WO2022026534A1 - Compositions immunogènes contre le sras-cov-2 et méthodes de protection contre les signes cliniques du sras-cov-2 - Google Patents

Compositions immunogènes contre le sras-cov-2 et méthodes de protection contre les signes cliniques du sras-cov-2 Download PDF

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WO2022026534A1
WO2022026534A1 PCT/US2021/043432 US2021043432W WO2022026534A1 WO 2022026534 A1 WO2022026534 A1 WO 2022026534A1 US 2021043432 W US2021043432 W US 2021043432W WO 2022026534 A1 WO2022026534 A1 WO 2022026534A1
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sars
cov
disclosure
sequence
seq
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PCT/US2021/043432
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Juergen Richt
David MEEKINS
Chester MCDOWELL
Igor Morozov
Natasha GAUDREAULT
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Kansas State University Research Foundation
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the field of the disclosure relates generally to immunogenic compositions and methods for protecting against clinical signs of Severe Acute Respiratory Syndrome-related Coronaviruses (“SARS-CoV” and “SARS-CoV-2”) infection.
  • Coronaviruses are enveloped single-stranded, positive-sense RNA viruses that belong to the order Nidovirales in the family Coronaviridae, subfamily Orthocoronavirinae, which are comprised of four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus .
  • Alphacoronavirus Alphacoronavirus
  • Betacoronavirus betacoronavirus
  • Gammacoronavirus Gammacoronavirus
  • Deltacoronavirus avirus
  • Many alpha- and betacoronaviruses origininate from bats, while gamma- and deltacoronaviruses tend to have their origin in birds.
  • SARS-CoV SARS-CoV-2 and the Middle East Respiratory Syndrome coronavirus (MERS-CoV) belong to the genus betacoronavirus.
  • Alpha- and betacoronaviruses infect only mammals and cause important diseases of humans, cattle, pigs, cats, dogs, horses, and camels.
  • coronaviruses cause respiratory, enteric, and systemic infections in humans and numerous animal hosts.
  • coronaviruses can easily cross-species barriers.
  • Bats have been identified as the reservoir species for many coronaviruses including those causing important human epidemics, namely SARS- CoV in 2002-2003 and MERS-CoV in 2012. Camels have since been shown to serve as the primary intermediate and reservoir host for MERS-CoV, causing continued zoonotic animal-to-human transmissions.
  • SARS-CoV epidemic infected domestic cats were identified from households of SARS-CoV positive patients, and both cats and ferrets were subsequently experimentally shown to be easily infected and transmit SARS-CoV.
  • SARS-CoV-2 is the cause of Coronavirus Disease 2019 (COVID-19) and responsible for the current global pandemic.
  • a zoonotic transmission event at a seafood and animal market in Wuhan, Hubei Province, China, is suspected to be the site of the disease origin in humans, with bats and/or pangolins being speculated as potential origin species based on the sequence homology of coronaviruses isolated from these animals.
  • SARS-CoV-2 has spread to more than 215 countries, has infected more than 10 million people and has caused more than 500,000 deaths. Since humans do not have pre-existing immunity against SARS-CoV-2, it is urgent to develop therapeutics and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19 in the future.
  • the present disclosure solves the problems inherent in the field and provides a distinct advance in the state of the art by providing immunogenic compositions for coronaviruses, together with methods of using such immunogenic compositions to treat, reduce the incidence of, and/or reduce the severity of clinical signs of infection by coronavirus. Additionally, the present disclosure provides methods for preventing or reducing the transmission of coronavirus between humans and other animal species.
  • the present disclosure provides a cat model for COVID-19.
  • Cats (Felis catus) are highly susceptible to SARS-CoV-2 infection, and are able to transmit the virus to naive contact cats. Inoculation of subadult cats (5 months old) with 10 6 TCID50 of the human SARS-CoV-2 isolate USA-WA1/2020 through the intranasal and oral routes resulted in virus replication in the upper and lower respiratory tract as well as the gastrointestinal tract. Both, infected and contact cats seroconverted, but no obvious clinical signs were observed. At necropsy on days 4 and 7 post infection, interstitial pneumonia and inflammation in nasal turbinates and the trachea were observed.
  • an immunogenic composition comprising the polypeptide sequence of SEQ ID NO. 1 is provided.
  • a composition may include SEQ ID NO. 1 as part of a larger sequence derived from SARS-CoV2.
  • the larger sequence comprises at least 50, 100, 150, 200, 215, or more polypeptides that flank either side of the SEQ ID NO. 1 sequence in the cat SARS-CoV-2 spike protein sequence are included.
  • sequences When such sequences are included, it is preferred that they have at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or even 100% sequence homology or sequence identity with the consensus sequence of human USA-WA1/2020 or the consensus sequence of the corresponding sequence after it has been recovered from a cat.
  • an inactivated immunogenic composition comprising inactivated SARS-CoV-2 having an insert encoding for SEQ ID NO. 1 therein.
  • the insert has the sequence of SEQ ID NO. 2.
  • the insert is at nucleotide position 645 corresponding to the SARS-CoV-2 sequence for isolate WA1/2020.
  • Such a composition may include SEQ ID NO. 2 as part of a larger sequence derived from SARS-CoV2.
  • the larger sequence comprises at least 50, 100, 150, 200, 215, or more nucleotides that flank either side of the SEQ ID NO. 2 sequence in the cat SARS-CoV-2 spike nucleotide sequence are included.
  • sequences When such sequences are included, it is preferred that they have at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or even 100% sequence homology or sequence identity with the consensus sequence of human USA-WA1/2020 or the consensus sequence of the corresponding sequence after it has been recovered from a cat.
  • a killed immunogenic composition comprising killed SARS-CoV-2 having an insert encoding for SEQ ID NO. 1 therein.
  • the insert has the sequence of SEQ ID NO. 2.
  • the insert is at nucleotide position 645 corresponding to the SARS-CoV-2 sequence for isolate WA1/2020.
  • an immunogenic composition comprising a vector having an insert corresponding to SEQ ID NOs 1 and/or 2 is provided.
  • an immunogenic composition comprising an attenuated SARS-CoV-2
  • the genome of the attenuated SARS-CoV-2 includes SEQ ID NO. 2 therein and encodes for a polypeptide that includes SEQ ID NO. 1.
  • an immunogenic composition comprising antibodies generated against a polypeptide comprising SEQ ID NO. 1 is provided.
  • the antibodies are recovered or harvested from an animal that has been infected with a SARS-CoV-2 that includes SEQ ID NO. 2 as an insert compared to a consensus SARS-CoV-2 sequence.
  • SARS-CoV-2 consensus sequence is for isolate WA1/2020.
  • an immunogenic composition is provided to an animal susceptible to infection by SARS-CoV-2.
  • the animal receiving the administration is one that does not show clinical signs of infection but is still infected with the virus.
  • Such administration is effected in order to prevent transmission of SARS-CoV-2 to other animals, including humans and to prevent signs of clinical infection in the animals that do exhibit clinical signs of infection.
  • the composition comprises the mRNA of SARS-CoV-2 with the insert of SEQ ID NO. 2 being present in the nucleotide sequence.
  • the mRNA encoding for SEQ ID NO. 1 is inserted into the mRNA sequence.
  • the insert is at position 645 when using SEQ ID NO. 3 as a reference sequence.
  • compositions and methods can be used for mammals, including companion animals and big cats such as lions and tigers. In some aspects, the compositions and methods can be used for humans, cattle, pigs, cats, dogs, horses, and camels.
  • Figure 1 is a visual depiction of the experiment described in
  • Fig. 2A is a graph illustrating body temperatures that were recorded daily over the course of the study
  • Fig. 2B is a graph illustrating the body weight of each cat recorded throughout the study
  • Fig. 2C is a graph illustrating serum biochemistry of alkaline phosphatase levels over the course of the study wherein treatment group averages and standard deviations are shown in each panel and shaded areas indicate normal range limits;
  • Fig. 3A is a graph illustrating qRT-PCR that was performed on nasal swabs collected over the course of the 21 -day study. Average viral copy number/mL with standard deviations are shown for each treatment group. Astrisks (*) indicate 1 ⁇ 2 of the sample qRT-PCR reactions were below the limit of detection;
  • Fig. 3B is a graph illustrating qRT-PCR that was performed on oropharyngeal swabs collected over the course of the 21 -day study. Average viral copy number/mL with standard deviations are shown for each treatment group. Astrisks (*) indicate 1 ⁇ 2 of the sample qRT-PCR reactions were below the limit of detection;
  • Fig. 3C is a graph illustrating qRT-PCR that was performed on rectal swabs collected over the course of the 21-day study. Average viral copy number/mL with standard deviations are shown for each treatment group. Astrisks (*) indicate 1 ⁇ 2 of the sample qRT-PCR reactions were below the limit of detection;
  • Figure 4A is a set of pictures illustrating gross pathological lesions in the lungs. Lungs were removed in toto from each animal, at 4, 7 and 21 DPC, to assess for gross pathological lesions. Representative lung are shown for a negative control animal, and one principal infected animal necropsied at each of the days indicated;
  • Figure 4B is a set of pictures illustrating the histopathology of trachea. Histological findings in the trachea of SARS-CoV-2 infected cats. At 4 and 7 dpi, multifocal submucosal glands and ducts are distended (ectatic), filled with a small amount of necrotic debris, and lined by an attenuated epithelium (arrowheads). The circumjacent interstitium is infiltrated by mild to moderate numbers of lymphocytes, histiocytes and scattered neutrophils that occasionally transmigrate through the lining epithelium. Inflammatory changes progress from minimal/mild to moderate between 4 and 7 dpi. No histologic changes are noted at 21 dpi. H&E. Total magnification: 200X;
  • Figure 4C is a set of pictures illustrating the histopathology of bronchi. Histological findings in main bronchi of SARS-CoV-2 infected cats. Histologic changes and their progression are similar to those observed in the trachea, with multifocal, widespread, mild to moderate lymphocytic and neutrophilic adenitis noted at 4 and 7 dpi. Necrotic debris within distorted submucosal glands are indicated with arrowheads and few transmigrating lymphocytes are indicated with an arrow. No histologic changes are noted at 21 dpi. H&E. Total magnification: 200X; and
  • FIG. 5 is an illustration representing the schematic of the tissue sampling of the airways and the lung lobes.
  • One aspect of the present disclosure provides a method of producing and/or recovering at least a portion of the mutated spike protein identified in cats after infection with the human SARS-CoV-2 isolate WA1/2020.
  • the mutated protein can be recovered from such cats and expanded for use in immunogenic compositions.
  • the mutated protein can be introduced into an expression vector for expression thereof.
  • recombinant SARS- CoV-2 mutated spike protein is inserted into an appropriate vector, and this vector is used to 1) infect a number of susceptible cells in culture with a recombinant viral vector encoding a SARS-CoV-2 mutated spike protein, 2) expressing SARS-CoV-2 mutated spike protein by the recombinant viral vector, 3) recovering the SARS-CoV-2 mutated spike protein, and, 4) separating cell debris from the expressed SARS-CoV-2 mutated spike protein via a separation step.
  • an inactivation step is preferred in order to inactivate the viral vector prior to recovery of SARS-CoV-2 mutated spike protein that will be used in an immunogenic or immunological composition such as a vaccine.
  • Such a step can be performed as step 5) in addition to steps 1-4 described above.
  • this inactivation is done either just before or just after the filtration or separation step. Any conventional inactivation method can be used for purposes of the present disclosure. Thus, inactivation can be performed by chemical and/or physical treatments.
  • One representative inactivation method includes the addition of cyclized binary ethylenimine (BEI).
  • the method described above may also include a neutralization step after step 5).
  • the inactivation agent is BEI
  • addition of sodium thiosulfate to an equivalent amount is preferred.
  • the sodium thiosulfate is added in equivalent amount as compared to the BEI added for inactivation.
  • an “immunogenic or immunological composition” refers to a composition of matter that comprises at least one antigen which elicits an immunological response in the host of a cellular and / or antibody-mediated immune response to the composition or vaccine of interest.
  • an “immunological response” includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or yd T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in the severity or prevalence of, up to and including a lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.
  • each lot or just selected lots of harvested SARS-CoV-2 mutated spike protein can be tested for inactivation.
  • an inactivation test for determining the effectiveness of the inactivation of the recombination viral vector, comprising the steps: 1) contacting at least a portion of the culture fluid containing the recombinant viral vector with an inactivating agent, 2) adding a neutralization agent to neutralize the inactivation agent, and 3) determining the residual infectivity.
  • the recombinant viral vector containing SARS-CoV-2 mutated spike protein DNA and expressing SARS-CoV-2 mutated spike protein used to infect the cells is generated by transfecting a transfer vector that has had a SARS-CoV-2 mutated spike protein gene cloned therein into a viral vector.
  • a transfer vector that has had a SARS-CoV-2 mutated spike protein gene cloned therein into a viral vector.
  • the portion of the transfer vector that contains the desired SARS- CoV-2 mutated spike protein DNA is transfected into the viral vector.
  • transfected into a viral vector means, and is used as a synonym for “introducing” or “cloning” a heterologous DNA into a viral vector, such as for example into a baculovirus vector.
  • a “transfer vector” means a DNA molecule, that includes at least one origin of replication, the heterologous gene, in the present case of SARS-CoV-2 mutated spike protein, DNA sequences which allow the cloning of said heterologous gene into the viral vector will be included.
  • the sequences which allow cloning of the heterologous gene into the viral vector are flanking the heterologous gene. Even more preferably, those flanking sequences are at least homologous in parts with sequences of the viral vector.
  • the methods of the present disclosure will begin with the isolation of SARS-CoV-2 mutated spike protein DNA.
  • Any SARS-CoV-2 mutated spike protein gene can be used for purposes of the present disclosure.
  • the SARS-CoV-2 mutated spike protein includes the KLRS (SEQ ID NO. 1) insertion described above.
  • the SARS-CoV-2 mutated spike protein DNA is preferably amplified using PCR methods. The resulting DNA is then cloned into the transfer vector.
  • a method for constructing a recombinant viral vector containing SARS-CoV-2 mutated spike protein DNA generally comprises the steps of: 1) cloning at least one recombinant SARS-CoV-2 mutated spike protein gene into a transfer vector; and 2) transfecting the portion of the transfer vector containing the recombinant SARS-CoV-2 mutated spike protein gene into a viral vector, to generate the recombinant viral vector.
  • the SARS-CoV-2 mutated spike protein DNA can be amplified prior to step 1) in vitro, wherein the flanking sequences of the SARS-CoV-2 mutated spike protein DNA are modified.
  • the flanking sequences of the SARS-CoV-2 mutated spike protein DNA are modified.
  • cloning in vitro amplified SARS-CoV-2 mutated spike protein DNA into a transfer vector and suitable transfer vectors are described above or known to a person skilled in the art.
  • the present disclosure relates to a method for constructing a recombinant viral vector containing SARS- CoV-2 mutated spike protein DNA and expressing a desired SARS-CoV-2 mutated spike protein comprising the steps of: 1) amplifying SARS-CoV-2 mutated spike protein DNA in vitro, wherein the flanking sequences of said SARS-CoV-2 mutated spike protein DNA are modified, 2) cloning the amplified SARS-CoV-2 mutated spike protein ORF2 DNA into a transfer vector; and 3) transfecting the transfer vector or a portion thereof containing the recombinant SARS-CoV-2 mutated spike protein DNA into a viral vector to generate the recombinant viral vector.
  • the modification of the flanking sequences of the SARS-CoV-2 mutated spike protein DNA is performed by introducing a 5’ Kozak’s sequence and /or an EcoR 1 site.
  • a further aspect of the present disclosure relates to a method for preparing a composition comprising SARS-CoV-2 mutated spike protein, and inactivated viral vector.
  • This method generally comprises the steps of: 1) cloning the amplified SARS-CoV-2 mutated spike protein DNA into a transfer vector; 2) transfecting the portion of the transfer vector containing the recombinant SARS-CoV- 2 mutated spike protein DNA into a virus; 3) infecting cells in media with the transfected viral vector; 4) causing the transfected viral vector to express the recombinant protein from the SARS-CoV-2 mutated spike protein DNA; 5) separating cells from the supemate; 6) recovering the expressed SARS-CoV-2 mutated spike protein; and 7) inactivating the recombinant viral vector.
  • a neutralization step step 8
  • step 8 the SARS-CoV-2 mutated spike protein DNA can be amplified in vitro, preferably with flanking sequences of the SARS-CoV-2 mutated spike protein DNA, as described above.
  • a method for preparing a composition for invoking an immune response against SARS-CoV-2 is provided.
  • this method includes the steps of transfecting a construct into a virus, wherein the construct comprises 1) recombinant DNA from SARS-CoV-2 mutated spike protein, 2) infecting cells in growth media with the transfected virus, 3) causing the virus to express the recombinant protein from SARS-CoV-2 mutated spike protein, 4) recovering the expressed recombinant protein, 5) and preparing the composition by combining the recovered protein with a suitable adjuvant and/or other pharmaceutically acceptable carrier.
  • the composition also includes at least a portion of the viral vector expressing said SARS-CoV-2 mutated spike protein, and/or a portion of the cell culture supemate.
  • adjuvants can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
  • the emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene oil resulting from theoligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters.
  • the oil is used in combination with emulsifiers to form the emulsion.
  • the emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121.
  • mannide e.g. anhydromannitol oleate
  • glycol glycol
  • polyglycerol propylene glycol and of oleic
  • isostearic ricinoleic or hydroxystearic acid
  • polyoxypropylene-polyoxyethylene copolymer blocks in particular the Pluronic products, especially L121.
  • a further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative.
  • Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U. S. Patent No.
  • 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms.
  • the preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups.
  • the unsaturated radicals may themselves contain other substituents, such as methyl.
  • the products sold under the name Carbopol ; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol.
  • Carbopol 974P, 934P and 97 IP there may be mentioned Carbopol 974P, 934P and 97 IP.
  • the copolymers of maleic anhydride and alkenyl derivative the copolymers EMA (Monsanto) which are copolymers of maleic anhydride and ethylene.
  • the dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.
  • Suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among many others.
  • the adjuvant is added in an amount of about 100 pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 100 pg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 pg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 750 pg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.
  • a method for preparing an immunogenic composition, such as a vaccine, for invoking an immune response against SARS-CoV-2 comprises the steps of 1) expressing and recovering SARS-CoV-2 mutated spike protein, and 2) admixing the recovered protein with a suitable adjuvant.
  • the expressing step 1) includes the steps as described for the preparation and recovery of SARS-CoV-2 mutated spike protein.
  • Another optional step for this method includes cloning the amplified SARS-CoV-2 mutated spike protein DNA into a first vector, excising the DNA from this first vector, and using this excised SARS-CoV-2 mutated spike protein DNA for cloning into the transfer vector.
  • the recovery step of this method also includes the step of separating the media from the cells and cell debris.
  • This can be done in any conventional manner, with one preferred manner comprising filtering the cells, cell debris, and growth media through a filter having pores ranging in size from about 0.45 mM to about 1.0 mM.
  • a neutralization step as described above.
  • the composition can include one or more pharmaceutical-acceptable or veterinary-acceptable carriers.
  • a pharmaceutical-acceptable carrier or “veterinary-acceptable carrier” includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like.
  • the composition provided herewith contains SARS- CoV-2 mutated spike protein recovered from in vitro cultured cells, wherein said cells were infected with a recombinant viral vector containing SARS-CoV-2 mutated spike protein DNA, preferably encoding the KLRS (SEQ ID NO.
  • this method can also include the addition of a protectant.
  • a protectant as used herein refers to an anti-microbiological active agent, such as for example Gentamycin, Merthiolate, and the like. In particular adding a protectant is most preferred for the preparation of a multi-dose composition. Those anti- microbiological active agents are added in concentrations effective to prevent the composition of interest from any microbiological contamination or for inhibition of any microbiological growth within the composition of interest.
  • the composition comprises at least one component selected from the group consisting of stabilizing agents, preservatives, antibacterial and antifungal agents, adjuvants, adsorption delaying agents, cell culture debris, and any combination thereof.
  • the methods of the present disclosure can also comprise the addition of any stabilizing agent, such as for example saccharides, trehalose, mannitol, saccharose and the like, to increase and/or maintain product shelf-life and/or to enhance stability.
  • any stabilizing agent such as for example saccharides, trehalose, mannitol, saccharose and the like, to increase and/or maintain product shelf-life and/or to enhance stability.
  • the present disclosure relates to a composition of matter comprising recombinantly expressed SARS-CoV-2 mutated spike protein.
  • this composition of matter also comprises an agent suitable for the inactivation of viral vectors.
  • Such products are useful as immunogenic compositions that induce an immune response and, more preferably, confers protective immunity against the clinical signs of SARS- CoV-2 mutated spike protein infection.
  • the composition generally comprises the polypeptide, or a fragment thereof, expressed by SARS-CoV-2 DNA encoding mutated spike protein, as the antigenic component of the composition.
  • SARS-CoV-2 mutated spike protein polypeptide used in an immunogenic composition in accordance with the present disclosure can be derived in any fashion including isolation and purification, standard protein synthesis, and recombinant methodology.
  • Clinical signs of SARS-CoV-2 infection include fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, diarrhea.
  • administration of a composition disclosed herein reduces the incidence of and/or the severity of one or more clinical signs of SARS-CoV-2 infection.
  • administration of a composition disclosed herein treats active SARS-CoV-2 infection by reducing the incidence, severity, or duration of one or more clinical signs of SARS-CoV-2 infection.
  • administration of a composition prevents one or more clinical signs of SARS- CoV2 infection.
  • any SARS-CoV-2 mutated spike protein would be effective as the source of the SARS-CoV-2 mutated spike protein DNA and/or polypeptide as used herein.
  • the antigenic characteristics of an immunological composition can be, for example, estimated by challenge experiments. Moreover, the antigenic characteristic of a modified antigen is still retained, when the modified antigen confers at least 70%, preferably 80%, more preferably 90% of the protective immunity as compared to the SARS-CoV-2 mutated spike protein, encoded by the polynucleotide sequence of SEQ ID NO. 2.
  • immunogenic portions of a SARS-CoV-2 mutated spike protein are used as the antigenic component in the composition.
  • immunogenic portion refers to truncated and/or substituted forms, or fragments of SARS-CoV-2 mutated spike protein and/or polynucleotide, respectively.
  • truncated and/or substituted forms, or fragments will comprise at least 4 contiguous amino acids from the full-length polypeptide with SEQ ID NO. 1 comprising these 4 contiguous amino acids.
  • the truncated or substituted forms, or fragments will have at least 8, more preferably 10, more preferably at least 15, and still more preferably at least 19 contiguous amino acids from the full-length polypeptide while still comprising SEQ ID NO. 1.
  • sequences may be a part of larger fragments or truncated forms.
  • such truncated or substituted forms, or fragments will comprise at least 12 contiguous nucleotides from the full-length nucleotide sequence, e.g. of SEQ ID NO. 2. More preferably, the truncated or substituted forms, or fragments will have at least 15, more preferably 18, still more preferably 24, even more preferably 30, more preferably at least 45, and still more preferably at least 57 contiguous nucleotides from the full-length nucleotide sequence.
  • Sequence Identity refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by -position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical.
  • Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M.
  • Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et ak, Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Alts chul, S. F.
  • BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et ak, NCVI NLM NIH Bethesda, MD 20894, Altschul, S. F. et ak, J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences.
  • nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% “sequence identity” to a reference nucleotide sequence it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%, preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5’ or 3’ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence.
  • a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence.
  • residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.
  • Sequence homology refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to “sequence identity”, conservative amino acid substitutions are counted as a match when determining sequence homology.
  • a polypeptide or polynucleotide having 95% sequence homology with a reference sequence 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence.
  • the homologous sequence comprises at least a stretch of 50, even more preferably 100, even more preferably 250, even more preferably 500 nucleotides.
  • a “conservative substitution” refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.
  • Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • an immunogenic composition effective for lessening the severity of clinical symptoms associated with SARS-CoV-2 infection comprising SARS-CoV-2 mutated spike protein
  • the SARS-CoV-2 mutated spike protein is selected from the group consisting of: 1) a polypeptide comprising the sequence of SEQ ID NO: 1 combination thereof; 2) any polypeptide that is at least 90% homologous to the polypeptide of 1 and still includes SEQ ID No.
  • these immunogenic portions will have the immunogenic characteristics of SARS-CoV-2 mutated spike protein that is encoded by the sequence of SEQ ID NO: 2.
  • SARS-CoV-2 mutated spike protein is provided in the immunological composition at an antigen inclusion level effective for inducing the desired immune response, namely reducing the incidence of or lessening the severity of clinical signs resulting from SARS-CoV-2 infection.
  • the SARS-CoV-2 mutated spike protein inclusion level is at least 0.2 pg antigen/ml of the final immunogenic composition (pg/ml), more preferably from about 0.2 to about 400 pg/ml.
  • the polypeptide is incorporated into a composition that can be administered to an animal susceptible to SARS-CoV-2 infection.
  • the composition may also include additional components known to those of skill in the art (see also Remington’s Pharmaceutical Sciences. (1990). 18th ed. Mack Pubk, Easton).
  • compositions herein may incorporate known injectable, physiologically acceptable, sterile solutions.
  • aqueous isotonic solutions such as e.g. saline or corresponding plasma protein solutions are readily available.
  • the immunogenic and vaccine compositions of the present disclosure can include diluents, isotonic agents, stabilizers, or adjuvants. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like.
  • Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others.
  • Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others. Suitable adjuvants, are those described above.
  • the immunogenic composition of the present disclosure further comprises a pharmaceutical acceptable salt, preferably a phosphate salt in physiologically acceptable concentrations.
  • a pharmaceutical acceptable salt preferably a phosphate salt in physiologically acceptable concentrations.
  • the pH of said immunogenic composition is adjusted to a physiological pH, meaning between about 6.5 and 7.5.
  • the immunogenic compositions described herein can further include one or more other immunomodulatory agents such as, e. g., interleukins, interferons, or other cytokines.
  • the immunogenic compositions can also include Gentamicin and Merthiolate.
  • the present disclosure contemplates vaccine compositions comprising from about lug/ml to about 60 pg/ml of antibiotics, and more preferably less than about 30 pg/ml of antibiotics.
  • compositions comprising recombinant SARS-CoV-2 mutated spike protein as provided herewith are very effective in reducing the severity of or incidence of clinical signs associated with SARS-CoV-2 infections up to and including the prevention of such signs.
  • kits include a container comprising at least one dose of the immunogenic composition of SARS-CoV-2 mutated spike protein as provided herewith, wherein one dose comprises at least 2 pg SARS-CoV-2 mutated spike protein.
  • Said container can comprise from 1 to 250 doses of the immunogenic composition.
  • the container contains 1, 10, 25, 50, 100, 150, 200, or 250 doses of the immunogenic composition of SARS-CoV-2 mutated spike protein.
  • each of the containers comprising more than one dose of the immunogenic composition of SARS-CoV-2 mutated spike protein further comprises an anti-microbiological active agent.
  • one aspect of the present disclosure relates to a container that comprises from 1 to 250 doses of the immunogenic composition of SARS-CoV-2 mutated spike protein, wherein one dose comprises at least 2 pg SARS-CoV-2 mutated spike protein ORF protein, and Gentamicin and/or Merthiolate, preferably from about 1 pg/ml to about 60 pg/ml of antibiotics, and more preferably less than about 30 pg/ml.
  • the kit also includes an instruction manual, including the information for the administration of at least one dose of the immunogenic composition of SARS-CoV-2 mutated spike protein into a susceptible animal, preferably selected from the group consisting of mammals, and still more preferably selected from the group consisting of humans, cattle, pigs, cats, dogs, horses, and camels to lessen the incidence and/or severity of clinical symptoms associated with SARS-CoV-2 infection.
  • said instruction manual comprises the information of a second or further administration(s) of at least one dose of the immunogenic composition of SARS-CoV-2 mutated spike protein, wherein the second administration or any further administration is at least 14 days beyond the initial or any former administration.
  • said instruction manual also includes the information, to administer an immune stimulant.
  • said immune stimulant shall be given at least twice.
  • at least 3, more preferably at least 5, and even more preferably at least 7 days are between the first and the second or any further administration of the immune stimulant.
  • the immune stimulant is given at least 10 days, preferably 15, even more preferably 20, and still even more preferably at least 22 days beyond the initial administration of the immunogenic composition of SARS-CoV-2 mutated spike protein. It is understood that any immune stimulant known to a person skilled in the art can also be used.
  • Immuno stimulant means any agent or composition that can trigger a general immune response, preferably without initiating or increasing a specific immune response, for example the immune response against a specific pathogen. It is further instructed to administer the immune stimulant in a suitable dose.
  • the kit may also comprise a second container, including at least one dose of the immune stimulant.
  • a further aspect relates to the use of any of the compositions provided herewith as a medicament, even more preferably as a vaccine.
  • the present disclosure also relates to the use of any of the compositions described herein, for the preparation of a medicament for lessening the severity of clinical symptoms associated with SARS-CoV-2 infection.
  • the medicament is for the prevention of a SARS-CoV-2 mutated spike protein infection in mammals, preferably humans, cattle, pigs, cats, dogs, horses, and camels.
  • a further aspect relates to a method for (1) the prevention of an infection, or re-infection with SARS-CoV-2 or (2) the reduction in incidence or severity of or elimination of clinical symptoms caused by SARS-CoV-2 in a subject, comprising administering any of the immunogenic compositions provided herewith to a subject in need thereof.
  • the subject is a mammal, and more preferably is selected from the group consisting of humans, cattle, pigs, cats, dogs, horses, and camels. It is understood that the reduction is in comparison to a subject that has not received an administration of a composition of the present disclosure.
  • one dose or at least two doses of the immunogenic composition is/are administered, wherein one dose preferably comprises at least about 2 pg SARS-CoV-2 mutated spike protein.
  • a further aspect relates to the method of treatment as described above, wherein a subsequent application of the immunogenic composition is administered.
  • the second administration is done with the same immunogenic composition, preferably having the same amount of SARS-CoV-2 mutated spike protein.
  • the second administration is done at least 14 days beyond the initial administration, even more preferably at least 4 weeks beyond the initial administration.
  • the method is effective after just a single dose of the immunogenic composition and does not require a second or subsequent administration(s) in order to confer the protective benefits upon the subject.
  • the immunological agent effective for reducing the incidence of or lessening the severity of SARS-CoV-2 infection is a SARS-CoV-2 mutated spike protein antigen.
  • said SARS-CoV-2 mutated spike protein antigen is a SARS-CoV-2 mutated spike protein as provided herewith, or any immunogenic composition as described above, that comprises SARS-CoV-2 mutated spike protein.
  • immunogenic protein refers to any amino acid sequence which elicits an immune response in a host against a pathogen comprising said immunogenic protein, immunogenic polypeptide or immunogenic amino acid sequence.
  • immunogenic fragment is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response against the relevant pathogen.
  • Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey.
  • linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc.
  • conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2- dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.
  • Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol.
  • an “immunological active component” as used herein means a component that induces or stimulates the immune response in an animal to which said component is administered. According to a preferred embodiment, said immune response is directed to said component or to a microorganism comprising said component. According to a further preferred embodiment, the immunological active component is an attenuated microorganism, including modified live virus (MLV), a killed-microorganism or at least an immunological active part of a microorganism.
  • MMV modified live virus
  • Immunological active part of a microorganism means a protein-, sugar-, and or glycoprotein containing fraction of a microorganism that comprises at least one antigen that induces or stimulates the immune response in an animal to which said component is administered. According to a preferred embodiment, said immune response is directed to said immunological active part of a microorganism or to a microorganism comprising said immunological active part.
  • biologically functional plasmids, viral vectors and the like that contain the new recombinant nucleic acid molecules described herein, suitable host cells transfected by the vectors comprising the molecular DNA clones and the immunogenic polypeptide expression products.
  • One particularly preferred immunogenic protein will have the amino acid sequence set forth in SEQ ID NO: 1.
  • the biologically active variants thereof are further encompassed by the disclosure.
  • One of ordinary skill in the art would know how to modify, substitute, delete, etc., amino acid(s) from the polypeptide sequence and produce biologically active variants that retain the same, or substantially the same, activity as the parent sequence without undue effort.
  • the process may include the following steps: growing, under suitable nutrient conditions, prokaryotic or eukaryotic host cells transfected with the new recombinant nucleic acid molecules described herein in a manner allowing expression of said polypeptide products, and isolating the desired polypeptide products of the expression of said nucleic acid molecules by standard methods known in the art. It is contemplated that the immunogenic proteins may be prepared by other techniques such as, for example, biochemical synthesis and the like.
  • Vaccines of the molecular DNA clones, and methods of using them are also included within the scope of the present disclosure. Inoculated subjects are protected from serious viral infection, and the clinical signs associated therewith that are described herein.
  • the novel method provides protection against viral infection, and the associated clinical signs by administering to the subject an immunologically effective amount of a vaccine according to the disclosure, such as, for example, a vaccine comprising an immunogenic amount of the polypeptide expression products comprising SEQ ID NO. 1, the recombinant SARS-CoV-2 mutated spike protein DNA, etc.
  • the immunogenic compositions can include an infectious SARS-CoV-2 molecular DNA clone comprising SEQ ID NO. 2, as described herein. In preferred forms, this results in an attenuated virus that includes the DNA of SEQ ID NO. 2 or that encodes the mutated spike protein of SEQ ID NO. 1.
  • the present disclosure provides an inactivated virus vaccine, preferably comprising a SARS-CoV-2 that includes the DNA sequence of SEQ ID NO. 2.
  • inactivated virus vaccines for instance, the virus propagation from the infectious DNA clone is done by methods known in the art or described herein. Serial virus inactivation is then optimized by protocols generally known to those of ordinary skill in the art.
  • Inactivated virus vaccines may be prepared by treating the isolated virus with inactivating agents such as formalin or hydrophobic solvents, acids, etc., by irradiation with ultraviolet light or X-rays, by heating, etc. Inactivation is conducted in a manner understood in the art. The virus is considered inactivated if it is unable to infect a cell susceptible to infection.
  • inactivating agents such as formalin or hydrophobic solvents, acids, etc.
  • the tissue culture adapted, live, pathogenic SARS-CoV-2 is first attenuated (rendered nonpathogenic or harmless) by inserting DNA encoding SEQ ID NO. 1 therein.
  • the DNA comprises SEQ ID NO. 2.
  • Further attenuation of pathogenic clones may also be made by gene deletions or viral-producing gene mutations. Then, the attenuated SARS-CoV-2 viruses may be administered to subjects in need thereof.
  • the present disclosure also relates to vaccines comprising a nucleotide sequence of the genome of SARS-CoV-2, such as SEQ ID NO. 2, that encodes the mutated spike protein, SEQ ID No. 1, or a homologue or fragment thereof, and an acceptable pharmaceutical or veterinary vehicle.
  • the nucleotide sequence is or comprises SEQ ID No. 2.
  • the vaccine further comprises an adjuvant.
  • a further aspect of the present disclosure relates to vaccines comprising a vector and an acceptable pharmaceutical or veterinary vehicle, the vector comprising a nucleotide sequence of the genome of SARS-CoV-2 that includes SEQ ID NO. 2.
  • the present disclosure also relates to vaccines and immunogenic compositions comprising a cell and an acceptable pharmaceutical or veterinary carrier, wherein the cell is transformed with a nucleotide sequence of the genome of SARS-CoV-2 that encodes the mutated spike protein of SEQ ID NO. 1.
  • the present disclosure relates to vaccines or immunogenic compositions comprising a pharmaceutically acceptable vehicle and a single polypeptide, wherein the single polypeptide consists of SEQ ID No. 1.
  • the present disclosure relates to methods of immunizing a mammal against SARS-CoV-2 comprising administering to a mammal an effective amount of a vaccine or immunogenic composition described above.
  • the present disclosure relates to nucleotide sequences of the genome of SARS-CoV-2 selected from the sequences that encode SEQ ID No. 1, including SEQ ID No. 2.
  • Nucleotide, polynucleotide or nucleic acid sequence will be understood according to the present disclosure as meaning both a double-stranded or single-stranded DNA in the monomeric and dimeric (so-called in tandem) forms and the transcription products of said DNAs.
  • the present disclosure does not relate to the genomic nucleotide sequences taken in their natural environment, that is to say in the natural state. It concerns sequences which it has been possible to isolate, purify or partially purify, starting from separation methods such as, for example, ion- exchange chromatography, by exclusion based on molecular size, or by affinity, or alternatively fractionation techniques based on solubility in different solvents, or starting from methods of genetic engineering such as amplification, cloning and subcloning, it being possible for the sequences of the disclosure to be carried by vectors.
  • separation methods such as, for example, ion- exchange chromatography, by exclusion based on molecular size, or by affinity, or alternatively fractionation techniques based on solubility in different solvents, or starting from methods of genetic engineering such as amplification, cloning and subcloning, it being possible for the sequences of the disclosure to be carried by vectors.
  • Complementary nucleotide sequence of a sequence of the disclosure is understood as meaning any DNA whose nucleotides are complementary to those of the sequence of the disclosure, and whose orientation is reversed (antiparallel sequence).
  • Hybridization under conditions of stringency with a nucleotide sequence according to the disclosure is understood as meaning a hybridization under conditions of temperature and ionic strength chosen in such a way that they allow the maintenance of the hybridization between two fragments of complementary DNA.
  • nucleotide sequences according to the disclosure those are likewise preferred which can be used as a primer or probe in methods allowing the homologous sequences according to the disclosure to be obtained, these methods, such as the polymerase chain reaction (PCR), nucleic acid cloning and sequencing, being well known to the person skilled in the art.
  • PCR polymerase chain reaction
  • nucleic acid cloning and sequencing being well known to the person skilled in the art.
  • nucleotide sequences according to the disclosure those are again preferred which can be used as a primer or probe in methods allowing the presence of SARS-CoV-2 mutated spike protein or one of its variants such as defined below to be diagnosed.
  • nucleotide sequences according to the disclosure capable of modulating, of inhibiting or of inducing the expression of SARS-CoV-2 mutated spike protein gene, and/or capable of modulating the replication cycle of SARS-CoV- 2 mutated spike protein in the host cell and/or organism are likewise preferred.
  • Replication cycle will be understood as designating the invasion and the multiplication of SARS-CoV-2, and its propagation from host cell to host cell in the host organism.
  • Modified nucleotide sequence will be understood as meaning any nucleotide sequence obtained by mutagenesis according to techniques well known to the person skilled in the art, and containing modifications with respect to the normal sequences according to the disclosure, for example mutations in the regulatory and/or promoter sequences of polypeptide expression, especially leading to a modification of the rate of expression of said polypeptide or to a modulation of the replicative cycle.
  • Modified nucleotide sequence will likewise be understood as meaning any nucleotide sequence coding for a modified polypeptide such as defined below.
  • the present disclosure relates to nucleotide sequences of SARS-CoV-2 mutated spike protein according to the disclosure, characterized in that it has or contains the sequence of SEQ ID No. 2.
  • the disclosure comprises the polypeptides encoded by a nucleotide sequence according to the disclosure, preferably a polypeptide whose sequence is represented by a fragment, especially a specific fragment, these four amino acid sequences corresponding to the polypeptides which can be encoded according to the sequence of SEQ ID No. 2.
  • the disclosure likewise relates to the polypeptides, characterized in that they contain or comprise a polypeptide of SEQ ID No. 1.
  • the polypeptide of amino acid sequence SEQ ID No. 1 is further capable of specifically recognizing the antibodies produced during infection by SARS-CoV-2.
  • This polypeptide thus has epitopes specific for the SARS-CoV-2 mutated spike protein and can thus be used in particular in the diagnostic field or as immunogenic agent to confer protection in subjects against infection by SARS-CoV-2.
  • polypeptide In the present description, the terms polypeptide, peptide and protein are interchangeable.
  • polypeptides in natural form, that is to say that they are not taken in their natural environment but that they can be isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or alternatively by chemical synthesis and that they can thus contain unnatural amino acids, as will be described below.
  • amino acids are replaced by “equivalent” amino acids.
  • the expression “equivalent” amino acid is directed here at designating any amino acid capable of being substituted by one of the amino acids of the base structure without, however, essentially modifying the biological activities of the corresponding peptides and such that they will be defined by the following.
  • These equivalent amino acids can be determined either by depending on their structural homology with the amino acids which they substitute, or on results of comparative tests of biological activity between the different polypeptides, which are capable of being carried out.
  • the specific homologous polypeptides likewise correspond to polypeptides encoded by the specific homologous nucleotide sequences such as defined above and thus comprise in the present definition the polypeptides which are mutated or correspond to variants which can exist in SARS-CoV-2 mutated spike protein, and which especially correspond to truncations, substitutions, deletions and/or additions of at least one amino acid residue.
  • Specific biologically active fragment of a polypeptide according to the disclosure will be understood in particular as designating a specific polypeptide fragment, such as defined above, having at least one of the characteristics of polypeptides according to the disclosure, especially in that it is: capable of inducing an immunogenic reaction directed against a SARS-CoV-2 mutated spike protein; and/or capable of being recognized by a specific antibody of a polypeptide according to the disclosure; and/or capable of linking to a polypeptide or to a nucleotide sequence of SARS-CoV-2 mutated spike protein; and/or capable of exerting a physiological activity, even partial, such as, for example, a dissemination or structural (capsid) activity; and/or capable of modulating, of inducing or of inhibiting the expression of SARS-CoV-2 mutated spike protein gene or one of its variants, and/or capable of modulating the replication cycle of SARS-CoV-2 in the cell and/or the host organism.
  • polypeptide fragments according to the disclosure can correspond to isolated or purified fragments naturally present in a SARS-CoV-2 mutated spike protein or correspond to fragments which can be obtained by cleavage of said polypeptide by a proteolytic enzyme, such as trypsin or chymotrypsin or collagenase, or by a chemical reagent, such as cyanogen bromide (CNBr) or alternatively by placing said polypeptide in a very acidic environment, for example at pH 2.5.
  • a proteolytic enzyme such as trypsin or chymotrypsin or collagenase
  • CNBr cyanogen bromide
  • Such polypeptide fragments can likewise just as easily be prepared by chemical synthesis, from hosts transformed by an expression vector according to the disclosure containing a nucleic acid allowing the expression of said fragments, placed under the control of appropriate regulation and/or expression elements.
  • Modified polypeptide of a polypeptide according to the disclosure is understood as designating a polypeptide obtained by genetic recombination or by chemical synthesis as will be described below, having at least one modification with respect to the normal sequence. These modifications will especially be able to bear on amino acids at the origin of a specificity, of pathogenicity and/or of virulence, or at the origin of the structural conformation, and of the capacity of membrane insertion of the polypeptide according to the disclosure. It will thus be possible to create polypeptides of equivalent, increased or decreased activity, and of equivalent, narrower, or wider specificity.
  • the modifications of the polypeptide will especially have as objective: to render it capable of modulating, of inhibiting or of inducing the expression of SARS-CoV-2 mutated spike protein gene and/or capable of modulating the replication cycle of SARS-CoV-2 in the cell and/or the host organism, of allowing its incorporation into vaccine compositions, and/or of modifying its bioavailability as a compound for therapeutic use.
  • the preceding modified polypeptides can be obtained by using combinatorial chemistry, in which it is possible to systematically vary parts of the polypeptide before testing them on models, cell cultures or microorganisms for example, to select the compounds which are most active or have the properties sought.
  • nucleotide sequences coding for a polypeptide according to the disclosure are likewise part of the disclosure.
  • the disclosure likewise relates to nucleotide sequences utilizable as a primer or probe, characterized in that said sequences are selected from the nucleotide sequences according to the disclosure.
  • the cloning and the sequencing of the SARS-CoV-2 mutated spike protein has allowed it to be identified, after comparative analysis with the nucleotide sequences of other SARS-CoV viruses, that, among the sequences of fragments of these nucleic acids, were those which are strictly specific to the SARS- CoV-2 mutated spike protein and those which correspond to a consensus sequence of SARS-CoV other than the SARS-CoV-2 mutated spike protein.
  • nucleotide sequences utilizable as a primer or probe specific to the whole of the other known and nonpathogenic coronaviruses.
  • the present disclosure likewise relates to specific polypeptides of known coronaviruses other than SARS-CoV-2, encoded by said consensus nucleotide sequences, capable of being obtained by purification from natural polypeptides, by genetic recombination or by chemical synthesis by procedures well known to the person skilled in the art and such as described in particular below.
  • the labeled or unlabeled mono- or polyclonal antibodies directed against said specific polypeptides encoded by said consensus nucleotide sequences are also part of the disclosure.
  • the disclosure additionally relates to the use of a nucleotide sequence according to the disclosure as a primer or probe for the detection and/or the amplification of nucleic acid sequences.
  • the nucleotide sequences according to the disclosure can thus be used to amplify nucleotide sequences, especially by the PCR technique (polymerase chain reaction) (Erlich, 1989; Innis et al., 1990; Rolfs et al., 1991; and White et al., 1997).
  • oligodeoxyribonucleotide or oligoribonucleotide primers advantageously have a length of at least 8 nucleotides, preferably of at least 12 nucleotides.
  • Other amplification techniques of the target nucleic acid can be advantageously employed as alternatives to PCR.
  • the nucleotide sequences of the disclosure can likewise be employed in other procedures of amplification of a target nucleic acid, such as: the TAS technique (Transcription- based Amplification System), described by Kwoh et al. in 1989; the 3 SR technique (Self-Sustained Sequence Replication), described by Guatelli et al. in 1990; the NASBA technique (Nucleic Acid Sequence Based Amplification), described by Kievitis et al. in 1991; the SDA technique (Strand Displacement Amplification) (Walker et al., 1992); the TMA technique (Transcription Mediated Amplification).
  • the polynucleotides of the disclosure can also be employed in techniques of amplification or of modification of the nucleic acid serving as a probe, such as: the LCR technique (Ligase Chain Reaction), described by Landegren et al. in 1988 and improved by Barany et al. in 1991, which employs a thermostable ligase; the RCR technique (Repair Chain Reaction), described by Segev in 1992; the CPR technique (Cycling Probe Reaction), described by Duck et al. in 1990; the amplification technique with Q-beta replicase, described by Miele et al. in 1983 and especially improved by Chu et al. in 1986, Lizardi et al. in 1988, then by Burg et al. as well as by Stone et al. in 1996.
  • LCR technique Liigase Chain Reaction
  • RCR technique Repair Chain Reaction
  • CPR technique Cycling Probe Reaction
  • the target polynucleotide to be detected is possibly an RNA, for example an MRNA
  • an enzyme of reverse transcriptase type in order to obtain a cDNA from the RNA contained in the biological sample.
  • the cDNA obtained will thus serve as a target for the primer(s) or the probe(s) employed in the amplification or detection procedure according to the disclosure.
  • the detection probe will be chosen in such a manner that it hybridizes with the target sequence or the amplicon generated from the target sequence.
  • a probe will advantageously have a sequence of at least 12 nucleotides, in particular of at least 20 nucleotides, and preferably of at least 100 nucleotides.
  • the disclosure also comprises the nucleotide sequences utilizable as a probe or primer according to the disclosure, characterized in that they are labeled with a radioactive compound or with a nonradioactive compound.
  • the unlabeled nucleotide sequences can be used directly as probes or primers, although the sequences are generally labeled with a radioactive element (32P, 35S, 3H, 1251) or with a nonradioactive molecule (biotin, acetylaminofluorene, digoxigenin, 5- bromodeoxyuridine, fluorescein) to obtain probes which are utilizable for numerous applications. Examples of nonradioactive labeling of nucleotide sequences are described, for example, in French Patent No.
  • the hybridization technique can be carried out in various manners (Matthews et al., 1988).
  • the most general method consists in immobilizing the nucleic acid extract of cells on a support (such as nitrocellulose, nylon, polystyrene) and in incubating, under well-defined conditions, the immobilized target nucleic acid with the probe. After hybridization, the excess of probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, of the fluorescence or of the enzymatic activity linked to the probe).
  • the disclosure likewise comprises the nucleotide sequences according to the disclosure, characterized in that they are immobilized on a support, covalently or noncovalently.
  • the latter can be used immobilized on a support and can thus serve to capture, by specific hybridization, the target nucleic acid obtained from the biological sample to be tested. If necessary, the solid support is separated from the sample and the hybridization complex formed between said capture probe and the target nucleic acid is then detected with the aid of a second probe, a so-called detection probe, labeled with an easily detectable element.
  • Another subject of the present disclosure is a vector for the cloning and/or expression of a sequence, characterized in that it contains a nucleotide sequence according to the disclosure.
  • the vectors according to the disclosure characterized in that they contain the elements allowing the expression and/or the secretion of said nucleotide sequences in a determined host cell, are likewise part of the disclosure.
  • the vector must then contain a promoter, signals of initiation and termination of translation, as well as appropriate regions of regulation of transcription. It must be able to be maintained stably in the host cell and can optionally have particular signals specifying the secretion of the translated protein. These different elements are chosen as a function of the host cell used.
  • the nucleotide sequences according to the disclosure can be inserted into autonomous replication vectors within the chosen host, or integrated vectors of the chosen host.
  • vectors will be prepared according to the methods currently used by the person skilled in the art, and it will be possible to introduce the clones resulting therefrom into an appropriate host by standard methods, such as, for example, lipofection, electroporation and thermal shock.
  • the vectors according to the disclosure are, for example, vectors of plasmid or viral origin.
  • the disclosure likewise comprises the host cells transformed by a vector according to the disclosure. These cells can be obtained by the introduction into host cells of a nucleotide sequence inserted into a vector such as defined above, then the culturing of said cells under conditions allowing the replication and/or expression of the transfected nucleotide sequence.
  • the host cell can be selected from prokaryotic or eukaryotic systems, such as, for example, bacterial cells (Olins and Lee, 1993), but likewise yeast cells (Buckholz, 1993), as well as animal cells, in particular the cultures of mammalian cells (Edwards and Aruffo, 1993), and especially Chinese hamster ovary (CHO) cells, but likewise the cells of insects in which it is possible to use procedures employing baculoviruses, for example (Luckow, 1993).
  • prokaryotic or eukaryotic systems such as, for example, bacterial cells (Olins and Lee, 1993), but likewise yeast cells (Buckholz, 1993), as well as animal cells, in particular the cultures of mammalian cells (Edwards and Aruffo, 1993), and especially Chinese hamster ovary (CHO) cells, but likewise the cells of insects in which it is possible to use procedures employing baculoviruses, for example (Luckow, 1993).
  • the disclosure likewise relates to animals comprising one of said transformed cells according to the disclosure.
  • the obtainment of transgenic animals according to the disclosure overexpressing one or more of the genes of SARS-CoV-2 mutated spike protein or part of the genes will be preferably carried out in rats, mice, cats, dogs, or rabbits according to methods well known to the person skilled in the art, such as by viral or nonviral transfections. It will be possible to obtain the transgenic animals overexpressing one or more of said genes by transfection of multiple copies of said genes under the control of a strong promoter of ubiquitous nature, or selective for one type of tissue.
  • transgenic animals by homologous recombination in embryonic cell strains, transfer of these cell strains to embryos, selection of the affected chimeras at the level of the reproductive lines, and growth of said chimeras.
  • the transformed cells as well as the transgenic animals according to the disclosure are utilizable in procedures for preparation of recombinant polypeptides.
  • the preparation procedures employing a vector, and/or a cell transformed by said vector and/or a transgenic animal comprising one of said transformed cells, containing a nucleotide sequence according to the disclosure coding for a polypeptide of SARS-CoV-2 mutated spike protein containing or comprising SEQ ID NO. 1 are preferred.
  • the recombinant polypeptides obtained as indicated above can just as well be present in glycosylated form as in nonglycosylated form and can or cannot have the natural tertiary structure.
  • a preferred variant consists in producing a recombinant polypeptide fused to a “carrier” protein (chimeric protein).
  • carrier chimeric protein
  • the disclosure relates to a procedure for preparation of a polypeptide of the disclosure comprising the following steps: a) culture of transformed cells under conditions allowing the expression of a recombinant polypeptide of nucleotide sequence according to the disclosure; b) if need be, recovery of said recombinant polypeptide.
  • the disclosure also relates to a polypeptide which is capable of being obtained by a procedure of the disclosure such as described previously.
  • the disclosure also comprises a procedure for preparation of a synthetic polypeptide, characterized in that it uses a sequence of amino acids of polypeptides according to the disclosure.
  • the disclosure likewise relates to a synthetic polypeptide obtained by a procedure according to the disclosure.
  • the polypeptides according to the disclosure can likewise be prepared by techniques which are conventional in the field of the synthesis of peptides. This synthesis can be carried out in homogeneous solution or in solid phase. For example, reference can be made to the technique of synthesis in homogeneous solution described by Houben-Weyl in 1974.
  • This method of synthesis consists in successively condensing, two by two, the successive amino acids in the order required, or in condensing amino acids and fragments formed previously and already containing several amino acids in the appropriate order, or alternatively several fragments previously prepared in this way, it being understood that it will be necessary to protect beforehand all the reactive functions carried by these amino acids or fragments, with the exception of amine functions of one and carboxyls of the other or vice-versa, which must normally be involved in the formation of peptide bonds, especially after activation of the carboxyl function, according to the methods well known in the synthesis of peptides.
  • recourse will be made to the technique described by Merrifield.
  • the disclosure additionally relates to hybrid polypeptides having at least one polypeptide according to the disclosure, and a sequence of a polypeptide capable of inducing an immune response in man or animals, preferably mammals as described herein.
  • the antigenic determinant is such that it is capable of inducing a humoral and/or cellular response. It will be possible for such a determinant to comprise a polypeptide according to the disclosure in glycosylated form used with a view to obtaining immunogenic compositions capable of inducing the synthesis of antibodies directed against multiple epitopes. Said polypeptides or their glycosylated fragments are likewise part of the disclosure.
  • hybrid molecules can be formed, in part, of a polypeptide carrier molecule or of fragments thereof according to the disclosure, associated with a possibly immunogenic part, in particular an epitope of the diphtheria toxin, the tetanus toxin, a surface antigen of the hepatitis B virus (patent FR 79 21811), the VP1 antigen of the poliomyelitis virus or any other viral or bacterial toxin or antigen.
  • the procedures for synthesis of hybrid molecules encompass the methods used in genetic engineering for constructing hybrid nucleotide sequences coding for the polypeptide sequences sought. It will be possible, for example, to refer advantageously to the technique for obtainment of genes coding for fusion proteins described by Minton in 1984.
  • hybrid nucleotide sequences coding for a hybrid polypeptide as well as the hybrid polypeptides according to the disclosure characterized in that they are recombinant polypeptides obtained by the expression of said hybrid nucleotide sequences are likewise part of the disclosure.
  • the disclosure likewise comprises the vectors characterized in that they contain one of said hybrid nucleotide sequences.
  • the host cells transformed by said vectors, the transgenic animals comprising one of said transformed cells as well as the procedures for preparation of recombinant polypeptides using said vectors, said transformed cells and/or said transgenic animals are, of course, likewise part of the disclosure.
  • polypeptides according to the disclosure, the antibodies according to the disclosure described below and the nucleotide sequences according to the disclosure can advantageously be employed in procedures for the detection and/or identification of SARS-CoV-2, or of a coronavirus other than a SARS-CoV-2, in a biological sample (biological tissue or fluid) capable of containing them.
  • a biological sample biological tissue or fluid
  • These procedures, according to the specificity of the polypeptides, the antibodies and the nucleotide sequences according to the disclosure which will be used, will in particular be able to detect and/or to identify a SARS-CoV-2 or a coronavirus other than a SARS-CoV-2 or other than the coronavirus SARS-CoV-2 having the mutated spike protein described herein.
  • the polypeptides according to the disclosure can advantageously be employed in a procedure for the detection and/or the identification of SARS-CoV-2 mutated spike protein in a biological sample (biological tissue or fluid) capable of containing them, characterized in that it comprises the following steps: a) contacting of this biological sample with a polypeptide or one of its fragments according to the disclosure (under conditions allowing an immunological reaction between said polypeptide and the antibodies possibly present in the biological sample); and b) demonstration of the antigen-antibody complexes possibly formed.
  • the biological sample is formed by a fluid, for example a subject serum, whole blood or biopsies. Any conventional procedure can be employed for carrying out such a detection of the antigen-antibody complexes possibly formed.
  • a preferred method brings into play immunoenzymatic processes according to the ELISA technique, by immunofluorescence, or radioimmunological processes (RIA) or their equivalent.
  • the disclosure likewise relates to the polypeptides according to the disclosure, labeled with the aid of an adequate label such as of the enzymatic, fluorescent or radioactive type.
  • Such methods comprise, for example, the following steps: 1) deposition of determined quantities of a polypeptide composition according to the disclosure in the wells of a microtiter plate; 2) introduction into said wells of increasing dilutions of serum, or of a biological sample other than that defined previously, having to be analyzed,; 3) incubation of the microplate; and 4) introduction into the wells of the microtiter plate of labeled antibodies directed against subject immunoglobulins, the labeling of these antibodies having been carried out with the aid of an enzyme selected from those which are capable of hydrolyzing a substrate by modifying the absorption of the radiation of the latter, at least at a determined wavelength, for example at 550 nm, detection, by comparison with a control test, of the quantity of hydrolyzed substrate.
  • the disclosure likewise relates to a kit or set for the detection and/or identification of SARS-CoV-2 having a mutated spike protein, characterized in that it comprises the following elements: 1) a polypeptide according to the disclosure; 2) if need be, the reagents for the formation of the medium favorable to the immunological or specific reaction; 3) if need be, the reagents allowing the detection of the antigen-antibody complexes produced by the immunological reaction between the polypeptide(s) of the disclosure and the antibodies possibly present in the biological sample, these reagents likewise being able to carry a label, or to be recognized in their turn by a labeled reagent, more particularly in the case where the polypeptide according to the disclosure is not labeled; 4) if need be, a biological reference sample (negative control) devoid of antibodies recognized by a polypeptide according to the disclosure; and 5) if need be, a biological reference sample (positive control) containing a predetermined quantity of antibodies recognized by a polypeptide according
  • the polypeptides according to the disclosure allow monoclonal or polyclonal antibodies to be prepared which are characterized in that they specifically recognize the polypeptides according to the disclosure. It will advantageously be possible to prepare the monoclonal antibodies from hybridomas according to the technique described by Kohler and Milstein in 1975. It will be possible to prepare the polyclonal antibodies, for example, by immunization of an animal, in particular a mouse, with a polypeptide or a DNA, according to the disclosure, associated with an adjuvant of the immune response, and then purification of the specific antibodies contained in the serum of the immunized animals on an affinity column on which the polypeptide which has served as an antigen has previously been immobilized.
  • the polyclonal antibodies according to the disclosure can also be prepared by purification, on an affinity column on which a polypeptide according to the disclosure has previously been immobilized, of the antibodies contained in the serum of an animal infected by a SARS-CoV-2 having the mutated spike protein described herein.
  • the disclosure likewise relates to mono- or polyclonal antibodies or their fragments, or chimeric antibodies, characterized in that they are capable of specifically recognizing a polypeptide according to the disclosure. It will likewise be possible for the antibodies of the disclosure to be labeled in the same manner as described previously for the nucleic probes of the disclosure, such as a labeling of enzymatic, fluorescent or radioactive type.
  • the disclosure is additionally directed at a procedure for the detection and/or identification of SARS-CoV-2 having the mutated spike protein, in a biological sample, characterized in that it comprises the following steps: a) contacting of the biological sample (biological tissue or fluid) with a mono- or polyclonal antibody according to the disclosure (under conditions allowing an immunological reaction between said antibodies and the polypeptides of SARS-CoV-2 mutated spike protein possibly present in the biological sample); and b) demonstration of the antigen-antibody complex possibly formed.
  • kits or set for the detection and/or the identification of SARS-CoV-2 having the mutated spike protein described herein characterized in that it comprises the following components: a) a polyclonal or monoclonal antibody according to the disclosure, if need be labeled; b) if need be, a reagent for the formation of the medium favorable to the carrying out of the immunological reaction; c) if need be, a reagent allowing the detection of the antigen-antibody complexes produced by the immunological reaction, this reagent likewise being able to carry a label, or being capable of being recognized in its turn by a labeled reagent, more particularly in the case where said monoclonal or polyclonal antibody is not labeled; and d) if need be, reagents for carrying out the lysis of cells of the sample tested.
  • the present disclosure likewise relates to a procedure for the detection and/or the identification of SARS-CoV-2 having the mutated spike protein in a biological sample, characterized in that it employs a nucleotide sequence according to the disclosure. More particularly, the disclosure relates to a procedure for the detection and/or the identification of SARS-CoV-2 having the mutated spike protein, in a biological sample, characterized in that it contains the following steps: a) if need be, isolation of the DNA from the biological sample to be analyzed; b) specific amplification of the DNA of the sample with the aid of at least one primer, or a pair of primers, according to the disclosure; and c) demonstration of the amplification products. These can be detected, for example, by the technique of molecular hybridization utilizing a nucleic probe according to the disclosure. This probe will advantageously be labeled with a nonradioactive (cold probe) or radioactive element.
  • DNA of the biological sample or “DNA contained in the biological sample” will be understood as meaning either the DNA present in the biological sample considered, or possibly the cDNA obtained after the action of an enzyme of reverse transcriptase type on the RNA present in said biological sample.
  • Another aim of the present disclosure consists in a procedure according to the disclosure, characterized in that it comprises the following steps: a) contacting of a nucleotide probe according to the disclosure with a biological sample, the DNA contained in the biological sample having, if need be, previously been made accessible to hybridization under conditions allowing the hybridization of the probe with the DNA of the sample; and b) demonstration of the hybrid formed between the nucleotide probe and the DNA of the biological sample.
  • the present disclosure also relates to a procedure according to the disclosure, characterized in that it comprises the following steps: a) contacting of a nucleotide probe immobilized on a support according to the disclosure with a biological sample, the DNA of the sample having, if need be, previously been made accessible to hybridization, under conditions allowing the hybridization of the probe with the DNA of the sample; b) contacting of the hybrid formed between the nucleotide probe immobilized on a support and the DNA contained in the biological sample, if need be after elimination of the DNA of the biological sample which has not hybridized with the probe, with a nucleotide probe labeled according to the disclosure; and c) demonstration of the novel hybrid formed in step b).
  • this is characterized in that, prior to step a), the DNA of the biological sample is first amplified with the aid of at least one primer according to the disclosure.
  • the disclosure is additionally directed at a kit or set for the detection and/or the identification of SARS-CoV-2 mutated spike protein, characterized in that it comprises the following elements: a) a nucleotide probe according to the disclosure; b) if need be, the reagents necessary for the carrying out of a hybridization reaction; and c) if need be, at least one primer according to the disclosure as well as the reagents necessary for an amplification reaction of the DNA.
  • the disclosure likewise relates to a kit or set for the detection and/or the identification of SARS-CoV-2 having the mutated spike protein or of coronavirus other than the SARS-CoV-2 having the mutated spike protein, characterized in that it comprises the following components: a) a nucleotide probe, called a capture probe, according to the disclosure; b) an oligonucleotide probe, called a revealing probe, according to the disclosure, and c) if need be, at least one primer according to the disclosure, as well as the reagents necessary for an amplification reaction of the DNA.
  • a nucleotide probe called a capture probe
  • an oligonucleotide probe called a revealing probe
  • the disclosure also relates to a kit or set for the detection and/or identification of SARS-CoV-2 having the mutated spike protein, characterized in that it comprises the following elements: a) at least one primer according to the disclosure; b) if need be, the reagents necessary for carrying out a DNA amplification reaction; and c) if need be, a component allowing the sequence of the amplified fragment to be verified, more particularly an oligonucleotide probe according to the disclosure.
  • the disclosure additionally relates to the use of a nucleotide sequence according to the disclosure, of a polypeptide according to the disclosure, of an antibody according to the disclosure, of a cell according to the disclosure, and/or of an animal transformed according to the disclosure, for the selection of an organic or inorganic compound capable of modulating, inducing or inhibiting the expression of genes, and/or of modifying the cellular replication of SARS-CoV-2 or capable of inducing or of inhibiting the pathologies including reducing the incidence of and severity of clinical signs linked to an infection by a SARS-CoV-2.
  • the disclosure likewise comprises a method of selection of compounds capable of binding to a polypeptide or one of its fragments according to the disclosure, capable of binding to a nucleotide sequence according to the disclosure, or capable of recognizing an antibody according to the disclosure, and/or capable of modulating, inducing or inhibiting the expression of genes, and/or of modifying the cellular replication of SARS-CoV-2 or capable of inducing or inhibiting the pathologies including reducing the incidence of and severity of clinical signs linked to an infection by a SARS-CoV-2, characterized in that it comprises the following steps: a) contacting of said compound with said polypeptide, said nucleotide sequence, or with a cell transformed according to the disclosure and/or administration of said compound to an animal transformed according to the disclosure; and b) determination of the capacity of said compound to bind to said polypeptide or said nucleotide sequence, or to modulate, induce or inhibit the expression of genes, or to modulate the growth or the replication of SARS-CoV-2,
  • the compounds capable of being selected can be organic compounds such as polypeptides or carbohydrates or any other organic or inorganic compounds already known, or novel organic compounds elaborated by molecular modelling techniques and obtained by chemical or biochemical synthesis, these techniques being known to the person skilled in the art. It will be possible to use said selected compounds to modulate the cellular replication of SARS-CoV-2 and thus to control infection by this virus, the methods allowing said modulations to be determined being well known to the person skilled in the art.
  • This modulation can be carried out, for example, by an agent capable of binding to a protein and thus of inhibiting or of potentiating its biological activity, or capable of binding to an envelope protein of the external surface of said virus and of blocking the penetration of said virus into the host cell or of favoring the action of the immune system of the infected organism directed against said virus.
  • This modulation can likewise be carried out by an agent capable of binding to a nucleotide sequence of a DNA of said virus and of blocking, for example, the expression of a polypeptide whose biological or structural activity is necessary for the replication or for the proliferation of said virus host cells to host cells in the host animal.
  • the disclosure relates to the compounds capable of being selected by a selection method according to the disclosure.
  • the disclosure likewise relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound selected from the following compounds: a) a nucleotide sequence, such as SEQ ID NO. 2, according to the disclosure; b) a polypeptide, such as SEQ ID NO. 1, according to the disclosure; c) a vector, a viral particle or a cell transformed according to the disclosure; d) an antibody according to the disclosure; and e) a compound capable of being selected by a selection method according to the disclosure; possibly in combination with a pharmaceutically acceptable carrier and, if need be, with one or more adjuvants of the appropriate immunity.
  • the disclosure also relates to an immunogenic and/or vaccine composition, characterized in that it comprises a compound selected from the following compounds: a) a nucleotide sequence according to the disclosure; b) a polypeptide according to the disclosure; c) a vector or a viral particle according to the disclosure; and d) a cell according to the disclosure.
  • the vaccine composition according to the disclosure is characterized in that it comprises a mixture of at least two of said compounds a), b), c) and d) above and in that one of the two said compounds is related to the SARS-CoV-2.
  • the vaccine composition is characterized in that it comprises at least one compound a), b), c), or d) above which is related to SARS-CoV-2.
  • the vaccine composition is characterized in that it comprises at least one compound a), b), c), or d) above which is related to SARS-CoV-2 having the mutated spike protein.
  • a compound related to the SARS-CoV-2 is understood here as respectively designating a compound obtained from the genomic sequence of the SARS-CoV-2 having the mutated spike protein.
  • the disclosure is additionally aimed at an immunogenic and/or vaccine composition, characterized in that it comprises at least one of the following compounds: 1) a nucleotide sequence SEQ ID No. 2; 2) a polypeptide of sequence SEQ ID No. 1; 3) a vector or a viral particle comprising a nucleotide sequence SEQ ID No. 2; 4) a transformed cell capable of expressing a polypeptide of sequence SEQ ID No. 1; or 5) a mixture of at least two of said compounds.
  • the disclosure also comprises an immunogenic and/or vaccine composition according to the disclosure, characterized in that it comprises said mixture of at least two of said compounds as a combination product for simultaneous, separate or protracted use for the prevention or the treatment of infection by a SARS-CoV-2.
  • the disclosure is likewise directed at a pharmaceutical composition according to the disclosure, for the prevention or the treatment of an infection by a SARS-CoV-2.
  • prevention includes the complete prevention of infection by a SARS-CoV-2, but also encompasses a reduction in the severity of or incidence of clinical signs associated with or caused by SARS-CoV-2. Such prevention is also referred to herein as a protective effect.
  • the disclosure likewise concerns the use of a composition according to the disclosure, for the preparation of a medicament intended for the prevention or the treatment of infection by a SARS-CoV-2.
  • the disclosure relates to a vector, a viral particle or a cell according to the disclosure, for the treatment and/or the prevention of a disease by gene therapy.
  • the disclosure comprises the use of a vector, of a viral particle or of a cell according to the disclosure for the preparation of a medicament intended for the treatment and/or the prevention of a disease by gene therapy.
  • polypeptides of the disclosure entering into the immunogenic or vaccine compositions according to the disclosure can be selected by techniques known to the person skilled in the art such as, for example, depending on the capacity of said polypeptides to stimulate the T cells, which is translated, for example, by their proliferation or the secretion of interleukins, and which leads to the production of antibodies directed against said polypeptides.
  • compositions according to the disclosure will contain an effective quantity of the compounds of the disclosure, that is to say in sufficient quantity of said compound(s) allowing the desired effect to be obtained, such as, for example, the modulation of the cellular replication of SARS-CoV-2.
  • the desired effect such as, for example, the modulation of the cellular replication of SARS-CoV-2.
  • the person skilled in the art will know how to determine this quantity, as a function, for example, of the age and of the weight of the individual to be treated, of the state of advancement of the pathology, of the possible secondary effects and by means of a test of evaluation of the effects obtained on a population range, these tests being known in these fields of application.
  • said vaccine combinations will preferably be combined with a pharmaceutically or veterinary acceptable carrier and, if need be, with one or more adjuvants of the appropriate immunity.
  • a pharmaceutically or veterinary acceptable carrier for protecting animals or man against infectious diseases: attenuated living microorganisms (M. bovis-BCG for tuberculosis), inactivated microorganisms (influenza virus), a cellular extracts (Bordetella pertussis for whooping cough), recombined proteins (surface antigen of the hepatitis B virus), polysaccharides (pneumococcal).
  • Vaccines prepared from synthetic peptides or genetically modified microorganisms expressing heterologous antigens are in the course of experimentation. More recently still, recombined plasmid DNAs carrying genes coding for protective antigens have been proposed as an alternative vaccine strategy. This type of vaccination is carried out with a particular plasmid originating from a plasmid of E. coli which does not replicate in vivo and which codes uniquely for the vaccinating protein. Animals have been immunized by simply injecting the naked plasmid DNA into the muscle. This technique leads to the expression of the vaccine protein in situ and to an immune response of cellular type (CTL) and of humoral type (antibody). This double induction of the immune response is one of the principal advantages of the vaccination technique with naked DNA.
  • CTL cellular type
  • antibody humoral type
  • the constitutive nucleotide sequence of the vaccine or immunogenic composition according to the disclosure can be injected into the host after having been coupled to compounds which favor the penetration of this polynucleotide into the interior of the cell or its transport to the cell nucleus.
  • the resultant conjugates can be encapsulated in polymeric microparticles, as described in the international application No. WO 94/27238 (Medisorb Technologies International).
  • the nucleotide sequence preferably a DNA
  • the nucleotide sequence is complexed with DEAE-dextran (Pagano et al., 1967) or with nuclear proteins (Kaneda et al., 1989), with lipids (Feigner et al., 1987) or encapsulated in liposomes (Fraley et al., 1980) or else introduced in the form of a gel facilitating its transfection into the cells (Midoux et al., 1993, Pastore et al., 1994).
  • the polynucleotide or the vector according to the disclosure can also be in suspension in a buffer solution or be combined with liposomes.
  • such a vaccine will be prepared according to the technique described by Tacson et al. or Huy gen et al. in 1996 or alternatively according to the technique described by Davis et al. in the international application No. WO 95/11307.
  • Such a vaccine can likewise be prepared in the form of a composition containing a vector according to the disclosure, placed under the control of regulation elements allowing its expression in man or animal.
  • these can comprise adjuvants of the appropriate immunity which are known to the person skilled in the art, such as, for example, those described above.
  • these compounds can be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal or subcutaneous route, or by the intranasal or oral route.
  • the vaccine composition comprising polypeptides according to the disclosure will be administered by the intramuscular route, through the food or by nebulization several times, staggered over time.
  • compositions can be determined according to the criteria generally taken into account in the establishment of a treatment adapted to an animal such as, for example, the age or the weight, the seriousness of its general condition, the tolerance to the treatment and the secondary effects noted.
  • the vaccine of the present disclosure is administered in an amount that is protective or provides a protective effect against SARS-CoV-2.
  • the polypeptide will be administered one time or several times, spread out over time, directly or by means of a transformed cell capable of expressing the polypeptide, in an amount of about 0.1 to 10 pg per kilogram weight of the animal, preferably about 0.2 to about 5 pg/kg, more preferably about 0.5 to about 2 pg/kg for a dose.
  • the present disclosure likewise relates to the use of nucleotide sequences of SARS-CoV-2 having the mutated spike protein according to the disclosure for the construction of autoreplicative retroviral vectors and the therapeutic applications of these, especially in the field of gene therapy in vivo.
  • the principle of gene therapy is to deliver a functional gene, called a gene of interest, of which the RNA or the corresponding protein will produce the desired biochemical effect in the targeted cells or tissues.
  • a gene of interest of which the RNA or the corresponding protein will produce the desired biochemical effect in the targeted cells or tissues.
  • the insertion of genes allows the prolonged expression of complex and unstable molecules such as RNAs or proteins which can be extremely difficult or even impossible to obtain or to administer directly.
  • the controlled insertion of the desired gene into the interior of targeted specific cells allows the expression product to be regulated in defined tissues. For this, it is necessary to be able to insert the desired therapeutic gene into the interior of chosen cells and thus to have available a method of insertion capable of specifically targeting the cells or the tissues chosen.
  • Some preferred genes of interest for the present disclosure are those that encode the mutated spike protein described herein.
  • a gene of interest in use in the disclosure can be obtained from a eukaryotic or prokaryotic organism or from a virus by any conventional technique. It is, preferably, capable of producing an expression product having a therapeutic effect and it can be a product homologous to the cell host or, alternatively, heterologous.
  • a gene of interest can code for an (1) intracellular or (2) membrane product present on the surface of the host cell or (3) secreted outside the host cell. It can therefore comprise appropriate additional elements such as, for example, a sequence coding for a secretion signal. These signals are known to the person skilled in the art.
  • a gene of interest can code for a protein corresponding to all or part of a native protein as found in nature. It can likewise be a chimeric protein, for example arising from the fusion of polypeptides of various origins or from a mutant having improved and/or modified biological properties. Such a mutant can be obtained, by conventional biological techniques, by substitution, deletion and/or addition of one or more amino acid residues.
  • the disclosure thus relates to the vectors characterized in that they comprise a nucleotide sequence of SARS-CoV-2 mutated spike protein according to the disclosure, preferably SEQ ID NO. 2, and in that they additionally comprise a gene of interest.
  • the present disclosure likewise relates to viral particles generated from said vector according to the disclosure. It additionally relates to methods for the preparation of viral particles according to the disclosure, characterized in that they employ a vector according to the disclosure, including viral pseudoparticles (VLP, virus-like particles).
  • VLP viral pseudoparticles
  • the disclosure likewise relates to animal cells transfected by a vector according to the disclosure.
  • animal cells especially mammalian, infected by a viral particle according to the disclosure.
  • Additional genetically engineered vaccines which are desirable in the present disclosure, are produced by techniques known in the art. Such techniques involve, but are not limited to, further manipulation of recombinant DNA, modification of or substitutions to the amino acid sequences of the recombinant proteins and the like. Genetically engineered vaccines based on recombinant DNA technology are made, for instance, by identifying alternative portions of the viral gene encoding proteins responsible for inducing a stronger immune or protective response in subjects (e.g., proteins derived from the modified spike protein). Such identified genes or immuno-dominant fragments can be cloned into standard protein expression vectors, such as the baculovirus vector, and used to infect appropriate host cells. The host cells are cultured, thus expressing the desired vaccine proteins, which can be purified to the desired extent and formulated into a suitable vaccine product.
  • standard protein expression vectors such as the baculovirus vector
  • the clones retain any undesirable natural abilities of causing disease, it is also possible to pinpoint the nucleotide sequences in the viral genome responsible for any residual virulence, and genetically engineer the virus avirulent through, for example, site-directed mutagenesis.
  • Site-directed mutagenesis is able to add, delete or change one or more nucleotides.
  • An oligonucleotide is synthesized containing the desired mutation and annealed to a portion of single stranded viral DNA. The hybrid molecule, which results from that procedure, is employed to transform bacteria.
  • double-stranded DNA which is isolated containing the appropriate mutation, is used to produce full-length DNA by ligation to a restriction fragment of the latter that is subsequently transfected into a suitable cell culture.
  • Ligation of the genome into the suitable vector for transfer may be accomplished through any standard technique known to those of ordinary skill in the art.
  • Transfection of the vector into host cells for the production of viral progeny may be done using any of the conventional methods such as calcium-phosphate or DEAE- dextran mediated transfection, electroporation, protoplast fusion and other well- known techniques.
  • the cloned virus then exhibits the desired mutation.
  • two oligonucleotides can be synthesized which contain the appropriate mutation. These may be annealed to form double-stranded DNA that can be inserted in the viral DNA to produce full-length DNA.
  • Genetically engineered proteins useful in vaccines, for instance, may be expressed in insect cells, yeast cells or mammalian cells.
  • the genetically engineered proteins which may be purified or isolated by conventional methods, can be directly inoculated into subjects to confer protection against clinical signs caused by SARS-CoV-2.
  • An insect cell line (like HI-FIVE) can be transformed with a transfer vector containing nucleic acid molecules obtained from the virus or copied from the viral genome which encodes one or more of the immuno-dominant proteins of the virus.
  • the transfer vector includes, for example, linearized baculovirus DNA and a plasmid containing the desired polynucleotides.
  • the host cell line may be co-transfected with the linearized baculovirus DNA and a plasmid in order to make a recombinant baculovirus.
  • An immunologically effective amount of the vaccines or immunogenic compositions of the present disclosure is administered to a subject in need of protection against clinical signs of infection from SARS-CoV2.
  • the immunologically effective amount or the immunogenic amount that inoculates the subject can be easily determined or readily titrated by routine testing.
  • An effective amount is one in which a sufficient immunological response to the vaccine is attained to protect the subject exposed to the virus which causes clinical signs of SARS-CoV2.
  • the subject is protected to an extent in which one to all of the adverse physiological symptoms or effects of the viral disease are significantly reduced, ameliorated or totally prevented.
  • the vaccine can be administered in a single dose or in repeated doses with single doses being preferred.
  • Single dose vaccines provide protection after a single dose without the need for any booster or subsequent dosages. Protection can include the complete prevention of clinical signs of infection, or a lessening of the severity, duration, or likelihood of the manifestation of one or more clinical signs of infection. Dosages may range, for example, from about 1 microgram to about 1,000 micrograms of the plasmid DNA containing the insert encoding for the mutated spike protein (dependent upon the concentration of the immuno-active component of the vaccine), preferably 100 to 200 micrograms of the DNA clone, but should not contain an amount of virus-based antigen sufficient to result in an adverse reaction or physiological symptoms of viral infection.
  • the infectious viral DNA clone encoding the mutated spike protein is used as a vaccine, or a live infectious virus can be generated in vitro and then the live virus is used as a vaccine.
  • TCID50 tissue culture infective dose
  • the vaccine is administered to a subject not yet exposed to the SARS-CoV2 virus.
  • the vaccine containing the DNA encoding for the mutant spike protein in an infectious DNA clone or other antigenic forms thereof can conveniently be administered intranasally, transdermally (i.e., applied on or at the skin surface for systemic absorption), parenterally, etc.
  • the parenteral route of administration includes, but is not limited to, intramuscular, intravenous, intraperitoneal, intradermal (i.e., injected or otherwise placed under the skin) routes and the like.
  • the present vaccine When administered as a liquid, the present vaccine may be prepared in the form of an aqueous solution, syrup, an elixir, a tincture and the like. Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems. Suitable carriers or solvents include, but are not limited to, water, saline, ethanol, ethylene glycol, glycerol, etc.
  • Typical additives are, for example, certified dyes, flavors, sweeteners and antimicrobial preservatives such as thimerosal (sodium ethylmercurithiosalicylate).
  • thimerosal sodium ethylmercurithiosalicylate
  • Such solutions may be stabilized, for example, by addition of partially hydrolyzed gelatin, sorbitol or cell culture medium, and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.
  • Liquid formulations also may include suspensions and emulsions that contain suspending or emulsifying agents in combination with other standard co-formulants. These types of liquid formulations may be prepared by conventional methods. Suspensions, for example, may be prepared using a colloid mill. Emulsions, for example, may be prepared using a homogenizer.
  • Parenteral formulations designed for injection into body fluid systems, require proper isotonicity and pH buffering to the corresponding levels of body fluids. Isotonicity can be appropriately adjusted with sodium chloride and other salts as needed. Suitable solvents, such as ethanol or propylene glycol, can be used to increase the solubility of the ingredients in the formulation and the stability of the liquid preparation. Further additives that can be employed in the present vaccine include, but are not limited to, dextrose, conventional antioxidants and conventional chelating agents such as ethylenediamine tetraacetic acid (EDTA). Parenteral dosage forms must also be sterilized prior to use.
  • EDTA ethylenediamine tetraacetic acid
  • Another aspect of the present disclosure is the preparation of the combination vaccine(s) or immunogenic compositions.
  • Such combinations can be between the different vaccine components described herein.
  • a vaccine of the present disclosure can include both protein portions and DNA portions of SARS-CoV-2 mutated spike protein , as described herein, which are administered concurrently or separately.
  • the combinations can be between the SARS- CoV-2 mutated spike protein vaccine components described herein and antigens of other disease-causing organisms, such as those described above.
  • the vaccine or immunogenic composition is first dehydrated. If the composition is first lyophilized or dehydrated by other methods, then, prior to vaccination, said composition is rehydrated in aqueous (e.g. saline, PBS (phosphate buffered saline)) or non-aqueous solutions (e.g. oil emulsion (mineral oil, or vegetable/metabolizable oil based/single or double emulsion based), aluminum-based, carbomer based adjuvant).
  • aqueous e.g. saline, PBS (phosphate buffered saline)
  • non-aqueous solutions e.g. oil emulsion (mineral oil, or vegetable/metabolizable oil based/single or double emulsion based
  • aluminum-based e.g. aluminum-based, carbomer based adjuvant
  • an effective amount of a combination vaccine administered to subjects provides effective immunity or a protective effect against microbiological infections caused by SARS-CoV-2 mutated spike protein and at least one further pathogen.
  • Preferred combinations of antigens for the treatment and prophylaxis of microbiological diseases in subjects are listed above.
  • the combination vaccine is administered to subjects in one or two doses at an interval of about 2 to 4 weeks.
  • the first administration is performed when the animal is about 2 to 3 weeks to about 8 weeks of age.
  • the second administration is performed about 1 to about 4 weeks after the first administration of the first vaccination.
  • revaccination is performed in an interval of 3 to 12 month after administration of the second dose.
  • Administration of subsequent vaccine doses is preferably done on a 6 month to an annual basis. In another preferred embodiment, animals vaccinated before the age of about 2 to 3 weeks should be revaccinated. Administration of subsequent vaccine doses is preferably done on an annual basis. In the event that one of the components of the combination vaccine is effective after just a single dose, such component needs to only be administered a single time with the other component(s) administered according to their preferred regimen.
  • the amount of combination vaccine that is effective depends on the ingredients of the vaccine and the schedule of administration. Typically, when an inactivated virus or a modified live virus preparation is used in the combination vaccine, an amount of the vaccine containing about 102 to about 109 TCID50 per dose, preferably about 103 to about 108 TCID50 per dose, more preferably, about 104 to about 108 TCID50 per dose. In general, inactivated antigen is normally used in higher amounts than live modified viruses.
  • the vaccine when bacterial antigen is used in the combination vaccine, the vaccine containing an amount of about 103 to about 109 colony forming units (CFU) per dose, preferably, about 104 to about 108 (CFU) per dose, more preferably about 105 to about 106 (CFU) per dose.
  • CFU colony forming units
  • Sub-unit vaccines are normally administered with an antigen inclusion level of at least 0.2 pg antigen per dose, preferably with about 0.2 to about 400 pg/dose, still more preferably with about 0.3 to about 200 pg/dose, even more preferably with about 0.35 to about 100 pg/dose, still more preferably with about 0.4 to about 50 pg/dose, still more preferably with about 0.45 to about 30 pg/dose, still more preferably with about 0.6 to about 15 pg/dose, even more preferably with about 0.75 to about 8 pg/dose, even more preferably with about 1.0 to about 6 pg/dose, and still more preferably with about 1.3 to about 3.0 mg/dose.
  • the antigen inclusion level of the SARS- CoV-2 mutated spike protein ORF2 antigen preferably of the SARS-CoV-2 mutated spike protein ORF2 protein as provided herewith, contains about 2 pg to about 150 pg, preferably about 2 pg to about 60 pg, even more preferably about 2pg to about 50 pg, even more preferably about 2pg to about 40 pg, even more preferably about 2pg to about 30 pg, even more preferably about 2pg to about 25 pg, even more preferably about 2pg to about 20 pg, even more preferably about 4 pg to about 20 pg, and even more preferably about 4pg to about 16 pg.
  • the composition according to the disclosure may be applied intradermally, intratracheally, or intravaginally.
  • the composition preferably may be applied intramuscularly or intranasally.
  • intravenous injection or by direct injection into target tissues.
  • intravenous, intravascular, intramuscular, intranasal, intraarterial, intraperitoneal, oral, or intrathecal routes are preferred.
  • a more local application can be effected subcutaneously, intradermally, intracutaneously, intracardially, intralobally, intramedullarly, intrapulmonarily or directly in or near the tissue to be treated (connective-, bone-, muscle-, nerve-, epithelial tissue).
  • the compositions according to the disclosure may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months, and in different dosages.
  • Vero E6 cells ATCC ® CRL-1586TM, American Type Culture Collection, Manassas, VA, USA
  • DMEM Dulbecco
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • the SARS-CoV-2 USA-WA1/2020 strain was acquired from Biodefense and Emerging Infection Research Resources Repository (BEI Resources, Manassas, VA, USA) and passaged 3 times in Vero E6 cells to establish a stock virus for inoculation of animals.
  • This stock virus was sequenced by next generation sequencing (NGS) using the Illumina MiSeq and its consensus sequence was found to be 100% homologous to the original USA-WA1/2020 strain (GenBank accession: MN985325.1).
  • NGS next generation sequencing
  • To determine infectious virus titer 10-fold serial dilutions were performed on 96-well plates of Vero E6 cells. The presence of cytopathic effect (CPE) after 3-5 days incubation was used to calculate the 50% tissue culture infective dose (TCID5o)/ml using the Spearman-Karber method (cit).
  • CPE tissue culture infective dose
  • TCID5o tissue culture infective dose
  • cit tissue culture infective dose
  • the prepared SARS-CoV- 2 stock has a titer of 1 x 10 6 TCIDso/ml; this virus was used for experimental infection of the cats.
  • Group 1 (principal infected animals) consisted of six cats (three cats/cage), and was inoculated via intranasal and oral routes simultaneously with a total dose of 1 x 10 6 TCID50 of SARS-CoV-2 in a total of 2 ml (0.5 ml per nostril and 1 ml oral).
  • DPC 1-day post challenge
  • the two cats in Group 2 were co-mingled with the principal infected animals in Group 1 (one cat per cage), and served as sentinel contact controls.
  • a full post mortem examination was performed for each cat at the indicated time-points and gross changes (if any) recorded. Tissues were collected either in formalin, or fresh and frozen at -80 °C.
  • a post mortem examination protocol was developed to collect the respiratory tract, gastrointestinal tract and central nervous system (brain and cerebral spinal fluid [CSF]) as well as accessory organs. For the lungs, the main bronchi were collected at the level of the bifurcation and at the entry point into the lung lobe. Lung lobes were scored based on gross pathology and collected separately. Bronchoalveolar lavage fluid (BALF), nasal wash, urine and feces were also collected during post mortem examination and stored at -80°C until analyzed.
  • BALF Bronchoalveolar lavage fluid
  • Fresh frozen tissue homogenates (10% w/v) were prepared by thawing frozen tissue and placing 200 mg of minced tissue in a tube containing 1 ml culture medium and a steel bead. Homogenization was performed with the TissueLyser LT (Qiagen) for 30 seconds at 30 hertz repeated 2-3 times. Supernatant was retained after centrifugation for RNA extraction and quantitative real-time reverse transcription PCR (RT-qPCR).
  • Serum chemistry was performed using an automated VetScan VS2 Chemistry Analyzer (Abaxis, Inc., Union City, CA) according to the manufacturer’s recommended protocol.
  • the Comprehensive Diagnostic Profile reagent rotor was used to perform complete chemistry and electrolyte analysis on 14 blood components. Briefly, 100 pi of serum was added to the sample port of the reagent rotor, which was subsequently run in the machine.
  • RNA extraction and quantitative real-time reverse transcription PCR RT-qPCR
  • RT-qPCR quantitative real time RT- qPCR
  • Virus neutralizing antibodies in sera were determined by incubating 2-fold serial dilutions of heat-inactivated (56°C for 30 minutes) sera samples, starting at a dilution of 1:10, in duplicate with 100 TCIDso of SARS-CoV-2 for 1 hour at 37°C, then culturing the mixture on VeroE6 cells. Results of virus neutralization were determined by the appearance of CPE, which is observed under a microscope at 72 and/or 96 hours post infection. The average of the duplicate results was used to determine the final titer. The neutralizing antibody titer is shown as the reciprocal of the average serum dilution at which no CPE breakthrough in any of the testing wells is observed.
  • Tissue samples from the respiratory tract (nasal turbinates, trachea and all 7 lung lobes), gastrointestinal tract (stomach, small and large intestine) and various other organs and tissues (spleen, kidney, liver, heart, lymph nodes, tonsils, olfactory bulb, bone marrow) were collected and either fixed in 10% neutral buffered formalin for histopathologic examination or frozen for RT-qPCR testing.
  • Tissues were routinely processed and stained with hematoxylin and eosin following standard procedures. Two independent veterinary pathologists examined the slides and were blinded to the treatment groups.
  • RNAscope ® in situ hybridization
  • RNAscope ® ISH an anti-sense probe targeting the spike (S, nt 21,563- 25,384) of SARS-CoV-2, WA1 strain (GenBank accession number MN985325.1) was designed (Advanced Cell Diagnostics [ACD], Newark, CA) and used as previously validated and described (cit).
  • ACD Advanced Cell Diagnostics
  • the RNAscope ® ISH assay was performed using the RNAscope 2.5 HD Red Detection Kit (ACD) as previously described (Carossino et al., 2019. PloS Pathogens. 15. el007950).
  • deparaffmized sections were incubated with a ready -to-use hydrogen peroxide solution for 10 minutes at room temperature and subsequently subjected to Target Retrieval for 15 minutes at 98-102 °C in IX Target Retrieval Solution.
  • Tissue sections were dehydrated in 100% ethanol for 10 minutes and treated with Protease Plus for 20 minutes at 40 °C in a HybEZTM oven (ACD).
  • Slides were subsequently incubated with a ready-to-use probe mixture for 2 hours at 40 °C in the HybEZTM oven, and the signal amplified using a specific set of amplifiers (AMP1-6 as recommended by the manufacturer).
  • the signal was detected using a Fast Red solution (Red B: Red A in a 1:60 ratio) for 10 minutes at room temperature. Slides were counterstained with 50% Gill hematoxylin I (Sigma Aldrich, St Louis, MO) for 2 min, and bluing performed with a 0.02% ammonium hydroxide in water. Slides were finally mounted with Ecomount ® (Biocare, Concord, CA). Sections from mock- and SARS-CoV-2-infected Vero cell pellets were used as negative and positive assay controls.
  • Red B Red A in a 1:60 ratio
  • IHC immunohistone deficiency virus
  • 4-micron sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged Superfrost ® Plus slides and subjected to IHC using a SARS-CoV-2-specific anti-nucleocapsid mouse monoclonal antibody (clone 6F10, BioVision, Inc., Milpitas, CA) as previously described (cit).
  • IHC was performed using the automated BOND-MAX and the Polymer Refine Red Detection kit (Leica Biosystems, Buffalo Grove, IL), as previously described.
  • HIER heat-induced epitope retrieval
  • WBC counts remained within normal limits for most animals during the course of the study; mildly increased WBC counts observed at -1 DPC and 1 DPC were attributed to stress early in the course of the study.
  • Thrombocytopenia was seen after 5 DPC in principal infected animals and after 10 DPC in sentinel contacts. Elevated ALP and phosphorus levels were observed in many of the animals starting at 5 DPC in the sentinels and at 10 DPC in the principal infected animals [Figure 2C]; this might indicate liver and kidney involvement during SARS-CoV-2 infection of cats. Some animals also had mildly elevated ALT at some points post infection (data not shown).
  • SARS-CoV-2 found throughout feline respiratory and gastrointestinal tract
  • SARS-CoV-2 RNA was detected in nasal swabs of the principal infected cats at 1 through 10 DPC, with maximal quantities observed from 1 through 5 DPC.
  • the nasal swabs of contact animals became RT-qPCR positive for SARS-CoV-2 RNA starting at day 2 DPCo (i.e. 3 DPC) and remained positive up to 9 DPCo/10 DPC, with a maximum on day 6 DPCo/7 DPC ( Figure 3A).
  • the oropharyngeal swabs were positive starting at 1 DPC through 10 DPC for the principals and 2 DPCo through 4 DPCo for the sentinels, with a maximum on 4 DPC and 4 DPCo, respectively (Figure 3B).
  • Rectal swabs were positive starting at 3 DPC for principals and 2 DPCo for sentinels and were found positive up to 14 DPC or 13 DPCo, respectively.
  • High levels of RNA shedding was maintained from 3 DPC or 2 DPCo throughout 14 DPC or 13 DPCo before animals became negative by 21 DPC or 20 DPCo for both principal infected and sentinel cats (Figure 3C).
  • RNA levels (copy number/mL) in cerebrospinal fluid, nasal cavity, trachea and bronchi.
  • RNA levels (copy number/mL) in lung and gastrointestinal tract tissues.
  • RNAscope ® ISH and IHC The cellular tropism, distribution and abundance of SARS-CoV-2 were subsequently investigated via detection of viral RNA and antigen by RNAscope ® ISH and IHC. Viral RNA and antigen correlated with the histological changes observed and were only detected within epithelial cells of multifocal submucosal glands and associated ducts at 4 DPC and 7 DPC. SARS-CoV-2 -positive submucosal glands were more frequently observed at 4 DPC compared to 7 DPC. Viral RNA and antigen were not detected at 21 DPC.
  • Identifying susceptible species and their capacity to transmit SARS-CoV-2 is critical for mitigating spread of the virus.
  • the development of animal models for COVID-19 is equally critical for studying the mechanisms of the disease and for evaluating the efficacy of potential vaccines, antiviral drugs and therapies.
  • In this example in-depth the infection, associated disease and transmission in domestic cats 4-5 -months of age was explored.
  • Detection of high levels of viral RNA from swab samples and various tissues, along with mild lung lesions and histologic changes and associated viral RNA and viral antigen in the airways and the development of SARS- CoV-2-specific antibodies demonstrates that cats were productively infected, without developing any obvious clinical signs.
  • the infected cats were able to transmit the virus to negative contact animals within 2 days of contact housing.
  • Significantly reduced viral shedding, absence of histologic changes within airways and seroconversion by 21 DPC suggests the cats were recovering from infection.

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Abstract

La présente invention concerne des compositions comprenant SEQ ID NO 1 et SEQ ID NO 2, ou des compositions comprenant un organisme ayant un génome qui inclut un insert comprenant SEQ ID NO 1 et SEQ ID NO 2, ainsi que des méthodes pour réduire l'incidence ou la gravité de signes cliniques provoqués par le SRAS-CoV-2 par l'administration de telles compositions à un animal en ayant besoin.
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