EP3955959A2 - Vaccin à virus sars-cov-2 inactivé - Google Patents

Vaccin à virus sars-cov-2 inactivé

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Publication number
EP3955959A2
EP3955959A2 EP21716442.5A EP21716442A EP3955959A2 EP 3955959 A2 EP3955959 A2 EP 3955959A2 EP 21716442 A EP21716442 A EP 21716442A EP 3955959 A2 EP3955959 A2 EP 3955959A2
Authority
EP
European Patent Office
Prior art keywords
cov
sars
vaccine
inactivated
particle
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21716442.5A
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German (de)
English (en)
Inventor
Andreas Meinke
Michael Möhlen
Robert Schlegl
Jürgen Heindl-Wruss
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Valneva Austria GmbH
Original Assignee
Valneva Austria GmbH
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Filing date
Publication date
Priority claimed from PCT/US2021/020313 external-priority patent/WO2021178318A1/fr
Application filed by Valneva Austria GmbH filed Critical Valneva Austria GmbH
Priority claimed from PCT/EP2021/058974 external-priority patent/WO2021204825A2/fr
Publication of EP3955959A2 publication Critical patent/EP3955959A2/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • 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
    • C12N2770/20061—Methods of inactivation or attenuation
    • C12N2770/20063—Methods of inactivation or attenuation by chemical treatment

Definitions

  • the disclosure relates to SARS-CoV-2 vaccines and compositions and methods for producing said vaccines and administering the vaccines to subjects for the generation of an anti-SARS-CoV-2 immune response.
  • SARS-CoV-2 (hereinafter the “virus”) was detected for the first time in China around November 2019. Since then, the virus has caused a global pandemic.
  • the natural reservoir are bats and the virus belongs to the Coronaviridae family, genus Betacoronavirus (betaCoV).
  • the virus has a ssRNA genome, 29,903 bp (Wuhan-Hu-1: GenBank Reference sequence: NC_045512.2) encoding for 9,860 amino acids, 25 non-structural protein and 4 structural proteins: spike (S), envelope (E), membrane (M), nucleocapside (N).
  • the virus has a variable size of between 60 to 140 nm in diameter. It is enveloped and sensitive to UV, heat, and lipid solvents.
  • SARS-CoV-2 presents a substantial public health threat.
  • the Imperial College COVID-19 (disease caused by SARS-CoV-2) Response Team published in March 16, 2020 a report wherein they evaluated all possible methods to stop or delay the spread of the virus leading ultimately to the break-down of the healthcare system and hundreds of thousands of deaths in the UK alone. They stated that only population-wide social distancing has a chance to reduce effects to manageable levels. These measures need to be upheld until a vaccine is available. This recommendation would mean for most of the population quarantine for at least 18 months. They concluded that a mass-producible vaccine is the only option to stop this pandemic other than willing to sacrifice the elderly population.
  • the present invention provides an inactivated SARS-CoV-2 vaccine.
  • SARS-CoV- 2 vaccine Whilst extensive effort has already been invested by research groups throughout the world in developing a SARS-CoV- 2 vaccine, most approaches have focused on subunit vaccines (e.g. encoding the SARS-CoV-2 S protein or fragments thereof), live attenuated vaccines or recombinant DNA or RNA vaccines encoding viral proteins.
  • subunit vaccines e.g. encoding the SARS-CoV-2 S protein or fragments thereof
  • live attenuated vaccines or recombinant DNA or RNA vaccines encoding viral proteins e.g. encoding the SARS-CoV-2 S protein or fragments thereof
  • live attenuated vaccines or recombinant DNA or RNA vaccines encoding viral proteins.
  • a successful inactivated SARS-CoV-2 vaccine has not yet been fully developed.
  • typical inactivating agents e.g. formaldehyde
  • alum under standard conditions may have drawbacks which hinder development of an effective vaccine candidate.
  • ADE antibody-dependent enhancement
  • the present invention aims to address these problems and thus to produce a safe and effective whole virus, inactivated SARS-CoV-2 vaccine that overcomes the drawbacks of the prior art.
  • the present invention provides a SARS-CoV-2 vaccine comprising a beta- propiolactone-inactivated SARS-CoV-2 particle; wherein the vaccine is capable of generating neutralizing antibodies against native SARS-CoV-2 particles in a human subject.
  • a native surface conformation of the SARS-CoV-2 particle is preserved in the vaccine.
  • the present invention provides a SARS-CoV-2 vaccine comprising an inactivated SARS-CoV-2 particle; wherein a native surface conformation of the SARS-CoV-2 particle is preserved in the vaccine, such that the vaccine is capable of generating neutralizing antibodies against native SARS-CoV-2 particles and/or other immunological responses in a human subject that are able to protect partly or fully more than 50%, preferably more than 60%, more than 70%, more than 80%, more than 90% of said vaccinated human subjects.
  • the present invention aims to provide optimally inactivated SARS-CoV-2 particles, which are incapable of replication and infection of human cells, but which retain immunogenic epitopes of viral surface proteins and are thus suitable for generating protective immunity in vaccinated subjects.
  • a novel vaccine composition can be obtained that preserves a native surface conformation of SARS-CoV-2 particles and which reduces the risk of negative effects such as
  • the invention aims to provide an optimal combination of optimally inactivated different SARS-CoV-2 particles, which are incapable of replication and infection of human cells, but which retain immunogenic epitopes of viral surface proteins and are thus suitable for generating protective immunity in vaccinated subjects.
  • an improved vaccine composition can be obtained that is capable of generating neutralizing antibodies against native SARS-CoV-2 particles and/or other immunological responses in a human subject that are able to protect partly or fully more than 50%, preferably more than 60%, more than 70%, more than 80%, more than 90% of said vaccinated human subjects.
  • Steps include cell buildup of Vero host cells, infection of Vero cells with SARS-CoV-2, virus harvest, DNA reduction, primary inactivation, purification, optional secondary inactivation and formulation with adjuvant.
  • Figure 3 A preferred set-up for the sucrose gradient centrifugation used as a polishing step for the SARS-CoV-2 vaccine of the invention.
  • Figure 4 Total IgG in response to SARS-CoV-2 vaccine. Coating antigens: SI (A), receptor binding domain of spike protein (B) and nucleoprotein (C). Endpoint titer: absorbance of 3 -fold the blank used as cut-off (dashed line).
  • FIG. 6 Production process delivers high density and intact spike proteins. Shown are electron micrographs of the SARS-CoV-2 inactivated drug substance produced according to Example 1. About 1-1.5 10 7 viral particles per AU.
  • FIG. 7 Comparison of Size-Exclusion-Chromatography and SDS-PAGE profiles of SARS-CoV-2 and JEV drug substance. High purity (>95%) according to SDS-PAGE (silver stain, reduced) and monomer virus (>95%) according to SE-HPLC. Difference in retention time due to different virus particle size (JEV (IXIARO) about 50nm, SARS-CoV2 about lOOnm)
  • FIG. 8 Study design forNHP challenge study. Three groups of 8 animals each; Two dose groups for SARS-CoV-2 vaccine (10 AU & 40 AU, formulated with 0.5 mg/dose Al 3+ and 1 mg Thl -stimulating adjuvant per dose added directly before administration) and a placebo group (DPBS).
  • the SARS-CoV- 2 challenge strain is BetaCoV/France/IDF/0372/2020 (Maisonmasse et ah, Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates, 2020, Nature 585:584-587).
  • SWABS viral load (qRT-PCR-genomic + subgenomic): nasal & tracheal swabs on d35, d49, d50, d51, d54, d57, d62; rectal swabs at baseline and on d2, d7, dl5.
  • Figure 9 Counts of residues within the footprints of 33 neutralizing mAbs, or respectively clusters 13, 4, 10, 2, 1, 3. Listed are residues within the footprint of neutralizing mAbs and/or which are lineage defining mutation positions for B.l.1.7, B.1.351 or P.l (marked “x”). E.g. K417 and E484 mutations which are amino acid positions in the S-protein are only to be found in the South African and Brazilian lineages.
  • FIG. 10 SDS-PAGE, silver stain, of two samples of SARS-CoV-2 candidates according to Example 1 (iCELLIS 500 bioreactor, protamine sulfate precipitated, BPL inactivated).
  • the bands could be clearly attributed to the three main viral proteins (Spike-protein, Membrane-protein, Nucleoprotein) as well as to background proteins from the host system.
  • Embodiments of the present invention are directed to a SARS-CoV-2 vaccine or immunogenic composition comprising inactivated SARS-CoV-2 particles.
  • the inactivated SARS-CoV-2 particles are whole virus, inactivated particles, i.e. the inactivated virus particles are derived from whole native SARS-CoV-2 particles that have been inactivated.
  • SARS-CoV-2 refers to the SARS-CoV-2 vims and “SARS-CoV-2 particles” typically refers to whole SARS-CoV-2 viral particles, i.e. virions.
  • the SARS-CoV-2 particles are inactivated without substantially modifying their surface structure.
  • a native surface conformation of the SARS-CoV-2 particles is retained in the inactivated vims particles.
  • an inactivated vims vaccine e.g. a beta-propiolactone-inactivated vims vaccine
  • the SARS-CoV-2 particles are inactivated by a method that preferentially targets viral RNA.
  • the inactivation step modifies viral RNA more than viral proteins.
  • the inactivated SARS-CoV-2 particles may comprise replication-deficient viral RNA, i.e. the viral RNA is modified in the inactivation step such that the inactivated particles are incapable of replicating.
  • the inactivation method spares viral (surface) proteins relative to viral RNA, e.g. the viral surface proteins (e.g. the spike (S) protein) may comprise fewer or more infrequent modifications resulting from the inactivation step compared to viral RNA.
  • the viral surface proteins e.g. the spike (S) protein
  • a lower proportion of amino acid residues in the viral surface proteins may be modified by the inactivation step compared to the proportion of modified nucleotide residues in the viral RNA.
  • the proportion of modified amino acid residues in the viral surface proteins (e.g. S protein) may be at least 5%, 10%, 20%, 30%, 50%, 70% or 90% lower than the proportion of modified nucleotide residues in the viral RNA.
  • modifications or “modified residues” it is meant to refer to non-native residues that are not present in the native SARS-CoV-2 particles, e.g. chemical (covalent) modifications of such residues resulting from the inactivation step.
  • the viral RNA is inactivated by alkylation and/or acylation, i.e. the modifications in the SARS-CoV-2 inactivated particles comprise alkylated and/or acylated nucleotide residues.
  • the modifications are preferentially targeted to purine (especially guanine) residues, e.g. the SARS-CoV-2 inactivated particles comprise one or more modified (e.g. alkylated or acylated) guanine residues.
  • the inactivation step may lead to cross-linking of viral RNA with viral proteins, e.g. via guanine residues in the viral RNA.
  • the inactivation step may also introduce nicks or strand breaks into viral RNA, e.g. resulting in fragmentation of the viral genome.
  • the inactivating agent comprises beta-propiolactone, i.e. the vaccine comprises beta-propiolactone-inactivated virus particles.
  • beta-propiolactone herein referred to also as “BPL”) treatment is particularly preferred according to the present invention, because it results in SARS-CoV- 2 particles, that are substantially inactive, but which retain high antigenicity and immunogenicity against neutralizing epitopes present in native SARS-CoV-2.
  • BPL beta-propiolactone
  • beta-propiolactone can be used to inactivate SARS-CoV-2 particles with a minimum number of protein modifications.
  • inactivation of SARS-CoV-2 particles using beta-propiolactone results in a much lower number of modifications of viral proteins compared to inactivation of influenza particles by beta-propiolactone.
  • beta- propiolactone-inactivated SARS-CoV-2 particles a native surface conformation of the viral particles can be preserved.
  • the viral RNA is inactivated in an optimized manner, i.e. such it is just sufficiently inactivated not to be infectious anymore but not “over”-inactivated so that numerous modification at different amino acids in particular at the S -protein occur.
  • the BPL inactivation not only sufficiently inactivates (but not over inactivates) the SARS-CoV-2 virus but also just sufficiently inactivates viruses that might be co enriched and co-cultured in the manufacturing process (see e.g. experimental part).
  • a particularly hard virus to inactivate that can co-culture and be co-enriched is PPV (porcine parvovirus) - see experimental part.
  • the concentration of beta-propiolactone in the inactivation step may be optimized to ensure complete inhibition of viral replication whilst preserving the conformation of surface proteins in the virus.
  • the concentration of beta-propiolactone in the inactivation step may be e.g. 0.01 to 1% by weight, preferably 0.01 to 0.1% by weight, more preferably about 0.03% by weight.
  • a preferred amount of BPL was found to be 500ppm where the SARS-CoV-2 virus but also other concerning viruses/impurities are inactivated whilst preserving (i.e. not modifying) most of the amino acids of the S-protein (i.e. only a few amino acids were shown to be modified at low probability).
  • the native SARS-CoV-2 particles may be contacted with beta-propiolactone for at least 5 hours, at least 10 hours, at least 24 hours or at least 4 days, e.g. 5 to 24 hours or longer such as 48 hours.
  • the inactivation step may be performed at about 0°C to about 25°C, preferably about 4°C or about 22°C, or e.g. 18 to 24°C.
  • the inactivation step e.g. with beta- propiolactone
  • the inactivation step may optionally and preferably be followed by a hydrolyzation step of the inactivating agent, as is known in the art (which may be performed e.g.
  • the inactivation step may be performed for e.g. the shortest time necessary in order to produce a fully inactivated virus particle.
  • the inactivated viral solution was in one embodiment immediately cooled down to 5 ⁇ 3°C and stored there until inactivation was confirmed by large volume plaque assay and serial passaging assay.
  • Beta-propiolactone inactivation of SARS-CoV-2 particles may preferentially modify cysteine, methionine and/or histidine residues.
  • the inactivated SARS-CoV-2 particle comprises one or more beta-propiolactone-modified cysteine, methionine and/or histidine residues.
  • the beta-propiolactone-inactivated SARS-CoV-2 particles show relatively few protein modifications.
  • an inactivated SARS-CoV-2 particle in the vaccine may comprise fewer than 200, 100, 50, 30, 20, 15, 10, 9, 8, 7 or 6 beta- propiolactone-modified amino acid residues.
  • a spike (S) protein of the inactivated SARS- CoV-2 particle comprises fewer than 100, 50, 30, 20, 15, 10, 9, 8, 7 or 6 beta-propiolactone-modified amino acid residues. More preferably the inactivated SARS-CoV-2 particle or spike protein thereof comprises 20 or fewer, 15 or fewer, 10 or fewer, or 5 or fewer beta-propiolactone-modified amino acid residues. Most preferably the inactivated SARS-CoV-2 particle or spike protein thereof comprises 1 to 100, 2 to 70, 3 to 50, 4 to 30, 5 to 25, 5 to 20, 10 to 20 or about 15 beta-propiolactone-modified amino acid residues.
  • fewer than 20%, 15%, 10%, 5% or 4% of SARS-CoV-2 polypeptides are beta- propiolactone-modified.
  • 0.1 to 10%, 1 to 8%, 2 to 7% or about 3%, 4%, 5% or 6% of SARS-CoV-2 polypeptides in the particle may be beta-propiolactone-modified.
  • Beta-propiolactone modification of residues and/or polypeptides in the vaccine may be detected by mass spectrometry, e.g. using liquid chromatography with tandem mass spectrometry (LC-MS-MS), for instance using a method as described in Examples 6 and 7.
  • LC-MS-MS liquid chromatography with tandem mass spectrometry
  • the SARS-CoV-2 particles may be digested in order to fragment proteins into SARS-CoV-2 polypeptides for LC-MS-MS analysis.
  • the digestion step may be performed by any suitable enzyme or combination of enzymes, e.g. by trypsin, chymotrypsin and/or PNGase F (peptide:N-glycosidase F), or by e.g. acid hydrolysis.
  • the percentage of BPL-modified polypeptides detected by LC-MS-MS following enzymatic digestion or acid hydrolysis is: (a) trypsin digestion, 1 to 5%, 2 to 4% or about 3%; (b) trypsin + PNGase F digestion, 1 to 5%, 2 to 4% or about 3%; (c) chymotrypsin, 1 to 10%, 3 to 8% or about 6% ; (d) acid hydrolysis, 1 to 6%, 2 to 5% or about 4%.
  • a “beta-propiolactone-modified” polypeptide means that the polypeptide comprises at least one beta-propiolactone modification, e.g. at least one beta- propiolactone-modified residue.
  • a spike (S) protein of the inactivated SARS-CoV-2 particle comprises a beta- propiolactone modification at one or more of the following residues: 49, 146, 166, 177, 207, 245, 379, 432, 519, 625, 1029, 1032, 1058, 1083, 1088, 1101, 1159 and/or 1271, e.g. in SEQ ID NO: 3, or a corresponding position in SEQ ID NO: 19, 21, 23, 25 or 27.
  • the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at one or more of the following residues: H49, H146, C166, M177, H207, H245, C432, H519, H625, M1029, H1058, H1083, H1088, HI 101, HI 159 and/or H1271, e.g. in SEQ ID NO: 3, or a corresponding position in SEQ ID NO: 19, 21, 23, 25 or 27.
  • the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at one or more of the following residues: H207, H245, C379, M1029 and/or C1032, e.g.
  • a corresponding position it is meant a position in SEQ ID NO: 19, 21, 23, 25 or 27 that aligns with position H207, H245, C379, M1029 and/or C1032 in SEQ ID NO: 3, e.g. when SEQ ID NO: 19, 21, 23, 25 or 27 is aligned with SEQ ID NO:3 using a program such as NCBI Basic Local Alignment Search Tool (BLAST).
  • BLAST NCBI Basic Local Alignment Search Tool
  • SEQ ID NO: 19 the positions in SEQ ID NO: 19, 21, 23, 25 or 27 corresponding to H207, H245, C379, M1029 and C1032 in SEQ ID NO: 3 are shown below:
  • a membrane (M) glycoprotein of the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at one or more of the following residues: 125, 154, 155, 159 and/or 210, preferably H154, H155, C159 and/or H210, e.g. in SEQ ID NO: 29.
  • a nucleocapsid (N) protein of the inactivated SARS-CoV-2 particle comprises a beta-propiolactone modification at M234, e.g. in SEQ ID NO: 28.
  • fewer than 30%, 20%, 10%, 5%, 3% or 1% of one or more of the following residues in the inactivated SARS-CoV-2 particles are beta-propiolactone modified: (i) in the spike (S) protein, e.g.
  • SEQ ID NO: 29 residues 125, 154, 155, 159 and/or 210; preferably H154, H155, C159 and/or H210; and/or (iii) M234 of the nucleocapsid (N) protein, e.g. in SEQ ID NO: 28.
  • fewer than 30%, 20%, 10%, 5%, 3% or 1% of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or each of the above residues in the inactivated SARS-CoV- 2 particles are beta-propiolactone modified.
  • the percentage of modified residues is intended to refer to the site occupancy, e.g. the ratio of modified to unmodified peptide for the same modification site normalized to the protein abundance as described in Examples 6 and/or 7 below.
  • the proportion of beta-propiolactone-modified residues (i.e. site occupancy) at the following positions in the inactivated SARS-CoV-2 particles is:
  • the spike (S) protein e.g. of SEQ ID NO: 3, or a corresponding position in SEQ ID NO: 19, 21, 23, 25 or 27:
  • H245 less than 10%, preferably 0.1 to 5%;
  • nucleocapsid (N) protein e.g. of SEQ ID NO: 28
  • (j) M234 less than 90%, less than 10% or less than 0.1%.
  • the proportion of beta-propiolactone-modified residues (i.e. site occupancy) at each of the following positions in the spike (S) protein (e.g. of SEQ ID NO: 3, or a corresponding position in SEQ ID NO: 19, 21, 23, 25 or 27) of the inactivated SARS-CoV-2 particles is:
  • residues M177, C432, H625 less than 30%, preferably 0.1 to 20%, more preferably 1 to 10%;
  • the proportion of beta-propiolactone-modified amino acid residues in the inactivated SARS-CoV-2 particle may be at least 5%, 10%, 20%, 30%, 50%, 70% or 90% lower than the proportion of modified residues in a beta-propiolactone-inactivated influenza particle (or hemagglutinin (HA) or neuraminidase (NA) protein thereof), e.g. in an influenza particle that has been inactivated under similar conditions to the SARS-CoV-2 particle.
  • HA hemagglutinin
  • NA neuraminidase
  • the viral RNA may be inactivated by treatment with ultraviolet (UV) light.
  • UV treatment can be used to preferentially target RNA (compared to polypeptides) in the viral particles, resulting in e.g. modified nucleotides and/or fragmentation.
  • UV treatment can be combined with beta-propiolactone treatment to improve inactivation of the virus, e.g. a beta-propiolactone treatment step can be followed by a UV treatment step or vice versa, or a UV treatment step can be performed at the same time as the beta-propiolactone treatment step.
  • the native SARS-CoV-2 particles may be inactivated using formaldehyde.
  • formaldehyde inactivation is typically less preferred in the present invention, as it is less suitable for preferentially targeting viral RNA and preserving immunogenic epitopes in the viral surface proteins.
  • the inactivation step(s) are performed under mild conditions in order to preserve surface antigen integrity, especially integrity of the S protein.
  • such a mild inactivation method comprises contacting a liquid composition comprising native SARS-CoV-2 particles with a chemical viral inactivating agent (such as e.g. any of the chemical inactivation agents as listed above or a combination, for instance formaldehyde or preferably beta-propiolactone) in a container, mixing the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles under conditions of laminar flow but not turbulent flow, and incubating the chemical viral inactivating agent and the liquid composition comprising SARS- CoV-2 particles for a time sufficient to inactivate the viral particles.
  • the mild inactivation step is optionally performed in a flexible bioreactor bag.
  • the mild inactivation step preferably comprises 5 or less container inversions during the period of inactivation.
  • the mixing of the chemical viral inactivating agent and the composition comprising native SARS-CoV-2 particles comprises subjecting the container to rocking, rotation, orbital shaking, or oscillation for not more than 10 minutes at not more than 10 rpm during the period of incubation.
  • the inactivation step substantially eliminates infectivity of mammalian (e.g. human) cells by the inactivated SARS-CoV-2 particle.
  • infectivity of mammalian cells may be reduced by at least 99%, 99.99% or 99.9999% as compared to a native SARS-CoV-2 particle, or infectivity of human cells by the inactivated A SARS-CoV-2 particle may be undetectable.
  • Standard assays may be used for determining residual infectivity and effective viral titer, e.g. plaque assays, determination of TCID50 (50% tissue culture infectious dose).
  • the mammalian cells may be MDCK, COS or Vero cells.
  • a native surface conformation of the SARS-CoV-2 particles is preserved in the inactivated virus particles.
  • e.g. one or more or all immunogenic (neutralizing) epitopes are retained in the inactivated virus particles, such that the inactivated particles are capable of generating neutralizing antibodies against native SARS-CoV-2 particles when administered to a human subject.
  • native surface conformation it is meant to refer to the surface conformation found in native SARS-CoV-2 particles, i.e. SARS-CoV-2 particles (virions) that have not been inactivated.
  • the property of the vaccine or inactivated SARS-CoV-2 particles in generating neutralizing antibodies in a subject may be determined using e.g. a plaque reduction neutralization test (PRNT assay), e.g. using a serum sample from the subject as known in the art.
  • PRNT assay plaque reduction neutralization test
  • the present invention comprises that a native conformation of (i) spike (S) protein; (ii) nucleocapsid (N) protein; (iii) membrane (M) glycoprotein; and/or (iv) envelope (E) protein is preserved in the inactivated viral particles.
  • the inactivated SARS-CoV-2 particle comprises a native conformation spike (S) protein.
  • the S (and/or N and/or M and/or E) protein in the inactivated SARS-CoV-2 particle preferably comprises one or more or all (intact) immunogenic (neutralizing) epitopes present in native SARS-CoV-2 particles.
  • the S (and/or N and/or M and/or E) protein in the inactivated viral particles is not modified, or not substantially modified by the inactivation step.
  • preservation of the surface conformation of the viral particles can be assessed using standard techniques. For instance, methods such as X-ray crystallography, MS analysis (shift of amino acid mass by modification) and cryo-electron microscopy may be used to visualize the virus surface.
  • the secondary and tertiary structures of proteins present on the surface of viral particles may also be analyzed by methods such as by circular dichroism (CD) spectroscopy (e.g. in the far (190-250 nm) UV or near (250-300 nm) UV range).
  • CD circular dichroism
  • preservation of a native surface conformation can be confirmed by using antibodies directed against epitopes present on the native viral surface, e.g. in the S protein. Cross-reaction of anti-SARS-CoV-2 antibodies between the inactivated and native virus particles can thus be used to demonstrate retention of potentially neutralizing epitopes in the vaccine.
  • SARS-CoV-2 virions and in particular the spike (S) protein is known, and has been published in several recent studies. See for instance Shang, J. et al. (Structural basis of receptor recognition by SARS-CoV-2. Nature https://doi.org/10.1038/s41586-020-2179-y (2020)), which describes the crystal structure of the SARS-CoV-2 receptor binding domain. In addition, Walls et al.
  • SARS-CoV-2 nucleocaspid (N) protein which has been confirmed as an important antigen in studies using convalescent sera (Zeng W et al. Biochemical characterization of SARS-CoV- 2 nucleocapsid protein. 2020 BBRC 527(3): 618-623). Further guidance with regard to potentially important SARS-CoV-2 epitopes is available in the COVIEdb database, a compilation of information from coronavirus epitope mapping studies (http://biopharm.ziu.edu.cn/coviedb/: Wu J COVIEdb: A Database for Potential Immune Epitopes of Coronaviruses. 2020 Front. Pharmacol. 11:572249; doi: 10.3389/fphar.2020.572249).
  • Monoclonal antibodies against SARS-CoV-2 surface epitopes are described in the literature (e.g. as mentioned above), available from commercial sources and/or can be generated using standard techniques, such as immunization of experimental animals.
  • SARS-CoV-2 surface epitopes are described in the literature (e.g. as mentioned above), available from commercial sources and/or can be generated using standard techniques, such as immunization of experimental animals.
  • MyBioSource, Inc. San Diego, CA
  • MBS857474747 see www.MvBioSource.com
  • At least 28 different antibodies against SARS-CoV-2 were available from Sino Biological US Inc., Wayne, PA (e.g. cat. no. 40150-D006, see https ://www .sinobiological .com/) .
  • a skilled person can detect preservation of a native surface conformation of SARS-CoV-2 (or e.g. the S or N protein thereof) via cross-reaction of such antibodies with the inactivated particles.
  • the inactivated particles bind specifically to one or more anti-SARS-CoV-2 antibodies directed against surface epitopes, preferably anti-S-protein antibodies, e.g. to antibodies generated against neutralizing epitopes in native SARS-CoV-2 virions.
  • the SARS-CoV-2 particles in the vaccine composition may be derived from any known strain of SARS- CoV-2, or variants thereof.
  • the virus may be a strain as defined in Figure 2, or may comprise a nucleotide or amino acid sequence as defined therein, or a variant sequence having at least e.g. 95% sequence identity thereto.
  • the SARS-CoV-2 particle comprises an RNA sequence corresponding to a DNA sequence (i) as defined in SEQ ID NO: 1 (which is also defined in NCBI Reference Sequence NC_045512.2).
  • corresponding to it will be understood that the defined DNA sequence is an equivalent of the viral RNA sequence, i.e.
  • the inactivation process may result in modification (e.g. alkylation or acylation) and/or fragmentation of viral RNA, and thus it will be understood that the inactivated viral particles may not comprise an intact RNA sequence as defined herein, but rather are derived from native viral particles which do comprise such a sequence.
  • the SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 Wuhan-Hu-1 lineage or also referred to as the reference lineage, e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 and/or NCBI Reference Sequence NC_045512.2.
  • the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus.
  • SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 South African lineage B.1.351, e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 18 and/or NCBI Reference Sequence MW598408.
  • the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS- CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus.
  • variants of the known SARS-CoV-2 South African lineage B.1.351 are given in Figure 2.
  • SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 Brazilian lineage P.1, e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 20 and/or NCBI Reference Sequence MW520923.
  • the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus.
  • variants of the known SARS-CoV-2 Brazilian lineage P.l are given in Figure 2.
  • variants of the known SARS-CoV-2 UK lineage B.l.1.7 may also comprise variants of the known SARS-CoV-2 UK lineage B.l.1.7, e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 22 and/or NCBI Reference Sequence MW422256.
  • the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus.
  • variants of the known SARS-CoV-2 UK lineage B.1.1.7 are given in Figure 2.
  • SARS-CoV-2 particles may also comprise variants of the known SARS-CoV-2 Californian lineages B.1.427 and B.1.429, e.g. sequences having at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 24 and/or SEQ ID NO: 26.
  • the variant sequence encodes an infectious SARS-CoV-2 particle, e.g. a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence that is able to pack a virulent SARS-CoV-2 virus.
  • variants of the known SARS-CoV-2 Californian lineages can be found in GenBank.
  • the SARS-CoV-2 particle comprises an S protein of the Wuhan lineage comprising or consisting of (i) an amino acid sequence as defined in SEQ ID NO: 3, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 3.
  • the SARS-CoV-2 particle comprises an S protein of the South African B 1.351 lineage comprising or consisting of (i) an amino acid sequence as defined in SEQ ID NO: 19, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 19.
  • the SARS-CoV-2 particle comprises an S protein of the Brazilian P.1 lineage comprising or consisting of (i) an amino acid sequence as defined in SEQ ID NO: 21, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 21.
  • the SARS-CoV-2 particle comprises an S protein of the UK B.l.1.7 lineage comprising or consisting of (i) an amino acid sequence as defined in SEQ ID NO: 23, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 23.
  • the SARS-CoV-2 particle comprises an S protein of the Californian B.1.427 lineage comprising or consisting of (i) an amino acid sequence as defined in SEQ ID NO: 25, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 25.
  • the SARS-CoV-2 particle comprises an S protein of the Californian B.1.429 lineage comprising or consisting of (i) an amino acid sequence as defined in SEQ ID NO: 27, or (ii) an amino acid sequence having at least 95%, at least 97% or at least 99% identity to SEQ ID NO: 27.
  • a combination of SARS-CoV-2 particles in the vaccine comprises or consists of at least two SARS-CoV-2 particles selected from the group consisting of i) the reference Wuhan_l lineage such as e.g. SEQ ID Nos: 1, 9, 12, 15; ii) the South African B.1.531 lineage such as e.g. SEQ ID NO: 18; the Brazilian P.l lineage such as e.g. SEQ ID NO: 20; the UK B.1.1.7 lineage such as e.g. SEQ ID NO: 22 and the Californian B.1.427 lineage such as e.g. SEQ ID NO: 24 or B.1.429 lineages such as e.g. SEQ ID NO: 26.
  • a preferred embodiment is a combination comprising i) a Wuhan l lineage such as e.g. SEQ ID NO: 9; and ii) a South African B.1.531 lineage such as e.g. SEQ ID NO: 18.
  • a combination of SARS-CoV-2 particles in the vaccine comprises or consists of at least three, e.g. three SARS-CoV-2 particles selected from the group consisting of i) the reference Wuhan_l lineage such as e.g. SEQ ID NOs 1, 9, 12, 15; ii) the South African B.1.531 lineage such as e.g. SEQ ID NO: 18; the Brazilian P.l lineage such as e.g. SEQ ID NO: 20; the UK B.1.1.7 lineage such as e.g. SEQ ID NO: 22 and the Californian B.1.427 such as e.g. SEQ ID NO: 24 or B.1.429 lineages such as e.g. SEQ ID NO: 26.
  • the reference Wuhan_l lineage such as e.g. SEQ ID NOs 1, 9, 12, 15
  • the South African B.1.531 lineage such as e.g. SEQ ID NO: 18
  • the Brazilian P.l lineage such as e.g. SEQ ID NO: 20
  • a preferred embodiment of such a trivalent vaccine is a combination comprising i) a Wuhan l lineage such as e.g. SEQ ID NO: 9; and ii) a South African B.1.531 lineage such as e.g. SEQ ID NO: 18; and iii) an UK B.1.1.7 lineage such as e.g. SEQ ID NO: 22.
  • Another preferred embodiment of such a trivalent vaccine is a combination comprising i) a Wuhan_l lineage such as e.g. SEQ ID NO: 9; and ii) a South African B.1.531 lineage such as e.g. SEQ ID NO: 18; and iii) a Brazilian P.l lineage such as e.g. SEQ ID NO: 20.
  • sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are.
  • homologs, orthologs, or variants of a polynucleotide or polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.
  • the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences.
  • the percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.
  • 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2.
  • the length value will always be an integer.
  • NCBI Basic Local Alignment Search Tool (Altschul et al., Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • NCBI National Center for Biotechnology Information
  • the BLAST and the BLAST 2.0 algorithms are also described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977.
  • the BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989).
  • Homologs and variants of a polynucleotide or polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over at least 50, 100, 150, 250, 500, 1000, 2000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence, over the full length of the reference sequence or over the full length alignment with the reference amino acid sequence of interest.
  • Polynucleotides or proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5: 151-153, 1989.
  • a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984).
  • reference to "at least 80% identity” refers to at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to a specified reference sequence, e.g. to at least 50, 100, 150, 250, 500, 1000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence or to the full length of the sequence.
  • reference to “at least 90% identity” refers to "at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity" to a specified reference sequence, e.g. to at least 50, 100, 150, 250, 500, 1000, 5000 or 10,000 nucleotide or amino acid residues of the reference sequence or to the full length of the sequence.
  • the inactivated SARS-CoV-2 particles are combined with an adjuvant in the vaccine.
  • the adjuvant is a Thl response-directing adjuvant.
  • Thl response-directing adjuvant By this it is meant that when the vaccine is administered to a subject, the adjuvant promotes the induction of a predominantly T helper type 1 (i.e. Thl) immune response in the subject (rather than a Th2 type response).
  • Thl T helper type 1
  • Th2 Th2 type response
  • the adjuvant comprises 3-0-desacyl-4'-monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a-tocopherol, a cationic peptide, a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN) and/or imiquimod.
  • MPL 3-0-desacyl-4'-monophosphoryl lipid A
  • CpG ODN CpG-containing oligodeoxynucleotide
  • squalene e.g., a CpG-containing oligodeoxynucleotide
  • DL-a-tocopherol a cationic peptide
  • I-ODN immunostimulatory oligodeoxynucleic acid molecule
  • suitable adjuvants may comprise: Adjuvant System 01 (AS01), which is a liposomal preparation comprising 3-0-desacyl-4'-monophosphoryl lipid A (MPL) and saponin QS-21; CpG 1018, a CpG ODN comprising the sequence 5’ TGACTGTGAACGTTCGAGATGA 3’ (SEQ ID NO: 4); Adjuvant System 03 (AS03), comprising squalene, DL-a-tocopherol and polysorbate 80; IC31, comprising a peptide comprising the sequence KLKLsKLK (SEQ ID NO: 5) and an I-ODN comprising oligo-d(IC)i 3 (SEQ ID NO: 6); or MF59, an oil-in-water emulsion comprising squalene, Tween 80 and Span 85.
  • Adjuvant System 01 AS01
  • MPL 3-0-desacyl-4'-monophosphoryl lipid A
  • SEQ ID NO: 4 Ad
  • the vaccine or adjuvant does not comprise a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the vaccine or adjuvant does not comprise CpG 1018, i.e. the vaccine or adjuvant does not comprise the sequence 5’ TGACTGTGAACGTTCGAGATGA 3’ (SEQ ID NO: 4).
  • the dosage of a Thl promoting adjuvant such as especially AS01, AS03, MF59, imiquimod or CpG, will be arrived at empirically. In some embodiments, the dosage of the Thl promoting adjuvant will be determined from previous studies.
  • the adjuvant may comprise an aluminium salt, e.g. aluminium oxide, aluminium hydroxide or aluminium phosphate.
  • a preferred aluminium salt is the aluminium hydroxide with reduced Cu content, e.g. lower than 1.25 ppb based on the weight of the vaccine composition, an adjuvant described in detail in WO2013/083726 or Schlegl et ah, Vaccine 33 (2015) 5989-5996.
  • an alum adjuvant is the only adjuvant in the vaccine composition.
  • the weight of the alum component refers to the weight of the Al 3+ in the solution, regardless of what type of aluminium salt is used.
  • 0.5 mg of Al 3+ corresponds to 1.5 mg alum.
  • the amount alum (Al 3+ ) present in the SARS-CoV-2 vaccine composition is between about 0.1 and 2 mg/mL, between about 0.2 and 1.5 mg/mL, between about 0.5 and 1.3 mg/mL, especially between about 0.8 to 1.2 mg/mL, most preferably about 1 mg/mL, i.e., 0.5 mg/dose.
  • the use of aluminium adjuvants alone is generally less preferred in the present invention, as they tend to direct a predominantly Th2 type immune response. Therefore in embodiments where the vaccine comprises an aluminium salt, it is particularly preferred that the vaccine further comprises a Thl- directing adjuvant, e.g. as described above.
  • the adjuvant may comprise an aluminium salt and a CpG ODN, e.g. CpG 1018 (SEQ ID NO: 4).
  • CpG 1018 can be adsorbed onto alum and, when used as a combinatorial adjuvant, has been shown to induce both Thl and Th2 responses (Tian. et al. 2017 Oncotarget 8(28)45951-45964); i.e. a more “balanced” immune response.
  • CpG when administered in combination with alum, CpG has been shown to increase the overall magnitude of the immune response and to reduce the Th2 bias that is induced by conventional adjuvants such as alum (X.P. Sicilnou et al.
  • CpG-containing oligodeoxynucleotides in combination with conventional adjuvants, enhance the magnitude and change the bias of the immune responses to a herpesvirus glycoprotein.
  • 2002 Vaccine 21: 127-137 The dose range for CpG in combination with alum may be anywhere between 10 pg and 3 mg.
  • the adjuvant is combined with the inactivated SARS-CoV-2 particles during manufacture of the vaccine product, i.e. the manufactured vaccine product comprises the adjuvant and is sold/distributed in this form.
  • the adjuvant may be combined with the inactivated SARS-CoV-2 particles at the point of use, e.g. immediately before clinical administration of the vaccine (sometimes referred to as “bedside mixing” of the components of the vaccine).
  • the present invention comprises both vaccine products comprising inactivated SARS-CoV-2 particles and an adjuvant as described herein, as well as kits comprising the individual components thereof (e.g. suitable for bedside mixing), and the combined use of the individual components of the vaccine in preventing or treating SARS-CoV-2 infection.
  • the SARS-CoV-2 vaccine may be produced by methods involving a step of inactivation of native SARS-CoV-2 particles, as described above.
  • the native SARS-CoV-2 particles may be obtained by standard culture methods, e.g. by in vitro production in mammalian cells, preferably using Vero cells.
  • the native SARS-CoV-2 particles may be produced using methods analogous to those described in e.g. WO 2017/109225 and/or WO 2019/057793, the contents of which are incorporated herein in their entirety, which describe methods for the production of Zika and Chikungunya viruses in Vero cells.
  • the steps such as passaging, harvesting, precipitation, dialysis, filtering and purification described in those documents are equally applicable to the present process for producing SARS-CoV-2 particles.
  • the method may comprise purifying the inactivated SARS-CoV-2 particles by one or more size exclusion methods such as (i) a sucrose density gradient centrifugation, (ii) a solid-phase matrix packed in a column comprising a ligand-activated core and an inactive shell comprising pores, wherein the molecular weight cut-off of the pores excludes the virus particles from entering the ligand-activated core, and wherein a molecule smaller than the molecular weight cut-off of the pores can enter the ligand-activated core and collecting the virus particles, and/or (iii) batch or size exclusion chromatography; to obtain purified inactivated SARS-CoV-2 particles.
  • size exclusion methods such as (i) a sucrose density gradient centrifugation, (ii) a solid-phase matrix packed in a column comprising a ligand-activated core and an inactive shell comprising pores, wherein the molecular weight cut-off of the pores excludes the virus particles from entering the ligand
  • the concentration of residual host cell DNA is less than 100 ng/mL; (ii) the concentration of residual host cell protein is less than 1 pg/mL; and (iii) the concentration of residual aggregates of infectious virus particles is less than 1 pg/mL.
  • the method may comprise a step of precipitating a harvested culture medium comprising SARS-CoV-2 particles, thereby producing native SARS-CoV-2 particles in a supernatant.
  • the precipitating step may comprise contacting the culture medium with protamine sulfate or benzonase.
  • a molecule smaller than the molecular weight cut-off of the pores e.g. the protamine sulfate
  • the residual host cell DNA of the obtained virus preparation or vaccine may be less than 1 pg/mL, especially less than 900, 800, 700, 600, 500, 400, 300 or 200 ng/mL, preferably less than 150 or 100 ng/mL.
  • the residual host cell DNA of the virus preparation or vaccine is less than 40 pg/mL.
  • the residual host cell protein of the virus preparation or vaccine is less than 10 pg/mL, especially less than 9, 8, 7, 6, 5, 4, 3 or 2 pg/mL, preferably less than 1 pg/mL.
  • the residual host cell protein of the virus preparation or vaccine is less than 150 ng/mL.
  • the residual non-infectious virus particles of the virus preparation or vaccine is less than 10 pg/mL, especially less than 9, 8, 7, 6, 5, 4, 3 or 2 pg/mL, preferably less than 1 mg/mL. In a preferred embodiment, the content of residual non-infectious virus particles of the virus preparation or vaccine is less than 100 ng/mL.
  • the vaccine and/or SARS-CoV-2 particles may comprise residual protamine (e.g. protamine sulfate), typically in trace amounts.
  • residual protamine (e.g. protamine sulfate) in the virus preparation or vaccine is less than 2 pg/mL or 1 pg/mL, especially less than 900, 800, 700, 600, 500, 400, 300 or 200 ng/mL, preferably less than 100 ng/mL, more preferably is below the detection limit of HPLC, in particular below the detection limit in the final drug substance.
  • the PS content is tested by HPLC or size exclusion chromatography (SEC).
  • HPLC is validated for PS determination in JEV sucrose gradient pool samples as a routine release assay and is very sensitive (i.e., limit of quantification (LOQ) 3 pg/mL; limit of detection (LOD) 1 pg/mL).
  • PS content in SARS-CoV-2 drug substance was ⁇ LOD.
  • the HPLC assessment of PS content can be performed on a Superdex Peptide 10/300GL column (GE: 17-5176-01) using 30% Acetonitrile, 0,1% Trifluoroacetic acid as solvent with a flow rate of 0.6 ml/min at 25°C and detection at 214 nm.
  • a more sensitive method of measurement for residual protamine in a purified virus preparation is mass spectrometry (MS).
  • MS mass spectrometry
  • the residual PS levels in a Zika virus preparation are tested by MS or other such highly sensitive method, e.g. nuclear magnetic resonance (NMR).
  • NMR nuclear magnetic resonance
  • residual PS, as well as fragments and/or break-down products of PS can be detected at trace amounts, such as levels as low as, for example, 10 6 , 10 7 or 10 8 molecules per typical sample load.
  • the PS levels are tested in the drug product.
  • the PS levels are tested in the drug substance.
  • an amount of the inactivating agent (e.g. beta-propiolactone) in the drug product or drug substance (e.g. vaccine composition) is very low, e.g. less than 100 ppm, less than 10 ppm, or less than
  • the SARS-CoV-2 vaccine may be administered to a subject, preferably a mammalian subject, more preferably a human subject.
  • a subject preferably a mammalian subject, more preferably a human subject.
  • the SARS-CoV-2 vaccine is administered to a subject at risk of SARS-CoV-2 infection, e.g. in order to prevent SARS-CoV-2 infection and/or to prevent SARS-CoV-
  • the subject is preferably (i) an elderly subject (e.g. older than 65 years, 70 years or 80 years) (ii) a pregnant subject (iii) an immunocompromised subject or (iv) a child (e.g. a person younger than 18 years, 16 years, 14 years, 12 years, 10 years, 8 years, 6 years, 4 years, 2 years or younger).
  • the SARS-CoV-2 vaccine described herein is advantageously capable of generating robust immune responses in subjects particularly susceptible or vulnerable to SARS-CoV-2 -mobidity or mortality, i.e. immunocompromised, pregnant or elderly subjects.
  • the SARS-CoV-2 vaccine may be administered to the subject in a single dose or two or more doses, e.g. separated by intervals of about 7, 14, 21 or 28 days.
  • the vaccine does not induce antibody- dependent enhancement (ADE) of SARS-CoV-2-associated disease (COVID-19).
  • ADE is a phenomenon by which virus-specific antibodies (e.g. as generated by vaccination) can enhance viral entry into host cells and/or viral replication. It is an advantage of the present invention that the inactivated SARS-CoV-2 vaccine described herein shows low or no ADE in human subjects, and can therefore be safely used for mass vaccination purposes. In particular, the vaccine described herein retains high quality immunogenic epitopes, which therefore results in high neutralizing antibody titers and diminishes the risk of ADE on administration to subjects. The risk of ADE development may be assessed in non-human primates, as described in the Examples (see also Luo F, etal. (2016), Virologica Sinica 33:201-204).
  • a vaccine e.g. a SARS-CoV vaccine
  • a Th2-type immunopathology e.g. a hypersensitivity response to SARS-CoV components in animals.
  • a Thl type response is favored, e.g. by use of a Thl-directing adjuvant (e.g. AS01 or another adjuvant as described herein).
  • a balanced Th2/Thl-type immune response is preferred, such as that induced by use of a Th2-stimulating adjuvant, e.g., alum, combined with a Thl -stimulating adjuvant.
  • a Th2-stimulating adjuvant e.g., alum
  • Thl -stimulating adjuvant e.g., alum
  • the risk of immunopathology developing may be assessed in animal models, e.g. as described in Tseng C.T. et al. (2012) PLoS ONE 7(4):e35421.
  • the vaccines of the invention show a shift in the Th2/Thl-type immune response to a Thl -type immune response compared to a vaccine adjuvanted with alum.
  • any of the SARS-CoV-2 vaccines or compositions described herein may be administered to a subject in a therapeutically effective amount or a dose of a therapeutically effective amount.
  • a “therapeutically effective amount” of vaccine is any amount that results in a desired response or outcome in a subject, such as those described herein, including but not limited to prevention of infection, an immune response or an enhanced immune response to SARS-CoV-2, or prevention or reduction of symptoms associated with SARS-CoV-2 disease. More specifically, a therapeutic amount of the SARS- CoV-2 vaccine of the invention may be a total viral protein mass of between about 0.05 and 50 pg, more preferably between about 0.5 to 10 pg.
  • the therapeutically effective amount of a SARS-CoV-2 vaccine or composition described herein is an amount sufficient to generate antigen-specific antibodies (e.g., anti-SARS-CoV- 2 antibodies). In some embodiments, the therapeutically effective amount is sufficient to seroconvert a subject with at least 70% probability. In some embodiments, the therapeutically effective amount is sufficient to seroconvert a subject with at least 75%, 80%, 85% 90%, 95%, 96%, 97%, 98%, or at least 99% probability. Whether a subject has seroconverted can be assessed by any method known in the art, such as obtaining a serum sample from the subject and performing an assay to detect anti-SARS-CoV- 2 antibodies.
  • a subject is seroconverted if a serum sample from the subject contains an amount of anti-SARS-CoV-2 antibodies that surpasses a threshold or predetermined baseline.
  • a subject is generally considered seroconverted if there is at least a 4-fold increase in anti- SARS-CoV-2 antibodies (i.e., anti-SARS-CoV-2 S protein IgG antibodies) present in a serum sample from the subject as compared to a serum sample previously taken from the same subject.
  • the dose of the inactivated SARS-CoV-2 virus in the vaccine composition of the current invention is between about 0.01 and 25 mAU (milli-absorption units x minutes as assessed by SEC-HPLC), preferably between about 0.05 and 10 mAU, more preferably between about 0.1 and 5 mAU, most preferably between about 0.25 and 2.5 mAU. In one embodiment, the dose is between about 0.05 and 50 pg total protein as measured by (p)BCA assay, between about 0.1 and 25 pg, between about 0.25 and 12.5 pg, preferably between about 0.5 and 5 pg total protein.
  • the dose of the inactivated SARS-CoV-2 virus in the vaccine composition is at least 2.5 pg total protein, at least 3.5 pg total protein or at least 2.5 pg total protein, e.g. the vaccine composition comprises 2.5 pg to 25 pg, 3.5 pg to 10 pg or 4 pg to 6 pg total protein/dose, preferably about 5 pg total protein/dose.
  • the dosage is determined by the total amount of S protein in the inactivated SARS-CoV- 2 formulation, as assessed by e.g. EUISA.
  • the mass of antigen may also be estimated by assessing the SE-HPLC peak area per dose equivalent (recorded as milli-absorption units x minutes; mAU), which is estimated to be approximately 2 pg/ml total surface protein and approximately 1 pg/mL S-protein.
  • the dose is between about 0.025 and 25 pg S-protein as measured by ELISA, between about 0.05 and 12.5 pg, between about 0.125 and 6.25 pg, preferably between about 0.25 and 2.5 pg S- protein.
  • the amount of antigen in the SARS-CoV-2 vaccine is determined by ELISA.
  • the ELISA measures a SARS-CoV-2 protein or portion of a protein, e.g., nucleocapsid (N), membrane (M) or spike (S) protein; i.e., the ELISA utilizes a coating antibody specific to a SARS-CoV-2 protein or portion of a protein.
  • the coating antibody is specific to the SARS-CoV-2 Spike protein SI subunit, e.g.
  • the ELISA readout is a mass per unit measure of the detected protein, e.g. pg/mL S-protein.
  • the standard used is a spike protein trimer and the results of the SARS-CoV-2 ELISA are reported as “antigen units” (AU), corresponding to the ACE-2 binding ability of the standard protein (determined by the manufacturer).
  • the amount of SARS-CoV-2 antigen administered to a subject is between about 1 to 100 AU/dose, preferably between about 2 to 75 AU/dose, preferably between about 3 and 60 AU/dose, more preferably between about 3 and 55 AU/dose, more preferably between about 3 and 53 AU/dose. In an even more preferred embodiment, the amount of SARS-CoV-2 antigen administered to a subject is 3 AU, 10 AU or 40 AU per dose, most preferred 40 AU per dose. In further preferred embodiments, the amount of SARS-CoV-2 antigen administered to a subject is at least 10 AU/dose, at least 20 AU/dose, at least 25 AU/dose or at least 30 AU/dose, e.g.
  • SARS-CoV- 2 antigen e.g. in AU/dose
  • the amount of SARS-CoV- 2 antigen may be assessed, for example, by a SARS-CoV-2 ELISA assay as described in Example 1. It is estimated that there are about 1 to 1.5 x 10 7 viral particles per AU, and the amounts of SARS-CoV-2 antigen described above may be construed accordingly.
  • the amount of SARS-CoV-2 antigen administered to a subject is between about 1.5 x 10 7 to 1.5 x 10 9 viral particles/dose, or between about 4.5 x 10 7 to 9.0 x 10 8 viral particles/dose, e.g. at least 1.5 x 10 8 viral particles/dose or at least 3.0 x 10 8 viral particles/dose, about 1.5 x 10 8 to 7.5 x 10 8 viral particles/dose or about 4.5 x 10 8 to 6.0 x 10 8 viral particles/dose.
  • PRNT plaque reduction neutralization test
  • the PRNT50 may be carried out using monolayers of Vero cells or any other cell type/line that can be infected with SARS-CoV-2.
  • Sera from subjects are diluted and incubated with live, non-inactivated SARS-CoV-2.
  • the serum/virus mixture may be applied to Vero cells and incubated for a period of time.
  • Plaques formed on the Vero cell monolayers are counted and compared to the number of plaques formed by the SARS-CoV-2 in the absence of serum or a control antibody.
  • a threshold of neutralizing antibodies of 1 : 10 dilution of serum in a PRNT50 is generally accepted as evidence of protection in the case of JEV (Hombach et. al. Vaccine (2005) 23:5205-5211).
  • the SARS-CoV-2 particles may be formulated for administration in a composition, such as a pharmaceutical composition.
  • pharmaceutical composition means a product that results from the mixing or combining of at least one active ingredient, such as an inactivated SARS-CoV-2, and one or more inactive ingredients, which may include one or more pharmaceutically acceptable excipient.
  • a preferred pharmaceutically acceptable excipient is human serum albumin (HSA), such as, especially recombinant HSA (rHSA).
  • the SARS- CoV-2 vaccine of the invention contains about 10 to 50 pg HSA/dose, preferably about 20 to 40 pg HS A/dose, more preferably about 25 to 35 pg HSA/dose.
  • compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art (see e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co. 20th ed. 2000; and Ingredients of Vaccines - Fact Sheet from the Centers for Disease Control and Prevention, e.g., adjuvants and enhancers as described above to help the vaccine improve its work, preservatives and stabilizers to help the vaccine remain unchanged (e.g., albumin, such as human serum albumin (HSA) or recombinant HSA (rHSA), phenols, glycine)).
  • HSA human serum albumin
  • rHSA recombinant HSA
  • glycine refers to an immunogenic composition, e.g.
  • the vaccine or composition capable of inducing an immune response in a (human) subject against an antigen (e.g. against a SARS-CoV-2 antigen).
  • the vaccine or composition may be capable of generating neutralizing antibodies against SARS-CoV-2.
  • the vaccine or composition is capable of generating antibodies (e.g. IgG) against SARS-CoV-2 S (spike) protein.
  • the vaccine or composition is capable of generating a T cell response against SARS-CoV-2 proteins or peptides, for instance a T cell response against a SARS-CoV-2 S-protein, membrane (M) protein and/or nucleocapsid (N) protein or peptides derived therefrom.
  • the vaccine or immunogenic composition generates neutralizing antibodies and a T cell response against SARS-CoV-2.
  • the vaccine or immunogenic composition is capable of inducing a protective effect against a disease caused by the antigen, e.g. a protective effect against SARS-CoV-2 infection (e.g. symptomatic and/or asymptomatic infection) and/or COVID-19 disease).
  • compositions are preferably manufactured under GMP conditions.
  • a therapeutically effective dose of the inactivated SARS-CoV-2 vaccine preparation is employed in the pharmaceutical composition of the invention.
  • the inactivated SARS-CoV-2 particles are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., the prophylactic response).
  • Dosages of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired pharmaceutical response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • the selected dosage level depends upon a variety of pharmacokinetic factors, including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors.
  • a physician, veterinarian or other trained practitioner can start dosing of the inactivated SARS-CoV-2 vaccine employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect (e.g., production of anti-SARS-CoV-2 virus antibodies) is achieved.
  • effective doses of the compositions of the present invention, for the prophylactic treatment of groups of people as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and the titer of anti- SARS-CoV-2 antibodies desired. Dosages need to be titrated to optimize safety and efficacy.
  • the dosing regimen entails subcutaneous or intramuscular administration of a dose of inactivated SARS-CoV-2 vaccine twice, once at day 0 and once at about day 7. In some embodiments, the dosing regimen entails subcutaneous administration of a dose of inactivated SARS-CoV-2 vaccine twice, once at day 0 and once at about day 14. In some embodiments, the dosing regimen entails subcutaneous administration of a dose of inactivated SARS-CoV-2 vaccine twice, once at day 0 and once at about day 28. In some embodiments, the inactivated SARS-CoV-2 vaccine is administered to the subject once. In a preferred embodiment, the SARS-CoV-2 vaccine is administered to the subject more than once, preferably two times. In a preferred embodiment, the vaccine is administered on day 0 and day 21. In another preferred embodiment, the vaccine is administered on day 0 and day 28.
  • a first (prime) dose of the inactivated SARS-CoV-2 vaccine is administered and a second (boost) dose of the inactivated SARS-CoV-2 vaccine is administered at least 28 days, at least 60 days, at least 70 days, at least 80 days or 90 days after the first dose.
  • the second dose of the inactivated SARS-CoV-2 vaccine is administered 30 to 120 days or 1 to 4 months (preferably about 3 months) after the first dose.
  • the inactivated SARS-CoV-2 vaccine is administered as a booster dose only, e.g. a first (prime) dose of a (different) SARS-CoV-2 vaccine is administered and then a second (boost) dose of the inactivated SARS-CoV-2 vaccine is administered, e.g. at least 7, 14, 21, 28, 60 or 90 days after the first dose.
  • the first (prime) dose of the SARS-CoV-2 vaccine may comprise any other vaccine or immunogenic composition that stimulates an immune response and/or a protective effect in subjects against SARS-CoV-2 virus.
  • the first dose of SARS-CoV-2 vaccine may comprise a recombinant viral vector or an mRNA sequence encoding one or more SARS-CoV-2 proteins and/or fragments thereof, e.g. a SARS-CoV-2 spike (S) protein.
  • the first dose of SARS-CoV-2 vaccine may comprise a subunit vaccine, e.g. comprising one or more SARS-CoV-2 proteins and/or fragments thereof, e.g. a SARS-CoV-2 spike (S) protein or fragment thereof.
  • kits for use in prophylactic administration to a subject for example to prevent or reduce the severity of SARS-CoV-2 infection.
  • kits can include one or more containers comprising a composition containing inactivated SARS-CoV-2, such as an inactivated SARS-CoV-2 vaccine.
  • the kit may further include one or more additional components comprising a second composition, such as a second vaccine, e.g. a second kind of SARS-CoV-2 vaccine that applies a different technology than in the first dose.
  • the second vaccine is a vaccine for an arbovirus.
  • the second vaccine is a Japanese encephalitis virus vaccine, a Zika virus vaccine, a Dengue virus vaccine and/or a Chikungunya virus vaccine.
  • the kit can comprise instructions for use in accordance with any of the methods described herein.
  • the included instructions can comprise a description of administration of the composition containing inactivated SARS-CoV-2 vaccine to prevent, delay the onset, or reduce the severity of SARS-CoV-2 infection.
  • the kit may further comprise a description of selecting a subject suitable for administration based on identifying whether that subject is at risk for exposure to SARS- CoV-2 or contracting a SARS-CoV-2 infection.
  • the instructions comprise a description of administering a composition containing inactivated SARS-CoV-2 vaccine to a subject at risk of exposure to SARS-CoV-2 or contracting SARS-CoV-2 infection.
  • the instructions relating to the use of the composition containing inactivated SARS-CoV-2 vaccine generally include information as to the dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi -dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions are also acceptable.
  • kits of the present disclosure are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as a syringe or an infusion device.
  • the container may have a sterile access port, for example the container may be a vial having a stopper pierceable by a hypodermic injection needle.
  • At least one active agent in the composition is an inactivated SARS-CoV- 2, as described herein.
  • the JEV process platform (Srivastava et ah, Vaccine 19 (2001) 4557-4565; US 6,309,650B1) was used as a basis, also taking into account improvements in the process as adapted to Zika virus purification as disclosed in WO2017/109223A1 (which is incorporated herein in its entirety). Briefly, non-infectious SARS-CoV-2 particle aggregates, host cell proteins and other low molecular weight impurities are removed by protamine sulfate precipitation or benzonase treatment and the resulting preparation is optionally further purified by sucrose gradient centrifugation. See Fig. 1 for an outline of the production process.
  • SARS-CoV-2 isolates from Italy, identified and characterized at the National Institute for Infectious Diseases “Lazzaro Spallanzani” IRCCS, Rome, Italy (Accession No: MT066156), the RNA sequence thereof corresponding to the DNA sequence provided by SEQ ID NO: 9, was used in all Examples disclosed herein.
  • Other novel coronavirus SARS-CoV-2 isolates may also be obtained from the following sources:
  • BetaCoV/France/IDF0372/2020 (Ref-SKU:014V-03890, https://www.european-virus- archive . com/virus/human-2019-ncov-O); 2019-nCoV/Italy-INMI 1 , (Ref-SKU:008V-03893, SEQ ID NO:
  • -BEI Resources Biodefense and Emerging Infections Research Resources: e.g. Isolate USA- WA1/2020, NIAID, NIH: SARS-Related Coronavirus 2, NR-52281 (GenBank accession MN985325).
  • Vero cells used in the methods described herein were the VERO (WHO) cell line, obtained from the Health Protection Agency general cell collection under catalogue number 88020401, from which a master cell bank was generated.
  • a research master seed bank (rMSB) of SARS-CoV-2 strain used 2019-nCoV/Italy-INMIl was prepared on Vero cells and the genomic sequence was checked by sequencing.
  • Vero cells were grown in Eagle's minimal essential medium (EMEM) containing 10% fetal bovine serum (FBS) and monolayers were infected with SARS-CoV-2 at a multiplicity of infection (moi) of 0.001 to 1, preferably 0.01, plaque forming units (pfii) per cell. After allowing virus adsorption, the cultures were washed 2-4 times with PBS, fed with serum-free EMEM and incubated at 35°C with 5% CO2 until the virus titer reaches a desired level.
  • EMEM Eagle's minimal essential medium
  • FBS fetal bovine serum
  • pfii plaque forming units
  • SARS-CoV-2 harvest The culture medium was harvested at days 2, 3, 5 and 7 and harvests were pooled and centrifuged in a standard centrifuge. The resulting supernatant was filtered, followed by TFF ultrafiltration to remove cell culture medium components and reduce batch volume. Host cell DNA and protein reduction as well as reduction of non-infectious virus aggregates in the concentrated material was achieved by precipitation with protamine sulfate. Protamine sulfate was added to the diafiltrated SARS-CoV-2 material to a final nominal concentration of ⁇ 2 mg/mL, while stirring, followed by incubation at 2-8°C for 30 minutes. Alternatively, the diafiltrated SARS-CoV-2 material was treated with benzonase.
  • SARS-CoV-2 virus was inactivated by treatment with beta- propiolactone directly after removal of virus-containing cell culture medium from Vero cells, in order to render the virus safe to handle at BSL2.
  • Inactivation is possible at any stage in the purification process, however, such as e.g., after centrifugation, before, during or after treatment with protamine sulfate or benzonase or before or after sucrose gradient centrifugation.
  • Inactivation is carried out by the use of a chemical inactivation agent such as formaldehyde (formalin); enzyme; beta-propiolactone; ethanol; trifluroacetic acid; acetonitrile; bleach; urea; guanidine hydrochloride; tri-n-butyl phosphate; ethylene-imine or a derivative thereof; an organic solvent, optionally Tween, Triton, sodium deoxycholate, or sulfobetaine; or a combination thereof.
  • a chemical inactivation agent such as formaldehyde (formalin); enzyme; beta-propiolactone; ethanol; trifluroacetic acid; acetonitrile; bleach; urea; guanidine hydrochloride; tri-n-butyl phosphate; ethylene-imine or a derivative thereof; an organic solvent, optionally Tween, Triton, sodium deoxycholate, or sulfobetaine; or a combination thereof.
  • Inactivation may also be achieved by pH changes (very high or very low pH), by heat treatment or by irradiation such as gamma irradiation or UV irradiation, particularly UV-C irradiation.
  • the SARS-CoV-2 virus is optionally inactivated by two separate inactivation steps, such as, e.g. beta-propiolactone treatment and UV-C irradiation.
  • BPL starting concentration for inactivation of a highly resistant model virus PPV
  • Porcine Parvovirus (PPV) was selected as a model virus to evaluate the inactivation capability of BPL in aqueous solution because of its high resistance to physico-chemical inactivation.
  • Three starting concentrations of BPL were evaluated: 300 ppm (1/3333), 500 ppm (1/2000) and 700 ppm (1/1429).
  • Virus solution was spiked with BPL at these concentrations and incubated at 5 ⁇ 2°C for 24 hours.
  • Kinetic samples were taken at 0.5, 2, 6, 24h and after the BPL hydrolyzation step and analysed for remaining infectivity. The results are shown in Table A.
  • BPL concentration 500 ppm (1/2000) was selected for the inactivation of SARS-CoV- 2 virus harvest material.
  • an incubation temperature of 5 ⁇ 3°C and an incubation time of 24 hours were selected to ensure enough BPL present throughout the whole inactivation.
  • the inactivation solution is transferred to a fresh container where the inactivation takes place under controlled conditions. This transfer excludes the possibility of virus particles in potential dead- spots during initial mixing not being in contact with BPL.
  • protamine sulfate (PS) treated concentrated harvest pre-cooled to 5 ⁇ 3°C is supplemented with 25 mM HEPES pH 7.4.
  • the solution is warmed to temperatures above 32°C for a total time of 2.5 hours ⁇ 0.5 hours in a temperature-controlled incubator set to 37 ⁇ 2°C.
  • the total time of the hydrolyzation step for the current process volume of about 1L was between 5 hours 15 minutes and 6 hours 15 minutes including the warming to and the incubation above 32°C.
  • the inactivated viral solution (IV S) was immediately cooled down to 5 ⁇ 3°C in a temperature-controlled fridge and stored there until inactivation was confirmed by large volume plaque assay and serial passaging assay which currently requires 18 days in total. Recovery of virus particles throughout the inactivation process was monitored by size-exclusion chromatography.
  • the inactivation step(s) are particularly gentle, in order to preserve surface antigen integrity, especially integrity of the S protein.
  • the gentle inactivation method comprises contacting a liquid composition comprising SARS-CoV-2 particles with a chemical viral inactivating agent (such as e.g. any of the chemical inactivation agents as listed above or a combination thereof, preferably beta-propiolactone) in a container, mixing the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles under conditions of laminar flow but not turbulent flow, and incubating the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles for a time sufficient to inactivate the viruses.
  • a chemical viral inactivating agent such as e.g. any of the chemical inactivation agents as listed above or a combination thereof, preferably beta-propiolactone
  • the gentle inactivation step is optionally performed in a flexible bioreactor bag.
  • the gentle inactivation step preferably comprises five or less container inversions during the period of inactivation.
  • the mixing of the chemical viral inactivating agent and the composition comprising SARS-CoV-2 particles comprises subjecting the container to rocking, rotation, orbital shaking, or oscillation for not more than 10 minutes at not more than 10 rpm during the period of incubation.
  • the material was immediately further processed by batch adsorption (also known herein as batch chromatography) with CaptoTM Core 700 or CC400 chromatography media at a final concentration of ⁇ 1% CC700 or CC400.
  • batch adsorption also known herein as batch chromatography
  • CaptoTM Core 700 or CC400 chromatography media at a final concentration of ⁇ 1% CC700 or CC400.
  • the material was incubated at 4°C for 15 minutes under constant agitation using a magnetic stirrer. After incubation, if used, the CC700 or CC400 solid matter was allowed to settle by gravity for 10 minutes and the SARS-CoV-2 material is removed from the top of the solution in order to avoid blockage of the filter by CaptoCore particles.
  • CaptoCore particles and DNA precipitate were then removed from the solution by filtration using a 0.2 pm Mini Kleenpak EKV filter capsule (Pall).
  • the pooled filtered harvest material was adjusted to a final concentration of 25 mM Tris pH 7.5 and 10% sucrose (w/w) using stock solutions of both components. This allowed for freezing the concentrated harvest at ⁇ -65°C if required.
  • the resulting filtrate is further processed by sucrose density gradient centrifugation (also known herein as batch centrifugation) for final concentration and polishing of the SARS-CoV-2 material.
  • sucrose density gradient centrifugation also known herein as batch centrifugation
  • PS concentrated protamine sulfate
  • benzonase preferred is PS
  • treated harvest was loaded on top of a solution consisting of three layers of sucrose with different densities. The volumes of individual layers for a centrifugation in 100 mL bottle scale are shown in Table la.
  • Table la Volumes for sucrose density centrifugation.
  • the sucrose gradient bottles were prepared by stratifying the individual sucrose layers by pumping the solutions into the bottom of the bottles, starting with the SARS-CoV-2 material with the lowest sucrose density (10% sucrose (w/w)), followed by the other sucrose solutions in ascending order.
  • the described setup is shown in Figure 3.
  • the prepared SG bottles were transferred into a rotor pre-cooled to 4°C and centrifuged at ⁇ 11,000 RCF max at 4°C for at least 20 hours, without brake/deceleration. After centrifugation, harvest of serial 2 mL fractions of the sucrose gradient is performed from the bottom up with a peristaltic pump. The fractions were immediately tested by SDS-PAGE / silver staining to identify virus-containing fractions with sufficiently high purity. Thus, identified fractions were pooled and further processed.
  • the purified SARS-CoV-2 was stored at ⁇ -65°C or immediately formulated.
  • SARS-CoV-2 Formulation of SARS-CoV-2 with adjuvant.
  • the SARS-CoV-2 particles were formulated with alum.
  • a Thl adjuvant was also added to the formulation or provided as a separate composition for bedside mixing.
  • SARS-CoV-2 ELISA Assay Inactivated SARS-CoV-2 antigen content (i.e. content of SI as the major antigenic protein) in preparations described herein was determined (quantified) by ELISA.
  • the SARS- CoV-2 ELISA used herein is a four-layer immuno-enzymatic assay with a SARS-CoV-2 spike antibody (AM001414; coating antibody) immobilized on a microtiter plate to which the SARS-CoV-2 sample is added. On binding of the antigen to the coating antibody, the plate was further treated with primary antibody (i.e. AbFlex®SARS-COV-2 spike antibody (rAb) (AM002414)).
  • the secondary antibody which is an enzyme linked conjugate antibody (i.e. Goat anti -Mouse IgG HRP Conjugate).
  • the plates were washed between various steps using a mild detergent solution (PBS-T) to remove any unbound proteins or antibodies.
  • PBS-T mild detergent solution
  • the plate was developed by addition of a tetramethyl benzidine (TMB) substrate.
  • TMB tetramethyl benzidine
  • the hydrolyzed TMB forms a stable colored conjugate that is directly proportional to the concentration of antigen content in the sample.
  • the antigen quantification was carried out by spectrophotometric detection at l450hth (l630hih reference) using the standard curve generated in an automated plate reader as a reference.
  • Standard concentrations 20 AU/mL, 10 AU/mL, 5 AU/mL, 2.5 AU/mL, 1.25 AU/mL, 0.625 AU/mL, 0.3125 AU/mL and 0.1263 AU/mL. Each dilution was tested in duplicate per plate.
  • An “antigen unit” of the spike trimer standard corresponds to its binding ability in a functional ELISA with Recombinant Human ACE-2 His-tag.
  • SARS-CoV-2 Spike Antibody SARS-CoV-2 Spike Antibody (AM001414)
  • Spike Trimer S1+S2
  • His-tag SARS-CoV-2
  • SARS-CoV-2 QC (e g. RSQC240920AGR)
  • SARS-CoV-2 drug substance according to the invention was highly pure (>95%) according to SDS-PAGE (silver stain, reduced) and free from aggregates (monomer virus (>95%) according to SE-HPLC (see Figure 7).
  • Example 2 In vitro and in vivo assessment of immunogenicity and protective capacity of inactivated SARS-CoV-2 virus compositions Immunogenicity. Prior to immunization, experimental groups of 10 Balb/c mice were bled and pre- immune sera are prepared. The mice were administered a dose titration of inactivated SARS-CoV-2 formulated with alum subcutaneously (see Table 2). At two different intervals after immunization (see below), blood was collected and immune sera prepared, spleens were collected at the final bleed. All animal experiments were carried out in accordance with Austrian law (BGB1 Nr. 501/1989) and approved by “Magistrats 58”.
  • Sera were assessed for total IgG and subclasses (IgGl/IgG2a) by ELISA and neutralizing antibodies by PRNT. Thl/Th2 responses were further assessed by IFN-g ELISpot and intracellular cytokine staining (CD4 + /CD8 + ).
  • mice/group 3 dosage groups: 0.2 - 2 pg total protein; number of experiments: 3.
  • the Thl adjuvant is added directly to the SARS-CoV-2/alum formulation before immunization of the mice.
  • Plaque reduction neutralization test PRNT. Each well of a twelve-well tissue culture plate was seeded with Vero cells and incubated 35°C with 5% CO2 for three days. Serial dilutions from pools of heat-inactivated sera from each treatment group are tested. Each serum preparation was incubated with approximately 50-80 pfii of SARS-CoV-2 at 35°C with 5% CO2 for 1 hour.
  • the cell culture medium was aspirated from the Vero cells and the SARS-CoV-2 /serum mixtures were added to each well.
  • the plates are gently rocked and then incubated for 2 hours at 35°C with 5% CO2.
  • 1 mL of a 2% methylcellulose solution containing EMEM and nutrients are added, and the plates were further incubated for 4 days at 35°C with 5% CO2.
  • the cells were then stained for 1 hour with crystal violet/5% formaldehyde and washed 3 times with deionized water.
  • the plates were air dried and the numbers of plaques in each well manually counted.
  • other methods such as e.g. TCID50 may be applied.
  • mice/group Immunization schedule as for Table 2, but in addition; interim bleeds 2, 6, 10, 14, 18 and 22 weeks after second immunization; end- bleed 26 weeks after second immunization; only with the preferred dose; only subcutaneous route; number of experiments: 1.
  • the Thl adjuvant was added directly to the SARS-CoV-2/alum formulation before immunization of the mice.
  • the protective capacity of inactivated SARS-CoV-2 is assessed using a SARS- susceptible transgenic mouse expressing a humanized ACE2 protein (Jackson Laboratory) (Tseng, C - T.K. et al., Severe Acute Respiratory Syndrome Coronavirus Infection of Mice Transgenic for the Human Angiotensin-Converting Enzyme 2 Virus Receptor (2007) J of Virol 81:1162-1173) or aNHP model developed for SARS-CoV-2 infection. Groups of animals are immunized subcutaneously (s.c.) with different dosages of inactivated SARS-CoV-2 with or without adjuvant or PBS as a negative control. Three weeks after the last dose, animals are challenged with SARS-CoV-2 and monitored for disease progression and survival. In addition, serum samples are taken in order to determine the neutralizing antibody titers induced by vaccination in a PRNT assay.
  • Table 3A Design of dosing experiment 4743 using SARS-CoV-2 ELISA-determined dosages.
  • mice Female Balb/c mice (10 mice/group) were immunized two times s.c. (100 pL) on days 0 and 21 with doses and adjuvants as outlined in Table 3A. The readouts from the experiment were total IgG and subclasses (IgGl/IgG2a) and virus neutralization (PRNT).
  • Vaccine formulation used in experiment 4743 purified inactivated SARS-CoV-2 produced from a research virus seed bank (rVSB) formulated in PBS with 17 pg Al 3+ (alum)/dose.
  • HRP-conjugated goat anti-mouse IgG was used and developed with ABTS and read at absorbance 405 nm. Wells were washed with PBS-T between each step. Endpoint titers were determined with a cut-off set to 3-fold the blank.
  • IgG subclass immune response Plates were coated with the SI part ( Figure 4A) of spike glycoprotein and sera taken on day 35 were analyzed. Subclass specific secondary antibodies (IgGl and IgG2a) conjugated with HRP were used for detection. As standard curves (4-paramater regression) for determination of the amount of the different IgG subclasses (IgGl and IgG2a), monoclonal antibodies with different subclasses were used (IgGl mAb clone 43 and IgG2a mAb clone CR3022). Bound HRP- conjugated secondary mAbs were developed with ABTS and read at absorbance 405 nm. Wells were washed with PBS-T between each step. The relative IgG subclass concentration is shown in Figure 5A and the ratio of IgG2a/IgGl in Figure 5B.
  • the alum-adjuvanted inactivated SARS-CoV-2 promoted an immune response shifted more towards a Th2 (IgGl) compared with a Thl (IgG2a) response as demonstrated by quantification of IgG subclasses by SI ELISA.
  • the total amounts of IgG2a and IgGl measured and the ratio of IgG2a:IgGl in the treatment groups are shown in Figs. 5A and 5B, respectively.
  • a shift in the immune response toward Thl (IgG2a) would likewise be expected by addition of a Thl -stimulating adjuvant to the SARS-CoV-2 vaccine composition.
  • Immune sera from inactivated SARS-CoV-2- vaccinated mice are assessed for hallmarks of enhanced disease in vitro.
  • Such assays are described by e.g. Wang, S.-F., el al. 2014 (Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins (2014) BBRC 451 :208-214). Briefly, susceptible cell types or cell lines are incubated with immune sera and subsequently infected with SARS-CoV-2. Cells are assessed for cytopathic effect and/or production of inflammatory markers.
  • mice are immunized twice at two-week intervals with inactivated SARS-Cov-2 formulated as described herein followed by challenge with SARS-CoV-2. SARS-CoV-2 titers and immune cell infdtration of the lung are tested.
  • Non-human primate model of ADE The risk of ADE development in non-human primates is assessed as described by Luo F, et al. (Evaluation of Antibody-Dependent Enhancement of SARS-CoV Infection in Rhesus Macaques Immunized with an Inactivated SARS-CoV Vaccine (2016) Virologica Sinica 33:201-204). Briefly, NHPs are immunized with inactivated SARS-CoV-2, followed by SARS-CoV-2 challenge and evaluation of symptoms and disease pathology.
  • Formulation of inactivated SARS-CoV-2 for Phase 1 trial The objective of the Phase 1 trial is to assess the safety of the vaccine, along with immunogenicity, and to determine an optimal dose and adjuvant(s). As such, three antigen doses are tested in clinical phase 1: High, Medium and Low, which are chosen to have a distance between each dose of approximately 3 -fold and a span covering about a 10-fold difference between the high and low doses. The dose range is selected in part to indicate any potential dose-sparing effect of a Thl adjuvant.
  • the SARS-CoV-2 virus as purified herein has a high purity of >90% as assessed by SDS-PAGE, SE- HPLC and/or SARS-CoV-2 ELISA (data not shown). Furthermore, preliminary studies have indicated that the incidence of genetic heterogeneities during passage of the virus is low and no particular individual mutations stand out (data not shown).
  • the SARS-CoV-2 virus as purified herein has a high purity of >90% as assessed by SDS-PAGE, SE- HPLC and/or SARS-CoV-2 ELISA (see, e.g., Fig. 7). Furthermore, preliminary studies have indicated that the incidence of genetic heterogeneities during passage of the virus is low and no particular individual mutations stand out (data not shown).
  • the SARS-CoV-2 virus was compared with JEV, specifically assessing SE- HPLC peak area per dose equivalent (recorded as milli-absorption units x minutes; mAU), the total amount of inactivated viral particles per dose and the total viral surface equivalent per dose (see Table 4).
  • SE- HPLC peak area per dose equivalent recorded as milli-absorption units x minutes; mAU
  • This assessment was based on the assumption of a similar surface antigen density between S (spike; SARS-CoV-2) and E (envelope; JEV) proteins.
  • Total protein was determined by pBCA assay (Table 4). Although the assay was variable, a correspondence of 1 mAU to ⁇ 2 pg total protein per mL was observed.
  • SARS-CoV-2 ELISA assay As described in Example 1, was developed and the doses of the vaccine formulations for entry into Phase 1 trials were determined using this assay.
  • the Phase 1 treatment groups are set forth in Table 5.
  • rHSA Human Serum Albumin
  • PBS Phosphate buffered saline
  • vaccinated subjects are challenged with an infectious dose of live SARS-CoV-2 virus (Asian and/or European lineage).
  • Treatment groups for Phase 1 testing of inactivated SARS-CoV-2 vaccine (low, medium and high doses are those provided in Table 4).
  • Example 5 Testing of Sera of vaccinated organism with a neutralization assay
  • Sera of vaccinated mice, hamsters, non-human primates or humans can be tested in neutralization assays such as e.g. described in “Szurgot, I., Hanke, L., Sheward, D.J. et al. DNA-launched RNA replicon vaccines induce potent anti-SARS-CoV-2 immune responses in mice. Sci Rep 11, 3125 (2021). https://doi.org/10.1038/s41598-021-82498-5”.
  • the read-out of the test gives an indication how well sera of vaccinated subjects can neutralize new variants and thus guides in the design of the vaccine.
  • Example 6 Liquid chromatography with tandem mass spectrometry (LC-MS-MS)analysis of inactivated SARS-CoV-2
  • the bands could be clearly attributed to the three main viral proteins (Spike-protein, Membrane- protein, Nucleoprotein) as well as to background proteins from the host system (see Figure 10).
  • SARS-CoV-2 ORF9b and the replicase polyprotein could also be detected, but these proteins were probably not well resolved on the gel due to their size.
  • the separation pattern on the gel was very similar for both samples with the exception of a host protein band (band 2.3), a slightly different S-protein pattern (bands 2.10-2.13), and an expected strong band of serum albumin in one of the samples (sample 2). Additionally, a number of typical lab contaminants of human origin (e.g. keratins) were detected in the background of both samples.
  • the processing of the Spike-protein (from full length to S 1, S2, and S2’) is difficult to resolve with the applied methodology but is most likely represented by the pattern in bands 9-13 in both samples.
  • BPU can react with up to 9 different amino acids (C,H,M,D,E,Y,K,E,S) depending on actual pH. In their studies higher conversions within the relevant pH range 7 to 9 were observed for Cysteine (>95%), Histidine (15-25%) and Methionine (36%) residues. The conversion rates for Aspartic Acid, Glutamic acid and Tyrosine were much lower in the range of approximately 3-15%. It was shown that disulfide groups in Cystine residues do not react. In BPL-inactivated SARS-CoV-2 particles, BPL modifications could be detected (mainly in the form of +72 Da) but at a low abundance.
  • M234 of the nucleoprotein has to be interpreted carefully, as that particular peptide sequence has problematic features which likely make the estimation for this particular peptide less accurate and reliable as compared to the other sites.
  • the FragPipe search revealed two other modifications (most likely acetaldehyde and acetylation) to occur in around 10% of the spectra. These modifications represent most likely artifacts introduced during gel staining and sample preparation, as they also occur on contaminant proteins.
  • Example 7 Further liquid chromatography with tandem mass spectrometry (LC-MSMS) analysis of inactivated SARS-CoV-2 Methodology:
  • the Coomassie-stained bands corresponding to spike protein (based on previous analysis) were subjected to in-gel digestion with trypsin or chymotrypsin or to acid hydrolysis. Trypsin digests were performed twice, once with and once without previous PNGase F (peptide:N-glycosidase F) digestion, to identify peptides masked by glycosylation.
  • PNGase F peptide:N-glycosidase F
  • Digested peptides were analysed by LC-MSMS essentially as described in Example 6.
  • the resulting peptides were analyzed with nano-liquid chromatography coupled to a high-resolution accurate mass spectrometer.
  • Peptides were identified from raw spectra using the MaxQuant software package and the UniProt reference databases for SARS-CoV-2 and Chlorocebus sabaeus in combination with a database of common lab contaminants.
  • BPL b-propiolactone
  • the degree of modification was globally estimated as the percentage of BPL-modified spectra identified, and on site-level by calculating site occupancies from the ratio of modified to unmodified peptides for each peptide/site separately.
  • Example 6 this confirms that the percentage of BPL-modified peptides is low regardless of the digestion method, e.g. less than 7%, 2 to 7% or around 2-5% on average.
  • BPL-modifications were detected at the positions in the spike (S) and membrane (M) proteins shown in Table 9 below.
  • the mean percentage occupancy at each site, as described in Example 6 above, is also shown in Table 9. Table 9. BPL-modified sites identified in S protein and their occupancy
  • the present invention provides:
  • a SARS-CoV-2 vaccine comprising an optimally (e.g. wherein the native surface of the S- protein is preserved) inactivated SARS-CoV-2 particle, wherein the SARS-CoV-2 particle is able to seroconvert a subject that is administered the SARS-CoV-2 vaccine with at least a 70% probability.
  • the SARS-CoV-2 vaccine of aspect Al wherein the SARS-CoV-2 particle is able to serocovert the subject that is administered the SARS-CoV-2 vaccine with at least a 80%, 85%, 90%, or 95% probability.
  • SEQ ID NO: 1 (see Genbank NC_045512.2), or a variant nucleic acid sequence that is at least 85% identical to SEQ ID NO: 1 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 9 (see NCBI MT066156), or a variant nucleic acid sequence that is at least 85% identical to SEQ ID NO: 1 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 18 (see NCBI MW598408). or a variant nucleic acid sequence that is at least 85% identical to SEQ ID NO: 18 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 20 (see NCBI MW520923). or a variant nucleic acid sequence that is at least 85% identical to SEQ ID NO: 20 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 22 (see NCBI MW422256). or a variant nucleic acid sequence that is at least 85% identical to SEQ ID NO: 22 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 24 (see NCBI MW493681). or a variant nucleic acid sequence that is at least 85% identical to SEQ ID NO: 24 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 26 (see NCBI MW306426). or a variant nucleic acid sequence that is at least 85% identical to SEQ ID NO: 26 and able to pack a virulent SARS-CoV-2.
  • A4 The vaccine of any one of aspects A1-A3, wherein the SARS-CoV-2 particle has an S protein as defined by the amino acid sequence
  • SEQ ID NO: 3 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 3 and able to pack a virulent SARS-CoV-2; or • SEQ ID NO: 11, or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 11 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 19 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 19 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 21 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 21 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 23 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 23 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 25 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 25 and able to pack a virulent SARS-CoV-2; or
  • SEQ ID NO: 27 or a variant amino acid sequence that is at least 95% identical to SEQ ID NO: 27 and able to pack a virulent SARS-CoV-2.
  • A4.1 The vaccine of any one of aspects A1-A4, comprising a second SARS-CoV-2 particle that is different to the first SARS-CoV-2 particle and is selected from the group consisting of SEQ ID NO: 1, 9, 18, 20, 22, 24 and 26.
  • A5. The vaccine of any one of aspects A1-A4 and A4.1, wherein the SARS-CoV-2 is inactivated by chemical inactivation, thermal inactivation, pH inactivation, or UV inactivation or radiation inactivation.
  • A6 The vaccine of aspect A5, wherein the chemical inactivation comprises contacting the SARS- CoV-2 particles with a chemical inactivation agent for longer than is required to completely inactivate the SARS-CoV-2 as measured by plaque assay or as measured by plaque assay plus one day.
  • the vaccine of aspect A6, wherein the chemical inactivation comprises contacting the SARS- CoV-2 particle with formaldehyde and/or beta-propiolactone, preferably beta-propiolactone.
  • the vaccine of aspect A7, wherein the formaldehyde and/or beta-propiolactone inactivation comprises contacting the SARS-CoV-2 particle with formaldehyde and/or beta-propiolactone for between 2-10 days.
  • A9 The vaccine of any one of aspects A5-A8, wherein the chemical activation is performed at about 4°C or about 22°C.
  • A10. The vaccine of any one of aspects A1-A9, further comprising an adjuvant.
  • the vaccine of aspect A 10 wherein the adjuvant is an aluminium salt adjuvant, optionally in combination with AS01, AS03, MF59, imiquimod and/or CpG 1018.
  • A13 The vaccine of any one of A10-A12, wherein the vaccine comprises or further comprises an adjuvant comprising a peptide and a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN).
  • I-ODN immunostimulatory oligodeoxynucleic acid molecule
  • A14 The vaccine of aspect A13, wherein the peptide comprises the sequence KLKL5KLK (SEQ ID NO: 5) and the I-ODN comprises oligo-d(IC)i3 (SEQ ID NO: 6).
  • A15 The vaccine of any one of aspects A1-A14, further comprising one or more pharmaceutically acceptable excipient.
  • kits comprising a SARS-CoV-2 vaccine of any one of aspects A1-A15.
  • kit of aspect B 1, further comprising a second vaccine.
  • kits of aspect B2 wherein the second vaccine is another SARS-CoV-2 virus vaccine (e.g. of another technology such as mRNA or adenovirus vectored), an influenza virus vaccine or a Chikungunya virus vaccine.
  • another SARS-CoV-2 virus vaccine e.g. of another technology such as mRNA or adenovirus vectored
  • influenza virus vaccine e.g. of a virus vaccine
  • Chikungunya virus vaccine e.g. of another technology such as mRNA or adenovirus vectored
  • a method comprising administering a first dose of a therapeutically effective amount of the SARS-CoV-2 vaccine of any one of aspects A1-A15 to a subject in need thereof.
  • a method of producing a SARS-CoV-2 vaccine comprising
  • D4 The method of any one of aspects D1-D3, further comprising (v) dialyzing the inactivated SARS-CoV-2 of (iv), thereby producing a dialyzed SARS-CoV-2.
  • D5. The method of aspect D4, further comprising a step (vi), comprising filtering the dialyzed SARS-CoV-2 of (v).
  • D6 The method of any one of aspects D1-D5, wherein the inactivating is by chemical inactivation, thermal inactivation, pH inactivation, or UV inactivation.
  • D12 The method of any one of aspects Dl-Dl 1, wherein the chemical inactivation is performed with BPL, preferably at a concentration of 300 to 700ppm, more preferably 500ppm and inactivated for about 1 to 48h, preferably 20 to 28h, most preferred 24 hours ⁇ 2 hours (such as also ⁇ 1 hour or ⁇ 0.5 hour) at 2°C to 8°C.
  • a pharmaceutical composition for use in the treatment and prevention of a SARS-CoV-2 infection wherein said pharmaceutical composition comprises the optimally inactivated SARS-CoV-2 vaccine of any one of aspects A1-A15.
  • aspect F3 The use of aspect F2, wherein the inactivated SARS-CoV-2 vaccine is administered in a second dose of a therapeutically effective amount to the subject.
  • aspect F3 wherein the second dose of the inactivated SARS-CoV-2 vaccine is administered about 7 days after the first dose of the SARS-CoV-2 vaccine.
  • F8 The use of any one of aspects F1-F6, wherein the administering results in production of SARS-CoV-2 neutralizing antibodies.
  • a SARS-CoV-2 vaccine comprising an effective amount of antigen, wherein said effective amount is able to seroconvert a subject that is administered the SARS-CoV-2 vaccine with at least a 70% probability.
  • the SARS-CoV-2 vaccine according to aspect Gl wherein said effective amount is able to seroconvert a subject that is administered the SARS-CoV-2 vaccine with at least 80%, 85%, 90%, or 95% probability.
  • SARS-CoV-2 vaccine according to aspect Gl or G2, wherein said effective amount is between about 1 to 100 AU/dose, preferably between about 2 to 75 AU/dose, preferably between about 3 and 60 AU/dose, more preferably between about 3 and 55 AU/dose, more preferably between about 3 and 53 AU/dose.
  • SARS-CoV-2 vaccine according to aspect G3, where said effective amount is determined by EUISA wherein the antigen units (AU) correspond to ACE-2 binding capacity of the spike protein used as a standard.
  • a SARS-CoV-2 vaccine comprising an inactivated SARS-CoV-2 particle; wherein a native surface conformation of the SARS-CoV-2 particle is preserved in the vaccine, such that the vaccine is capable of generating neutralizing antibodies against native SARS-CoV-2 particles in a human subject.
  • a SARS-CoV-2 vaccine according to aspect HI wherein viral RNA in the inactivated SARS- CoV-2 particle is replication-deficient.
  • a SARS-CoV-2 vaccine according to aspect HI or H2 wherein viral RNA in the inactivated SARS-CoV-2 particle (i) is alkylated and/or acylated (ii) comprises one or more modified purine (preferably guanine) residues or strand breaks and/or (iii) is cross-linked with one or more viral proteins.
  • a SARS-CoV-2 vaccine according to any preceding aspect wherein the inactivated SARS-CoV- 2 particle is a beta-propiolactone-inactivated SARS-CoV-2 particle, preferably at a concentration of 300 to 700ppm, more preferably 500ppm and inactivated for about 1 to 48h, preferably 20 to 28h, most preferred 24 hours ⁇ 2 hours (such as also ⁇ 1 hour or ⁇ 0.5 hour) at 2°C to 8°C, followed optionally by a hydrolyzation for 2.5 hours ⁇ 0.5 hours at 35°C to 39°C, preferably around 37°C.
  • the inactivated SARS-CoV- 2 particle is a beta-propiolactone-inactivated SARS-CoV-2 particle, preferably at a concentration of 300 to 700ppm, more preferably 500ppm and inactivated for about 1 to 48h, preferably 20 to 28h, most preferred 24 hours ⁇ 2 hours (such as also ⁇ 1 hour or ⁇ 0.5 hour) at 2°C to 8°
  • UV ultraviolet
  • a SARS-CoV-2 vaccine according to any preceding aspect wherein surface proteins in the inactivated SARS-CoV-2 particle comprise reduced modifications compared to viral RNA in the inactivated SARS-CoV-2 particle, preferably wherein surface proteins comprise a reduced proportion of modified residues compared to viral RNA in the inactivated SARS-CoV-2 particle; said modifications being with respect to a native SARS-CoV-2 particles, preferably wherein said modifications comprise alkylated and/or acylated nucleotide or amino acid residues.
  • a SARS-CoV-2 vaccine according to any preceding aspect, wherein the inactivated SARS-CoV- 2 particle comprises a native conformation of (i) spike (S) protein; (ii) nucleocapsid (N) protein; (iii) membrane (M) glycoprotein; and/or (iv) envelope (E) protein; preferably wherein the inactivated SARS-CoV-2 particle comprises a native conformation spike (S) protein.
  • a SARS-CoV-2 vaccine according to any preceding aspect wherein infectivity of mammalian cells by the inactivated SARS-CoV-2 particle is reduced by at least 99%, 99.99% or 99.9999% compared a native SARS-CoV-2 particle, or wherein infectivity of mammalian cells by the inactivated A SARS-CoV-2 particle is undetectable.
  • a SARS-CoV-2 vaccine according to any preceding aspect further comprising one or more pharmaceutically acceptable excipients, such as e.g., human serum albumin (HSA). H10.
  • a SARS-CoV-2 vaccine according to any preceding aspect further comprising an adjuvant.
  • Thl response-directing adjuvant comprises 3-0-desacyl-4'-monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a-tocopherol, a cationic peptide, a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN) and/or imiquimod.
  • MPL 3-0-desacyl-4'-monophosphoryl lipid A
  • saponin QS-21 a CpG-containing oligodeoxynucleotide
  • CpG ODN CpG-containing oligodeoxynucleotide
  • squalene DL-a-tocopherol
  • a cationic peptide a deoxyinosine-containing immunostimulatory oligodeoxynucleic acid molecule (I-ODN) and/or imi
  • a liposomal preparation comprising 3-0-desacyl-4'-monophosphoryl lipid A (MPL) and saponin QS-21, preferably Adjuvant System 01;
  • a CpG ODN comprising the sequence 5’ TGACTGTGAACGTTCGAGATGA 3’ (SEQ ID NO:4), preferably CpG 1018;
  • H16 The SARS-CoV-2 vaccine according to any preceding aspect, wherein the vaccine is able to seroconvert a subject that is administered the SARS-CoV-2 vaccine with at least a 70% probability.
  • H17 The SARS-CoV-2 vaccine according to aspect H16, wherein the SARS-CoV-2 vaccine is able to seroconvert the subject that is administered the SARS-CoV-2 vaccine with at least an 80%, 85%, 90%, or 95% probability.
  • the SARS-CoV-2 vaccine according to any one of the preceding aspects, wherein the SARS- CoV-2 particle comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 9; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 9; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2.
  • RNA sequence and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 9; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least
  • the SARS-CoV-2 vaccine according to any one of the preceding aspects, wherein the said vaccine comprises an additional SARS-CoV-2 particle that comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 18; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 18; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2.
  • an additional SARS-CoV-2 particle that comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 18; or (ii) having at least
  • the SARS-CoV-2 vaccine according to any one of the preceding aspects, wherein the said vaccine comprises an additional SARS-CoV-2 particle that comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 22; or (ii) having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 22; preferably wherein a native (non-inactivated) SARS-CoV-2 particle comprising the RNA sequence is able to pack a virulent SARS-CoV-2.
  • an additional SARS-CoV-2 particle that comprises an RNA sequence (and/or fragments thereof, optionally comprising modified (preferably alkylated or acylated) nucleotide residues) corresponding to a DNA sequence (i) as defined by SEQ ID NO: 22; or (ii) having at least
  • SARS-CoV-2 vaccine according to any preceding aspect, wherein, upon administration to a human subject, the vaccine (i) does not induce antibody-dependent enhancement (ADE) of SARS-CoV-2-associated disease (COVID-19); and/or (ii) does not induce immunopathology in the subject.
  • ADE antibody-dependent enhancement
  • COVID-19 SARS-CoV-2-associated disease
  • COVID-19 SARS-CoV-2-associated disease
  • prophylactically or therapeutically effective amount of the SARS-CoV-2 vaccine per dose is defined as about 1 to 100 AU/dose, preferably between about 2 to 75 AU/dose, preferably between about 3 and 60 AU/dose, more preferably between about 3 and 55 AU/dose, more preferably between about 3 and 53 AU/dose, as assessed by EUISA, even more preferably between about 3 and 40 AU/dose such as e.g. 40 AU/dose.
  • prophylactically or therapeutically effective amount per dose of the SARS-CoV-2 vaccine is defined as about 0.05 to 50 pg total protein, about 0.1 to 25 pg, about 0.25 to 12.5 pg, preferably about 0.5 to 5 pg total protein, as measured by (p)BCA.
  • H29 The method according to any one of aspects H22 to H28, wherein the administering results in production of SARS-CoV-2 neutralizing antibodies.
  • H30 A method of producing a SARS-CoV-2 vaccine, comprising:
  • the inactivation step comprises (i) alkylating and/or acylating viral RNA (ii) modifying purine (preferably guanine) residues or introducing strand breaks into viral RNA and/or (iii) cross-linking viral RNA with one or more viral proteins.
  • a concentration of beta-propiolactone in the inactivation step is 0.01 to 1% by weight, preferably 0.05 to 0.5% by weight, more preferably about 0.1% by weight.
  • H37 The method according to aspect H35 or H36, wherein the native SARS-CoV-2 particles are contacted with beta-propiolactone for at least 5 hours, at least 10 hours, at least 24 hour or at least 4 days.
  • step (a) comprises one or more of the following steps:
  • the inactivation step comprises contacting a liquid composition comprising native SARS-CoV-2 particles with a chemical viral inactivating agent in a container, mixing the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles under conditions of laminar flow but not turbulent flow, and incubating the chemical viral inactivating agent and the liquid composition comprising SARS-CoV-2 particles for a time sufficient to inactivate the viral particles.
  • H49 The method according to any one of aspects H30 or H33 to H48, further comprising purifying the inactivated SARS-CoV-2 particles by one or more methods selected from (i) batch chromatography and/or (ii) sucrose density gradient centrifugation.
  • step (c) comprises combining the inactivated SARS-CoV-2 particles with an adjuvant.
  • the adjuvant comprises 3-0-desacyl-4'- monophosphoryl lipid A (MPL), saponin QS-21, a CpG-containing oligodeoxynucleotide (CpG ODN), squalene, DL-a-tocopherol and/or imiquimod.
  • MPL 3-0-desacyl-4'- monophosphoryl lipid A
  • saponin QS-21 saponin QS-21
  • CpG ODN CpG-containing oligodeoxynucleotide
  • squalene DL-a-tocopherol and/or imiquimod.
  • a SARS-CoV-2 vaccine obtained or obtainable by the method of any one of aspects H30 or H33 to H52.
  • SARS-CoV-2 vaccine of any one of aspects HI to H22 or H53 for the treatment or prevention of a SARS-CoV-2 infection in a subject.
  • a pharmaceutical composition for use in the prevention or treatment of a SARS-CoV-2 infection in a subject wherein said pharmaceutical composition is the inactivated SARS-CoV-2 vaccine as defined in any one of aspects HI to H22 or H53, optionally in combination with one or more pharmaceutically acceptable excipients and/or adjuvants.
  • H56 The SARS-CoV-2 vaccine as defined in any one of aspects HI to H22 or H53 for use as a medicament.
  • H57 A vaccine, method, use or pharmaceutical composition according to any preceding aspect, wherein the subject is (i) an elderly subject, preferably a subject over 65, over 70 or over 80 years of age; (ii) an immunocompromised subject; or (iii) a pregnant subject.
  • ADE antibody- dependent enhancement
  • COVID-19 SARS-CoV-2-associated disease
  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate Wuhan-Hu-1, complete genome (GenBank: MN908947; Wu, F., et al. A new coronavirus associated with human respiratory disease in China (2020) Nature 579:265-269)
  • AAAG AAAAAG CTT G ATG G CTTT ATG G GTAG AATT CG ATCTGTCT ATCCAGTT G CGTCACCAAATG AAT G CAACCAAAT GT
  • AAAA AT CT CT AT G AC AAACTT GTTT CAAG CTTTTT G G AAAT G AAG AG TG AAA AG C AAG TTG AAC AAAAG ATCG CTG AG AT
  • CT C AGTTTT G CAACAACT CAG AGT AG AAT CAT CAT CT AAATT GTG G G CT CAAT GTGTCCAGTT ACACAAT G ACATT CT CTT A
  • CT ACAAG CT G GTAAT G CAACAG AAGT G CCTG CCAATT CAACT GT ATT AT CTTT CTGTG CTTTT G CTGTAG ATG CTG CT AAA
  • CAG G CAAT AACAGTTAC ACCG G AAG CCAAT ATG GAT CAAG AAT CCTTT G GTG GTG CATCGT GTTGTCTGT ACT G CCGTTG C
  • CT AACAAT GTTG CTTTT CAAACT GT CAAACCCG GTAATTTT AACAAAG ACTT CT AT G ACTTT G CTGTGTCT AAG G GTTTCTTT
  • AAATT GTT G ACACT GTG AGTG CTTT G GTTTAT GAT AAT AAG CTT AAAG CACAT AAAG ACAAAT CAG CT CAAT G CTTT AAAA
  • AAACAT G ATG GTG G CAGTTTGTAT GT AAAT AAACAT G CATT CC ACACACCAG CTTTT GAT AAAAGT G CTTTT GTT AATTT AAAACAATT
  • GAGTAAATTTCCCCTT AAATT AAGGGGTACTGCTGTT ATGTCTTT AAAAGAAGGTCAAATCAATG AT ATG ATTTT ATCTCTT
  • CTACTG C AACG AT ACCG AT AC AAG CCT CACT CCCTTT CG G ATG G CTT ATT GTTGGCGTTG CACTT CTT G CTGTTTTT CAG AG
  • Severe acute respiratory syndrome coronavirus 2 orflab polyprotein of isolate Wuhan-Hu-1 (GenBank: QHD43415)
  • Severe acute respiratory syndrome coronavirus 2 surface glycoprotein GenBank: QHD434136
  • AAAG GTTTAT ACCTT CCC AG GT AACAAACCAACCAACTTT CG ATCT CTT GTAG ATCTGTTCT CT AAACG AACTTT AAAA
  • AAAG AAAAAG CTT G ATG G CTTT ATG G GTAG AATT CG ATCTGTCT ATCCAGTT G CGTCACCAAATG AAT G CAACCAAAT GT
  • CAG G CAAT AACAGTTAC ACCG G AAG CCAAT ATG G AT CAAG AAT CCTTT G GTG GTG CATCGT GTTGTCTGT ACT G CCGTTG C
  • CT AACAAT GTTG CTTTT CAAACT GT CAAACCCG GTAATTTT AACAAAG ACTT CT AT G ACTTT G CTGTGTCT AAG G GTTTCTTT
  • AAATT GTT G ACACT GTG AGTG CTTT G GTTTAT GAT AAT AAG CTT AAAG CACAT AAAG ACAAAT CAG CT CAAT G CTTT AAAA
  • AAACAT G ATG GTG G CAGTTTGTAT GT AAAT AAACAT G CATT CC ACACACCAG CTTTT GAT AAAAGT G CTTTT GTT AATTT AAAACAATT
  • GAGTAAATTTCCCCTT AAATT AAGGGGTACTGCTGTT ATGTCTTT AAAAGAAGGTCAAATCAATG AT ATG ATTTT ATCTCTT

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Abstract

L'invention concerne des vaccins contre le SARS-CoV-2 et des compositions et procédés de production et d'administration desdits vaccins à des sujets en ayant besoin.
EP21716442.5A 2020-04-06 2021-04-06 Vaccin à virus sars-cov-2 inactivé Pending EP3955959A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
EP20168324 2020-04-06
EP20202118 2020-10-15
EP20211853 2020-12-04
EP21154647 2021-02-01
PCT/US2021/020313 WO2021178318A1 (fr) 2020-03-01 2021-03-01 Vaccins à coronavirus comprenant un agoniste de tlr9
EP21160913 2021-03-05
PCT/EP2021/058974 WO2021204825A2 (fr) 2020-04-06 2021-04-06 Vaccin à virus sars-cov-2 inactivé

Publications (1)

Publication Number Publication Date
EP3955959A2 true EP3955959A2 (fr) 2022-02-23

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EP21716442.5A Pending EP3955959A2 (fr) 2020-04-06 2021-04-06 Vaccin à virus sars-cov-2 inactivé

Country Status (14)

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US (1) US20240293531A1 (fr)
EP (1) EP3955959A2 (fr)
JP (1) JP2023520521A (fr)
KR (1) KR20220164500A (fr)
CN (1) CN115768469A (fr)
AU (1) AU2021253605A1 (fr)
BR (1) BR112022020100A2 (fr)
CA (1) CA3168784A1 (fr)
CL (1) CL2022002365A1 (fr)
CO (1) CO2022013715A2 (fr)
EC (1) ECSP22072590A (fr)
IL (1) IL296072A (fr)
MX (1) MX2022012447A (fr)
ZA (1) ZA202209826B (fr)

Also Published As

Publication number Publication date
IL296072A (en) 2022-11-01
KR20220164500A (ko) 2022-12-13
ZA202209826B (en) 2023-05-31
AU2021253605A1 (en) 2022-10-06
US20240293531A1 (en) 2024-09-05
MX2022012447A (es) 2022-10-27
JP2023520521A (ja) 2023-05-17
BR112022020100A2 (pt) 2022-11-29
ECSP22072590A (es) 2022-10-31
CL2022002365A1 (es) 2023-02-03
CO2022013715A2 (es) 2022-12-30
CN115768469A (zh) 2023-03-07
CA3168784A1 (fr) 2021-10-14

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