna viva y recombinante contra el Sars-Cov-2 que tiene una elevadísima probabilidad de éxito sustentada en la estabilidad de la proteína recombinante, sencillez y facilidad en la aplicación que se ofrecería a la salud pública nacional e internacional utilizando como vector una cepa de SE atenuada. Estas ventajas se pueden resumir en: (1 ) atenuación severa gracias a varios procesos de mutagénesis dirigida mediante un mecanismo de intercambio alélico que evita la reversión y convierten a la cepa resultante en un vector vacunal totalmente seguro; [013] In this sense, the present invention provides the development of a live and recombinant vaccine against Sars-Cov-2 that has a very high probability of success based on the stability of the recombinant protein, simplicity and ease of application that is would offer national and international public health using an attenuated SE strain as a vector. These advantages can be summarized as: (1) severe attenuation thanks to several directed mutagenesis processes through an allelic exchange mechanism that prevents reversion and converts the resulting strain into a totally safe vaccine vector;
(2) la cantidad de expresión del antígeno RBD que puede ser regulada mediante manipulación genética; (2) the amount of RBD antigen expression that can be regulated by genetic manipulation;
(3) puede administrarse por rutas de inoculación intradermal, oral e intranasal;(3) can be administered by intradermal, oral, and intranasal inoculation routes;
(4) estimula fuertemente el sistema inmune innato y adaptativo; y, (4) strongly stimulates the innate and adaptive immune system; and,
(5) brindaría a su vez protección contra SE. El desarrollo de vacunas empleando vectores bacterianos ha sido y es actualmente objeto de numerosas investigaciones, pero a la fecha no existen vacunas comerciales con licencia basados en SE. Por todo ello, el desarrollo y registro de esta vacuna, marcaría un buen precedente en el ámbito de investigación y desarrollo en la industria de biológicos nacional y no tendría competencia en el mercado nacional e internacional. (5) would in turn provide protection against SE. The development of vaccines using bacterial vectors has been and is currently the subject of numerous investigations, but to date there are no licensed commercial vaccines based on SE. For all these reasons, the development and registration of this vaccine would set a good precedent in the field of research and development in the national biological industry and would not have competition in the national and international market.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
[014] La figura 1 ¡lustra la electroforesis SDS-PAGE con tinción de coomassie realizada con anti-HIS de los extractos totales obtenidos. [014] Figure 1 illustrates SDS-PAGE electrophoresis with coomassie staining performed with anti-HIS of the total extracts obtained.
[015] La figura 2 ¡lustra la electroforesis SDS-PAGE con tinción de coomassie einmunodetección con anti-SARS-CoV-2 de los extractos proteicos totales (Western Blot). [015] Figure 2 illustrates SDS-PAGE electrophoresis with coomassie staining and anti-SARS-CoV-2 immunodetection of total protein extracts (Western Blot).
[016] La figura 3 muestra los resultados de la prueba de aglutinación que detecta los anticuerpos contra el antígeno particulado. [016] Figure 3 shows the results of the agglutination test that detects antibodies against the particulate antigen.
[017] La figura 4 enseña fotografías de cajas de Peth con muestras de heces de individuos inmunizados con la vacuna de acuerdo con la presente invención. [017] Figure 4 shows photographs of Peth dishes with stool samples from individuals immunized with the vaccine according to the present invention.
[018] La figura 5 muestra los resultados de la respuesta de los individuos inmunizados con el antígeno de Salmonella enterítidis que expresa RBD del SARs_CoV-2 (Covid- 19). [018] Figure 5 shows the results of the response of individuals immunized with the Salmonella enteritidis antigen expressing RBD of SARs_CoV-2 (Covid-19).
[019] La figura 6 muestra los resultados de células inmunológicas procedentes del bazo de ratones inmunizados con la vacuna oral, de acuerdo con la presente invención, donde muestra una gran actividad inmunológica de células T productoras de citocinas funcionales como IFN-y (interferon gamma) entre otras. BREVE DESCRIPCIÓN DE LA INVENCIÓN [019] Figure 6 shows the results of immune cells from the spleen of mice immunized with the oral vaccine, according to the present invention, where it shows a great immunological activity of T cells that produce functional cytokines such as IFN-y (interferon gamma ) among other. BRIEF DESCRIPTION OF THE INVENTION
[020] La presente invención se refiere a una vacuna viva recombinante basada en una cepa de Salmonella enterítidis que expresa la proteína S del virus Sars-2019-Cov-2 en donde la mejor expresión se ha logrado cuando la inserción se encuentra en el plásmido en vez del cromosoma. Asimismo, se refiere a una vacuna que precisa de dicha cepa. [020] The present invention relates to a live recombinant vaccine based on a strain of Salmonella enteritidis that expresses the S protein of the Sars-2019-Cov-2 virus where the best expression has been achieved when the insert is in the plasmid instead of the chromosome. Likewise, it refers to a vaccine that requires said strain.
[021] También, la presente invención hace referencia al uso de una cepa de Salmonella enterítidis 3934 (depositada en la Colección Española de Cultivos Tipo (CECT) con el número de acceso CECT9332) para el tratamiento de SARS-Cov-2 y al método para controlar la infección del SARS-CoV-2 mediante la administración a mamíferos de una vacuna viva recombinante [021] Also, the present invention refers to the use of a strain of Salmonella enteritidis 3934 (deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT9332) for the treatment of SARS-Cov-2 and to the method to control SARS-CoV-2 infection by administering a live recombinant vaccine to mammals
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
[022] La presente invención se refiere al desarrollo de una vacuna contra el SARS- Cov-2-2019 causante de la pandemia COVID-19, para lo cual se construyó el cassette <PR-LppOmpA-RBD-6XHis-TTterminator > mediante PCR sobrelapante de dos fragmentos de 670 y 671 de pares de bases. Se incluyó un linker (GGGSGGGS) entre el fragmento LppOmpA y el fragmento RBD. Finalmente, el fragmento obtenido de 1348 pb fue subclonado en un plásmido de expresión específicamente diseñado para la inserción de secuencias en el locus attTn7, el cual se usó para transformar la cepa rugosa de Salmonella enterítidis 3934 AXII ArpoS Awaal (3934VacR) mediante electroporación. Las colonias transformantes fueron seleccionadas en presencia de ampicilina. [022] The present invention relates to the development of a vaccine against SARS-Cov-2-2019 causing the COVID-19 pandemic, for which the <PR-LppOmpA-RBD-6XHis-TTterminator> cassette was constructed by PCR overlapping of two fragments of 670 and 671 base pairs. A linker (GGGSGGGS) was included between the LppOmpA fragment and the RBD fragment. Finally, the 1348 bp fragment obtained was subcloned into an expression plasmid specifically designed for the insertion of sequences in the attTn7 locus, which was used to transform the rough strain of Salmonella enteritidis 3934 AXII ArpoS Awaal (3934VacR) by electroporation. Transformant colonies were selected in the presence of ampicillin.
[023] Para la expresión del antígeno de la proteína S del virus Sars-Cov-2-2019 Covid- 19 se ha diseñado y construido un “cassette" de expresión, el cual se caracteriza porque incluye: [023] For the expression of the protein S antigen of the Sars-Cov-2-2019 Covid-19 virus, an expression "cassette" has been designed and built, which is characterized in that it includes:
• Región promotora (PR) • Promoter Region (PR)
• Secuencia para la exportación a membrana del antígeno RBD (LppOmpA) • Sequence for membrane export of the RBD antigen (LppOmpA)
• Linker o enlazante (GGGSGGGS) • Linker or linker (GGGSGGGS)
• Región de 6 residuos de histidina para la detección de la expresión (6XHis) • Region of 6 histidine residues for expression detection (6XHis)
• Fragmento de proteína RBD de S1 de la Spike de Corona 223 aminoácidos y • Terminador (TT). • RBD protein fragment of S1 of the Corona Spike 223 amino acids and • Terminator (TT).
[024] Continuando con el proceso, con el fin de integrar en el cromosoma y plásmido, se realizó un abordaje por recombinación homologa de un cassette de expresión que permitiese la producción del antígeno heterólogo RBD de la proteína S del virus Sars- Cov-2-2019 en la cepa de SE. En la presente invención, el plásmido contiene, a parte de las regiones flanqueantes de recombinación, un cassette de expresión que queda integrado en el cromosoma. [024] Continuing with the process, in order to integrate into the chromosome and plasmid, an approach was carried out by homologous recombination of an expression cassette that would allow the production of the heterologous antigen RBD of the protein S of the Sars-Cov-2 virus. -2019 in the SE strain. In the present invention, the plasmid contains, apart from the recombination framework regions, an expression cassette that is integrated into the chromosome.
[025] La cepa resultante a la que se refiere esta invención es una cepa mutante modificada de Salmonella enterítidis que porta una deleción del gen waaL, y presenta por tanto un fenotipo rugoso, y expresa un gen que codifica el dominio de la proteína RBD de la proteína S del Sars-Cov-2-2019, causante de la pandemia Covid-19. [025] The resulting strain to which this invention refers is a modified mutant strain of Salmonella enteritidis that carries a deletion of the waaL gene, and therefore presents a rough phenotype, and expresses a gene that encodes the RBD protein domain of the S protein of Sars-Cov-2-2019, causing the Covid-19 pandemic.
[026] Para efectos de la presente invención Salmonella enterítidis es abreviatura de Salmonella entérica serovar Enterítidis y la cepa SE3934-Cap ha sido depositada con el nombre SALVAC CIRCO en el depositario internacional de acuerdo con los lineamientos del tratado de BUDAPEST. Por tanto, a lo largo del documento técnico, dichas denominaciones pueden ser intercambiables. [026] For the purposes of the present invention Salmonella enteritidis is an abbreviation for Salmonella enterica serovar Enteritidis and the strain SE3934-Cap has been deposited under the name SALVAC CIRCO in the international depositary in accordance with the guidelines of the BUDAPEST treaty. Therefore, throughout the technical document, these names may be interchangeable.
[027] Como se indicó anteriormente, una vacuna viva o vector vacunal debe ser seguro y eficaz, con un genotipo y fenotipo totalmente controlados, que eviten el riesgo de reversión a virulencia. Además, la cepa debe mantener un equilibrio entre el grado de atenuación e inmunogenicidad, permaneciendo en el organismo del hospedador el tiempo suficiente para dar lugar a una respuesta inmune protectora frente a antígenos homólogos y/o heterólogos. [027] As stated above, a live vaccine or vaccine vector must be safe and effective, with fully controlled genotype and phenotype, avoiding the risk of reversion to virulence. In addition, the strain must maintain a balance between the degree of attenuation and immunogenicity, remaining in the host organism long enough to give rise to a protective immune response against homologous and/or heterologous antigens.
[028] En la presente invención, la vacuna viva recombinante comprende la cepa de Salmonella enterítidis 3934 generada que presenta como características primordiales una drástica atenuación de la enfermedad, en donde es una cepa registrada y aceptada para la presente invención. Además, la cepa Salmonella enterítidis 3934 es incapaz de formar biopelículas y tiene una supervivencia muy reducida en el ambiente, evitando cualquier riesgo asociado al período en el que los mamíferos, tales como animales vacunados puedan excretar esta cepa. De manera que también se estima que puede tener un potencial uso en seres humanos. A diferencia de la mayoría de las vacunas comerciales, su genotipo y fenotipo están totalmente controlados y no posee genes de resistencia a antibióticos. La inocuidad de bacterias vivas y atenuadas ha sido verificada en otros modelos como la vacuna 9R, cuyos reportes descartan científicamente una potencial reversión a la forma virulenta original (Okamoto., et al 2010 Revista Brasilera de Ciencia Avícola). [028] In the present invention, the live recombinant vaccine comprises the strain of Salmonella enteritidis 3934 generated, which has as its main characteristics a drastic attenuation of the disease, where it is a registered and accepted strain for the present invention. In addition, the Salmonella enteritidis 3934 strain is unable to form biofilms and has a very low survival rate in the environment, avoiding any risk associated with the period in which mammals, such as vaccinated animals, can excrete this strain. So it is also estimated that it may have a potential use in humans. Unlike most vaccines commercial, its genotype and phenotype are fully controlled and it does not have antibiotic resistance genes. The safety of live and attenuated bacteria has been verified in other models such as the 9R vaccine, whose reports scientifically rule out a potential reversion to the original virulent form (Okamoto., et al 2010 Brazilian Journal of Poultry Science).
[029] En este sentido, la presente invención involucra además el uso de una cepa de Salmonella enterítidis 3934 (depositada en la Colección Española de Cultivos Tipo (CECT) con el número de acceso CECT9332) a la cual, por técnicas de ingeniería genética, se le han delecionado los doce genes que codifican enzimas diguanilatociclasas, el gen rpoS y el gen waaL. Esta última mutación se realizó con el objetivo de obtener una cepa vacunal de fenotipo rugoso (Salmonella enterítidis 3934vac DwaaL) que confiera protección a animales mamíferos, incluyendo seres humanos. [029] In this sense, the present invention also involves the use of a strain of Salmonella enteritidis 3934 (deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT9332) to which, by genetic engineering techniques, The twelve genes encoding diguanylate cyclase enzymes, the rpoS gene and the waaL gene have been deleted. This last mutation was made with the aim of obtaining a vaccine strain with a rough phenotype (Salmonella enteritidis 3934vac DwaaL) that confers protection to mammalian animals, including humans.
[030] Asimismo, otro aspecto de la presente invención comprende una cepa vacunal de fenotipo rugoso que porta un cassette de expresión para el antígeno heterólogo RBD de la proteína S del virus Sars-Cov2-2019 causante de la pandemia del coronavirus 2019, la cual confiere inmunidad contra la infección viral causada por virus en ciertos animales. Es decir, la cepa CECT-9932 ha sido modificada para expresar una proteína del antígeno heterólogo RBD de la proteína S del virus SARS-Cov-2-2019. [030] Likewise, another aspect of the present invention comprises a vaccine strain with a rough phenotype that carries an expression cassette for the heterologous antigen RBD of the protein S of the Sars-Cov2-2019 virus that causes the 2019 coronavirus pandemic, which it confers immunity against viral infection caused by viruses in certain animals. That is, the CECT-9932 strain has been modified to express a protein of the heterologous RBD antigen of the protein S of the SARS-Cov-2-2019 virus.
[031] Con el objetivo de reducir la toxicidad y mantener la inmunogenicidad de las cepas basadas en el fondo genético 3934vac, se procedió a un doble proceso de atenuación, es decir se retiró 13 genes (AXIII) (cepa S. Enterítidis) llamado AXII, llevando a cabo mutaciones en los doce genes que codifican las proteínas del dominio GGDEF y el gen rpoS, Latasa 2016) y luego se hace rugosa a fin de hacerla más propensa al ataque inmune. [031] In order to reduce the toxicity and maintain the immunogenicity of the strains based on the 3934vac genetic background, a double attenuation process was carried out, that is, 13 genes (AXIII) were removed (strain S. Enteritidis) called AXII , carrying out mutations in the twelve genes that encode the GGDEF domain proteins and the rpoS gene, Latase 2016) and then becomes rough in order to make it more prone to immune attack.
[032] La metodología empleada para la generación de los mutantes rugosos utiliza de manera general los siguientes pasos: a) Construcción del vector integrativo pKO:waaL b) Integración del vector suicida pKO:waaL (primera recombinación) c) Excusión del vector integrativo pKO:waaL (segunda recombinación) d) Verificación de los mutantes por PCR Transformación del cassette de expresión <PR-LppOmoA-RBD-6XHis-TT> en la cepa 3934: [032] The methodology used to generate the rough mutants generally uses the following steps: a) Construction of the integrative vector pKO:waaL b) Integration of the suicide vector pKO:waaL (first recombination) c) Exclusion of the integrative vector pKO :waaL (second recombination) d) Verification of the mutants by PCR Transformation of the expression cassette <PR-LppOmoA-RBD-6XHis-TT> in strain 3934:
[033] Para la construcción del casette de expresión <PR-SEM-6XHis-RBD-TT> Se utilizó la cepa rugosa de Salmonella enteritidis 3934 AXI I ArpoS Awaal (3934vacR) como vehículo para la expresión del antígeno de la proteína de la superficie RBD del virus del SARS-CoV-2. [033] For the construction of the <PR-SEM-6XHis-RBD-TT> expression cassette, the rough strain of Salmonella enteritidis 3934 AXI ArpoS Awaal (3934vacR) was used as a vehicle for the expression of the surface protein antigen RBD of the SARS-CoV-2 virus.
Verificación de la expresión mediante Western Blot: Verification of expression by Western Blot:
[034] Para corroborar la expresión de la fusión <PR-LppOmpA-RBD-6XHis-TT> de las cepas 3934Vac con el cassette att7n7;:PR-LppOmpA-RBD-6XHis-TT integrado, se prepararon extractos proteicos totales a partir de cultivos planctónicos crecidos en medio LB a 32eC. Dichos extractos se separaron mediante electroforesis en gel de poliacrilamida y SDS (SDS-PAGE). Una vez separadas, las proteínas se electro- transfirieron a una membrana de nitrocelulosa (fig . 1 y 2) y se incubaron con anticuerpos anti-histidinas conjugados con Horseradish Peroxidase (HRP) (fig. 1 ). El experimento de inmunodetección demostró la presencia de una banda específica de aproximadamente 35-38 KDa (que se corresponde con el tamaño proteico esperado) tanto en las cepas en las que la expresión provenía desde plásmido (líneas 2, 3 y 4) como en las cepas con el cassette de expresión integrado en el cromosoma (líneas 5, 6, 7 y 8). Como control negativo se han utilizado extractos proteicos de la cepa 3934Vac salvaje (WT), en la cual, no se detecta expresión del antígeno (línea 9). También se incluyeron extractos procedentes de un clon de E. coi! conteniendo el vector de expresión (línea 1 ). La banda es reconocida tanto por el anticuerpo Anti-His, como por el Anti-SARS-CoV- 2. (fig. 2). [034] To corroborate the expression of the <PR-LppOmpA-RBD-6XHis-TT> fusion of the 3934Vac strains with the integrated att7n7;:PR-LppOmpA-RBD-6XHis-TT cassette, total protein extracts were prepared from planktonic cultures grown in LB medium at 32 ° C. These extracts were separated by polyacrylamide gel electrophoresis and SDS (SDS-PAGE). Once separated, the proteins were electrotransferred to a nitrocellulose membrane (fig. 1 and 2) and incubated with anti-histidine antibodies conjugated with Horseradish Peroxidase (HRP) (fig. 1). The immunodetection experiment showed the presence of a specific band of approximately 35-38 KDa (which corresponds to the expected protein size) both in the strains in which the expression came from the plasmid (lines 2, 3 and 4) and in those strains with the expression cassette integrated in the chromosome (lanes 5, 6, 7 and 8). Protein extracts from the wild type 3934Vac strain (WT) were used as negative control, in which no expression of the antigen was detected (line 9). Extracts from a clone of E. coi! containing the expression vector (line 1). The band is recognized by both the Anti-His and the Anti-SARS-CoV-2 antibodies (Fig. 2).
[035] Las composiciones de vacuna de acuerdo con la presente invención se pueden administrar por cualquier vía convencional, que incluye inyección, oral, aerosol intranasal por inhalación, por ejemplo, un aerosol o gotas nasales, o por infusión gradual en el tiempo. La administración puede ser, por ejemplo, oral, intravenosa, intraperitoneal, intramuscular, intracavitaha, subcutánea o transdérmica. En una modalidad preferida de la invención la vacuna viva recombinante se administra vía oral. [036] Las composiciones de vacuna de la presente invención pueden ser administradas en cantidades eficaces. Una "cantidad eficaz" es aquella cantidad de una composición de vacuna que, sola o junto con dosis adicionales, produce la respuesta inmune deseada. [035] Vaccine compositions according to the present invention may be administered by any conventional route, including injection, oral, inhalation intranasal aerosol, eg, a nasal spray or drops, or by gradual infusion over time. Administration can be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous, or transdermal. In a preferred embodiment of the invention, the live recombinant vaccine is administered orally. [036] The vaccine compositions of the present invention may be administered in effective amounts. An "effective amount" is that amount of a vaccine composition which, alone or in conjunction with additional doses, produces the desired immune response.
[037] La presentación de las dosis y/o empaques para la vacuna viva recombinante para SARS-CoV 2 basada en salmonella enteritidis recombinante, puede ser cualquier forma conocida, por ejemplo, es decir, el empaque primario, esto es, el empaque que está directamente en contacto con la vacuna, puede ser, pero no se limita a ampolletas de vidrio o de plástico estériles, shachets (sobres), bien sea en frascos unidosis o multidosis. En otra modalidad de la invención, si se desea, la vacuna puede estar en un frasco y el adyuvante o diluyente en otro frasco separado, en donde el volumen de la vacuna y el diluyente en cada frasco se determina de acuerdo con la práctica normal del fabricante y pueden estar en empaques secundarios, es decir, una caja que contiene vahos envases primarios de la vacuna y/o del adyuvante o diluyente. Estos empaques pueden estar a temperatura entre -5°C y 10°C, preferiblemente entre 5°C y 8°C; por lo que es una ventaja adicional de la vacuna viva recombinante de la presente invención, ya que no requiere de dispositivos especiales de almacenamiento y/o de transporte para mantener temperaturas bajas extremas. [037] The presentation of the doses and/or packaging for the recombinant live vaccine for SARS-CoV 2 based on recombinant salmonella enteritidis, can be any known form, for example, that is, the primary packaging, that is, the packaging that is in direct contact with the vaccine, it can be, but is not limited to, sterile glass or plastic ampoules, shachets (envelopes), either in single-dose or multi-dose vials. In another embodiment of the invention, if desired, the vaccine may be in one vial and the adjuvant or diluent in a separate vial, wherein the volume of vaccine and diluent in each vial is determined in accordance with normal practice. manufacturer and may be in secondary packaging, that is, a box containing several primary containers of the vaccine and/or the adjuvant or diluent. These packages can be at a temperature between -5°C and 10°C, preferably between 5°C and 8°C; Therefore, it is an additional advantage of the recombinant live vaccine of the present invention, since it does not require special storage and/or transport devices to maintain extreme low temperatures.
[038] En una modalidad preferida de la presente invención, las composiciones de vacuna comprenden un adyuvante y/o excipiente, en donde los adyuvantes farmacéuticamente aceptables se definen como sustancias que aumentan las respuestas inmunitahas específicas a los antígenos modulando la actividad de las células inmunitahas. Ejemplos de adyuvantes que pueden ser empleados en la presente invención para la vacuna viva recombinante incluyen, pero no se limitan a saponinas, anticuerpos agonistas para moléculas coestimuladoras, adyuvante de Freund, muramil dipéptido (MPD), DNA bacteriano (oligo CpG), lipo-polisacáridos (LPS), MPL (Mozilla Public license) y derivados sintéticos, I ipopéptidos y liposomas, entre otros. El adyuvante es un inmunomodulador. En una modalidad de la invención, otros adyuvantes preferidos pueden ser escualeno, Quillaja saponaria y surfactantes. [038] In a preferred embodiment of the present invention, the vaccine compositions comprise an adjuvant and/or excipient, wherein pharmaceutically acceptable adjuvants are defined as substances that increase antigen-specific immune responses by modulating immune cell activity. . Examples of adjuvants that can be used in the present invention for the recombinant live vaccine include, but are not limited to saponins, agonist antibodies to co-stimulatory molecules, Freund's adjuvant, muramyl dipeptide (MPD), bacterial DNA (oligo CpG), lipo- polysaccharides (LPS), MPL (Mozilla Public license) and synthetic derivatives, ipopeptides and liposomes, among others. The adjuvant is an immunomodulator. In one embodiment of the invention, other preferred adjuvants may be squalene, Quillaja saponaria, and surfactants.
[039] La vacuna de acuerdo con la presente invención, comprende una cepa de Salmonella Enteritidis modificada de acuerdo con la presente invención la cual expresa proteínas de la región RBD del dominio S1 del SARS CoV-2, agua estéril y opcionalmente, adyuvantes tales como el escualeno, quillaja saponaria y surfactantes, [040] Las dosis de vacuna o concentraciones de la misma de acuerdo con la presente invención se formulan y administran en dosis entre un intervalo de 105 UFC y 1015 UFC; de preferencia, en un intervalo entre 108 UFC y 1012 UFC de acuerdo con cualquier procedimiento estándar en la técnica. La administración puede realizarse en una, dos, tres o más tomas en periodos recomendados. [039] The vaccine according to the present invention comprises a strain of Salmonella Enteritidis modified according to the present invention which expresses proteins of the RBD region of the S1 domain of SARS CoV-2, sterile water and optionally, adjuvants such as squalene, quillaja saponaria and surfactants, [040] The vaccine doses or concentrations thereof according to the present invention are formulated and administered in doses between a range of 10 5 CFU and 10 15 CFU; preferably, in a range between 10 8 CFU and 10 12 CFU according to any standard procedure in the art. The administration can be carried out in one, two, three or more doses in recommended periods.
[041] Un experto en la técnica conocerá otros protocolos para la administración de las composiciones de vacuna, en los que la cantidad de dosis, el programa de inyecciones, los lugares de las inyecciones, el modo de administración y similares podrían variar de acuerdo con la práctica recomendada. La administración de las composiciones de vacuna a mamíferos distintos de los seres humanos (por ejemplo, con fines de prueba o con fines terapéuticos veterinarios) se lleva a cabo sustancialmente en las mismas condiciones que las descritas anteriormente. Un sujeto, como se usa en este documento, es un mamífero, preferiblemente un ser humano, e incluye un primates, bovinos, equinos, porcinos, ovinos, felinos y roedores. [041] Other protocols for administration of vaccine compositions will be known to one skilled in the art, in which the number of doses, schedule of injections, sites of injections, mode of administration, and the like may vary according to recommended practice. Administration of the vaccine compositions to mammals other than humans (eg, for testing purposes or for veterinary therapeutic purposes) is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and includes primate, bovine, equine, porcine, ovine, feline, and rodent.
[042] Las composiciones de vacuna de acuerdo con la presente invención, también pueden contener, opcionalmente, conservantes adecuados, tales como: cloruro de benzalconio; clorobutanol, parabenos y tiomerosal, entre otros; inactivantes como los que se usan formaldehido, glutaraldehído, propiolactona y beta-propiolactona en una cantidad a nivel de trazas. [042] Vaccine compositions according to the present invention may also optionally contain suitable preservatives, such as: benzalkonium chloride; chlorobutanol, parabens and thiomerosal, among others; inactivating agents such as formaldehyde, glutaraldehyde, propiolactone and beta-propiolactone are used in a trace level amount.
[043] Las composiciones de vacuna adecuadas para el caso de la administración parenteral comprenden convenientemente una preparación de vacuna acuosa o no acuosa estéril, que preferiblemente es ¡sotónica con la sangre del receptor. Estas vacunas se pueden formular de acuerdo con métodos conocidos usando agentes dispersantes o humectantes y agentes de suspensión adecuados. La preparación inyectable estéril también puede ser una solución o suspensión inyectable estéril en un diluyente o solvente no tóxico parenteralmente aceptable, por ejemplo, como una solución en 1 ,3-butanodioL Entre los vehículos y disolventes aceptables que se pueden emplear se encuentran el agua, la solución de Ringer y la solución ¡sotónica de cloruro de sodio. Además, convencionalmente se emplean aceites fijos estériles como disolvente o medio de suspensión. Para este propósito, se puede emplear cualquier aceite fijo suave incluyendo mono o diglicéridos sintéticos. Además, los ácidos grasos como el ácido oleico pueden usarse en la preparación de inyectables. Se puede encontrar una formulación de vehículo adecuada para administraciones subcutáneas, intravenosas, intramusculares, orales. Para el caso de vacunas vivas recombinantes de acuerdo con la presente invención, se puede tener como adyuvantes, vehículos y/o diluyentes, agua estéril, levaduras, almidones, gelatina, albúmina, sacarosa, lactosa, glutamato sódico y glicina en cantidades farmacéuticamente aceptables. [043] Vaccine compositions suitable for parenteral administration conveniently comprise a sterile aqueous or nonaqueous vaccine preparation, which is preferably isotonic with the blood of the recipient. These vaccines can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanedioL Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any Mild fixed oil including synthetic mono or diglycerides. Also, fatty acids such as oleic acid can be used in the preparation of injectables. A suitable carrier formulation can be found for subcutaneous, intravenous, intramuscular, oral administrations. In the case of live recombinant vaccines according to the present invention, sterile water, yeasts, starches, gelatin, albumin, sucrose, lactose, sodium glutamate and glycine in pharmaceutically acceptable amounts can be used as adjuvants, vehicles and/or diluents.
EJEMPLO DE LA MEJOR FORMA DE LLEVAR A CABO LA INVENCÓN EXAMPLE OF THE BEST WAY TO CARRY OUT THE INVENTION
Inmunización de ratones Immunization of mice
[044] Los ratones Balb/c fueron divididos en 7 grupos de 6 ratones cada uno. Se colectó una muestra de sangre pre-inmune, mediante un corte en la vena caudal. Posteriormente, se administraron por cada grupo por vía oral, 60 mL de medio de cultivo con ampicilina, medio con Salmonella “wild type”, medio con Salmonella modificada con el inserto PL-1 , así como un tratamiento con la modificación de PL-1 1 , PL-12, PL-13 y un adicional con una combinación de todos los insertos. Todos los grupos recibieron una concentración de 108 UFC/mL. Luego se administró 2 refuerzos adicionales cada 15 días, previa toma de muestra de sangre. [044] Balb/c mice were divided into 7 groups of 6 mice each. A pre-immune blood sample was collected through a cut in the tail vein. Subsequently, each group was administered orally, 60 mL of culture medium with ampicillin, medium with Salmonella "wild type", medium with Salmonella modified with the PL-1 insert, as well as a treatment with the modification of PL-1 1, PL-12, PL-13 and one additional with a combination of all inserts. All groups received a concentration of 10 8 CFU/mL. Then 2 additional reinforcements were administered every 15 days, after taking a blood sample.
[045] Para saber y medir la respuesta inmune de los animales, así como título de anticuerpos, a los 28 días se realizó la prueba inmunológica ELISA indirecta. (Figura 5). [046] En la figura 5 se muestran resultados donde se observa la respuesta de los individuos inmunizados con el antígeno de Salmonella enterítidis que expresa RBD. Obsérvese que la respuesta incrementa con los días y sobresale la muestra antigénica cromosoma 30 a los 35 días, mientras que la respuesta del microorganismo cromosoma 23, es menor. [045] To know and measure the immune response of the animals, as well as the antibody titer, the indirect ELISA immunological test was performed at 28 days. (Figure 5). [046] Figure 5 shows results where the response of individuals immunized with the Salmonella enteritidis antigen expressing RBD is observed. Note that the response increases with days and the chromosome 30 antigenic sample stands out at 35 days, while the response of the chromosome 23 microorganism is lower.
Ensayo de aglutinación Agglutination assay
[047] La aglutinación en lámina o placa es una prueba serológica empleada para detectar anticuerpos contra un antígeno particulado. Es una prueba rápida y sencilla, donde se deposita sobre una lámina portabjetos el antígeno particulado, en este caso la Salmonella enterítidis modificada, en cuya superficie se expresa la molécula viral recombinante RBD. Sobre el antígeno se deposita una cantidad de suero inmune del individuo estimulado con el antígeno. Durante 2-3 minutos, la lámina es rotada y/o agitada para favorecer la unión entre el antígeno con el anticuerpo a fin de formar complejos antígeno-anticuerpo. En una muestra positiva, las moléculas de inmunoglobulina unidas con las partículas bacterianas serán visibles a simple vista pues tienen un aspecto grumoso como se muestra en la figura 3 (B). Si en la muestra no hubiese anticuerpos contra el antígeno, Salmonella enterítídís, no se observará la aglutinación tal como se observa en la misma figura 3 (A). Si hubiese dificultad o dudas en la observación, la aglutinación se comprueba con la ayuda del microscopio. En nuestro caso, los sueros de los individuos inmunizados resultaron positivos en la prueba de aglutinación en lámina, pues se observó aglutinación (Fig. 3), en donde A es una muestra control seronegativa donde se observa la ausencia de aglutinación. No hay aglutinación pues no tiene anticuerpos contra la bacteria y en B se observa la reacción positiva del suero de un individuo vacunado que contiene anticuerpos contra la Salmonella enterítídís. Por haber sido expuesto al antígeno microbiano que posee el antígeno RBD en la superficie, el anticuerpo reconocerá al antígeno y ocurrirá la reacción antígeno anticuerpo que será notoria como se nota en la reacción de aglutinación en B (figura 3). [047] Slide or plate agglutination is a serological test used to detect antibodies against a particulate antigen. It is a quick and simple test, where the particulate antigen is deposited on a slide, in this case the modified Salmonella enteritidis, on whose surface the recombinant viral molecule RBD is expressed. An amount of immune serum from the antigen is deposited on the antigen. antigen-stimulated individual. For 2-3 minutes, the slide is rotated and/or shaken to promote binding between the antigen and the antibody to form antigen-antibody complexes. In a positive sample, the immunoglobulin molecules bound with the bacterial particles will be visible to the naked eye as they have a clumpy appearance as shown in Figure 3(B). If there were no antibodies against the antigen, Salmonella enterítídís, in the sample, agglutination will not be observed, as can be seen in the same figure 3 (A). If there is difficulty or doubt in the observation, the agglutination is checked with the help of the microscope. In our case, the sera of the immunized individuals were positive in the slide agglutination test, since agglutination was observed (Fig. 3), where A is a seronegative control sample where the absence of agglutination is observed. There is no agglutination because it does not have antibodies against the bacteria and in B the positive reaction of the serum of a vaccinated individual that contains antibodies against Salmonella enterítídís is observed. Due to having been exposed to the microbial antigen that has the RBD antigen on the surface, the antibody will recognize the antigen and the antigen-antibody reaction will occur, which will be noticeable as noted in the agglutination reaction in B (figure 3).
Análisis de las heces de los individuos inmunizados con Salmonella enterítídís Analysis of feces from individuals immunized with Salmonella enteritidis
[048] Como se puede apreciar en la figura 4, en las placas Petri conteniendo el medio de cultivo Agar SS estéril, se cultivaron muestras de heces obtenidas en diferentes días luego de la inmunización de los individuos (ratones). Obsérvese que la presencia de colonias de Salmonella enterítídís -positivos- (puntos oscuros) van disminuyendo a medida que transcurren los días post-inmunización. Del resultado se puede concluir que comparando el cuarto y quinto, al sexto día, el cultivo de la muestra de heces es negativo, lo cual significa que los individuos inmunizados de acuerdo con la vacuna de la presente invención excretan la totalidad de la bacteria luego del 6to día de la inmunización. Esta característica del comportamiento de la inmunización con la presente vacuna, permite de manera ventajosa que en ambientes insalubres o de muy baja salubridad, ocurra una inmunización indirecta de otros individuos. [048] As can be seen in figure 4, in the Petri dishes containing the sterile SS Agar culture medium, stool samples obtained on different days after the immunization of the individuals (mice) were cultured. Note that the presence of -positive- Salmonella enteritidis colonies (dark dots) decreases as the post-immunization days go by. From the result it can be concluded that comparing the fourth and fifth, on the sixth day, the culture of the stool sample is negative, which means that the individuals immunized according to the vaccine of the present invention excrete all the bacteria after the 6th day of immunization. This characteristic of the behavior of the immunization with the present vaccine advantageously allows an indirect immunization of other individuals to occur in unhealthy or very poor environments.
Salmonella Respuesta inmune celular Interferon Gamma [049] Ratones hembra de 5 semanas recibieron la vacuna oral de acuerdo con la presente invención a los 0 y 15 días con 108 UFC/ml. Luego de vacunarlos a los 15 y 30 DPV con Salmonella se les practicó la eutanasia y se les extrajo asépticamente el bazo. Los órganos fueron desagregados, la suspensión celular filtrada y colocada en un tubo de centrífuga que contenía 2 mL de Histopaque 1077 (Sigma), obteniéndose células mononucleares. Éstas fueron cultivadas en una placa ELISPOT de 96 pozos, precubiertas con el anticuerpo anti-ratón IFN-y (clon RMMG-1 , Merck) y bloqueadas con 1 % de albúmina sérica bovina (BSA, Sigma) y estimuladas con la rRBD del SARS-CoV- 2, durante 24 horas a 37°C y 5% de CO2. Como control positivo fue utilizada Concanavalina A (Sigma). Las células fueron eliminadas por lavado y los pozos incubados con el anti-ratón biotinilado IFN-y (policlonal, Abeam; o clon R4-6A2, Biolegend) durante 2 horas a temperatura ambiente. Previo lavado, los pozos fueron incubados con fosfatasa alcalina ligada a estreptavidina (SAV-ALP, Sigma) durante una hora a temperatura ambiente. Seguido de un lavado exhaustivo, fue utilizado el sustrato cromogénico, azul de nitro tetrazolio NBT / BCIP (Abeam). (Fig. 6) Salmonella Cellular immune response Interferon Gamma [049] 5 week old female mice received the oral vaccine according to the present invention at 0 and 15 days with 10 8 CFU/ml. After vaccinating them at 15 and 30 DPV with Salmonella, they were euthanized and their spleens were aseptically removed. The organs were disaggregated, the cell suspension filtered and placed in a centrifuge tube containing 2 mL of Histopaque 1077 (Sigma), obtaining mononuclear cells. These were cultured in a 96-well ELISPOT plate, precoated with the anti-mouse IFN-γ antibody (clone RMMG-1, Merck) and blocked with 1% bovine serum albumin (BSA, Sigma) and stimulated with SARS rRBD. -CoV- 2, for 24 hours at 37°C and 5% CO2. Concanavalin A (Sigma) was used as a positive control. Cells were washed away and wells incubated with biotinylated anti-mouse IFN-γ (polyclonal, Abeam; or clone R4-6A2, Biolegend) for 2 hours at room temperature. After washing, the wells were incubated with streptavidin-linked alkaline phosphatase (SAV-ALP, Sigma) for one hour at room temperature. Following an exhaustive wash, the chromogenic substrate, nitro blue tetrazolium NBT / BCIP (Abeam) was used. (Fig.6)
[050] La presente invención no está limitada al ámbito de los microorganismos depositados en la patente dado que estos representan una ilustración puntual de un aspecto de la invención. Cualquier microorganismo o plásmido que sea funcionalmente equivalente a los descritos en la invención se incluyen dentro de la invención. [050] The present invention is not limited to the scope of the microorganisms deposited in the patent since these represent a specific illustration of an aspect of the invention. Any microorganism or plasmid that is functionally equivalent to those described in the invention are included within the invention.

Claims

REIVINDICACIONES
1. Una vacuna viva recombinante para SARS-CoV-2 de salmonella enteritidis recombinante caracterizada porque comprende una cantidad eficaz de una cepa mutante modificada de salmonella enteritidis 3934 Vac Dwaal en donde dicha cepa de salmonella enteritidis comprende un fragmento de proteína RBD de S1 de la Spike de Corona 223 aminoácidos. 1. A recombinant live vaccine for SARS-CoV-2 of recombinant salmonella enteritidis characterized in that it comprises an effective amount of a modified mutant strain of salmonella enteritidis 3934 Vac Dwaal where said strain of salmonella enteritidis comprises an RBD protein fragment of S1 of the Corona Spike 223 amino acids.
2. La vacuna viva recombinante para para SARS-CoV-2 de salmonella enteritidis recombinante de acuerdo con la reivindicación 1 caracterizada porque la concentración de la salmonella enteritidis recombinante se encuentra en un intervalo de 105 UFC y 1015 UFC. 2. The recombinant live vaccine for SARS-CoV-2 of recombinant salmonella enteritidis according to claim 1, characterized in that the concentration of recombinant salmonella enteritidis is in a range of 10 5 CFU and 10 15 CFU.
3. La vacuna viva recombinante para para SARS-CoV-2 de salmonella enteritidis recombinante de acuerdo con la reivindicación 1 caracterizada porque comprende, además un adyuvantes, excipientes o vehículos farmacéuticamente aceptables. 3. The live recombinant vaccine for SARS-CoV-2 of recombinant salmonella enteritidis according to claim 1, characterized in that it also comprises pharmaceutically acceptable adjuvants, excipients or vehicles.
4. La vacuna viva recombinante para para SARS-CoV-2 de salmonella enteritidis recombinante de acuerdo con la reivindicación 3 caracterizada porque los adyuvantes se seleccionan del grupo que consiste de saponinas, anticuerpos agonistas para moléculas coestimuladoras, adyuvante de Freund, muramil dipéptido (MPD), DNA bacteriano (oligo CpG), lipo-polisacáridos (LPS), MPL (Mozilla Public license) y derivados sintéticos, lipopéptidos, liposomas, escualeno, Quillaja y surfactantes. 4. The recombinant live vaccine for SARS-CoV-2 of recombinant salmonella enteritidis according to claim 3, characterized in that the adjuvants are selected from the group consisting of saponins, agonist antibodies for costimulatory molecules, Freund's adjuvant, muramyl dipeptide (MPD ), bacterial DNA (oligo CpG), lipopolysaccharides (LPS), MPL (Mozilla Public license) and synthetic derivatives, lipopeptides, liposomes, squalene, Quillaja and surfactants.
5. La vacuna viva recombinante para para SARS-CoV-2 de salmonella enteritidis recombinante de acuerdo con la reivindicación 3 caracterizada porque el vehículo farmacéuticamente aceptable es agua estéril, solución de Ringer y solución ¡sotónica de cloruro de sodio. 5. The recombinant live vaccine for SARS-CoV-2 of recombinant salmonella enteritidis according to claim 3, characterized in that the pharmaceutically acceptable vehicle is sterile water, Ringer's solution and isotonic sodium chloride solution.
6. La vacuna viva recombinante para para SARS-CoV-2 de salmonella enteritidis recombinante de acuerdo con la reivindicación 3 caracterizada porque además comprende conservantes tales como, cloruro de benzalconio; clorobutanol, parabenos y tiomerosal. 6. The recombinant live vaccine for SARS-CoV-2 of recombinant salmonella enteritidis according to claim 3 characterized in that in addition comprises preservatives such as, benzalkonium chloride; chlorobutanol, parabens and thiomerosal.
7. La vacuna viva recombinante para para SARS-CoV-2 de salmonella enterítidis recombinante de acuerdo con la reivindicación 3 caracterizada porque además comprende inactivantes tales como formaldehido, glutaraldehído, propiolactona y beta-propiolactona en una cantidad a nivel de trazas. 7. The live recombinant vaccine for SARS-CoV-2 of recombinant salmonella enteritidis according to claim 3, characterized in that it also comprises inactivating agents such as formaldehyde, glutaraldehyde, propiolactone and beta-propiolactone in a trace level amount.
8. La vacuna viva recombinante para para SARS-CoV-2 de salmonella enterítidis recombinante de acuerdo con la reivindicación 1 caracterizada porque es una vacuna inyectable, oral, aerosol intranasal por inhalación o por infusión gradual en el tiempo. 8. The live recombinant vaccine for SARS-CoV-2 of recombinant salmonella enteritidis according to claim 1, characterized in that it is an injectable, oral, intranasal aerosol vaccine by inhalation or by gradual infusion over time.
9. La vacuna viva recombinante para para SARS-CoV-2 de salmonella enterítidis recombinante de acuerdo con la reivindicación 1 caracterizada porque la vacuna es una vacuna oral. 9. The live recombinant vaccine for SARS-CoV-2 of recombinant salmonella enteritidis according to claim 1, characterized in that the vaccine is an oral vaccine.
10. Uso de una cepa de Salmonella enterítidis 3934 (depositada en la Colección Española de Cultivos Tipo (CECT) con el número de acceso CECT9332) para el tratamiento de SARS-Cov-2. 10. Use of a strain of Salmonella enteritidis 3934 (deposited in the Spanish Collection of Type Cultures (CECT) with the access number CECT9332) for the treatment of SARS-Cov-2.
1 1 . Un método para controlar la infección causada por el SARS-CoV-2 mediante la administración a mamíferos de una vacuna viva recombinante de acuerdo con cualquiera de las reivindicaciones 1 a 9. eleven . A method for controlling the infection caused by SARS-CoV-2 by administering to mammals a recombinant live vaccine according to any of claims 1 to 9.
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WO2018231078A2 (en) * 2017-06-12 2018-12-20 Farmacologicos Veterinarios Sac Production of rough-type salmonella enteritidis and the genetic modifications thereof for use as an avian vaccine

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