WO2023150838A1 - Coronavirus vaccination regimen - Google Patents

Coronavirus vaccination regimen Download PDF

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Publication number
WO2023150838A1
WO2023150838A1 PCT/AU2023/050093 AU2023050093W WO2023150838A1 WO 2023150838 A1 WO2023150838 A1 WO 2023150838A1 AU 2023050093 W AU2023050093 W AU 2023050093W WO 2023150838 A1 WO2023150838 A1 WO 2023150838A1
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coronavirus
spike
rbd
antigen
vaccine
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PCT/AU2023/050093
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French (fr)
Inventor
Dale Ian GODFREY
Georgia DELIYANNIS
Nicholas Anthony GHERARDIN
David Charles Jackson
Damian Francis John Purcell
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The University Of Melbourne
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Priority claimed from AU2022900288A external-priority patent/AU2022900288A0/en
Application filed by The University Of Melbourne filed Critical The University Of Melbourne
Publication of WO2023150838A1 publication Critical patent/WO2023150838A1/en

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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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—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
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to novel prime-boost regimens for immunisation against coronavirus infections.
  • a significant bottleneck in vaccine efficacy is the ability to induce a strong and effective immune response that is maintained for a period of time.
  • Conventional approaches to provide a vaccination regimen that produces a sufficiently strong, effective and/or long-lasting immune response and/or protection is to either: (i) administer the vaccine composition in increased dosages; or (ii) additionally administer one or more subsequent vaccinations (so called "boost” vaccinations) after the initial (so called “prime”) vaccination.
  • boost subsequent vaccinations
  • Coronaviruses constitute a group of phylogenetically diverse enveloped viruses that encode the largest plus strand RNA genomes and replicate efficiently in most mammals.
  • Coronaviruses belong to the Coronaviridae family in the Nidovirales order, are minute in size (65-125 nm in diameter) and contain a single-stranded RNA as a nucleic material, size ranging from 26 to 32kbs in length.
  • the subgroups of coronaviruses family are alpha (a), beta ( ), gamma (y) and delta (5) coronavirus.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • ALI acute lung injury
  • ARDS acute respiratory distress syndrome
  • SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
  • ARDS acute respiratory distress syndrome
  • MERS-CoV Middle Eastern Respiratory Syndrome Coronavirus
  • COVID-19 manifestations range from mild to severe life-threatening with a substantial mortality rate.
  • the present invention is based on the surprising finding by the inventors that administration of a Spike protein receptor binding domain (RBD) based vaccine to a subject, who has been naturally infected by a coronavirus or who has received a prime and boost of a coronavirus whole Spike protein based vaccine, leads to an increase in humoral immunity including (a) enhancing the level of RBD specific antibodies against wildtype (WT) RBD and a number of RBD variants, (b) enhancing the neutralising activity as determined by inhibition of interaction between RBD antigens and human ACE2, and (c) enhancing the level of RBD neutralising antibodies against the WT RBD and variants, including the beta, delta and omicron variants (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
  • WT wildtype
  • RBD variants enhancing the neutralising activity as determined by inhibition of interaction between RBD antigens and human ACE2
  • the present invention provides a method for raising an immune response in a subject who has been exposed to a coronavirus whole Spike protein, for example in the form of a prior infection or a prior vaccination, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby raising an immune response in the subject.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention provides a method for increasing an immune response in a subject who has been exposed to a coronavirus whole Spike protein, for example in the form of a prior infection or a prior vaccination, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby increasing an immune response in the subject.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention provides a method for providing cross coronavirus variant immunity in a subject who has been exposed to a coronavirus whole Spike protein, for example in the form of a prior infection or a prior vaccination, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby providing cross coronavirus variant immunity in the subject.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the coronavirus Spike receptor binding domain (RBD) antigen may be a chimeric or fusion protein as described herein.
  • the nucleic acid encoding a coronavirus Spike RBD antigen may encode a chimeric or fusion protein as described herein.
  • the present invention provides a method for raising an immune response in a subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein as described herein or nucleic acid encoding a chimeric or fusion protein as described herein, or a pharmaceutical composition as described herein, thereby raising an immune response in the subject.
  • the present invention provides a method for increasing an immune response in a subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein as described herein or nucleic acid encoding a chimeric or fusion protein as described herein, or a pharmaceutical composition as described herein, thereby increasing an immune response in the subject.
  • the present invention provides a method for immunizing a subject against a coronavirus infection, the method comprising a step of administering to a subject a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, wherein the subject has been exposed to a coronavirus whole Spike protein, thereby immunizing a subject against a coronavirus infection.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention provides a method for immunizing a subject against a coronavirus infection, the method comprising the steps of:
  • the present invention provides a method of inducing, or increasing, a humoral immune response to a coronavirus in a subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby inducing, or increasing, a humoral immune response to a coronavirus in the subject.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention provides a method for reducing or minimising the severity of a symptom associated with an infection with coronavirus, comprising:
  • a vaccine comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen
  • a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen.
  • the present invention provides a method for reducing or minimising the severity of a symptom associated with an infection with coronavirus in subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby reducing or minimising the severity of a symptom associated with an infection with coronavirus.
  • the symptoms of a coronavirus infection are selected from the group consisting of fever, dry cough, tiredness, aches and pains, sore throat, diarrhoea, nausea and vomiting, conjunctivitis, headache, loss of taste or smell, a rash on skin, or discolouration of fingers or toes, difficulty breathing or shortness of breath, chest pain or pressure, and loss of speech or movement.
  • a method of the invention further comprises a step of identifying a subject at risk of coronavirus infection (for example at risk of infection with any coronavirus described herein, including SARS-CoV-2 or any variant thereof such as those described herein) or who has been exposed to a whole Spike protein through a naturally acquired infection of a coronavirus or by immunisation with a coronavirus whole Spike based vaccine that is either a protein based vaccine that comprises a whole Spike protein or a nucleic acid (e.g. DNA, RNA, preferably mRNA) based vaccine that encodes a whole Spike protein.
  • a subject at risk of coronavirus infection for example at risk of infection with any coronavirus described herein, including SARS-CoV-2 or any variant thereof such as those described herein
  • a coronavirus whole Spike based vaccine that is either a protein based vaccine that comprises a whole Spike protein or a nucleic acid (e.g. DNA, RNA, preferably
  • the present invention provides for use of a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen in the preparation of a medicament or vaccine for treating and/or preventing (a) a disease associated with, or caused by, a coronavirus, or (b) a coronavirus infection in a subject in need thereof who has been exposed to a coronavirus whole Spike protein.
  • the invention also provides for use of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen for inhibiting or reducing the amount of coronavirus particles in a tissue or organ in a subject who has been exposed to a coronavirus whole Spike protein.
  • the tissue or organ may be all or part of any tissue or organ described herein, or that is known to have detectable coronavirus particles.
  • the tissue or organ may be all, or part of, the upper respiratory tract (URT) or lower respiratory tract (LRT).
  • An inhibition or reduction in the amount of coronavirus particles in a tissue or organ may be determined by any means described herein, and may involve determining the amount of coronavirus particles in a sample of the tissue or organ or a bodily fluid that has originated from or is in contact with the tissue or organ.
  • the invention provides a method of inhibiting or reducing the amount of coronavirus particles in a tissue or organ of a subject (e.g. upper respiratory tract), the method comprising administering a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby inhibiting or reducing the amount of coronavirus particles in the tissue or organ of the subject (e.g. upper respiratory tract).
  • a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the invention provides a method of inhibiting, delaying or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of a subject, the method comprising administering a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby inhibiting, delaying or reducing the progression of the coronavirus particle from the upper respiratory tract to the lungs of the subject.
  • the invention further provides for use of a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen in the preparation of a medicament for inhibiting, delaying or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of a subject.
  • the invention provides for use of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen for inhibiting, delaying or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of a subject.
  • a subject may have been, or still be, exposed to a whole Spike protein through a naturally acquired infection of a coronavirus, or by immunisation with a vaccine comprising a coronavirus whole Spike antigen that is either a protein based vaccine that comprises a whole Spike protein or a nucleic acid (e.g., DNA or RNA, preferably mRNA) based vaccine that encodes a whole Spike antigen.
  • a protein based vaccine that comprises a whole Spike protein or a nucleic acid (e.g., DNA or RNA, preferably mRNA) based vaccine that encodes a whole Spike antigen.
  • a subject exposed to a whole Spike protein as described herein has been exposed by immunisation with a vaccine comprising a coronavirus whole Spike antigen that is either a protein based vaccine that comprises a whole Spike protein or a nucleic acid (e.g., DNA or RNA, preferably mRNA) based vaccine that encodes a whole Spike protein.
  • the nucleic acid that encodes a whole spike protein may be provided in a vector (e.g. a viral vector, such as a chimp- or human- adenoviral vector or adenovirus-associated virus (e.g. recombinant replication-incompetent adenovirus type 26 vectors (Ad26)) or a lipid nanoparticle formulation.
  • a subject has received a protein or nucleic acid (e.g. DNA or RNA) based vaccine (e.g. where the vaccine comprises an antigen that is protein or encodes for an antigen, respectively) that comprises (or consists of), or encodes for, a coronavirus whole Spike protein
  • the subject has typically been immunised with at least two doses of a vaccine.
  • a prime e.g. first dose
  • boost immunisation e.g. second or further dose
  • the prime and boost immunisation may be with the same vaccine (e.g. same vaccine platform, for example as shown in Table 2) or different vaccines (e.g. different vaccine platform, for example as shown in Table 2).
  • the subject may have received a first, or prime, dose or immunisation with a vaccine comprising a coronavirus whole Spike protein antigen and a subsequent second, or boost, dose or immunisation with a vaccine comprising a coronavirus whole Spike protein antigen, where the whole Spike protein antigen in the first or prime immunisation has a different amino acid sequence to the whole Spike protein antigen in the second or boost immunisation.
  • the subject may have received a first, or prime, dose or immunisation with a vaccine comprising a coronavirus whole Spike protein antigen and a subsequent second, or boost, dose or immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid (e.g. DNA or RNA).
  • a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid (e.g. DNA or RNA) and a subsequent second, or boost, dose or immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid (e.g. DNA or RNA).
  • the subject may have received a first, or prime, immunisation with a coronavirus whole Spike RNA based vaccine and a subsequent second, or boost, immunisation with a coronavirus whole Spike protein based vaccine.
  • the subject may have received a first, or prime, immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in a viral vector and a subsequent second, or boost, immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in a viral vector.
  • the subject may have received a first, or prime, immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in a viral vector and a subsequent second, or boost, immunisation with a coronavirus whole Spike RNA based vaccine (i.e. a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in the form of mRNA).
  • a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in the form of mRNA
  • the subject may have received a first, or prime, immunisation with a vaccine comprising a coronavirus whole Spike antigen in the form of an inactivated virus and a subsequent second, or boost, immunisation with a vaccine comprising a coronavirus whole Spike antigen in the form of an inactivated virus.
  • the subject may have received any combination of vaccine as described herein with one, two, three, four or more further boosters, i.e a third, fourth, fifth, sixth immunisation, with a vaccine comprising a coronavirus whole Spike antigen, coronavirus whole Spike antigen-encoding nucleic acid, including nucleic acid in a viral vector, or a coronavirus whole Spike antigen in the form of an inactivated virus.
  • a vaccine comprising a coronavirus whole Spike antigen, coronavirus whole Spike antigen-encoding nucleic acid, including nucleic acid in a viral vector, or a coronavirus whole Spike antigen in the form of an inactivated virus.
  • the vaccines used may be the same or different.
  • the vaccines used for the prime and boost may be the same or different.
  • the vaccines may contain the same or different adjuvants, may contain the same or different amino acid (for protein based) or nucleotide (for RNA based) sequences provided.
  • the vaccines may comprise, or encode for, the same or different Spike variants, or be derived from the same or different coronavirus (preferably SARS-CoV-2) strains.
  • an antigen includes both a protein antigen or a nucleic acid encoding a protein antigen.
  • the subject may have been, or still be, exposed to any variant or strain of a coronavirus, preferably any variant or strain of a coronavirus described herein.
  • the subject may have had, or still have, a coronavirus infection where the coronavirus is from any of the genera Alpha-coronavirus, Betacoronavirus, Gamma-coronavirus or Delta-coronavirus.
  • the coronavirus is from one of the Alpha-coronavirus subgroup clusters 1 a and 1 b or one of the Betacoronavirus subgroup clusters 2a, 2b, 2c, and 2d.
  • the coronavirus may be any coronavirus that infects humans.
  • coronaviruses are SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1 , although the coronavirus may be any one as described herein or genotypic decedents thereof.
  • the coronavirus infection is an infection with SARS-CoV-2, for example any of the SARS-CoV-2 variants described herein including beta, delta and omicron (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
  • a subject who may have had, or still have, a coronavirus infection is determined by the presence of coronavirus-specific seroconversion (e.g. the presence of coronavirus-specific antibodies in serum).
  • the subject has significantly higher antibodies that bind and/or neutralise coronavirus (e.g. SARS-CoV-2) compared to uninfected and/or unvaccinated subjects.
  • the coronavirus-specific antibodies bind the Spike protein and are below a level that confers protection against coronavirus infection.
  • the coronavirus Spike receptor binding domain (RBD) based vaccine and/or the coronavirus whole Spike based vaccine may be formulated or adapted for administration subcutaneously, intramuscularly or via any other route described herein.
  • the method comprises administering the coronavirus Spike receptor binding domain (RBD) based vaccine and/or the coronavirus whole Spike based vaccine subcutaneously, intramuscularly or via any other route described herein.
  • the prime (or first), boost (or second) and further doses or immunisations are administered by the same route.
  • the route of administration is intramuscular.
  • Any vaccine described herein may include an adjuvant.
  • the adjuvant may be any one known in the art or described herein.
  • any method reduces or prevents dissemination of the coronavirus from the upper respiratory tract to the lungs.
  • a method of the invention may be useful to treat or prevent a disease or condition associated with, or caused by, a coronavirus.
  • the disease is a respiratory disease.
  • the vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen may be anyone described herein.
  • the vaccine comprises, consists essentially of or consists of a chimeric or fusion protein as described herein or a nucleic acid that encodes a chimeric or fusion protein as described herein.
  • the present invention also provides a method for obtaining an antibody directed to a coronavirus, the method comprising performing a method of the invention as described herein, and subsequent to administering vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, obtaining a sample from the subject that contains antibodies directed to a coronavirus.
  • the sample is a sample of blood or blood-derived components such as plasma or serum.
  • the method further comprises purifying the antibodies from the subject, preferably purifying the antibodies directed to, or neutralising for, a coronavirus.
  • the present invention also provides an antibody preparation comprising an antibody directed to a coronavirus, wherein the antibody preparation is obtained or obtainable from a subject on whom a method of the invention as described herein has been performed.
  • FIG. 1 Total ancestral or wildtype (WT) SARS-Cov-2 strain RBD specific antibodies in mice following primary and secondary boost of different antigen combinations.
  • ELISA titres are expressed as the reciprocal of the antibody dilution (Iog10) giving an absorbance of 0.3. This represents at least five times the background level of binding.
  • Figure 2 Total anti-RBD antibody titres specific for WT, Beta and Delta RBD monomers in mice following primary and secondary boost of different antigen combinations.
  • ELISA titres are expressed as the reciprocal of the antibody dilution (logio) giving an absorbance of 0.3. This represents at least five times the background level of binding.
  • FIG. 1 IgG antibody responses to WT, Beta and Delta RBD monomers in mice vaccinated with 2 doses of WT Spike and boosted with WT RBD-hFc, Beta RBD-hFc, WT-spike or Beta spike.
  • the multiplex bead-based assay was used to assess RBD binding activity of secondary (day 56) sera collected 5 weeks following the second immunisation (2°) and tertiary sera collected 16 days (day 86) following the third immunisation (3°) from groups of 5 mice immunised intramuscularly with: (A) WT-spike/ WT-spike/ WT RBD-hFc, (B) WT-spike/ WT-spike/Beta RBD-hFc, (C) WT-spike/WT- spike/WT-spike or (D) WT-spike/WT-spike/Beta spike. All vaccinations were administered in the presence of MF59®.
  • Antibody binding levels against the 3 RBD antigens were also assessed in day 35 plasma samples from 5 humans vaccinated on day 0 and 21 with Comirnaty (Pfizer mRNA) vaccine (E).
  • Half-maximal effective dilution (EDso) for each test sample against WT-RBD, Beta-RBD and Delta RBD are displayed. Bars depict geometric mean and geometric SD.
  • Figure 4 Binding of serum antibodies to RBD variants in a multiplex binding assay. Assessing RBD binding activity of secondary (day 56) sera collected 5 weeks following the second immunisation (2°) and tertiary sera collected 16 days (day 86) following the third immunisation (3°) from groups of 5 mice immunised intramuscularly with (A) WT-spike/ WT-spike/ WT RBD-hFc, (B) WT-spike/ WT- spike/Beta RBD-hFc (C) WT-spike/WT-spike/WT-spike or (D) WT-spike/WT-spike/Beta spike. All vaccinations were administered in the presence of MF59.
  • ED50 Half-maximal effective dilution
  • WT RBD and 8 RBD Variants including alpha (a), Beta (0), Gamma (y), Delta (5) and Kappa (K). Bars depict geometric mean and geometric SD.
  • FIG. 1 IgG antibody responses to nine RBD monomers in mice vaccinated with 2 doses of WT Spike and boosted with WT RBD-hFc, Beta RBD- hFc, WT-spike or Beta spike.
  • ED50 Half-maximal effective dilution
  • Figure 6 Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or Beta RBD-hFc + MF59®.
  • ID50 half-maximal inhibitory dilution
  • CPE cytopathic effect
  • Figure 7 Neutralising Ab responses against WT, Beta and Delta RBDs in sera of mice vaccinated with 2 doses of WT spike + MF59® and boosted with RBD- hFc + MF59® or whole spike protein + MF59®.
  • ID50 Half-maximal Inhibitory dilution
  • Figure 8 Neutralising Ab responses against 9 RBD antigens in sera of mice vaccinated with 2 doses of WT spike + MF59® and boosted with RBD-hFc + MF59® or whole spike protein + MF59®.
  • FIG. 9 Mutations in the SARS-CoV-2 spike protein and neutralization of SARS-CoV-2 variants by human convalescent plasma samples (hCov) and sera from mice vaccinated with RBD-mouse lgG1-Fc dimer (RBD). Spike coding mutations and (frequency) with >10 local (Victoria, Australia) cases, mutations in the RBD region are shown in red.
  • FIG. 10 Mutations in the Beta variant SARS-CoV-2 spike protein.
  • Figure 12 Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or WT RBD-hFc + MF59® or Beta RBD-hFc + MF59®, or Beta Spike + MF59®.
  • the three vaccinations were administered on days 0, 21 and 70 in the presence of MF59®.
  • the half-maximal inhibitory dilution (ID50) was calculated based on the reciprocal dilution of serum that completely prevented cytopathic effect (CPE) in 50% of the wells and was calculated by the Reed-Muench formula.
  • LOD limit of detection
  • MNS mouse non-immune serum
  • WT-Spike wild type spike
  • WT RBD-hFc wild type RBD-human Fc dimer
  • beta-RBD-hFc beta variant RBD-human Fc dimer
  • beta-spike beta variant spike.
  • the three vaccinations were administered on days 0, 21 and 70 in the presence of MF59®.
  • the half-maximal inhibitory dilution (ID50) was calculated based on the reciprocal dilution of serum that completely prevented cytopathic effect (CPE) in 50% of the wells and was calculated by the Reed-Muench formula.
  • LOD limit of detection
  • MNS mouse non-immune serum
  • WT-Spike wild type spike
  • WT RBD-hFc wild type RBD-human Fc dimer
  • beta-RBD- hFc beta variant RBD-human Fc dimer
  • beta-spike beta variant spike).
  • Figure 14 Neutralising Ab responses against SARS-CoV-2 variants in sera of mice vaccinated with 2 doses of WT spike + MF59® and boosted with RBD- hFc + MF59® or whole spike protein + MF59®.
  • Figure 15 Neutralising Ab responses against BANAL-52, BANAL-236 and GD-1 coronavirus types in sera of mice vaccinated with 2 doses of WT spike + MF59® and boosted with RBD-hFc + MF59® or whole spike protein + MF59®.
  • the present invention is based on the surprising finding by the inventors that administration of a Spike protein receptor binding domain (RBD) based vaccine to an individual/subject, who has been naturally infected by a coronavirus or who has received a prime and boost of a coronavirus whole Spike protein based vaccine, leads to a boost in humoral immunity including (a) enhancing the level of RBD specific antibodies against the ancestral or wildtype (WT) SARS-CoV-2 RBD and a number of RBD variants, (b) enhancing the neutralising activity as determined by inhibition of interaction between RBD antigens and human ACE2, and (c) enhancing the level of RBD neutralising antibodies against the WT RBD and the beta and delta variants.
  • WT ancestral or wildtype
  • RBD variants enhancing the neutralising activity as determined by inhibition of interaction between RBD antigens and human ACE2
  • enhancing the level of RBD neutralising antibodies against the WT RBD and the beta and delta variants enhancing the level of RBD neutralising antibodies against
  • coronavirus refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV as well as endemic coronaviruses such as HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1.
  • SARS-CoV-2 refers to the newly emerged coronavirus which was identified as the cause of the serious outbreak starting in Wuhan, China, and which has rapidly spread to other areas of the globe.
  • SARS-CoV-2 has also been known as 2019-nCoV and Wuhan coronavirus. It binds via the viral spike protein to human host cell receptor angiotensinconverting enzyme 2 (ACE2). The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for membrane fusion of the virus.
  • ACE2 human host cell receptor angiotensinconverting enzyme 2
  • the subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales International Committee on Taxonomy of Viruses.
  • This subfamily consists of four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus, on the basis of their phylogenetic relationships and genomic structures.
  • Subgroup clusters are labeled as 1 a and 1 b for the Alphacoronavirus and 2a, 2b, 2c, and 2d for the Betacoronavirus.
  • the alphacoronaviruses and betacoronaviruses infect only mammals.
  • the gammacoronaviruses and deltacoronaviruses infect birds, but some of them can also infect mammals.
  • Alphacoronaviruses and betacoronaviruses usually cause respiratory illness in humans and gastroenteritis in animals.
  • the three highly pathogenic viruses, SARS-CoV, MERS- CoV and SARS-CoV-2, cause severe respiratory syndrome in humans, and the other four human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1 ) induce only mild upper respiratory diseases in immunocompetent hosts, although some of them can cause severe infections in infants, young children and elderly individuals/subjects.
  • Alphacoronaviruses and betacoronaviruses can pose a heavy disease burden on livestock; these viruses include porcine transmissible gastroenteritis virus, porcine enteric diarrhoea virus (PEDV) and the recently emerged swine acute diarrhoea syndrome coronavirus (SADS-CoV).
  • porcine transmissible gastroenteritis virus porcine enteric diarrhoea virus (PEDV)
  • SADS-CoV porcine enteric diarrhoea virus
  • SADS-CoV swine acute diarrhoea syndrome coronavirus
  • the coronaviruses include antigenic groups I, II, and III.
  • Nonlimiting examples of coronaviruses include SARS coronavirus, MERS coronavirus, transmissible gastroenteritis virus (TGEV), human respiratory coronavirus, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, rabbit coronavirus, murine hepatitis virus, sialodacryoadenitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus, as well as any others described herein, and including those referred to in Cui, et al. Nature Reviews Microbiology volume 17, pages181-192 (2019), and Shereen et al. Journal of Advanced Research, Volume 24, July 2020 (published online 16 March 2020), Pages 91 -98.
  • Non-limiting examples of a subgroup 1 a coronavirus include FCov.FIPV.79.1146. VR.2202 (GenBank Accession No. NV_007025), transmissible gastroenteritis virus (TGEV) (GenBank Accession No. NC_002306; GenBank Accession No. Q811789.2; GenBank Accession No. DQ811786.2; GenBank Accession No. DQ81 1788.1 ; GenBank Accession No. DQ811785.1 ; GenBank Accession No. X52157.1 ; GenBank Accession No. AJ011482.1 ; GenBank Accession No. KC962433.1 ; GenBank Accession No. AJ271965.2; GenBank Accession No.
  • JQ693060.1 GenBank Accession No. KC609371.1 ; GenBank Accession No. JQ693060.1 ; GenBank Accession No. JQ693059.1 ; GenBank Accession No. JQ693058.1 ; GenBank Accession No.
  • PRRSV porcine reproductive and respiratory syndrome virus
  • Non-limiting examples of a subgroup 1 b coronavirus include
  • BtCoV.1 A.AFCD62 GenBank Accession No. NC_010437
  • BtCoV.1 B.AFCD307 GenBank Accession No. NC_010436
  • BtCov.HKU8.AFCD77 GenBank Accession No. NC_010438
  • BtCoV.512.2005 GenBank Accession No. DQ648858
  • porcine epidemic diarrhea virus PEDV.CV777 GenBank Accession No. NC_003436, GenBank Accession No. DQ355224.1 , GenBank Accession No. DQ355223.1 , GenBank Accession No.
  • FJ687462.1 GenBank Accession No. FJ687461.1 , GenBank Accession No. FJ687460.1 , GenBank Accession No. FJ687459.1 , GenBank Accession No. FJ687458.1 , GenBank Accession No. FJ687457.1 , GenBank Accession No. FJ687456.1 , GenBank Accession No.
  • BtCoV.HKU2.GD.430.2006 GenBank Accession No. EF203064
  • any other subgroup 1 b coronavirus now known e.g., as can be found in the GenBank® Database
  • any combination thereof e.g., as can be found in the GenBank® Database
  • Non-limiting examples of a subgroup 2a coronavirus include HCoV.HKU1 -C.N5 (GenBank Accession No. DQ339101 ), MHV.A59 (GenBank Accession No. NC 001846), PHEV.VW572 (GenBank Accession No. NC 007732), HCoV.OC43.ATCC.VR.759 (GenBank Accession No. NC_005147), bovine enteric coronavirus (BCoV.ENT) (GenBank Accession No. NC_003045), as well as any other subgroup 2a coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
  • Non-limiting examples of subgroup 2b coronaviruses include Bat SARS CoV (GenBank Accession No. FJ211859), SARS CoV (GenBank Accession No. FJ211860), SARS-CoV-2 (GenBank Accession No. NC_045512.2), BtSARS.HKU3.1 (GenBank Accession No. DQ022305), BtSARS.HKU3.2 (GenBank Accession No. DQ084199), BtSARS.HKU3.3 (GenBank Accession No. DQ084200), BtSARS.Rml (GenBank Accession No. DQ412043), BtCoV.279.2005 (GenBank Accession No.
  • Non-limiting examples of subgroup 2c coronaviruses include: Middle East Respiratory Syndrome coronavirus isolate Riyadh_2_2012 (GenBank Accession No. KF600652.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_18_2013 (GenBank Accession No. KF600651.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_17_2013 (GenBank Accession No. KF600647.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_15_2013 (GenBank Accession No. KF600645.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_16_2013 (GenBank Accession No.
  • Middle East respiratory syndrome coronavirus isolate AI-Hasa_21_2013 GenBank Accession No. KF600634
  • Middle East respiratory syndrome coronavirus isolate AI-Hasa_19_2013 GenBank Accession No. KF600632
  • Middle East respiratory syndrome coronavirus isolate Buraidah_1_2013 GenBank Accession No. KF600630.1
  • Middle East respiratory syndrome coronavirus isolate Hafr-AI-Batin_1_2013 GenBank Accession No. KF600628.1
  • Middle East respiratory syndrome coronavirus isolate AI-Hasa_12_2013 GenBank Accession No.
  • KF186566.1 Middle East respiratory syndrome coronavirus isolate AI-Hasa_4_2013 (GenBank Accession No. KF186564.1 ), Middle East respiratory syndrome coronavirus (GenBank Accession No. KF192507.1 ), Betacoronavirus England 1 -N1 (GenBank Accession No. NC-019843), MERS-CoV_SA-N1 (GenBank Accession No.
  • GenBank Accession No: KF600656.1 GenBank Accession No: KF600655.1 , GenBank Accession No: KF600654.1 , GenBank Accession No: KF600649.1
  • GenBank Accession No: KF600648.1 GenBank Accession No: KF600646.1
  • GenBank Accession No: KF600643.1 GenBank Accession No: KF600642.1
  • GenBank Accession No: KF600640.1 GenBank Accession No: KF600639.1
  • GenBank Accession No: KF600638.1 GenBank Accession No: KF600637.1
  • GenBank Accession No: KF600636.1 GenBank Accession No: KF600635.1
  • GenBank Accession No: KF600631.1 GenBank Accession No: KF600626.1
  • GenBank Accession No: KF600625.1 GenBank Accession No: KF600624.1
  • BtCoV.HKU4.1 GenBank Accession No. NC_009019
  • BtCoV.HKU4.3 GenBank Accession No. EF065507
  • BtCoV.HKU4.4 GenBank Accession No. EF065508
  • BtCoV 133.2005 GenBank Accession No. NC 008315
  • BtCoV.HKU5.5 GenBank Accession No. EF065512
  • BtCoV. HKU5.1 GenBank Accession No. NC_009020
  • BtCoV.HKU5.2 GenBank Accession No. EF065510
  • BtCoV.HKU5.3 GenBank Accession No.
  • Pipistrellus bat coronavirus HKU4 isolates (GenBank Accession No:
  • KC522063.1 or GenBank Accession No: KC522062.1 , as well as any other subgroup 2b coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
  • Non-limiting examples of a subgroup 2d coronavirus include BtCoV.HKU9.2 (GenBank Accession No. EF065514), BtCoV.HKU9.1 (GenBank Accession No.
  • Non-limiting examples of a subgroup 3 coronavirus include IBV.Beaudette.IBV.p65 (GenBank Accession No. DQ001339), as well as any other subgroup 3 coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
  • the coronavirus may be any variant of SARS- CoV-2, including those described herein such as those in Table 3.
  • the coronavirus may be any virus that comprises a receptor binding domain of a Spike protein that includes one or more of the mutations as shown in Figure 9. Subjects
  • a subject or individual in need of treatment according to any aspect of the invention, or requiring administration of any composition described herein may be a subject or individual who is displaying a symptom of a coronavirus infection or who has been diagnosed with a coronavirus infection. Further, the subject or individual may be one who has been clinically or biochemically determined to be infected with a coronavirus.
  • a subject may be in a stage of coronavirus infection before end stage-organ failure has developed.
  • a subject in need thereof may be anyone with a coronavirus infection from the onset of clinical progression, before end-organ failure has developed.
  • the subject has had coronavirus infection symptoms for less than or equal to 12 days, and who does not have life-threatening organ dysfunction or organ failure.
  • the subject is early in the course of the disease, for example, before day 14 from symptom onset, or during the viremic and seronegative stage.
  • a “subject” or “individual” can also be any animal that is susceptible to infection by coronavirus and/or susceptible to diseases or disorders caused by coronavirus infection.
  • a subject of this invention can be a mammal and in particular embodiments is a human, which can be an infant, a child, an adult or an elderly adult.
  • a “subject at risk of infection by a coronavirus” or a “subject at risk of coronavirus infection” is any subject who may be or has been exposed to a coronavirus. “Subject” or “individual” includes any human or non-human animal.
  • the compounds of the present invention may also be useful for veterinary treatment of mammals, including companion animals and farm animals, such as, but not limited to dogs, cats, horses, cows, sheep, and pigs, or any animal that can be infected by coronavirus.
  • the coronavirus may be any coronavirus described herein, including SARS-CoV-2 and any variant thereof such as those described herein.
  • the subjects at risk include, but are not limited to, an immunocompromised person, an elderly adult (more than 65 years of age), children younger than 2 years of age, healthcare workers, adults or children in close contact with a person(s) with confirmed or suspected coronavirus infection, and people with underlying medical conditions such as pulmonary infection, heart disease or diabetes, primary or secondary immunodeficiency.
  • Coronavirus whole Spike based vaccine an immunocompromised person, an elderly adult (more than 65 years of age), children younger than 2 years of age, healthcare workers, adults or children in close contact with a person(s) with confirmed or suspected coronavirus infection, and people with underlying medical conditions such as pulmonary infection, heart disease or diabetes, primary or secondary immunodeficiency.
  • Coronavirus whole Spike based vaccine include, but are not limited to, an immunocompromised person, an elderly adult (more than 65 years of age), children younger than 2 years of age, healthcare workers, adults or children in close contact with a person(s) with confirmed or suspected coronavirus infection, and people with
  • a coronavirus whole Spike based vaccine or a vaccine comprising a whole Spike antigen refers to a composition that comprises (or consists essentially of or consists of) a protein that comprises, consists essentially of or consists of an amino acid sequence of a full-length Spike protein of a coronavirus, or a nucleic acid (preferably DNA or RNA, more preferably mRNA) that comprises, consists essentially of or consists of a nucleotide sequence encoding a full- length Spike protein.
  • a nucleic acid encoding a whole Spike antigen may be a viral vector, such as chimp- or human- adenovirus, that contains a nucleotide sequence that encodes a full-length Spike protein.
  • coronavirus whole Spike based vaccine may comprise an inactivated virus or any other form as outlined in Table 2 below.
  • CoV-S also called “S” or “S protein” or “spike protein” of the coronavirus and can refer to specific S proteins such as SARS-CoV-2-S, MERS-CoV S, and SARS-CoV S or other members of the coronavirus family.
  • SARS-CoV-2 spike protein is a 1273 amino acid type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle.
  • the protein has two essential functions, (1) host receptor binding and (2) membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein.
  • the initial attachment of the CoV virion to the host cell is initiated by interactions between the S protein and its receptor.
  • the sites of receptor binding domains (RBD) within the S1 region of a corona virus S protein vary depending on the virus, with some having the RBD at the N-terminus of S1 (MHV) while others (SARS-CoV) have the RBD at the C-terminus of S1 .
  • the S-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and governs the tissue tropism of the virus.
  • Many coronaviruses utilize peptidases as their cellular receptor. It is unclear why peptidases are used, as entry occurs even in the absence of the enzymatic domain of these proteins.
  • SARS-CoV aminopeptidase N
  • SARS-CoV-2 SARS-CoV-2
  • HCoV-NL63 use angiotensin-converting enzyme 2 (ACE2) as their receptor
  • ACE2 angiotensin-converting enzyme 2
  • MHV MHV enters through CEACAMI
  • DPP4 dipeptidyl-peptidase 4
  • the subject is infected with SARS-CoV-2, which causes the COVID-19 disease.
  • the amino acid sequence of the full-length SARS-CoV-2 spike protein is exemplified by the amino acid sequence provided in SEQ ID NO: 1 . Examples of whole spike based vaccines are described in Table 2.
  • SARS-CoV-2 vaccines comprising the whole spike protein in phase IV clinical trials (WHO) and some variant whole spike protein vaccines
  • CoV-S includes protein variants of CoV spike protein isolated from different CoV spike protein or a fragment thereof.
  • the term also encompasses CoV spike protein or a fragment thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as ROR1 .
  • the Spike protein may be from a variant or strain as described herein, including in Table 3 below.
  • a coronavirus Spike receptor binding domain (RBD) based vaccine or a vaccine comprising a coronavirus Spike RBD antigen refers to a composition that comprises (or consists essentially of or consists of) a protein that comprises, consists essentially of or consists of an amino acid sequence of a Spike protein RBD of a coronavirus, or a nucleic acid (preferably RNA, more preferably mRNA) that comprises, consists essentially of or consists of a nucleotide sequence encoding a Spike protein RBD of a coronavirus, however neither the protein nor the nucleic acid comprises, or encodes for, a full length coronavirus Spike protein.
  • a nucleic acid that encodes a coronavirus Spike RBD antigen may be DNA or RNA, and may be part of a viral vector (e.g. adenoviral vector).
  • the coronavirus Spike RBD is a SARS-CoV-2 Spike RBD.
  • SARS- CoV-2 RBD based vaccines have been previously described in the art, including SARS- CoV-2 RBD-dimers (Yang et al., Lancet Infectious Diseases, 2021 , 21 (8): 1107-1119), SARS-CoV-2 RBD-Fc-dimers (Liao et al., Emerging Microbes & Infections, 2021 , 10(1 ): 1589-1597; Alieva et al., Vaccine, 2021 , 39(45): 6601 -6613), SARS-CoV-2 RBD multimers (Tan et al., Nat Common, 2021 , 12: 542; Saunders et al., 2021 , Nature, 594: 553-559; Li et al., bioRxiv, 2022.01 .26.477915), and SARS-CoV-2 RBD monomers (Tan et al., Nat Commun, 2021 , 12: 140
  • the coronavirus Spike RBD antigen (also referred to as RBD antigen) comprises, consists essentially of or consists of an amino acid sequence of a Spike protein RBD of a coronavirus however it does not comprise an amino acid sequence of a full length coronavirus Spike protein.
  • the RBD antigen comprises an amino acid sequence of a RBD of a coronavirus and additional amino acid sequence of one or more domains of the Spike protein.
  • the one or more domains of the Spike protein include N-terminal domain, Subdomain 1 , Subdomain 2, fusion peptide, Heptad repeat 1 , Central helix, Connector domain or Heptad repeat 2.
  • the RBD antigen does not comprise part of, or all of, one or more of the following domains of the Spike protein N-terminal domain, Subdomain 1 , Subdomain 2, fusion peptide, Heptad repeat 1 , Central helix, Connector domain or Heptad repeat 2.
  • the RBD antigen consists of amino acid sequence of all or part of the S1 subunit of the Spike protein.
  • the RBD antigen comprises an amino acid sequence of an RBD of a coronavirus and additional amino acid sequence of all of, or part of, one or more domains of the S1 subunit of the Spike protein.
  • the RBD antigen does not contain any amino acid sequence with identity to, or that is derived from, the S2 subunit of the Spike protein of a coronavirus.
  • the present invention provides a RBD antigen that is highly conserved across SARS coronaviruses and current SARS-CoV-2 variants.
  • RBD antigen may comprise the amino acids sequence of, or equivalent to, N334- P527 of a SARS-CoV-2 spike protein, for example as defined by GenBank accession NC_045512.2.
  • the RBD antigen can include additional structures (e.g. amino acids, sugar side chains) that may stabilise the protein conformation.
  • the RBD antigen is effective, particularly in the presence of an adjuvant, for use as or in an immunogenic composition (e.g., a vaccine), and/or for achieving immunological effects as described herein (e.g., generation of coronavirus neutralising antibodies, and/or T cell responses (e.g., CD4+ and/or CD8+ T cell responses)).
  • an adjuvant for use as or in an immunogenic composition (e.g., a vaccine), and/or for achieving immunological effects as described herein (e.g., generation of coronavirus neutralising antibodies, and/or T cell responses (e.g., CD4+ and/or CD8+ T cell responses)).
  • the present invention provides a nucleic acid (e.g., DNA, RNA, preferably mRNA) comprising an open reading frame encoding a polypeptide that comprises, consists essentially of or consists of an RBD antigen as described herein, which nucleic acid is suitable for intracellular expression of the polypeptide.
  • SARS-CoV-2 RBD has been observed in an "open” conformation, wherein the RBD is present in a homotrimer of RBD monomers and the RBD of at least one RBD monomer of the trimer points upward relative to the other two RBDs (“open” conformation), away from the C-terminal end of the RBD, and also in a "closed” conformation, where none of the three RBDs of a surface glycoprotein trimer point upward, ie they are downward.
  • the RBD is comprised in a trimer thereof.
  • one or two RBDs of the trimer is in an open conformation (partially open).
  • all RBDs of the trimer are in an open conformation (fully open).
  • two or three RBDs of the trimer are in a closed conformation.
  • any of the RBDs of the trimer are in an intermediate conformation, ie not fully up- or downward.
  • a receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 2 or 12, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 2 or 12.
  • the amino acid sequence may include one or more of the mutations as shown in Figure 9, Table 3 or described herein in relation to a SARS-CoV-2 variant, such as an alpha, beta, gamma, kappa, delta, delta plus, lambda, mu, iota or omicron strain (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
  • a SARS-CoV-2 variant such as an alpha, beta, gamma, kappa, delta, delta plus, lambda, mu, iota or omicron strain (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
  • a receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 2 or 12, or any variant described in Table 3, having 0 to 16 amino acid insertions, deletions, substitutions or additions (or a combination thereof).
  • the relevant amino acid sequence may have from 0 to 16, preferably from 0 to 15, preferably from 0 to 14, preferably from 0 to 13, preferably from 0 to 12, preferably from 0 to 11 , preferably from 0 to 10, preferably from 0 to 9, preferably from 0 to 8, preferably from 0 to 7, preferably from 0 to 6, preferably from 0 to 5, preferably from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1 amino acid insertions, deletions, substitutions or additions (or a combination thereof), wherein the amino acid insertions, deletions, substitutions or additions (or a combination thereof) are located at the N- and/or C-terminus.
  • an immune response may comprise generation of a binding antibody titre against the receptor binding domain (RBD) of the coronavirus spike protein.
  • the RBD is a SARS- CoV-2 RBD.
  • a receptor binding domain may be from a SARS-CoV-2 variant, such as those described in Table 3 below.
  • SARS-CoV-2 variants have evolved from the original WT (“Wuhan”) strain of the virus (genome reference sequence: GenBank accession NC_045512.2).
  • WT WT
  • alpha, beta, gamma, kappa, delta, delta plus, lambda, mu, iota or omicron strain Table 3
  • the receptor binding domain may be N334- P527 of a WT (“Wuhan”), alpha, beta, gamma, kappa, delta, lambda, mu, iota or omicron strains and others that may emerge such as delta plus, for example Y.1 , AY.2, AY.3 and AY.4.2 and omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5.
  • the alpha variant carries 1 mutation in the RBD compared to the original WT (“Wuhan”) strain: N501 Y.
  • the beta variant carries three mutations in the RBD compared to the original WT (“Wuhan”) strain: N501Y, E484K and K417N.
  • the gamma variant carries three mutations in the RBD compared to the original WT (“Wuhan”) strain: K417T, E484K and N501 Y.
  • the kappa variant carries two mutations in the RBD compared to the original WT (“Wuhan”) strain: L452R and E484Q.
  • the delta variant carries two mutations in the RBD compared to the original WT (“Wuhan”) strain: L452R and T478K.
  • the lambda variant carries two mutations in the RBD compared to the original WT (“Wuhan”) strain: L452Q and F490S.
  • the iota variant carries 1 mutation in the RBD compared to the original WT (“Wuhan”) strain: E484K.
  • the mu variant carries two mutations in the RBD compared to the original WT (“Wuhan”) strain: E484K and N501 Y.
  • the omicron subvariant BA.1 carries fifteen mutations in the RBD compared to the original WT (“Wuhan”) strain: G339D, S371 L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501 Y and Y505H.
  • the beta RBD (with mutations (N501Y, K417N and E484K) is closer to omicron than a wildtype (“Wuhan”) RBD because it shares with omicron the RBD mutations N501Y and K417N (two important mutations known to influence binding to ACE2 (N501Y) and immune evasion (K417N)).
  • the beta RBD is closer to gamma (N501 Y, E484K (immune evasion) and K417T), mu (N501 Y and E484K) and iota (E484K).
  • a beta RBD should therefore drive superior responses against gamma, mu, iota and omicron.
  • Table 3 Summary of SARS-CoV-2 variants or strains and RBD mutations
  • peptides, polypeptides or polynucleotides encoding peptides or polypeptides as described herein may contain substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this invention.
  • sequence tags or amino acids such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation.
  • amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
  • Certain amino acids e.g., C-terminus and/or N-terminus residues
  • sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like may be substituted with alternative sequences that achieve the same or a similar function.
  • cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids.
  • buried hydrogen bond networks may be replaced with hydrophobic resides to improve stability.
  • glycosylation sites may be removed and replaced with appropriate residues.
  • sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminus and/or C-terminus ends) that may be deleted, for example, prior to use in the preparation of a vaccine composition.
  • protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of coronavirus RBD antigens of interest.
  • any protein fragment meaning a polypeptide sequence at least one amino acid residue shorter than a reference antigen sequence but otherwise identical
  • an antigen includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of the sequences provided or referenced herein.
  • a receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 12 or an SARS-CoV-2 variant that has evolved from the original WT (“Wuhan”) strain of the virus (e.g.
  • genome reference sequence GenBank accession NC_045512.2), including those described in Table 3, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 12 or a SARS-CoV-2 variant that has evolved from the original WT (“Wuhan”) strain of the virus (e.g. genome reference sequence: GenBank accession NC_045512.2), including those described in Table 3.
  • WT original WT
  • the amino acid sequence may include one or more of the mutations as shown in Figure 9, Table 3 or described herein in relation to a SARS-CoV-2 variant, such as an alpha, beta, gamma, kappa, delta, delta plus, lambda, mu, iota and omicron strain (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
  • a SARS-CoV-2 variant such as an alpha, beta, gamma, kappa, delta, delta plus, lambda, mu, iota and omicron strain (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
  • a receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 12 or a SARS-CoV-2 variant that has evolved from the original WT (“Wuhan”) strain of the virus (e.g genome reference sequence: GenBank accession NC_045512.2), including those described in Table 3, having 0 to 16 amino acid insertions, deletions, substitutions or additions (or a combination thereof).
  • WT WT
  • the relevant amino acid sequence may have from 0 to 16, preferably from 0 to 15, preferably from 0 to 14, preferably from 0 to 13, preferably from 0 to 12, preferably from 0 to 11 , preferably from 0 to 10, preferably from 0 to 9, preferably from 0 to 8, preferably from 0 to 7, preferably from 0 to 6, preferably from 0 to 5, preferably from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1 amino acid insertions, deletions, substitutions or additions (or a combination thereof), wherein the amino acid insertions, deletions, substitutions or additions (or a combination thereof) are located at the N- and/or C-terminus.
  • a particularly preferred coronavirus Spike receptor binding domain (RBD) based vaccine comprises, consists essentially of or consists of a chimeric or fusion protein as described herein, particularly this section.
  • the coronavirus Spike receptor binding domain (RBD) antigen may be a chimeric or fusion protein as described herein.
  • the nucleic acid encoding a coronavirus Spike RBD antigen may encode a chimeric or fusion protein as described herein.
  • the chimeric or fusion protein comprises 2 or more polypeptides comprising or consisting of an amino acid sequence of a receptor binding domain (RBD) from a Spike protein of a coronavirus linked to an Fc region of an antibody.
  • RBD receptor binding domain
  • the chimeric or fusion protein comprises 2 or more polypeptides each comprising or consisting of an amino acid sequence of a receptor binding domain from a Spike protein of a coronavirus linked to a polypeptide comprising an Fc receptor binding domain.
  • the chimeric or fusion protein comprises a dimer of receptor binding domains from a Spike protein of a coronavirus linked to an Fc region of an antibody.
  • the chimeric or fusion protein comprises a dimer of receptor binding domains from a Spike protein of a coronavirus linked to a polypeptide comprising an Fc receptor binding domain.
  • the chimeric or fusion protein is a single chain dimer wherein a contiguous polypeptide chain comprises two RBD chain sequences that are covalently linked.
  • the chimeric or fusion protein comprises comprises, consists essentially of or consists of a sequence as set forth in any one of SEQ ID NO: 2, 4, 5, 7, 8, 9, 12, 13 or 18.
  • the term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody.
  • the Fc region comprises two heavy chain fragments, preferably the CH2 and CH3 domains of said heavy chain.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, s, Y, and p.
  • the RBD-Fc dimer does not exhibit any effect function or any detectable effector function.
  • “Effector functions” or “effector activities” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity).
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991 ).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wl).
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998).
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et aL, J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101 :1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929 Al).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581 ).
  • an antibody variant may comprise an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues).
  • substitutions are L234A and L235A (LALA) (See, e.g., WO 2012/130831 ).
  • alterations may be made in the Fc region that result in altered (i.e., diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551 , WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • the Fc region includes mutations to the complement (C1q) and/or to Fc gamma receptor (FcyR) binding sites.
  • such mutations can render the fusion protein incapable of antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • Fc region also includes native sequence Fc regions and variant Fc regions.
  • the Fc region may include the carboxyl-terminus of the heavy chain.
  • Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • Amino acid sequence variants of the Fc region of an antibody may be contemplated.
  • Amino acid sequence variants of an Fc region of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the Fc region of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., inducing or supporting an anti-inflammatory response.
  • the Fc region of the antibody may be an Fc region of any of the classes of antibody, such as IgA, IgD, IgE, IgG, and IgM.
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the antibody may be an Fc region of an IgG.
  • the Fc region of the antibody may be an Fc region of an IgG 1 , an lgG2, an lgG3 or an lgG4.
  • the fusion protein of the present invention comprises an IgG of an Fc region of an antibody.
  • the Fc region of the antibody is an Fc region of an IgG, preferably lgG1.
  • the Fc region is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen.
  • An Fc receptor binding domain is any protein or polypeptide that binds to the Fc receptor on the surface of a cell.
  • the Fc receptor binding domain may be an antigen binding domain of an antibody.
  • the Fc receptor binding domain also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.
  • the herein provided fusion proteins may comprise a linker (or “spacer”).
  • linker or “spacer”.
  • the 2 or more polypeptides comprising or consisting of an amino acid sequence of a receptor binding domain, or the dimer of receptor binding domains from a spike protein of a coronavirus, is fused via a linker at the C-terminus to the Fc region or Fc receptor binding domain.
  • a linker is usually a peptide having a length of up to 20 amino acids.
  • the term “linked”, “linked to” or “fused to” refers to a covalent bond, e.g., a peptide bond, formed between two moieties. Accordingly, in the context of the present invention the linker may have a length of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids.
  • the herein provided fusion protein may comprise a linker between the 2 or more polypeptides comprising or consisting of an amino acid sequence of a receptor binding domain, or the dimer of receptor binding domains from a spike protein of a coronavirus, and the Fc region of the antibody, such as between the N-terminus of the Fc regions/FcR binding domains and the C-terminus of the receptor binding domain polypeptide.
  • linkers have the advantage that they can make it more likely that the different polypeptides of the fusion protein fold independently and behave as expected.
  • the 2 or more polypeptides comprising or consisting of an amino acid sequence of a receptor binding domain, or the dimer of receptor binding domains from a spike protein of a coronavirus, and the Fc region of an antibody or Fc receptor binding domain may be comprised in a single covalently associated multi-functional polypeptide.
  • the fusion protein of the present invention includes a peptide linker.
  • the peptide linker can include the amino acid sequence Gly- Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS) or Gly-Gly-Gly-Gly-Ser (GGGGS).
  • the peptide linker can include the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer.
  • the linker may a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS)n where n is any number from 1 to 15 or more.
  • the linker may be (GS)3 (i.e., GSGSGS) or longer (GS)11 or longer.
  • n can be any number including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. Fusion proteins having linkers of such length are included within the scope of the present invention. Additionally, fusion proteins that have no linker are included within the scope of the present invention.
  • the present invention provides a vaccine compositions that may also include a pharmaceutically acceptable adjuvant in addition to the peptides as defined herein.
  • Adjuvants are added in order to enhance the immunogenicity of the vaccine composition.
  • a single vaccine may include two or more of said adjuvants.
  • the adjuvants for use in the present invention may be modulators and/or agonists of Toll-Like Receptors (TLR).
  • TLR Toll-Like Receptors
  • they may be agonists of one or more of the human TLR1 , TLR2, TLR3, TLR4, TLR7, TLR8, and/or TLR9 proteins.
  • Preferred agents are agonists of TLR7 (e.g. imidazoquinolines) and/or TLR9 (e.g. CpG oligonucleotides). These agents are useful for activating innate immunity pathways.
  • Suitable adjuvants are known in the art and include any one described herein, preferably a TLR2-agonist, more preferably a Pam-2-Cys containing molecule such as PEG-R4-Pam-2-Cys, or preferably a stimulator of NKT cells, more preferably alpha- Galactosylceramide (also referred to herein as “a-GalCer”), alpha-glucosylceramide, alpha-glucosyldiacylglycerol, alpha-galactosyldiacylglycerol, beta-mannosylceramide, or other NKT cell-stimulatory lipid molecules and analogues thereof comprising variations in acyl and sphingosine chain lengths, saturation, and variations in polar head group composition.
  • TLR2-agonist preferably a Pam-2-Cys containing molecule such as PEG-R4-Pam-2-Cys
  • a stimulator of NKT cells more preferably alpha- Galacto
  • the TLR2-agonist can be selected from the group consisting of Pam3CSK4, PEG-R4-Pam-2-Cys, MALP-2, lipoteichoic acid, OspA, Porin, LcrV, lipomannan, Lysophosphatidylserine, Lipophosphoglycan (LPG), Glycophosphatidylinositol (GPI) and Zymosan.
  • the adjuvant can be selected from the group consisting of is selected from the group consisting of poly-l:C, CpG, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, B(C, CP-870,893, CpG7909, ASO3, ASO4, MatrixM, CyaA, dSLIM, GM-CSF, IC30, IC31 , Imiquimod, 3dMPL, ImuFact IMP321 , IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59®, AddaVaxTM, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA V, Montanide ISA-51 , OK- 432, OM-174, OM-197-MP-EC, ONTAK.
  • PEPTEL vector system
  • PLGA microparticles resiquimod
  • SRL172 Virosomes and other Virus-like particles
  • YF-17D VEGF trap
  • R848 beta-glucan, Pam3Cys, and Aquila's QS21 stimulon.
  • Oil-in-water emulsions have been found to be particularly suitable for use in adjuvanting viral vaccines.
  • Various such emulsions are known, and they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible.
  • the oil droplets in the emulsion are generally less than 5 pm in diameter, and may even have a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions. Droplets with a size less than 220 nm are preferred as they can be subjected to filter sterilization.
  • the invention can be used with oils such as those from an animal (such as fish) or vegetable source.
  • Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils.
  • Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used.
  • 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils.
  • Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention.
  • the procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.
  • Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein.
  • a number of branched chain oils are synthesized biochemically in 5- carbon isoprene units and are generally referred to as terpenoids.
  • Shark liver oil contains a branched, unsaturated terpenoids known as squalene, 2,6,10,15,19,23- hexamethyl-2,6,10,14,18,22-tetracosahexaene, which is particularly preferred herein.
  • Squalane the saturated analog to squalene
  • Fish oils, including squalene and squalane are readily available from commercial sources or may be obtained by methods known in the art. Other preferred oils are the tocopherols (see below). Mixtures of oils can be used.
  • Surfactants can be classified by their ‘HLB’ (hydrophile/lipophile balance).
  • Preferred surfactants for vaccine composition have a HLB of at least 10, preferably at least 15, and more preferably at least 16.
  • the invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1 ,2-ethanediyl) groups, with octoxynol- 9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)poly
  • Non-ionic surfactants are preferred.
  • Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.
  • Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures.
  • a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable.
  • Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
  • Preferred amounts of surfactants are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.
  • polyoxyethylene sorbitan esters such as Tween 80
  • octyl- or nonylphenoxy polyoxyethanols such as Triton X-100, or other detergents in the Triton series
  • polyoxyethylene ethers such as laureth 9
  • the adjuvant comprises a metabolizable oil and an emulsifying agent (such as a detergent or surfactant).
  • the oil and the emulsifying agent are present in the form of an oil-in-water emulsion having oil droplets substantially all of which are less than 1 micron in diameter.
  • Exemplary metabolizable oils and emulsifying agents are described in US6,299,884 and US6,086,901 .
  • the adjuvant comprises an oil-in-water emulsion.
  • the oil is squalene.
  • the aqueous phase is a citrate buffer (for example 10mM at pH 6.5).
  • the adjuvant comprises squalene in an oil-in-water emulsion.
  • the adjuvant further comprises TWEEN® 80 (polyoxyethylenesorbitan monooleate) and Span® 85 (sorbitan trioleate).
  • the adjuvant may comprise 4.3% squalene, 0.5% TWEEN® 80 (polyoxyethylenesorbitan monooleate), 0.5% Span® 85 (sorbitan trioleate), optionally with 400 pig/ml MTP-PE (N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1 ,2-dipalmitoyl-sn-glycero-3- 3(hydroxyphosphoryl-oxy)]ethylamide)
  • the composition comprises 50%vol/vol adjuvant, preferably the adjuvant is MF59®.
  • MF59® is an oil-in-water emulsion of squalene oil. Squalene, a naturally occurring substance found in humans, animals and plants, is highly purified for the vaccine manufacturing process.
  • MF59 adjuvant (MF59C.1 ) is an oil-in-water emulsion with a squalene internal oil phase and a citrate buffer external aqueous phase. See, e.g., U.S. Pat. Nos. 6,299,884 and 6,086,901 ; Ott et al.
  • MF59 Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines
  • Vaccine Design The Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, pp. 277-296 (1995).
  • the safety of the MF59 adjuvant has been demonstrated in animals and in humans in combination with a number of antigens.
  • MF59 is 4.3% squalene, 0.5% TWEEN® 80 (polyoxyethylenesorbitan monooleate), 0.5% Span® 85 (sorbitan trioleate), optionally with 400 pg/ml MTP-PE (N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1 ,2-dipalmitoyl-sn-glycero-3- 3(hydroxyphosphoryl-oxy)]ethylamide).
  • An exemplary composition of MF59 comprises citrate buffer pH 6.5 (10mM citrate, 140mM NaCI, 0.02% PS80, pH 6.5) and 39mg/ml Squalene, 4.7mg/ml Polysorbate 80, 4.7mg/ml Sorbitan Trioleate, 2.65mg/ml Sodium Citrate, 0.17mg/ml Citric Acid monohydrate.
  • the adjuvant is an emulsion of squalene, a tocopherol, and Tween 80.
  • the emulsion may include phosphate buffered saline. It may also include Span 85 (e.g. at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene:tocopherol is preferably ⁇ 1 as this provides a more stable emulsion. Squalene and Tween 80 may be present volume ratio of about 5:2.
  • One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-a-tocopherol and 5 ml squalene), then microfluidising the mixture.
  • the resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250 nm, preferably about 180 nm.
  • An emulsion of squalene, a tocopherol, and a Triton detergent e.g. Triton X- 100.
  • the emulsion may also include a 3d-MPL (see below).
  • the emulsion may contain a phosphate buffer.
  • An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an a-tocopherol succinate).
  • the emulsion may include these three components at a mass ratio of about 75:11 :10 (e.g. 750 pg/ml polysorbate 80, 110 pg/ml Triton X-100 and 100 pg/ml a-tocopherol succinate), and these concentrations should include any contribution of these components from antigens.
  • the emulsion may also include squalene.
  • the emulsion may also include a 3d-MPL (see below).
  • the aqueous phase may contain a phosphate buffer.
  • An emulsion of squalane, polysorbate 80 and poloxamer 401 (“PluronicTM L121 ”).
  • the emulsion can be formulated in phosphate buffered saline, pH 7.4.
  • This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the “SAF-1” adjuvant (Allison & Byars, Res Immuno , 1992, 143:519- 25) (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80).
  • An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or ‘Span 80’).
  • the emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm (US2007/014805)
  • the emulsion may also include one or more of alditol; a cryoprotective agent (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or an alkylpolyglycoside. Such emulsions may be lyophilized.
  • An emulsion having from 0.5-50% of an oil, 0.1 -10% of a phospholipid, and 0.05-5% of a non-ionic surfactant.
  • preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.
  • Additives may be included, such as Qui1 A saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in US6,080,725, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyldioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis(2- hydroxyethyl)propanediamine.
  • a non-metabolisable oil such as light mineral oil
  • surfactant such as lecithin, Tween 80 or Span 80.
  • Additives may be included, such as Qui1 A saponin, cholesterol, a saponin-lipophile conjugate (
  • An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer) (W02006/113373).
  • An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer) (W02006/113373).
  • the emulsions may be mixed with antigen extemporaneously, at the time of delivery.
  • the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use.
  • the antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids.
  • the volume ratio of the two liquids for mixing can vary (e.g. between 5:1 and 1 :5) but is generally about 1 :1 .
  • compositions include a tocopherol
  • 3, y, 5, tocopherols can be used, but a-tocopherols are preferred.
  • the tocopherol can take several forms e.g. different salts and/or isomers. Salts include organic salts, such as succinate, acetate, nicotinate, etc. D-a-tocopherol and DL-a-tocopherol can both be used.
  • Tocopherols are advantageously included in vaccines for use in elderly patients (e.g. aged 60 years or older) because vitamin E has been reported to have a positive effect on the immune response in this patient group. They also have antioxidant properties that may help to stabilize the emulsions.
  • a preferred a-tocopherol is DL-a- tocopherol, and the preferred salt of this tocopherol is the succinate.
  • the succinate salt has been found to cooperate with TNF-related ligands in vivo.
  • a-tocopherol succinate is known to be compatible with influenza vaccines and to be a useful preservative as an alternative to mercurial compounds.
  • the adjuvants known as aluminium hydroxide and aluminium phosphate may be used. These names are conventional, but are used for convenience only, as neither is a precise description of the actual chemical compound which is present (e.g. see chapter 9 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)).
  • the invention can use any of the “hydroxide” or “phosphate” adjuvants that are in general use as adjuvants.
  • aluminium hydroxide typically aluminium oxyhydroxide salts, which are usually at least partially crystalline.
  • Aluminium oxyhydroxide which can be represented by the formula AIO(OH)
  • IR infrared
  • the degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes.
  • WHH diffraction band at half height
  • the surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption.
  • a fibrous morphology e.g. as seen in transmission electron micrographs
  • the pl of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1 .8-2.6 mg protein per mg Al +++ at pH 7.4 have been reported for aluminium hydroxide adjuvants.
  • the adjuvants known as “aluminium phosphate” are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PCU/AI molar ratio between 0.3 and 1 .2. Hydroxyphosphates can be distinguished from strict AlPC by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm -1 (e.g. when heated to 200° C.) indicates the presence of structural hydroxyls.
  • the PC /AI 3+ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1 .2, preferably between 0.8 and 1 .2, and more preferably 0.95+0.1 .
  • the aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts.
  • a typical adjuvant is amorphous aluminium hydroxyphosphate with PO4/AI molar ratio between 0.84 and 0.92, included at 0.6 mg Al 3+ /ml.
  • the aluminium phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5-20 pm (e.g. about 5-10m) after any antigen adsorption.
  • Aluminium phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.
  • Suspensions of aluminium salts used to prepare vaccine compositions may contain a buffer (e.g. a phosphate or a histidine or a Tris buffer), but this is not always necessary.
  • the suspensions are preferably sterile and pyrogen-free.
  • a suspension may include free aqueous phosphate ions e.g. present at a concentration between 1 .0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.
  • the suspensions may also comprise sodium chloride.
  • the invention can use a mixture of both an aluminium hydroxide and an aluminium phosphate.
  • there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2:1 e.g. ⁇ 5:1 , ⁇ 6:1 , ⁇ 7:1 , ⁇ 8:1 , ⁇ 9:1 , etc.
  • the concentration of Al +++ in a composition for administration to a patient is preferably less than 10 mg/ml e.g. ⁇ 5 mg/ml, ⁇ 4 mg/ml, ⁇ 3 mg/ml, ⁇ 2 mg/ml, ⁇ 1 mg/ml, etc.
  • a preferred range is between 0.3 and 1 mg/ml.
  • a maximum of 0.85 mg/dose is preferred.
  • Immunostimulatory oligonucleotides can include nucleotide modifications/analogs such as phosphorothioate modifications and can be doublestranded or (except for RNA) single-stranded. References Kandimalla et al.
  • a CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT.
  • the CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN (oligodeoxynucleotide), or it may be more specific for inducing a B cell response.
  • the CpG is a CpG-A ODN.
  • the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form “immunomers”.
  • a useful CpG adjuvant is CpG7909, also known as ProMuneTM (Coley Pharmaceutical Group, Inc.).
  • TpG sequences can be used WO01/22972. These oligonucleotides may be free from unmethylated CpG motifs.
  • the immunostimulatory oligonucleotide may be pyrimidine-rich.
  • it may comprise more than one consecutive thymidine nucleotide (e.g. TTTT, as disclosed in ref. 158), and/or it may have a nucleotide composition with >25% thymidine (e.g. >35%, >40%, >50%, >60%, >80%, etc.).
  • it may comprise more than one consecutive cytosine nucleotide (e.g. CCCC), and/or it may have a nucleotide composition with >25% cytosine (e.g. >35%, >40%, >50%, >60%, >80%, etc.).
  • These oligonucleotides may be free from unmethylated CpG motifs.
  • Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They may comprise fewer than 100 nucleotides.
  • the adjuvant is 3dMPL (also known as 3 de-O-acylated monophosphoryl lipid A or 3-0-desacyl-4'-monophosphoryl lipid A).
  • 3dMPL is an adjuvant in which position 3 of the reducing end glucosamine in monophosphoryl lipid A has been de-acylated.
  • 3dMPL has been prepared from a heptoseless mutant of Salmonella minnesota, and is chemically similar to lipid A but lacks an acid-labile phosphoryl group and a base-labile acyl group.
  • 3dMPL 3dMPL
  • 3dMPL can form micellar aggregates or particles with different sizes e.g. with a diameter ⁇ 150 nm or >500 nm. Either or both of these can be used with the invention, and the better particles can be selected by routine assay. Smaller particles (e.g. small enough to give a clear aqueous suspension of 3dMPL) are preferred for use according to the invention because of their superior activity. Preferred particles have a mean diameter less than 220 nm, more preferably less than 200 nm or less than 150 nm or less than 120 nm, and can even have a mean diameter less than 100 nm. In most cases, however, the mean diameter will not be lower than 50 nm.
  • Particle diameter can be assessed by the routine technique of dynamic light scattering, which reveals a mean particle diameter. Where a particle is said to have a diameter of x nm, there will generally be a distribution of particles about this mean, but at least 50% by number (e.g. 60%, 170%, 180%, 190%, or more) of the particles will have a diameter within the range x ⁇ 25%.
  • 3dMPL can advantageously be used in combination with an oil-in-water emulsion. Substantially all of the 3dMPL may be located in the aqueous phase of the emulsion.
  • the 3dMPL can be used on its own, or in combination with one or more further compounds.
  • 3dMPL in combination with the QS21 saponin (including in an oil-in-water emulsion), with an immunostimulatory oligonucleotide, with both QS21 and an immunostimulatory oligonucleotide, with aluminium phosphate, with aluminium hydroxide, or with both aluminium phosphate and aluminium hydroxide.
  • the adjuvant is any one described herein, preferably PEG- R4-Pam-2-Cys, alpha-Galactosylceramide (also referred to herein as “a-GalCer”) or MF59.
  • the present invention provides the use of a vaccine composition that comprises (or consists essentially of or consists of) a protein that comprises, consists essentially of or consists of an amino acid sequence of a Spike protein RBD of a coronavirus, or a nucleic acid (preferably RNA, more preferably mRNA) that comprises, consists essentially of or consists of a nucleotide sequence encoding a Spike protein RBD of a coronavirus, however neither the protein nor the nucleic acid comprises, or encodes for, a full length coronavirus Spike protein.
  • a vaccine composition that comprises (or consists essentially of or consists of) a protein that comprises, consists essentially of or consists of an amino acid sequence of a Spike protein RBD of a coronavirus, or a nucleic acid (preferably RNA, more preferably mRNA) that comprises, consists essentially of or consists of a nucleotide sequence encoding a Spike protein RBD of a cor
  • Vaccine compositions as described herein are pharmaceutically acceptable and are typically in aqueous form. They may include components in addition to the antigen or nucleic acid encoding the antigen (and, where applicable, the adjuvant) e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s).
  • a pharmaceutically acceptable excipient after administered to a subject, does not cause undesirable physiological effects.
  • the excipient in the pharmaceutical composition must be "acceptable” also in the sense that it is compatible with mRNA or protein or polypeptide and can be capable of stabilizing it.
  • One or more excipients e.g., solubilizing agents
  • Examples of a pharmaceutically acceptable excipients include, but are not limited to, biocompatible vehicles (e.g., LNPs), carriers, adjuvants, additives, and diluents to achieve a composition usable as a dosage form.
  • biocompatible vehicles e.g., LNPs
  • examples of other excipients include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical excipients, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.
  • the vaccine is formulated using one or more excipients to: (1 ) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo.
  • excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
  • compositions comprising mRNA or protein does not include an adjuvant (they are adjuvant free).
  • Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free.
  • General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
  • Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
  • preparatory methods include the step of bringing the mRNA or protein into association with an excipient (e.g., a mixture of lipids and/or a lipid nanoparticle), and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • Relative amounts of the mRNA or protein, the pharmaceutically-acceptable excipient, and/or any additional ingredients in a composition in accordance with the disclosure may vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • Compositions may include one or more buffers.
  • Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20 mM range.
  • the pH of a composition will generally be between 5.0 and 8.1 , and more typically between 6.0 and 8.0 e.g. between 6.5 and 7.5, between 7.0 and 7.8.
  • a process of vaccine composition may therefore include a step of adjusting the pH of the bulk vaccine prior to packaging.
  • the composition is preferably sterile.
  • the composition is preferably non- pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
  • the composition is preferably gluten free.
  • the composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a ‘multidose’ kit).
  • a preservative is preferred in multidose arrangements.
  • the compositions may be contained in a container having an aseptic adaptor for removal of material.
  • the present invention provides methods comprising administering vaccine compositions to a subject in need thereof.
  • the exact amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
  • the phrase “therapeutically effective amount” generally refers to an amount of a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen as described herein that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein, when applied in a method or use of the invention.
  • Undesirable effects e.g., side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount”.
  • a spike based vaccine composition described herein is administered to subjects of age 16 or older (including, e.g., 16-85 years). In some such embodiments, a spike based vaccine composition described herein is administered to subjects of age 18-55. In some such embodiments, a spike based vaccine composition escribed herein is administered to subjects of age 56-85. In some embodiments, a spike based vaccine composition described herein is administered (e.g., by intramuscular injection) as a single dose.
  • the vaccination regimen comprises a RBD based booster vaccination wherein a subject has received at least two doses of a vaccine described herein, e.g., two doses of the whole spike based vaccine as described herein or wherein the subject has been previously infected with coronavirus and optionally has had at least one dose of a vaccine described herein, e.g., one dose of the whole spike based vaccine as described herein.
  • a vaccine described herein e.g., two doses of the whole spike based vaccine as described herein or wherein the subject has been previously infected with coronavirus and optionally has had at least one dose of a vaccine described herein, e.g., one dose of the whole spike based vaccine as described herein.
  • an effective amount of a RBD antigen described herein, for example a chimeric or fusion protein described herein, for a human subject lies in the range of about 0.25 nmoles/kg body weight/dose to 0.0001 nmoles/kg body weight/dose.
  • the range is about 0.25 nmoles/kg body weight/dose to 0.0001 nmoles/kg body weight/dose. More preferably, the range is about 0.002 nmoles/kg body weight/dose to 0.001 nmoles/kg body weight/dose.
  • the body weight/dose range is about 0.25 nmoles/kg, to 0.001 nmoles/kg, about 0.1 nmoles/kg to 0.001 nmoles/kg, about 0.025 nmoles/kg to 0.001 nmol/kg, about 0.01 nmoles/kg to 0.001 nmoles/kg, or about 0.005 nmoles/kg to 0.001 nmoles/kg body weight/dose.
  • the amount is at, or about, 0.25 nmoles, 0.1 nmoles, 0.05 nmoles, 0.01 nmoles, 0.005 nmoles, 0.002 nmoles, or 0.001 nmoles/kg body weight/dose of the RBD antigen, chimeric or fusion protein as described herein. Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic dose.
  • an effective amount of a RBD antigen described herein, for example a chimeric or fusion protein as described herein for a human subject lies in the range of about 0.01 pg to 100 pg per dose.
  • the range is about 0.1 pg to 50 pg per dose. More preferably, the range is about 0.1 pg to 20 pg per dose.
  • the dose is about 1 pg to 100 pg, about 5 pg to 50 pg, about 10 pg to 45 pg, about 10 pg to 25 pg or about 10 pg to 20 pg per dose.
  • the dose is at, or about, 0.01 pg.
  • the dose is 15 pg.
  • an effective amount of nucleic acid preferably DNA or RNA, more preferably mRNA
  • an RBD antigen for example a chimeric or fusion protein as described herein for a human subject
  • the mRNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the mRNA may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective amount, prophylactically effective, or appropriate imaging dose level for any particular subject may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the effective amount of the RNA may range from about 25 pg - 500 pg, administered as a single dose or as multiple booster doses.
  • a single dose of a vaccine composition e.g., administered once, twice, three times, or more
  • a single dose of a vaccine composition comprises about 25 pg mRNA.
  • a single dose of a vaccine composition e.g., administered once, twice, three times, or more
  • a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 250 pg mRNA.
  • a total amount of mRNA administered to a subject is about 5 pg, 10 pg, 25 pg, 50 pg, about 100 pg, or about 200 pg. In some embodiments, a total amount of mRNA administered to a subject is about 25 pg. In some embodiments, a total amount of mRNA administered to a subject is about 50 pg. In some embodiments, a total amount of mRNA administered to a subject is about 100 pg. In some embodiments, a total amount of mRNA administered to a subject is about 200 pg-
  • the dose administered to a subject is any dose that reduces viral load.
  • RBD antigen or nucleic acid encoding thereof
  • chimeric or fusion protein or vaccine compositions thereof can be administered using immunisation schemes known by persons of ordinary skill in the art to induce protective immune responses.
  • the present invention provides for single or multiple immunisations in a boosting strategy in subjects who have previously received one or more coronavirus vaccine compositions, and/or previously been infected with coronavirus.
  • the coronavirus vaccine composition is a whole spike based vaccine.
  • a single or multiple boosting immunisation is provided to a subject who has received one, two or more doses of a whole spike based vaccine.
  • a boosting immunisation or third dose of a coronavirus Spike receptor binding domain (RBD) based vaccine in a vaccination schedule of the invention can be administered at a time after a subject has received a second dose immunisation or has been previously infected with coronavirus that is days, weeks, months or even years after the prime immunisation.
  • a booster dose of a vaccine composition as described herein is administered following a second dose immunisation.
  • the second dose immunisation is provided by a whole (or full length) spike based coronavirus vaccine.
  • a booster dose is a dose that is given at a certain interval after completion of a second dose or after a subject is infected with a coronavirus that is intended to boost immunity to, and therefore prolong protection against, the disease that is to be prevented.
  • the time between administration of a second dose of a vaccine composition, or infection of subject with coronavirus, and a booster dose may be, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 1 year, 2 years, 3 years, 4 years, 5 years or 10 years.
  • the time between administration of a second dose of a vaccine composition, or infection of subject with coronavirus, and a booster dose of a coronavirus Spike receptor binding domain (RBD) based vaccine is 1 to 6 months. In some embodiments, the time between administration of an initial dose of a vaccine and a booster dose is less than 1 month. In certain embodiments, a boosting immunisation is administered 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or 12 months or more after a subject has received a second dose immunisation or has been previously infected with coronavirus.
  • RBD coronavirus Spike receptor binding domain
  • boost immunisations can be administered weekly, every other week, monthly, every other month, every third month, every sixth month, or more.
  • the boost immunisation is administered every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, or every 24 weeks.
  • boosting immunisation can continue until a protective anti-coronavirus antibody titre is seen in the subject’s serum.
  • a subject is given one boost immunisation, two boost immunisations, three boost immunisations, or four or more boost immunisations, as needed to obtain and maintain a protective antibody titre.
  • the booster dose or administration of a coronavirus Spike RBD based vaccine may be administered by intramuscular injection.
  • a booster dose may be administered in the deltoid muscle.
  • a provided composition is established to achieve elevated antibody titres, and/or B cell and/or T-cell titres (e.g., specific for a relevant portion of a coronavirus spike protein) for a period of time longer than about 3 weeks; in some such embodiments, a whole Spike based vaccine dosing regimen (the administrations prior to the Spike RBD based vaccine) may involve only a single dose, or may involve two or more doses, which may, in some embodiments, be separated from one another by a period of time that is longer than about 21 days or three weeks.
  • such period of time may be about 4 weeks, 5 weeks, 6 weeks 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or more, or about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10, months, 11 months, 12 months or more, or in some embodiments about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years or more.
  • a first dose and a second dose of a whole Spike based vaccine may be administered by intramuscular injection.
  • a first dose and a second dose may be administered in the deltoid muscle. In some embodiments, a first dose and a second dose may be administered in the same arm. In some embodiments, a whole spike based vaccine composition described herein is administered (e.g., by intramuscular injection) as a series of two doses (e.g., 0.3 ml each) 21 days part.
  • composition as described herein can be administered by any convenient route as described herein, such as via the intramuscular, dermal, intranasal, subcutaneous, intravenous, intraperitoneal or oral routes.
  • composition as described herein is formulated for or adapted for administration by any convenient route as described herein, such as via the intramuscular, intranasal, subcutaneous, dermal, intravenous, intraperitoneal or oral routes.
  • the upper respiratory tract may include the following regions: nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords).
  • the lower respiratory tract includes the following regions: portion of the larynx below the vocal folds, trachea, bronchi and bronchioles.
  • the lungs can be included in the lower respiratory tract and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.
  • administration to the URT may be administration to the nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords). Also contemplated is administration to any one or more regions of the URT provided that the compound is retained in the URT or does not contact a region of the LRT.
  • the composition as described herein may be formulated for administration to the URT only. Limitation to the URT may be achieved by an amount, particularly volume and composition of form i.e. particle size, physical form whether dry powder or solution droplet, of composition that would otherwise be administered to the LRT or TRT.
  • the vaccine composition as described herein may be administered via a device that ensures retention in the URT only.
  • composition as described herein may be formulated for intranasal administration, including dry powder, sprays, mists, or aerosols. This may be particularly preferred for treatment of coronavirus infection.
  • the compound can be formulated into a solution, e.g., water or isotonic saline, buffered or unbuffered, or as a suspension, for intranasal administration as drops or as a spray.
  • a solution e.g., water or isotonic saline, buffered or unbuffered, or as a suspension
  • such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0.
  • Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers.
  • a representative nasal decongestant is described as being buffered to a pH of about 6.2 (Remington's, Id. at page 1445).
  • a suitable saline content and pH for an innocuous aqueous carrier for nasal and/or upper respiratory administration is described as being buffered to a pH of about 6.2 (Remington's, Id. at page 1445).
  • the ordinary artisan can readily determine a suitable saline content and pH for an innocuous aqueous carrier for nasal and/or upper respiratory administration.
  • ingredients such as art known preservatives, colorants, lubricating or viscous mineral or vegetable oils, perfumes, natural or synthetic plant extracts such as aromatic oils, and humectants and viscosity enhancers such as, e.g., glycerol, can also be included to provide additional viscosity, moisture retention and a pleasant texture and odour for the formulation.
  • various devices are available in the art for the generation of drops, droplets and sprays.
  • a vaccine composition described herein can be administered into the nasal passages by means of a simple dropper (or pipet) that includes a glass, plastic or metal dispensing tube from which the contents are expelled drop by drop by means of air pressure provided by a manually powered pump, e.g., a flexible rubber bulb, attached to one end.
  • a simple dropper or pipet
  • a manually powered pump e.g., a flexible rubber bulb
  • a suitable pharmaceutically acceptable ophthalmic solution can be readily provided by the ordinary artisan as a carrier for the vaccine composition described herein to be delivered and can be administered to the orbit of the eye in the form of eye drops to provide for both ophthalmic and intranasal administration.
  • a premeasured unit dosage dispenser that includes a dropper or spray device containing a solution or suspension for delivery as drops or as a spray is prepared containing one or more doses of the drug to be administered.
  • the invention also includes a kit containing one or more unit dehydrated doses of vaccine composition, together with any required salts and/or buffer agents, preservatives, colorants and the like, ready for preparation of a solution or suspension by the addition of a suitable amount of water.
  • the water may be sterile or nonsterile, although sterile water is generally preferred.
  • composition may be administered via a skin patch.
  • Vaccine Efficacy
  • the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein are administered in effective amounts to induce an immune response to a coronavirus.
  • an immune response to a vaccine composition is the development in a subject of a humoral response to a (one or more) coronavirus protein(s) present in the composition.
  • a "humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules.
  • an immune response is assessed by determining [protein] antibody titre in the subject.
  • the ability of serum or antibody from an immunized subject is tested for its ability to neutralise viral uptake or reduce viral transformation of human cells.
  • the ability to promote a robust T cell response(s) is measured.
  • an antigen-specific immune response is characterized by measuring an anti-antigen antibody titre produced in a subject administered a composition as provided herein, wherein the antigen is a SARS-CoV-2 S protein (e.g., prefusion stabilized S protein).
  • An antibody titre is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen.
  • Antibody titre is typically expressed as the inverse of the greatest dilution that provides a positive result.
  • a variety of serological tests can be used to measure antibody against encoded antigen of interest, for example, SARS-CoV-2 virus or SARS-CoV-2 viral antigen, e.g., SARS-CoV-2 spike or S protein, of domain thereof.
  • SARS-CoV-2 virus or SARS-CoV-2 viral antigen e.g., SARS-CoV-2 spike or S protein, of domain thereof.
  • These tests include the hemagglutination-inhibition test, complement fixation test, fluorescent antibody test, enzyme-linked immunosorbent assay (ELISA), microneutralisation test, pseudovirus neutralisation test, surrogate virus neutralisation tests (RBD-ACE2 binding inhibition assays) and plaque reduction neutralisation test (PRNT). These tests measures antibody activities in a range of different settings.
  • neutralising assays such as a plaque reduction neutralisation test, or PRNT (e.g., PRNT50 or PRNT80), or microneutralisation assays, areis used as a serological correlates of protection. These assays measure the biological parameter of in vitro virus neutralisation and are the most serologically virus-specific test among certain classes of viruses, correlating well to serum levels of protection from virus infection.
  • the basic design of the PRNT allows for virus-antibody interaction to occur in a test tube or microtitre plate, and then measuring antibody effects on viral infectivity by plating the mixture on virus-susceptible cells, preferably cells of mammalian origin.
  • virus-susceptible cells preferably cells of mammalian origin.
  • the cells are overlaid with a semi-solid media that restricts spread of progeny virus.
  • Each virus that initiates a productive infection produces a localized area of infection (a plaque), that can be detected in a variety of ways. Plaques are counted and compared back to the starting concentration of virus to determine the percent reduction in total virus infectivity.
  • the serum sample being tested is usually subjected to serial dilutions prior to mixing with a standardized amount of virus.
  • the concentration of virus is held constant such that, when added to susceptible cells and overlaid with semi-solid media, individual plaques can be discerned and counted. In this way, PRNT end-point titres can be calculated for each serum sample at any selected percent reduction of virus activity.
  • the serum sample dilution series for antibody titration should ideally start below the "seroprotective" threshold titre.
  • the "seroprotective" threshold titre remains unknown; but a seropositivity threshold of 1 : 10 can be considered a seroprotection threshold in certain embodiments.
  • PRNT end-point titres are expressed as the reciprocal of the last serum dilution showing the desired percent reduction in plaque counts.
  • the PRNT titre can be calculated based on a 50% or greater reduction in plaque counts (PRNT50).
  • a PRNT50 titre is preferred over titres using higher cut-offs (e.g., PRNT80) for vaccine sera, providing more accurate results from the linear portion of the titration curve.
  • PRNT titres There are several ways to calculate PRNT titres. The simplest and most widely used way to calculate titres is to count plaques and report the titre as the reciprocal of the last serum dilution to show >50% reduction of the input plaque count as based on the back-titration of input plaques. Use of curve fitting methods from several serum dilutions may permit calculation of a more precise result. There are a variety of computer analysis programs available for this (e.g., SPSS or GraphPad Prism).
  • an antibody titre is used to assess whether a subject has had an infection or to determine whether immunisations are required. In some embodiments, an antibody titre is used to determine the strength of an autoimmune response, to determine whether a booster immunisation is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. In accordance with the present invention, an antibody titre may be used to determine the strength of an immune response induced in a subject by a vaccine composition.
  • antibody-mediated immunogenicity in a subject is assessed at one or more time points.
  • Methods of assessing antibody-mediated immunogenicity include geometric mean concentration (GMC) of antibody to antigen, geometric mean fold rise (GMFR) in serum antibody, geometric mean titre (GMT), median, minimum, maximum, 95% confidence interval (Cl), geometric mean ratio (GMR) of post-baseline/ baseline titres, and seroconversion rate.
  • the GMC is the average antibody concentration for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data.
  • GMT is the average antibody titre for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data.
  • a control in some embodiments, is an anti-antigen antibody titre produced in a subject who has not been administered a vaccine composition, or who has been administered a saline placebo (an unvaccinated subject).
  • an immune response may comprise generation of a neutralising antibody titre against coronavirus protein (including, e.g., a stabilized prefusion spike trimer in some embodiments) or a fragment thereof.
  • an immune response may comprise generation of a neutralising antibody titre against the receptor binding domain (RBD) of the coronavirus spike protein.
  • RBD receptor binding domain
  • a provided immunogenic composition has been established to achieve a neutralising antibody titre in an appropriate system (e.g., in a human infected with coronavirus and/or a population thereof, and/or in a model system thereof).
  • a neutralising antibody titre is a titre that is (e.g., that has been established to be) sufficient to reduce viral infection relative to that observed for an appropriate control. In some such embodiments, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.
  • induction of a neutralising antibody titre may be characterized by an elevation in the number of B cells, which in some embodiments may include plasma cells, class-switched lgG1 - and lgG2-positive B cells, and/or germinal center B cells.
  • induction of a neutralising antibody titre may be characterized by a reduction in the number of circulating B cells in blood.
  • the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein generates antibodies to, preferably neutralising antibodies, any one or more of the coronavirus strains described herein, particularly those in Table 3.
  • the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein provides a therapeutic or prophylactic treatment for any or more of the coronavirus strains described herein, particularly those in Table 3.
  • reference to “a coronavirus” also includes reference to one or more strains of coronavirus as described herein, including but not limited to those in Table 3, for example: WT, alpha and beta strains; WT, alpha, beta and VIC2089 strains; WT, alpha, beta, and delta strains; WT, alpha, beta, delta, delta plus, gamma, lambda, mu, kappa, iota and omicron strains (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
  • the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein may provide a therapeutic or prophylactic benefit against SARS-CoV-2 strains that are different from which the RBD amino acid sequence in the RBD antigen, chimeric or fusion protein was derived.
  • Exemplary strains against which the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein provides a therapeutic or prophylactic benefit are shown in the Examples.
  • the present invention provides a method of treating and/or preventing a disease associated with a coronavirus, the method comprising administering to the subject in need thereof, a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby treating and/or preventing a disease associated with a coronavirus, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention provides a method of treating and/or preventing a disease associated with, or caused by, a coronavirus, the method comprising administering to a subject in need thereof a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby treating and/or preventing a disease associated with, or caused by, a coronavirus, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention provides a method of treating and/or preventing a respiratory disease or condition associated with a coronavirus infection, the method comprising administering to a subject in need thereof, a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby treating and/or preventing a respiratory disease or condition associated with a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention provides a method for reducing airway inflammation associated with, or caused by, a coronavirus, the method comprising administering to a subject in need thereof, a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby reducing airway inflammation associated with, or caused by, a coronavirus, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention also provides a method of improving the ability of a subject to control a respiratory disease or condition during a coronavirus infection, the method comprising administering to a subject in need thereof, a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby improving the ability of a subject to control a respiratory disease or condition during a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen
  • the present invention provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for raising an immune response in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the present invention provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for increasing an immune response in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine
  • RBD coronavirus Spike receptor binding domain
  • the present invention provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for treating and/or preventing a disease caused by a coronavirus in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the present invention further provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for treating and/or preventing a respiratory disease or condition associated with a coronavirus infection in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the present invention further provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for treating and/or preventing a coronavirus infection in a subject who has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the present invention further provides use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for reducing airway inflammation in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the present invention further provides use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for improving the ability of a subject to control a respiratory disease or condition during a coronavirus infection preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the present invention provides for a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in raising an innate immune response in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the present invention provides for a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in increasing an innate immune response in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine
  • RBD coronavirus Spike receptor binding domain
  • the present invention provides for a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in treating and/or preventing a disease caused by a coronavirus in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the present invention provides for a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in treating and/or preventing a respiratory disease or condition associated with a coronavirus infection in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the invention provides a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in reducing airway inflammation in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • the invention provides a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in controlling a respiratory disease or condition during a coronavirus infection in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein is administered to the subject before any clinically or biochemically detectable symptoms of viral infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • RBD coronavirus Spike receptor binding domain
  • administering reduces viral load in the subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
  • the viral load is reduced in the respiratory tract, for example the upper and/or lower respiratory tract.
  • the viral load is reduced in the nasal cavity and pharynx (i.e. throat).
  • the viral load may be in the gastrointestinal tract, in the peripheral circulation, in the heart, liver, kidney, spleen or other organ known to be susceptible to infection with coronavirus, preferably to infection with SARS-CoV-2.
  • coronavirus infection refers to infection with a coronavirus such as SARS-CoV-2, MERS-CoV, or SARS-CoV.
  • coronavirus respiratory tract infections often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhoea, organ failure (kidney failure and renal dysfunction), septic shock, and death in severe cases.
  • a reduction in coronavirus infection may be determined using any method known in the art or described herein, including measuring viral load in a sample from the subject after treatment and comparing it to viral load in a sample from the same subject before treatment.
  • the sample may be any biological sample obtained from the subject, and may include blood, saliva, urine, faeces, nasal wash, sputum, and mucous secretions.
  • the sample may be taken from the respiratory tract, preferably the upper respiratory tract, for example the nose or pharynx (i.e. throat).
  • 'respiratory disease' or 'respiratory condition' refers to any one of several ailments that involve inflammation and affect a component of the respiratory system including the upper (including the nasal cavity, pharynx and larynx) and lower respiratory tract (including trachea, bronchi and lungs).
  • the inflammation in the upper and lower respiratory tract may be associated with or caused by viral infection.
  • a symptom of respiratory disease may include cough, excess sputum production, a sense of breathlessness or chest tightness with audible wheeze.
  • a parameter measured may be the presence or degree of lung function, signs and symptoms of obstruction; exercise tolerance; night time awakenings; days lost to school or work; bronchodilator usage; Inhaled corticosteroid (ICS) dose; oral glucocorticoid (GC) usage; need for other medications; need for medical treatment; hospital admission.
  • ICS Inhaled corticosteroid
  • GC oral glucocorticoid
  • the term respiratory infection means an infection anywhere in the respiratory tract.
  • respiratory infection include but are not limited to colds, sinusitis, throat infection, tonsillitis, laryngitis, bronchitis, pneumonia, or bronchiolitis.
  • the respiratory infection is a cold.
  • An individual or subject may be identified as having a respiratory tract infection by viral testing and may exhibit systems of itchy watery eyes, nasal discharge, nasal congestion, sneezing, sore throat, cough, headache, fever, malaise, nausea, vomiting, fatigue and weakness.
  • a subject having a respiratory infection may not have any other respiratory condition.
  • Detection of the presence or amount of virus, preferably coronavirus may be by PCR/sequencing of RNA isolated from clinical samples (nasal wash, sputum, BAL) or serology.
  • respiratory infection means an infection by a coronavirus, preferably by SARS-CoV-2, anywhere in the respiratory tract.
  • An individual or subject may be identified as having a respiratory tract infection by viral testing and may exhibit symptoms of itchy watery eyes, nasal discharge, nasal congestion, sneezing, sore throat, cough, headache, fever, malaise, nausea, vomiting, fatigue and weakness.
  • treatment includes the application or administration of an RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein to a subject (or application or administration of an RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein to a cell or tissue from a subject) with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition.
  • treating refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.
  • a positive response to vaccination may include any amelioration or improvement of symptoms experienced by the subject.
  • a positive response to vaccination may be a reduction in general levels of fatigue, muscle pain, headache and/or lethargy in the subject.
  • a positive response may also include a reduction in fever, and a return to afebrile state in the subject.
  • a positive response to vaccination may also be prevention or attenuation of worsening of respiratory symptoms following a respiratory virus infection. This could be assessed by comparison of the mean change in disease score from baseline to end of study period, for example, based on a questionnaire, and could also assess lower respiratory symptom score (LRSS - symptoms of chest tightness, wheeze, shortness or breath and cough) daily following infection/onset of cold symptoms. Change from baseline lung function (peak expiratory flow PEF) could also be assessed and a positive response to therapy could be a significant attenuation in reduced PEF.
  • LRSS lower respiratory symptom score
  • a positive response to vaccination may also be a reduction in the presence of ground-glass type opacities in the lung periphery or near the pleura (for example, as determined using chest CT imaging techniques).
  • a positive response to vaccination may also include an increase or return to normal levels of blood oxygenation levels.
  • a positive response to vaccination may also include an improvement in cardiovascular disorders such as alterations in blood pressure and increased presence of clotting factors.
  • Protective immune responses can include humoral immune responses and cellular immune responses. Protection against SARS-CoV-2 is believed to be conferred through serum neutralising antibodies (humoral immune response) directed to the spike protein, with mucosal IgA antibodies and cell-mediated immune responses also playing a role. Cellular immune responses are useful in protection against SARS-CoV-2 infection with CD4+ and CD8+ T cell responses and memory B cell responses being particularly important. CD8+ immunity is of particular importance in killing virally infected cells. Natural killer cells and NKT cells may also be important for killing and/or clearance of virally infected cells.
  • Beta variant RBD vaccine could perform as well as, or better than, a Beta-specific whole spike protein vaccine in its ability to boost immunity in general, and whether it can overcome imprinting, in particular in the WT strain spike primed mice - compare the immune responses elicited by WT or the beta variant targeted boosts against other variant SARS-CoV-2 coronaviruses including alpha, beta, gamma, delta, kappa and omicron.
  • the resulting PCR product was cloned into the mammalian expression vector pXC-17.4 using the restriction enzymes sites Hindlll and EcoRI to produce the polynucleotide sequence as set forth in SEQ ID NO: 14.
  • the RBD-human IgG 1 Fc fusion protein was expressed by transient transfection of Expi293S cells using ExpiFectamine 293 Transfection Kits as per manufacturer’s instruction (ThermoFisher Scientific). RBD-Fc protein was harvested on day six. RBD-Fc protein was purified from supernatants by Protein A Sepharose (CL- 4B, Cytiva). The RBD-Fc protein was further purified by gel filtration size exclusion chromatography using a Superdex-200 column (Cytiva). The RBD-Fc protein was sterile filtered and stored at -80°C prior to use.
  • a research cell bank (RCB) for a stable pool expressing the beta RBD-hFc protein was developed at the National Biologies Facility (QLD node). Cells from a single vial were revived and expanded in shake flasks and the protein produced in a fed batch shaker flask production over 12 days according to the Lonza GSv9TM recommendations. The conditioned media was harvested by depth filtration and 0.2 pm filtration. The antigen was captured on a protein A resin, MabSelect PrismA (Cytiva) with elution at pH 4.0. The antigen was subjected to low pH viral inactivation, with acidification to pH 3.5, holding for > 60 min, then neutralisation to pH 5.5. The antigen was buffer exchanged to pH 6.5 citrate buffer and formulated with PS80 to 0.02 % and diluted to 1 .06 mg/mL before being aseptically filled to vials.
  • QLD node National Biologies Facility
  • Enzyme-linked immunosorbent assay for measurement of RBD-specific antibody responses
  • RBD-specific antibody titres were determined by ELISA.
  • Flat bottom 96 well maxisorp plates (ThermoFisher Scientific) were coated with 50 pl/well of RBD monomer at a concentration of 2 pg/ml in Dulbecco’s phosphate buffered saline (DPBS; Gibco Life Technologies). Plates were incubated overnight at 4°C in a humidified atmosphere. Unbound antibody was removed, and wells were blocked with 100 pl/well of 1 % bovine serum albumin (BSA fraction V, Invitrogen Corporation, Gibco) in PBS for 1 -2 hours before washing 3 times with PBS containing 0.05% v/v Tween-20 (PBST).
  • BSA fraction V bovine serum albumin
  • a Labsystems Multiskan microplate reader (Labsystems, Finland) was used to measure the optical density (OD) of each well at wavelengths of 450 nm and 540 nm.
  • the titres of Ab are expressed as the reciprocal of the highest dilution of serum required to achieve an OD of 0.3.
  • SARS-CoV-2 isolates used in the microneutralisation assay were propagated in Vero cell cultures and stored at -80°C.
  • Flat-bottom 96-well plates were seeded with Vero cells at 2 x 10 4 cells/well the day before assay.
  • Serial 2-fold dilutions of heat- inactivated sera were incubated with 100 TCIDso (50% tissue culture infectious dose) of SARS-CoV-2 for 1 hour and residual virus infectivity was assessed in quadruplicate wells of Vero cells. Plates were incubated at 37°C and viral cytopathic effect was read on day 5.
  • the neutralising antibody titre was calculated using the Reed/Muench method.
  • Example 2 Administering a booster dose of RBD-hFc dimer + MF59® to mice previously vaccinated with WT Spike vaccines to assess serological responses and to evaluate immunological imprinting.
  • mice Groups of 5 C57BL/6 mice were inoculated via the intramuscular route with 4.5pg of WT Spike protein, 10pg of WT RBD-hFc, 10pg of Beta RBD-hFc or 4.5pg of Beta spike protein in the presence of MF59®. Mice were primed on day 0 and boosted on day 21 . Mice were bled prior to the first inoculation (pre-bleed), 21 days after the first immunisation (1° bleeds), and 2 weeks (day 35) and 5 weeks (day 56) following the second immunisation (2° bleeds). [0272] Humoral responses were evaluated after the first and second immunisation and included anti-RBD antibody responses assessed by ELISA and measured against WT RBD monomer, Beta variant RBD monomer and Delta variant RBD monomer.
  • Example 3 IgG antibody responses to WT, Beta and Delta RBD monomers in mice vaccinated with 2 doses of WT Spike and boosted with WT RBD-hFc, Beta RBD-hFc, WT-spike or Beta spike.
  • mice previously vaccinated with 2 doses of WT-Spike protein and boosted with WT RBD-hFc ( Figure 4A, Figure 5A) or Beta RBD-hFc ( Figure 4B, Figure 5B) developed the highest mean Ab binding levels against all the RBD antigens compared to the levels observed in mice vaccinated with 2 doses of WT-Spike protein and boosted with WT-spike ( Figure 4C, Figure 5C) or Beta spike ( Figure 4D, Figure 5D).
  • Plasma samples from the 5 adults vaccinated with Comirnaty exhibited the lowest Ab binding levels against all the RBD antigens assessed (Figure 5E).
  • Example 4 Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or Beta RBD-hFc + MF59®
  • Beta-RBD-hFc + MF59® boost provided enhanced mean nAb responses not only against the WT virus but even more prominently against the Beta virus strain.
  • the boost provided by the Beta- RBD + MF59® was equal to, or better than, the boost provided by the third dose of WT spike + MF59® vaccine.
  • nAb responses were assessed by examining the ability of antibodies in 2° and 3° bleeds to inhibit the interaction between RBD antigens and human ACE2 using the RBD-ACE2 multiplex inhibition assay (Figure 7). Strong neutralising activity against the WT RBD, Beta RBD and Delta RBD (indicated as the half-maximal inhibitory dilution (ID50)) was observed in secondary sera from all groups of mice.
  • ID50 half-maximal inhibitory dilution
  • Example 5 Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or WT RBD-hFc + MF59®’ or Beta RBD-hFc + MF59®, or Beta Spike + MF59®.
  • An in vitro micro-neutralisation assay measured the level of SARS-CoV-2- specific nAb in sera of immunised mice.
  • Stocks of SARS-CoV-2 WT VIC01 used in the microneutralisation assay were propagated and assayed in Vero cell cultures.
  • Stocks of the SARS-CoV-2 omicron variant were propagated and assayed in VeroE6/TMPRSS-2 cells (Matsuyama et al. Proc Natl Acad Sci U S A.
  • TMPRSS2 human transmembrane serine protease
  • TMPRSS2 Cell Bank Australia JCRB1819 http://www.cellbankaustralia.com/veroe6-tmprss2.html.
  • Viral stocks were stored at -80°C.
  • Flat-bottom 96-well plates were seeded with Vero or VeroE6/TMPRSS2 cells at 2 x 10 4 cells/well the day before assay.
  • Serial 2-fold dilutions of heat-inactivated sera were incubated with 10 TCID50 (50% tissue culture infectious dose) of SARS-CoV-2 for 1 hour and residual virus infectivity was assessed in quadruplicate wells.
  • Figure 12 shows nAb responses of experimental replicates against infection of
  • WT VIC01 (open circle) in Vero cells or omicron variant BA.1 (filled diamond) in VeroE6/TMPRSS2 cells with the tertiary sera (3°) collected 50 days following the third immunisation from groups of 5 mice immunised intramuscularly with WT-spike/WT- spike/WT-spike (groupl ), or WT-spike/WT-spike/WT- RBD-hFc (group 2), or WT-spike/ WT-spike/Beta RBD-hFc (group 3), or WT-spike/WT-spike/Beta-spike (group 4) on day 70.
  • the three vaccinations were administered on day 0, 21 and 70 in the presence of MF59®.
  • the half-maximal inhibitory dilution (ID50) was calculated based on the reciprocal dilution of serum that completely prevented cytopathic effect (CPE) in 50% of the wells and was calculated by the Reed-Muench formula.
  • LOD limit of detection
  • MNS mouse non-immune serum
  • WT-Spike wild type spike
  • WT RBD-hFc wild type RBD-human Fc dimer
  • beta-RBD-hFc beta variant RBD-human Fc dimer
  • beta-spike beta variant spike).
  • Group 1 WT spike x 3; Average titre 112
  • Group 2 WT spike x 2, WT RBD x 1 ; Ave titre 203
  • Group 3 WT spike x 2, beta RBD x 1 ; Ave titre 290
  • Group 4 WT spike x 2, beta spike x 1 ; Ave titres 51 .
  • mice were next tested serum samples from these mice for their ability to neutralise the omicron variants-of-concern (using a later bleed collected 50 days after the third dose).
  • the inventors performed the micro-neutralisation assay using 100 TCID50 virus units/well of omicron BA.1 virus. All samples from mice immunised with 3 doses of WT Spike vaccine showed no neutralising activity above the lower limit of detection ( Figure 13) noting the 10-fold higher TCID50 dose used in this assay compared to the previous assay ( Figure 12). Similarly, all but one sample from beta spike vaccine boosted mice had undetectable nAb. In contrast, 2/5 samples from WT RBD vaccine boosted mice and 4/5 from beta RBD vaccine boosted mice had clearly detectable nAb titres against omicron BA.1 virus.
  • the inventors tested serum samples from the heterologous third dose boost experiments described above, in a different RBD-ACE2 binding inhibition (sVNT) bead assay carrying a broad panel of CoV-derived RBDs of CoV-derived RBDs including some of the SARS-CoV-2 variants-of-concern RBDs described above, as well as omicron BA.2, plus bat CoV BANAL-52 and BANAL-236 and the pangolin CoV GD-1 .
  • sVNT RBD-ACE2 binding inhibition
  • samples were collected 30 days (day 100) following the third immunisation from groups of 5 mice immunised intramuscularly with WT-spike/WT-spike/WT-spike (group 1 ), or WT-spike/WT-spike/WT- RBD-hFc (group 2), or WT-spike/ WT-spike/Beta RBD-hFc (group 3), or WT-spike/WT-spike/Beta-spike (group 4).
  • Vaccinations were administered on days 0, 21 and 70, all in the presence of MF59®.
  • the results described herein suggest that the RBD-hFc vaccine works well not only as a prime and boost vaccine, but also as a booster vaccine for mice that have previously been primed and boosted with WT spike vaccines. Furthermore, the response to the RBD vaccines (WT and beta) were stronger than those from a third dose of whole spike vaccine (WT and beta), which suggests the third dose RBD vaccine boost is focussing the immune response on the critical part of the spike, being the RBD, for enhancing the nAb response.
  • beta RBD-Fc vaccine enhanced nAb responses across all variant-RBDs tested, including the original WT, lambda, gamma, mu and importantly, the delta, delta plus and omicron variants (including subvariants BA.1 and BA.2).

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Abstract

The present invention relates to novel prime-boost and heterologous boost regimens for immunisation against coronavirus infections. The method involves immunization of a subject or increasing an immune response of a subject previously exposed to coronavirus, comprising administering a therapeutically effective amount of a vaccine comprising a coronavirus spike receptor binding domain (RBD) antigen or a nucleic acid encoding the RBD antigen.

Description

Coronavirus vaccination regimen
Related application
[0001] This application claims the benefit of priority to Australian provisional application no. 2022900288 filed 11 February 2022, Australian provisional application no. 2022900729 filed 23 March 2022, Australian provisional application no. 2022902026 filed 20 July 2022 and Australian provisional application no. 2022903640 filed 30 November 2022, the entire contents of each are incorporated herein by reference.
Field of the invention
[0002] The present invention relates to novel prime-boost regimens for immunisation against coronavirus infections.
Background of the invention
[0003] A significant bottleneck in vaccine efficacy is the ability to induce a strong and effective immune response that is maintained for a period of time. Conventional approaches to provide a vaccination regimen that produces a sufficiently strong, effective and/or long-lasting immune response and/or protection is to either: (i) administer the vaccine composition in increased dosages; or (ii) additionally administer one or more subsequent vaccinations (so called "boost" vaccinations) after the initial (so called "prime") vaccination. However, such conventional approaches are limited for a number of reasons, including where increased amounts of the vaccine composition are needed or where subsequent vaccinations do not significantly increase the immunity.
[0004] Emerging respiratory coronaviruses offer a considerable threat to the health of global populations and the economy. Coronaviruses (CoVs) constitute a group of phylogenetically diverse enveloped viruses that encode the largest plus strand RNA genomes and replicate efficiently in most mammals. Human CoV (HCoVs-229E, OC43, NL63, and HKU 1 ) infections typically result in mild to severe upper and lower respiratory tract disease.
[0005] Coronaviruses, belong to the Coronaviridae family in the Nidovirales order, are minute in size (65-125 nm in diameter) and contain a single-stranded RNA as a nucleic material, size ranging from 26 to 32kbs in length. The subgroups of coronaviruses family are alpha (a), beta ( ), gamma (y) and delta (5) coronavirus. The severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) cause acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) which leads to pulmonary failure and result in fatality.
[0006] Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emerged in 2002-2003 causing acute respiratory distress syndrome (ARDS) with 10% mortality overall and up to 50% mortality in aged individuals. Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) emerged in the Middle East in April of 2012, manifesting as severe pneumonia, acute respiratory distress syndrome (ARDS) and acute renal failure.
[0007] In since late in 2019, the world has been faced with an extreme situation of a highly infectious coronavirus (2019-nCoV; SARS-CoV2) encountered by a global immunologically naive population, manifesting as a disease termed “COVID-19”.
COVID-19 manifestations range from mild to severe life-threatening with a substantial mortality rate.
[0008] While vaccines to increase immunity against coronavirus infection have been developed, there is an emergence of data showing waning humoral immunity levels and SARS-CoV-2 variants tending to reduce vaccine efficacy. Therefore, there is a need to provide a vaccination regimen that can boost immunity and preferably minimise imprinting.
[0009] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.
Summary of the invention
[0010] The present invention is based on the surprising finding by the inventors that administration of a Spike protein receptor binding domain (RBD) based vaccine to a subject, who has been naturally infected by a coronavirus or who has received a prime and boost of a coronavirus whole Spike protein based vaccine, leads to an increase in humoral immunity including (a) enhancing the level of RBD specific antibodies against wildtype (WT) RBD and a number of RBD variants, (b) enhancing the neutralising activity as determined by inhibition of interaction between RBD antigens and human ACE2, and (c) enhancing the level of RBD neutralising antibodies against the WT RBD and variants, including the beta, delta and omicron variants (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
[0011] In one aspect, the present invention provides a method for raising an immune response in a subject who has been exposed to a coronavirus whole Spike protein, for example in the form of a prior infection or a prior vaccination, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby raising an immune response in the subject.
[0012] In one aspect, the present invention provides a method for increasing an immune response in a subject who has been exposed to a coronavirus whole Spike protein, for example in the form of a prior infection or a prior vaccination, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby increasing an immune response in the subject.
[0013] In another aspect, the present invention provides a method for providing cross coronavirus variant immunity in a subject who has been exposed to a coronavirus whole Spike protein, for example in the form of a prior infection or a prior vaccination, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby providing cross coronavirus variant immunity in the subject.
[0014] In one embodiment, the coronavirus Spike receptor binding domain (RBD) antigen may be a chimeric or fusion protein as described herein. In one embodiment, the nucleic acid encoding a coronavirus Spike RBD antigen may encode a chimeric or fusion protein as described herein.
[0015] In one embodiment, the present invention provides a method for raising an immune response in a subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein as described herein or nucleic acid encoding a chimeric or fusion protein as described herein, or a pharmaceutical composition as described herein, thereby raising an immune response in the subject.
[0016] In one embodiment, the present invention provides a method for increasing an immune response in a subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein as described herein or nucleic acid encoding a chimeric or fusion protein as described herein, or a pharmaceutical composition as described herein, thereby increasing an immune response in the subject.
[0017] In another aspect, the present invention provides a method for immunizing a subject against a coronavirus infection, the method comprising a step of administering to a subject a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, wherein the subject has been exposed to a coronavirus whole Spike protein, thereby immunizing a subject against a coronavirus infection.
[0018] In another aspect, the present invention provides a method for immunizing a subject against a coronavirus infection, the method comprising the steps of:
(i) administering a first and second dose of a vaccine comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen; and
(ii) subsequently to (i), administering a dose of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby immunizing the subject against a coronavirus infection.
[0019] In another aspect, the present invention provides a method of inducing, or increasing, a humoral immune response to a coronavirus in a subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby inducing, or increasing, a humoral immune response to a coronavirus in the subject.
[0020] In another aspect, the present invention provides a method for reducing or minimising the severity of a symptom associated with an infection with coronavirus, comprising:
- administering one or two doses of a vaccine comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen; and
- subsequently, administering one or more doses of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen.
[0021] In another aspect, the present invention provides a method for reducing or minimising the severity of a symptom associated with an infection with coronavirus in subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby reducing or minimising the severity of a symptom associated with an infection with coronavirus.
[0022] In any aspect or embodiment, the symptoms of a coronavirus infection are selected from the group consisting of fever, dry cough, tiredness, aches and pains, sore throat, diarrhoea, nausea and vomiting, conjunctivitis, headache, loss of taste or smell, a rash on skin, or discolouration of fingers or toes, difficulty breathing or shortness of breath, chest pain or pressure, and loss of speech or movement.
[0023] In any aspect, a method of the invention further comprises a step of identifying a subject at risk of coronavirus infection (for example at risk of infection with any coronavirus described herein, including SARS-CoV-2 or any variant thereof such as those described herein) or who has been exposed to a whole Spike protein through a naturally acquired infection of a coronavirus or by immunisation with a coronavirus whole Spike based vaccine that is either a protein based vaccine that comprises a whole Spike protein or a nucleic acid (e.g. DNA, RNA, preferably mRNA) based vaccine that encodes a whole Spike protein. [0024] In another aspect, the present invention provides for use of a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen in the preparation of a medicament or vaccine for treating and/or preventing (a) a disease associated with, or caused by, a coronavirus, or (b) a coronavirus infection in a subject in need thereof who has been exposed to a coronavirus whole Spike protein.
[0025] In any embodiment, the invention also provides for use of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen for inhibiting or reducing the amount of coronavirus particles in a tissue or organ in a subject who has been exposed to a coronavirus whole Spike protein. In any aspect or embodiment, the tissue or organ may be all or part of any tissue or organ described herein, or that is known to have detectable coronavirus particles. For example, the tissue or organ may be all, or part of, the upper respiratory tract (URT) or lower respiratory tract (LRT). An inhibition or reduction in the amount of coronavirus particles in a tissue or organ may be determined by any means described herein, and may involve determining the amount of coronavirus particles in a sample of the tissue or organ or a bodily fluid that has originated from or is in contact with the tissue or organ.
[0026] In a further aspect, the invention provides a method of inhibiting or reducing the amount of coronavirus particles in a tissue or organ of a subject (e.g. upper respiratory tract), the method comprising administering a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby inhibiting or reducing the amount of coronavirus particles in the tissue or organ of the subject (e.g. upper respiratory tract).
[0027] In another aspect, the invention provides a method of inhibiting, delaying or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of a subject, the method comprising administering a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby inhibiting, delaying or reducing the progression of the coronavirus particle from the upper respiratory tract to the lungs of the subject.
[0028] In another aspect, the invention further provides for use of a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen in the preparation of a medicament for inhibiting, delaying or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of a subject.
[0029] In another aspect, the invention provides for use of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen for inhibiting, delaying or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of a subject.
[0030] In any aspect or embodiment described herein, a subject may have been, or still be, exposed to a whole Spike protein through a naturally acquired infection of a coronavirus, or by immunisation with a vaccine comprising a coronavirus whole Spike antigen that is either a protein based vaccine that comprises a whole Spike protein or a nucleic acid (e.g., DNA or RNA, preferably mRNA) based vaccine that encodes a whole Spike antigen. In a preferred embodiment, a subject exposed to a whole Spike protein as described herein has been exposed by immunisation with a vaccine comprising a coronavirus whole Spike antigen that is either a protein based vaccine that comprises a whole Spike protein or a nucleic acid (e.g., DNA or RNA, preferably mRNA) based vaccine that encodes a whole Spike protein. In any aspect or embodiment, the nucleic acid that encodes a whole spike protein may be provided in a vector (e.g. a viral vector, such as a chimp- or human- adenoviral vector or adenovirus-associated virus (e.g. recombinant replication-incompetent adenovirus type 26 vectors (Ad26)) or a lipid nanoparticle formulation.
[0031] Where a subject has received a protein or nucleic acid (e.g. DNA or RNA) based vaccine (e.g. where the vaccine comprises an antigen that is protein or encodes for an antigen, respectively) that comprises (or consists of), or encodes for, a coronavirus whole Spike protein, the subject has typically been immunised with at least two doses of a vaccine. For example, a prime (e.g. first dose) and boost immunisation (e.g. second or further dose). The prime and boost immunisation may be with the same vaccine (e.g. same vaccine platform, for example as shown in Table 2) or different vaccines (e.g. different vaccine platform, for example as shown in Table 2). For example, the subject may have received a first, or prime, dose or immunisation with a vaccine comprising a coronavirus whole Spike protein antigen and a subsequent second, or boost, dose or immunisation with a vaccine comprising a coronavirus whole Spike protein antigen, where the whole Spike protein antigen in the first or prime immunisation has a different amino acid sequence to the whole Spike protein antigen in the second or boost immunisation. Alternatively, the subject may have received a first, or prime, dose or immunisation with a vaccine comprising a coronavirus whole Spike protein antigen and a subsequent second, or boost, dose or immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid (e.g. DNA or RNA). Alternatively, the subject may have received a first, or prime, dose or immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid (e.g. DNA or RNA) and a subsequent second, or boost, dose or immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid (e.g. DNA or RNA). Or, the subject may have received a first, or prime, immunisation with a coronavirus whole Spike RNA based vaccine and a subsequent second, or boost, immunisation with a coronavirus whole Spike protein based vaccine. Or, the subject may have received a first, or prime, immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in a viral vector and a subsequent second, or boost, immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in a viral vector. Or, the subject may have received a first, or prime, immunisation with a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in a viral vector and a subsequent second, or boost, immunisation with a coronavirus whole Spike RNA based vaccine (i.e. a vaccine comprising a coronavirus whole Spike antigen encoding nucleic acid in the form of mRNA). Or, the subject may have received a first, or prime, immunisation with a vaccine comprising a coronavirus whole Spike antigen in the form of an inactivated virus and a subsequent second, or boost, immunisation with a vaccine comprising a coronavirus whole Spike antigen in the form of an inactivated virus. In any embodiment, the subject may have received any combination of vaccine as described herein with one, two, three, four or more further boosters, i.e a third, fourth, fifth, sixth immunisation, with a vaccine comprising a coronavirus whole Spike antigen, coronavirus whole Spike antigen-encoding nucleic acid, including nucleic acid in a viral vector, or a coronavirus whole Spike antigen in the form of an inactivated virus.
[0032] Where a subject has received at least two doses of a vaccine, the vaccines used may be the same or different. For example, where a subject has received a prime and boost with a vaccine comprising coronavirus whole Spike protein based vaccine or a prime and boost with a coronavirus whole Spike RNA based vaccine, the vaccines used for the prime and boost may be the same or different. For example, the vaccines may contain the same or different adjuvants, may contain the same or different amino acid (for protein based) or nucleotide (for RNA based) sequences provided. The vaccines may comprise, or encode for, the same or different Spike variants, or be derived from the same or different coronavirus (preferably SARS-CoV-2) strains.
[0033] As used herein, where reference to a vaccine comprising a coronavirus whole Spike antigen is made, also contemplated in that context is a whole Spike antigen based vaccine or a composition comprising a whole Spike antigen.
[0034] As used herein, where reference to a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen is made, also contemplated in that context is a coronavirus Spike receptor binding domain (RBD) based vaccine or a composition comprising a coronavirus Spike receptor binding domain (RBD) antigen.
[0035] As used herein, unless the context dictates otherwise, reference to an antigen includes both a protein antigen or a nucleic acid encoding a protein antigen.
[0036] In any aspect or embodiment herein, the subject may have been, or still be, exposed to any variant or strain of a coronavirus, preferably any variant or strain of a coronavirus described herein. The subject may have had, or still have, a coronavirus infection where the coronavirus is from any of the genera Alpha-coronavirus, Betacoronavirus, Gamma-coronavirus or Delta-coronavirus. Preferably, the coronavirus is from one of the Alpha-coronavirus subgroup clusters 1 a and 1 b or one of the Betacoronavirus subgroup clusters 2a, 2b, 2c, and 2d. The coronavirus may be any coronavirus that infects humans. Exemplary coronaviruses are SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1 , although the coronavirus may be any one as described herein or genotypic decedents thereof. Most preferably, the coronavirus infection is an infection with SARS-CoV-2, for example any of the SARS-CoV-2 variants described herein including beta, delta and omicron (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
[0037] In some embodiments, a subject who may have had, or still have, a coronavirus infection is determined by the presence of coronavirus-specific seroconversion (e.g. the presence of coronavirus-specific antibodies in serum). In one embodiment, the subject has significantly higher antibodies that bind and/or neutralise coronavirus (e.g. SARS-CoV-2) compared to uninfected and/or unvaccinated subjects. In one embodiment, the coronavirus-specific antibodies bind the Spike protein and are below a level that confers protection against coronavirus infection.
[0038] In accordance with the ‘protective neutralisation classification model’ (Khoury et al., Nature Med 27, 1205-1211 (2021 ), levels of neutralising antibody achieving a titre of 54 international units per ml against SARS-CoV-2 historical, prevailing and newly emergent strains will provide protection of 50% of vaccinees from infection resulting in detectable COVID-19 disease.
[0039] In any aspect, the coronavirus Spike receptor binding domain (RBD) based vaccine and/or the coronavirus whole Spike based vaccine may be formulated or adapted for administration subcutaneously, intramuscularly or via any other route described herein. In any aspect, the method comprises administering the coronavirus Spike receptor binding domain (RBD) based vaccine and/or the coronavirus whole Spike based vaccine subcutaneously, intramuscularly or via any other route described herein.
[0040] In any aspect or embodiment, the prime (or first), boost (or second) and further doses or immunisations are administered by the same route. Preferably, the route of administration is intramuscular.
[0041] Any vaccine described herein may include an adjuvant. The adjuvant may be any one known in the art or described herein.
[0042] Preferably, any method reduces or prevents dissemination of the coronavirus from the upper respiratory tract to the lungs.
[0043] In any aspect or embodiment, a method of the invention may be useful to treat or prevent a disease or condition associated with, or caused by, a coronavirus. Preferably, the disease is a respiratory disease.
[0044] In any aspect or embodiment of the invention described herein, the vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen may be anyone described herein. Preferably, the vaccine comprises, consists essentially of or consists of a chimeric or fusion protein as described herein or a nucleic acid that encodes a chimeric or fusion protein as described herein. [0045] In another aspect, the present invention also provides a method for obtaining an antibody directed to a coronavirus, the method comprising performing a method of the invention as described herein, and subsequent to administering vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, obtaining a sample from the subject that contains antibodies directed to a coronavirus. Preferably, the sample is a sample of blood or blood-derived components such as plasma or serum. Preferably the method further comprises purifying the antibodies from the subject, preferably purifying the antibodies directed to, or neutralising for, a coronavirus.
[0046] In another aspect, the present invention also provides an antibody preparation comprising an antibody directed to a coronavirus, wherein the antibody preparation is obtained or obtainable from a subject on whom a method of the invention as described herein has been performed.
[0047] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.
[0048] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.
Brief description of the drawings
[0049] Figure 1. Total ancestral or wildtype (WT) SARS-Cov-2 strain RBD specific antibodies in mice following primary and secondary boost of different antigen combinations. Total WT RBD-specific antibody titres in primary (1 °, day 21 ) and secondary (2°, day 35) sera from mice vaccinated intramuscularly on day 0 and 21 with the antigens indicated above each column. All antigens were administered with MF59®. ELISA titres are expressed as the reciprocal of the antibody dilution (Iog10) giving an absorbance of 0.3. This represents at least five times the background level of binding.
[0050] Figure 2: Total anti-RBD antibody titres specific for WT, Beta and Delta RBD monomers in mice following primary and secondary boost of different antigen combinations. Total antibody titres to WT RBD, Beta variant (0) RBD and Delta variant (5) RBD in secondary (day 56) sera from mice vaccinated intramuscularly on days 0 and 21 with the antigens indicated above each column. All antigens were administered with MF59®. ELISA titres are expressed as the reciprocal of the antibody dilution (logio) giving an absorbance of 0.3. This represents at least five times the background level of binding.
[0051] Figure 3. IgG antibody responses to WT, Beta and Delta RBD monomers in mice vaccinated with 2 doses of WT Spike and boosted with WT RBD-hFc, Beta RBD-hFc, WT-spike or Beta spike. The multiplex bead-based assay was used to assess RBD binding activity of secondary (day 56) sera collected 5 weeks following the second immunisation (2°) and tertiary sera collected 16 days (day 86) following the third immunisation (3°) from groups of 5 mice immunised intramuscularly with: (A) WT-spike/ WT-spike/ WT RBD-hFc, (B) WT-spike/ WT-spike/Beta RBD-hFc, (C) WT-spike/WT- spike/WT-spike or (D) WT-spike/WT-spike/Beta spike. All vaccinations were administered in the presence of MF59®. Antibody binding levels against the 3 RBD antigens were also assessed in day 35 plasma samples from 5 humans vaccinated on day 0 and 21 with Comirnaty (Pfizer mRNA) vaccine (E). Half-maximal effective dilution (EDso) for each test sample against WT-RBD, Beta-RBD and Delta RBD are displayed. Bars depict geometric mean and geometric SD.
[0052] Figure 4. Binding of serum antibodies to RBD variants in a multiplex binding assay. Assessing RBD binding activity of secondary (day 56) sera collected 5 weeks following the second immunisation (2°) and tertiary sera collected 16 days (day 86) following the third immunisation (3°) from groups of 5 mice immunised intramuscularly with (A) WT-spike/ WT-spike/ WT RBD-hFc, (B) WT-spike/ WT- spike/Beta RBD-hFc (C) WT-spike/WT-spike/WT-spike or (D) WT-spike/WT-spike/Beta spike. All vaccinations were administered in the presence of MF59. Half-maximal effective dilution (ED50) for each serum sample against WT RBD and 8 RBD Variants including alpha (a), Beta (0), Gamma (y), Delta (5) and Kappa (K). Bars depict geometric mean and geometric SD.
[0053] Figure 5. IgG antibody responses to nine RBD monomers in mice vaccinated with 2 doses of WT Spike and boosted with WT RBD-hFc, Beta RBD- hFc, WT-spike or Beta spike. Tertiary (day 86) sera collected from groups of 5 mice immunised intramuscularly with (A) WT-spike/WT-spike/WT RBD-hFc, (B) WT- spike/WT-spike/Beta RBD-hFc (C) WT-spike/WT-spike/WT-spike or (D) WT-spike/WT- spike/Beta-spike, in the presence of MF59® were assessed using the multiplex beadbased assay for their ability to bind to WT RBD and 8 RBD Variants. Half-maximal effective dilution (ED50) is indicated for each serum sample. For comparison ED50 levels measured in sera from human vaccinees 2 weeks following a day 21 boost with the Comirnaty (Pfizer mRNA) vaccine (E) are also displayed. This Figure is an alternate representation to Figure 4. Bars depict geometric mean and geometric SD.
[0054] Figure 6. Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or Beta RBD-hFc + MF59®. Neutralising antibody (nAb) responses against (A) WT VIC01 or (B) Beta variant B.1 .351 in secondary sera (2°) collected 5 weeks (day 56) following the second immunisation and tertiary sera (3°) collected 16 days (day 86) following the third immunisation from groups of 5 mice immunised intramuscularly with WT-spike/WT-spike/WT-spike or WT-spike/ WT-spike/Beta RBD-hFc. All vaccinations were administered in the presence of MF59®. The half-maximal inhibitory dilution (ID50) was calculated based on the reciprocal dilution of serum that completely prevented cytopathic effect (CPE) in 50% of the wells and was calculated by the Reed-Muench formula. (WT-S = wild type spike, [3 RBD-hFc = Beta RBD-hFc).
[0055] Figure 7. Neutralising Ab responses against WT, Beta and Delta RBDs in sera of mice vaccinated with 2 doses of WT spike + MF59® and boosted with RBD- hFc + MF59® or whole spike protein + MF59®. Secondary (day 56) sera collected 5 weeks following the second immunisation (2°) and tertiary sera collected 16 days (day 86) following the third immunisation (3°) from groups of 5 mice immunised intramuscularly with (A) WT-spike/ WT-spike/ WT RBD-hFc, (B) WT-spike/ WT- spike/Beta RBD-hFc (C) WT-spike/WT-spike/WT-spike or (D) WT-spike/WT-spike/Beta- spike were assessed using the RBD-ACE2 multiplex inhibition assay for their ability to inhibit the binding of ACE2 to WT-RBD, Beta-RBD and Delta RBD. Half-maximal Inhibitory dilution (ID50) is indicated for each serum sample. For comparison, ID50 levels measured in sera from human vaccinees 2 weeks following a day 21 boost with the Comirnaty (Pfizer mRNA S vaccine) (E) are also displayed. Bars depict geometric mean and geometric SD.
[0056] Figure 8. Neutralising Ab responses against 9 RBD antigens in sera of mice vaccinated with 2 doses of WT spike + MF59® and boosted with RBD-hFc + MF59® or whole spike protein + MF59®. Tertiary (day 86) sera collected from groups of 5 mice immunised intramuscularly with (A) WT-spike/ WT-spike/WT RBD-hFc, (B) WT-spike/WT-spike/Beta RBD-hFc (C) WT-spike/WT-spike/WT-spike or (D) WT- spike/WT-spike/Beta spike, in the presence of MF59® were assessed using the RBD- ACE2 multiplex inhibition assay for their ability to inhibit the binding of ACE2 to WT RBD and 8 RBD Variants. Half-maximal Inhibitory dilution (IDso) is indicated for each serum sample. For comparison IDso levels measured in sera from human vaccinees 2 weeks following a day 21 boost with the Comirnaty (Pfizer mRNA S vaccine) (E) are also displayed.
[0057] Figure 9. Mutations in the SARS-CoV-2 spike protein and neutralization of SARS-CoV-2 variants by human convalescent plasma samples (hCov) and sera from mice vaccinated with RBD-mouse lgG1-Fc dimer (RBD). Spike coding mutations and (frequency) with >10 local (Victoria, Australia) cases, mutations in the RBD region are shown in red.
[0058] Figure 10. Mutations in the Beta variant SARS-CoV-2 spike protein.
[0059] Figure 11. Mutations in the Omicron variant SARS-CoV-2 spike protein.
[0060] Figure 12. Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or WT RBD-hFc + MF59® or Beta RBD-hFc + MF59®, or Beta Spike + MF59®.
NAb responses of experimental replicates against infection of 10 TCID50 virus units/well of (A) WT VIC01 (open circle) in Vero cells or (B) omicron variant BA.1 (filled diamond) in VeroE6/TMPRSS2 cells with tertiary sera (3°) collected 50 days following the third immunisation from groups of 5 mice immunised intramuscularly with WT- spike/WT-spike/WT-spike (group 1 ), or WT-spike/WT-spike/WT- RBD-hFc (group 2), or WT-spike/ WT-spike/Beta RBD-hFc (group 3), or WT-spike/WT-spike/Beta-spike (group 4). The three vaccinations were administered on days 0, 21 and 70 in the presence of MF59®. The half-maximal inhibitory dilution (ID50) was calculated based on the reciprocal dilution of serum that completely prevented cytopathic effect (CPE) in 50% of the wells and was calculated by the Reed-Muench formula. (LOD = limit of detection, MNS = mouse non-immune serum, WT-Spike = wild type spike, WT RBD-hFc = wild type RBD-human Fc dimer, beta-RBD-hFc = beta variant RBD-human Fc dimer and beta-spike = beta variant spike). [0061] Figure 13. Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or WT RBD-hFc + MF59® or Beta RBD-hFc + MF59®, or Beta Spike + MF59®.
NAb responses of experimental replicates against infection of 100 TCID50 virus units/well of WT VIC01 in Vero cells or omicron variant BA.1 in VeroE6/TMPRSS2 cells with tertiary sera (3°) collected 50 days following the third immunisation from groups of 5 mice immunised intramuscularly with WT-spike/WT-spike/WT-spike (group 1 ), or WT- spike/WT-spike/WT- RBD-hFc (group 2), or WT-spike/ WT-spike/Beta RBD-hFc (group 3), or WT-spike/WT-spike/Beta-spike (group 4). The three vaccinations were administered on days 0, 21 and 70 in the presence of MF59®. The half-maximal inhibitory dilution (ID50) was calculated based on the reciprocal dilution of serum that completely prevented cytopathic effect (CPE) in 50% of the wells and was calculated by the Reed-Muench formula. (LOD = limit of detection, MNS = mouse non-immune serum, WT-Spike = wild type spike, WT RBD-hFc = wild type RBD-human Fc dimer, beta-RBD- hFc = beta variant RBD-human Fc dimer and beta-spike = beta variant spike).
[0062] Figure 14. Neutralising Ab responses against SARS-CoV-2 variants in sera of mice vaccinated with 2 doses of WT spike + MF59® and boosted with RBD- hFc + MF59® or whole spike protein + MF59®. Tertiary (day 100) sera collected from groups of 5 mice immunised intramuscularly with WT-spike/WT-spike/WT-spike (groupl ), or WT-spike/WT-spike/WT- RBD-hFc (group 2), or WT-spike/ WT-spike/Beta RBD-hFc (group 3), or WT-spike/WT-spike/Beta-spike (group 4) on days 0, 21 and 70, in the presence of MF59® were assessed using the RBD-ACE2 multiplex inhibition assay for their ability to inhibit the binding of ACE2 to WT RBD and 8 RBD Variants. Half-maximal Inhibitory dilution (ID50) is indicated for each serum sample.
[0063] Figure 15. Neutralising Ab responses against BANAL-52, BANAL-236 and GD-1 coronavirus types in sera of mice vaccinated with 2 doses of WT spike + MF59® and boosted with RBD-hFc + MF59® or whole spike protein + MF59®.
Tertiary (day 100) sera collected from groups of 5 mice immunised intramuscularly with WT-spike/WT-spike/WT-spike (groupl ), or WT-spike/WT-spike/WT- RBD-hFc (group 2), or WT-spike/ WT-spike/Beta RBD-hFc (group 3), or WT-spike/WT-spike/Beta-spike (group 4) on days 0,21 and 70, in the presence of MF59® were assessed using the RBD-ACE2 multiplex inhibition assay for their ability to inhibit the binding of ACE2 to 2 bat CoV RBDs BANAL-52 and BANAL-236, and the pangolin CoV GD-1 RBD. Half- maximal Inhibitory dilution (ID50) is indicated for each serum sample.
Description of the sequences
Table 1 : Sequences of coronavirus Spike proteins, RBD domains and vaccine constructs
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Detailed description of the embodiments
[0064] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
[0065] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.
[0066] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.
[0067] All of the patents and publications referred to herein are incorporated by reference in their entirety.
[0068] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.
[0069] The general chemical terms used in the formulae herein have their usual meaning.
[0070] The present invention is based on the surprising finding by the inventors that administration of a Spike protein receptor binding domain (RBD) based vaccine to an individual/subject, who has been naturally infected by a coronavirus or who has received a prime and boost of a coronavirus whole Spike protein based vaccine, leads to a boost in humoral immunity including (a) enhancing the level of RBD specific antibodies against the ancestral or wildtype (WT) SARS-CoV-2 RBD and a number of RBD variants, (b) enhancing the neutralising activity as determined by inhibition of interaction between RBD antigens and human ACE2, and (c) enhancing the level of RBD neutralising antibodies against the WT RBD and the beta and delta variants.
Coronavirus
[0071] The term “coronavirus” or “CoV” refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV as well as endemic coronaviruses such as HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1. SARS-CoV-2 refers to the newly emerged coronavirus which was identified as the cause of the serious outbreak starting in Wuhan, China, and which has rapidly spread to other areas of the globe. SARS-CoV-2 has also been known as 2019-nCoV and Wuhan coronavirus. It binds via the viral spike protein to human host cell receptor angiotensinconverting enzyme 2 (ACE2). The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for membrane fusion of the virus.
[0072] The subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales (International Committee on Taxonomy of Viruses). This subfamily consists of four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus, on the basis of their phylogenetic relationships and genomic structures. Subgroup clusters are labeled as 1 a and 1 b for the Alphacoronavirus and 2a, 2b, 2c, and 2d for the Betacoronavirus. The alphacoronaviruses and betacoronaviruses infect only mammals. The gammacoronaviruses and deltacoronaviruses infect birds, but some of them can also infect mammals. Alphacoronaviruses and betacoronaviruses usually cause respiratory illness in humans and gastroenteritis in animals. The three highly pathogenic viruses, SARS-CoV, MERS- CoV and SARS-CoV-2, cause severe respiratory syndrome in humans, and the other four human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43 and HKU1 ) induce only mild upper respiratory diseases in immunocompetent hosts, although some of them can cause severe infections in infants, young children and elderly individuals/subjects. Alphacoronaviruses and betacoronaviruses can pose a heavy disease burden on livestock; these viruses include porcine transmissible gastroenteritis virus, porcine enteric diarrhoea virus (PEDV) and the recently emerged swine acute diarrhoea syndrome coronavirus (SADS-CoV). On the basis of current sequence databases, all human coronaviruses have animal origins: SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63 and HCoV-229E are considered to have originated in bats; HCoV-OC43 and HKU1 likely originated from rodents.
[0073] The coronaviruses include antigenic groups I, II, and III. Nonlimiting examples of coronaviruses include SARS coronavirus, MERS coronavirus, transmissible gastroenteritis virus (TGEV), human respiratory coronavirus, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, rabbit coronavirus, murine hepatitis virus, sialodacryoadenitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus, as well as any others described herein, and including those referred to in Cui, et al. Nature Reviews Microbiology volume 17, pages181-192 (2019), and Shereen et al. Journal of Advanced Research, Volume 24, July 2020 (published online 16 March 2020), Pages 91 -98.
[0074] Non-limiting examples of a subgroup 1 a coronavirus include FCov.FIPV.79.1146. VR.2202 (GenBank Accession No. NV_007025), transmissible gastroenteritis virus (TGEV) (GenBank Accession No. NC_002306; GenBank Accession No. Q811789.2; GenBank Accession No. DQ811786.2; GenBank Accession No. DQ81 1788.1 ; GenBank Accession No. DQ811785.1 ; GenBank Accession No. X52157.1 ; GenBank Accession No. AJ011482.1 ; GenBank Accession No. KC962433.1 ; GenBank Accession No. AJ271965.2; GenBank Accession No. JQ693060.1 ; GenBank Accession No. KC609371.1 ; GenBank Accession No. JQ693060.1 ; GenBank Accession No. JQ693059.1 ; GenBank Accession No. JQ693058.1 ; GenBank Accession No.
JQ693057.1 ; GenBank Accession No. JQ693052.1 ; GenBank Accession No.
JQ693051 .1 ; GenBank Accession No. JQ693050.1 ), porcine reproductive and respiratory syndrome virus (PRRSV) (GenBank Accession No. NC_001961.1 ; GenBank Accession No. DQ811787), as well as any other subgroup 1 a coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
[0075] Non-limiting examples of a subgroup 1 b coronavirus include
BtCoV.1 A.AFCD62 (GenBank Accession No. NC_010437), BtCoV.1 B.AFCD307 (GenBank Accession No. NC_010436), BtCov.HKU8.AFCD77 (GenBank Accession No. NC_010438), BtCoV.512.2005 (GenBank Accession No. DQ648858), porcine epidemic diarrhea virus PEDV.CV777 (GenBank Accession No. NC_003436, GenBank Accession No. DQ355224.1 , GenBank Accession No. DQ355223.1 , GenBank
Accession No. DQ355221.1 , GenBank Accession No. JN601062.1 , GenBank Accession No. N601061.1 , GenBank Accession No. JN601060.1 , GenBank Accession No.
JN601059.1 , GenBank Accession No. JN601058.1 , GenBank Accession No.
JN601057.1 , GenBank Accession No. JN601056.1 , GenBank Accession No.
JN601055.1 , GenBank Accession No. JN601054.1 , GenBank Accession No.
JN601053.1 , GenBank Accession No. JN601052.1 , GenBank Accession No.
JN400902.1 , GenBank Accession No. JN547395.1 , GenBank Accession No. FJ687473.1 , GenBank Accession No. FJ687472.1 , GenBank Accession No. FJ687471.1 , GenBank Accession No. FJ687470.1 , GenBank Accession No. FJ687469.1 , GenBank Accession No. FJ687468.1 , GenBank Accession No. FJ687467.1 , GenBank Accession No. FJ687466.1 , GenBank Accession No. FJ687465.1 , GenBank Accession No. FJ687464.1 , GenBank Accession No. FJ687463.1 , GenBank Accession No. FJ687462.1 , GenBank Accession No. FJ687461.1 , GenBank Accession No. FJ687460.1 , GenBank Accession No. FJ687459.1 , GenBank Accession No. FJ687458.1 , GenBank Accession No. FJ687457.1 , GenBank Accession No. FJ687456.1 , GenBank Accession No.
FJ687455.1 , GenBank Accession No. FJ687454.1 , GenBank Accession No. FJ687453 GenBank Accession No. FJ687452.1 , GenBank Accession No. FJ687451.1 , GenBank Accession No. FJ687450.1 , GenBank Accession No. FJ687449.1 , GenBank Accession No. AF500215.1 , GenBank Accession No. KF476061.1 , GenBank Accession No. KF476060.1 , GenBank Accession No. KF476059.1 , GenBank Accession No. KF476058.1 , GenBank Accession No. KF476057.1 , GenBank Accession No. KF476056.1 , GenBank Accession No. KF476055.1 , GenBank Accession No. KF476054.1 , GenBank Accession No. KF476053.1 , GenBank Accession No. KF476052.1 , GenBank Accession No. KF476051.1 , GenBank Accession No. KF476050.1 , GenBank Accession No. KF476049.1 , GenBank Accession No.
KF476048.1 , GenBank Accession No. KF177258.1 , GenBank Accession No. KF177257.1 , GenBank Accession No. KF177256.1 , GenBank Accession No. KF177255.1 ), HCoV.229E (GenBank Accession No. NC_002645), HCoV.NL63. Amsterdam. I (GenBank Accession No. NC_005831 ), BtCoV.HKU2.HK.298.2006 (GenBank Accession No. EF203066), BtCoV.HKU2.HK.33.2006 (GenBank Accession No. EF203067), BtCoV.HKU2.HK.46.2006 (GenBank Accession No. EF203065),
BtCoV.HKU2.GD.430.2006 (GenBank Accession No. EF203064), as well as any other subgroup 1 b coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
[0076] Non-limiting examples of a subgroup 2a coronavirus include HCoV.HKU1 -C.N5 (GenBank Accession No. DQ339101 ), MHV.A59 (GenBank Accession No. NC 001846), PHEV.VW572 (GenBank Accession No. NC 007732), HCoV.OC43.ATCC.VR.759 (GenBank Accession No. NC_005147), bovine enteric coronavirus (BCoV.ENT) (GenBank Accession No. NC_003045), as well as any other subgroup 2a coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
[0077] Non-limiting examples of subgroup 2b coronaviruses include Bat SARS CoV (GenBank Accession No. FJ211859), SARS CoV (GenBank Accession No. FJ211860), SARS-CoV-2 (GenBank Accession No. NC_045512.2), BtSARS.HKU3.1 (GenBank Accession No. DQ022305), BtSARS.HKU3.2 (GenBank Accession No. DQ084199), BtSARS.HKU3.3 (GenBank Accession No. DQ084200), BtSARS.Rml (GenBank Accession No. DQ412043), BtCoV.279.2005 (GenBank Accession No. DQ648857), BtSARS.Rfl (GenBank Accession No. DQ412042), BtCoV.273.2005 (GenBank Accession No. DQ648856), BtSARS.Rp3 (GenBank Accession No. DQ071615), SARS CoV.A022 (GenBank Accession No. AY686863), SARSCoV.CUHK-W1 (GenBank Accession No. AY278554), SARSCoV.GDOI (GenBank Accession No. AY278489), SARSCoV.HC.SZ.61 .03 (GenBank Accession No. AY515512), SARSC0V.SZI 6 (GenBank Accession No. AY304488), SARSCoV.Urbani (GenBank Accession No. AY278741 ), SARSCoV.civetOI 0 (GenBank Accession No. AY572035), and SARSCoV.MA.15 (GenBank Accession No. DQ497008), Rs SHC014 (GenBank® Accession No. KC881005), Rs3367 (GenBank® Accession No. KC881006), WiV1 S (GenBank® Accession No. KC881007) as well as any other subgroup 2b coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
[0078] Non-limiting examples of subgroup 2c coronaviruses include: Middle East Respiratory Syndrome coronavirus isolate Riyadh_2_2012 (GenBank Accession No. KF600652.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_18_2013 (GenBank Accession No. KF600651.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_17_2013 (GenBank Accession No. KF600647.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_15_2013 (GenBank Accession No. KF600645.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_16_2013 (GenBank Accession No. KF600644.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_21_2013 (GenBank Accession No. KF600634), Middle East respiratory syndrome coronavirus isolate AI-Hasa_19_2013 (GenBank Accession No. KF600632), Middle East respiratory syndrome coronavirus isolate Buraidah_1_2013 (GenBank Accession No. KF600630.1 ), Middle East respiratory syndrome coronavirus isolate Hafr-AI-Batin_1_2013 (GenBank Accession No. KF600628.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_12_2013 (GenBank Accession No.
KF600627.1 ), Middle East respiratory syndrome coronavirus isolate Bisha_1_2012 (GenBank Accession No. KF600620.1 ), Middle East respiratory syndrome coronavirus isolate Riyadh_3_2013 (GenBank Accession No. KF600613.1 ), Middle East respiratory syndrome coronavirus isolate Riyadh_1_2012 (GenBank Accession No. KF600612.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_3_2013 (GenBank Accession No. KF186565.1 ), Middle East respiratory syndrome coronavirus isolate Al- Hasa_1_2013 (GenBank Accession No. KF186567.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_2_2013 (GenBank Accession No. KF186566.1 ), Middle East respiratory syndrome coronavirus isolate AI-Hasa_4_2013 (GenBank Accession No. KF186564.1 ), Middle East respiratory syndrome coronavirus (GenBank Accession No. KF192507.1 ), Betacoronavirus England 1 -N1 (GenBank Accession No. NC-019843), MERS-CoV_SA-N1 (GenBank Accession No. KC667074), following isolates of Middle East Respiratory Syndrome Coronavirus (GenBank Accession No: KF600656.1 , GenBank Accession No: KF600655.1 , GenBank Accession No: KF600654.1 , GenBank Accession No: KF600649.1 , GenBank Accession No: KF600648.1 , GenBank Accession No: KF600646.1 , GenBank Accession No: KF600643.1 , GenBank Accession No: KF600642.1 , GenBank Accession No: KF600640.1 , GenBank Accession No: KF600639.1 , GenBank Accession No: KF600638.1 , GenBank Accession No: KF600637.1 , GenBank Accession No: KF600636.1 , GenBank Accession No: KF600635.1 , GenBank Accession No: KF600631.1 , GenBank Accession No: KF600626.1 , GenBank Accession No: KF600625.1 , GenBank Accession No: KF600624.1 , GenBank Accession No: KF600623.1 , GenBank Accession No: KF600622.1 , GenBank Accession No: KF600621 .1 , GenBank Accession No: KF600619.1 , GenBank Accession No: KF600618.1 , GenBank Accession No: KF600616.1 , GenBank Accession No: KF600615.1 , GenBank Accession No: KF600614.1 , GenBank Accession No: KF600641.1 , GenBank Accession No: KF600633.1 , GenBank Accession No: KF600629.1 , GenBank Accession No: KF600617.1 ), Coronavirus Neoromicia/PML- PHE1/RSA/2011 GenBank Accession: KC869678.2, Bat Coronavirus Taper/CII_KSA_287/Bisha/Saudi Arabia/GenBank Accession No: KF493885.1 , Bat coronavirus Rhhar/CII_KSA_003/Bisha/Saudi Arabia/2013 GenBank Accession No: KF493888.1 , Bat coronavirus Pikuh/CII_KSA_001/Riyadh/Saudi Arabia/2013 GenBank Accession No: KF493887.1 , Bat coronavirus Rhhar/CII_KSA_002/Bisha/Saudi Arabia/2013 GenBank Accession No: KF493886.1 , Bat Coronavirus Rhhar/CII_KSA_004/Bisha/Saudi Arabia/2013 GenBank Accession No: KF493884.1 , BtCoV.HKU4.2 (GenBank Accession No. EF065506), BtCoV.HKU4.1 (GenBank Accession No. NC_009019), BtCoV.HKU4.3 (GenBank Accession No. EF065507), BtCoV.HKU4.4 (GenBank Accession No. EF065508), BtCoV 133.2005 (GenBank Accession No. NC 008315), BtCoV.HKU5.5 (GenBank Accession No. EF065512); BtCoV. HKU5.1 (GenBank Accession No. NC_009020), BtCoV.HKU5.2 (GenBank Accession No. EF065510), BtCoV.HKU5.3 (GenBank Accession No. EF065511), human betacoronavirus 2c Jordan-N3/2012 (GenBank Accession No. KC776174.1 ; human betacoronavirus 2c EMC/2012 (GenBank Accession No. JX869059.2), Pipistrellus bat coronavirus HKU5 isolates (GenBank Accession No: KC522089.1 , GenBank Accession No: KC522088.1 , GenBank Accession No: KC522087.1 , GenBank
Accession No: KC522086.1 , GenBank Accession No: KC522085.1 , GenBank
Accession No: KC522084.1 , GenBank Accession No: KC522083.1 , GenBank
Accession No: KC522082.1 , GenBank Accession No: KC522081.1 , GenBank
Accession No: KC522080.1 , GenBank Accession No: KC522079.1 , GenBank
Accession No: KC522078.1 , GenBank Accession No: KC522077.1 , GenBank
Accession No: KC522076.1 , GenBank Accession No: KC522075.1 , GenBank
Accession No: KC522104.1 , GenBank Accession No: KC522104.1 , GenBank
Accession No: KC522103.1 , GenBank Accession No: KC522102.1 , GenBank
Accession No: KC522101.1 , GenBank Accession No: KC522100.1 , GenBank
Accession No: KC522099.1 , GenBank Accession No: KC522098.1 , GenBank
Accession No: KC522097.1 , GenBank Accession No: KC522096.1 , GenBank
Accession No: KC522095.1 , GenBank Accession No: KC522094.1 , GenBank
Accession No: KC522093.1 , GenBank Accession No: KC522092.1 , GenBank
Accession No: KC522091.1 , GenBank Accession No: KC522090.1 , GenBank
Accession No: KC522119.1 GenBank Accession No: KC522118.1 GenBank Accession
No: KC522117.1 GenBank Accession No: KC522116.1 GenBank Accession No:
KC522115.1 GenBank Accession No: KC522114.1 GenBank Accession No:
KC522113.1 GenBank Accession No: KC522112.1 GenBank Accession No:
KC522111.1 GenBank Accession No: KC522110.1 GenBank Accession No:
KC522109.1 GenBank Accession No: KC522108.1 , GenBank Accession No:
KC522107.1 , GenBank Accession No: KC522106.1 , GenBank Accession No:
KC522105.1 ) Pipistrellus bat coronavirus HKU4 isolates (GenBank Accession No:
KC522048.1 , GenBank Accession No: KC522047.1 , GenBank Accession No:
KC522046.1 , GenBank Accession No: KC522045.1 , GenBank Accession No:
KC522044.1 , GenBank Accession No: KC522043.1 , GenBank Accession No:
KC522042.1 , GenBank Accession No: KC522041 .1 , GenBank Accession No:
KC522040.1 GenBank Accession No: KC522039.1 , GenBank Accession No:
KC522038.1 , GenBank Accession No: KC522037.1 , GenBank Accession No:
KC522036.1 , GenBank Accession No: KC522048.1 GenBank Accession No:
KC522047.1 GenBank Accession No: KC522046.1 GenBank Accession No:
KC522045.1 GenBank Accession No: KC522044.1 GenBank Accession No:
KC522043.1 GenBank Accession No: KC522042.1 GenBank Accession No:
KC522041.1 GenBank Accession No: KC522040.1 , GenBank Accession No: KC522039.1 GenBank Accession No: KC522038.1 GenBank Accession No:
KC522037.1 GenBank Accession No: KC522036.1 , GenBank Accession No:
KC522061.1 GenBank Accession No: KC522060.1 GenBank Accession No:
KC522059.1 GenBank Accession No: KC522058.1 GenBank Accession No:
KC522057.1 GenBank Accession No: KC522056.1 GenBank Accession No:
KC522055.1 GenBank Accession No: KC522054.1 GenBank Accession No:
KC522053.1 GenBank Accession No: KC522052.1 GenBank Accession No:
KC522051.1 GenBank Accession No: KC522050.1 GenBank Accession No:
KC522049.1 GenBank Accession No: KC522074.1 , GenBank Accession No:
KC522073.1 GenBank Accession No: KC522072.1 GenBank Accession No:
KC522071.1 GenBank Accession No: KC522070.1 GenBank Accession No:
KC522069.1 GenBank Accession No: KC522068.1 GenBank Accession No:
KC522067.1 , GenBank Accession No: KC522066.1 GenBank Accession No:
KC522065.1 GenBank Accession No: KC522064.1 , GenBank Accession No:
KC522063.1 , or GenBank Accession No: KC522062.1 , as well as any other subgroup 2b coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
[0079] Non-limiting examples of a subgroup 2d coronavirus include BtCoV.HKU9.2 (GenBank Accession No. EF065514), BtCoV.HKU9.1 (GenBank Accession No.
NC-009021 ), BtCoV.HkU9.3 (GenBank Accession No. EF065515), BtCoV.HKU9.4 (GenBank Accession No. EF065516), as well as any other subgroup 2d coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
[0080] Non-limiting examples of a subgroup 3 coronavirus include IBV.Beaudette.IBV.p65 (GenBank Accession No. DQ001339), as well as any other subgroup 3 coronavirus now known (e.g., as can be found in the GenBank® Database) or later identified, and any combination thereof.
[0081] In any aspect or embodiment, the coronavirus may be any variant of SARS- CoV-2, including those described herein such as those in Table 3.
[0082] The coronavirus may be any virus that comprises a receptor binding domain of a Spike protein that includes one or more of the mutations as shown in Figure 9. Subjects
[0083] A subject or individual in need of treatment according to any aspect of the invention, or requiring administration of any composition described herein, may be a subject or individual who is displaying a symptom of a coronavirus infection or who has been diagnosed with a coronavirus infection. Further, the subject or individual may be one who has been clinically or biochemically determined to be infected with a coronavirus.
[0084] A subject may be in a stage of coronavirus infection before end stage-organ failure has developed. A subject in need thereof may be anyone with a coronavirus infection from the onset of clinical progression, before end-organ failure has developed. In one embodiment, the subject has had coronavirus infection symptoms for less than or equal to 12 days, and who does not have life-threatening organ dysfunction or organ failure. Preferably the subject is early in the course of the disease, for example, before day 14 from symptom onset, or during the viremic and seronegative stage.
[0085] A “subject” or “individual” can also be any animal that is susceptible to infection by coronavirus and/or susceptible to diseases or disorders caused by coronavirus infection. A subject of this invention can be a mammal and in particular embodiments is a human, which can be an infant, a child, an adult or an elderly adult. A “subject at risk of infection by a coronavirus” or a “subject at risk of coronavirus infection” is any subject who may be or has been exposed to a coronavirus. “Subject” or “individual” includes any human or non-human animal. Thus, in addition to being useful for human treatment, the compounds of the present invention may also be useful for veterinary treatment of mammals, including companion animals and farm animals, such as, but not limited to dogs, cats, horses, cows, sheep, and pigs, or any animal that can be infected by coronavirus. The coronavirus may be any coronavirus described herein, including SARS-CoV-2 and any variant thereof such as those described herein.
[0086] The subjects at risk include, but are not limited to, an immunocompromised person, an elderly adult (more than 65 years of age), children younger than 2 years of age, healthcare workers, adults or children in close contact with a person(s) with confirmed or suspected coronavirus infection, and people with underlying medical conditions such as pulmonary infection, heart disease or diabetes, primary or secondary immunodeficiency. Coronavirus whole Spike based vaccine
[0087] As used herein, a coronavirus whole Spike based vaccine or a vaccine comprising a whole Spike antigen (or a nucleic acid encoding a whole Spike antigen, for example including nucleic acids inserted into viral vectors) refers to a composition that comprises (or consists essentially of or consists of) a protein that comprises, consists essentially of or consists of an amino acid sequence of a full-length Spike protein of a coronavirus, or a nucleic acid (preferably DNA or RNA, more preferably mRNA) that comprises, consists essentially of or consists of a nucleotide sequence encoding a full- length Spike protein. As used herein, “whole” or “full length” may be used interchangeably. A nucleic acid encoding a whole Spike antigen may be a viral vector, such as chimp- or human- adenovirus, that contains a nucleotide sequence that encodes a full-length Spike protein. Further, coronavirus whole Spike based vaccine may comprise an inactivated virus or any other form as outlined in Table 2 below.
[0088] The term “CoV-S” also called “S” or “S protein” or “spike protein” of the coronavirus and can refer to specific S proteins such as SARS-CoV-2-S, MERS-CoV S, and SARS-CoV S or other members of the coronavirus family. The SARS-CoV-2 spike protein is a 1273 amino acid type I membrane glycoprotein which assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle. The protein has two essential functions, (1) host receptor binding and (2) membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein. The initial attachment of the CoV virion to the host cell is initiated by interactions between the S protein and its receptor. The sites of receptor binding domains (RBD) within the S1 region of a corona virus S protein vary depending on the virus, with some having the RBD at the N-terminus of S1 (MHV) while others (SARS-CoV) have the RBD at the C-terminus of S1 . The S-protein/receptor interaction is the primary determinant for a coronavirus to infect a host species and governs the tissue tropism of the virus. Many coronaviruses utilize peptidases as their cellular receptor. It is unclear why peptidases are used, as entry occurs even in the absence of the enzymatic domain of these proteins. Many alpha-coronaviruses utilize aminopeptidase N (APN) as their receptor, SARS-CoV, SARS-CoV-2, and HCoV-NL63 use angiotensin-converting enzyme 2 (ACE2) as their receptor, MHV enters through CEACAMI, and the recently identified MERS-CoV binds to dipeptidyl-peptidase 4 (DPP4) to gain entry into human cells. [0089] In some embodiments, the subject is infected with SARS-CoV-2, which causes the COVID-19 disease. The amino acid sequence of the full-length SARS-CoV-2 spike protein is exemplified by the amino acid sequence provided in SEQ ID NO: 1 . Examples of whole spike based vaccines are described in Table 2.
[0090] Table 2. SARS-CoV-2 vaccines comprising the whole spike protein in phase IV clinical trials (WHO) and some variant whole spike protein vaccines
Figure imgf000037_0001
Figure imgf000038_0001
[0091] The term “CoV-S” includes protein variants of CoV spike protein isolated from different CoV spike protein or a fragment thereof. The term also encompasses CoV spike protein or a fragment thereof coupled to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as ROR1 .
[0092] In any embodiment, the Spike protein may be from a variant or strain as described herein, including in Table 3 below.
Coronavirus Spike receptor binding domain (RBD) based vaccine
[0093] As used herein, a coronavirus Spike receptor binding domain (RBD) based vaccine or a vaccine comprising a coronavirus Spike RBD antigen (or a nucleic acid that encodes a coronavirus Spike RBD antigen) refers to a composition that comprises (or consists essentially of or consists of) a protein that comprises, consists essentially of or consists of an amino acid sequence of a Spike protein RBD of a coronavirus, or a nucleic acid (preferably RNA, more preferably mRNA) that comprises, consists essentially of or consists of a nucleotide sequence encoding a Spike protein RBD of a coronavirus, however neither the protein nor the nucleic acid comprises, or encodes for, a full length coronavirus Spike protein. A nucleic acid that encodes a coronavirus Spike RBD antigen may be DNA or RNA, and may be part of a viral vector (e.g. adenoviral vector). Preferably, the coronavirus Spike RBD is a SARS-CoV-2 Spike RBD. SARS- CoV-2 RBD based vaccines have been previously described in the art, including SARS- CoV-2 RBD-dimers (Yang et al., Lancet Infectious Diseases, 2021 , 21 (8): 1107-1119), SARS-CoV-2 RBD-Fc-dimers (Liao et al., Emerging Microbes & Infections, 2021 , 10(1 ): 1589-1597; Alieva et al., Vaccine, 2021 , 39(45): 6601 -6613), SARS-CoV-2 RBD multimers (Tan et al., Nat Common, 2021 , 12: 542; Saunders et al., 2021 , Nature, 594: 553-559; Li et al., bioRxiv, 2022.01 .26.477915), and SARS-CoV-2 RBD monomers (Tan et al., Nat Commun, 2021 , 12: 1403) the contents of which are incorporated by reference in their entirety.
[0094] As used herein, the coronavirus Spike RBD antigen (also referred to as RBD antigen) comprises, consists essentially of or consists of an amino acid sequence of a Spike protein RBD of a coronavirus however it does not comprise an amino acid sequence of a full length coronavirus Spike protein. In one embodiment, the RBD antigen comprises an amino acid sequence of a RBD of a coronavirus and additional amino acid sequence of one or more domains of the Spike protein. The one or more domains of the Spike protein include N-terminal domain, Subdomain 1 , Subdomain 2, fusion peptide, Heptad repeat 1 , Central helix, Connector domain or Heptad repeat 2.
[0095] In one embodiment, the RBD antigen does not comprise part of, or all of, one or more of the following domains of the Spike protein N-terminal domain, Subdomain 1 , Subdomain 2, fusion peptide, Heptad repeat 1 , Central helix, Connector domain or Heptad repeat 2.
[0096] In one embodiment, the RBD antigen consists of amino acid sequence of all or part of the S1 subunit of the Spike protein. Preferably, the RBD antigen comprises an amino acid sequence of an RBD of a coronavirus and additional amino acid sequence of all of, or part of, one or more domains of the S1 subunit of the Spike protein. In other words, the RBD antigen does not contain any amino acid sequence with identity to, or that is derived from, the S2 subunit of the Spike protein of a coronavirus.
[0097] In some embodiments, the present invention provides a RBD antigen that is highly conserved across SARS coronaviruses and current SARS-CoV-2 variants. Such an RBD antigen may comprise the amino acids sequence of, or equivalent to, N334- P527 of a SARS-CoV-2 spike protein, for example as defined by GenBank accession NC_045512.2. The RBD antigen can include additional structures (e.g. amino acids, sugar side chains) that may stabilise the protein conformation.
[0098] In any aspect or embodiment, the RBD antigen is effective, particularly in the presence of an adjuvant, for use as or in an immunogenic composition (e.g., a vaccine), and/or for achieving immunological effects as described herein (e.g., generation of coronavirus neutralising antibodies, and/or T cell responses (e.g., CD4+ and/or CD8+ T cell responses)). In some embodiments, the present invention provides a nucleic acid (e.g., DNA, RNA, preferably mRNA) comprising an open reading frame encoding a polypeptide that comprises, consists essentially of or consists of an RBD antigen as described herein, which nucleic acid is suitable for intracellular expression of the polypeptide.
RBD Trimers
[0099] As described herein, SARS-CoV-2 RBD has been observed in an "open" conformation, wherein the RBD is present in a homotrimer of RBD monomers and the RBD of at least one RBD monomer of the trimer points upward relative to the other two RBDs (“open” conformation), away from the C-terminal end of the RBD, and also in a "closed" conformation, where none of the three RBDs of a surface glycoprotein trimer point upward, ie they are downward.
[0100] In some embodiments, the RBD is comprised in a trimer thereof. In some embodiments, one or two RBDs of the trimer is in an open conformation (partially open). In other embodiments all RBDs of the trimer are in an open conformation (fully open). In some embodiments, two or three RBDs of the trimer are in a closed conformation. In some embodiments any of the RBDs of the trimer are in an intermediate conformation, ie not fully up- or downward.
[0101] In any aspect, a receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 2 or 12, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 2 or 12. In one embodiment, the amino acid sequence may include one or more of the mutations as shown in Figure 9, Table 3 or described herein in relation to a SARS-CoV-2 variant, such as an alpha, beta, gamma, kappa, delta, delta plus, lambda, mu, iota or omicron strain (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
[0102] In any aspect, a receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 2 or 12, or any variant described in Table 3, having 0 to 16 amino acid insertions, deletions, substitutions or additions (or a combination thereof). In some embodiments, the relevant amino acid sequence may have from 0 to 16, preferably from 0 to 15, preferably from 0 to 14, preferably from 0 to 13, preferably from 0 to 12, preferably from 0 to 11 , preferably from 0 to 10, preferably from 0 to 9, preferably from 0 to 8, preferably from 0 to 7, preferably from 0 to 6, preferably from 0 to 5, preferably from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1 amino acid insertions, deletions, substitutions or additions (or a combination thereof), wherein the amino acid insertions, deletions, substitutions or additions (or a combination thereof) are located at the N- and/or C-terminus. In some embodiments, an immune response may comprise generation of a binding antibody titre against the receptor binding domain (RBD) of the coronavirus spike protein. In a preferred embodiment, the RBD is a SARS- CoV-2 RBD.
[0103] In any embodiment, a receptor binding domain may be from a SARS-CoV-2 variant, such as those described in Table 3 below. SARS-CoV-2 variants have evolved from the original WT (“Wuhan”) strain of the virus (genome reference sequence: GenBank accession NC_045512.2). As described herein, a receptor binding domain may be from a WT (“Wuhan”), alpha, beta, gamma, kappa, delta, delta plus, lambda, mu, iota or omicron strain (Table 3) and other strains that are expected to emerge with mutations in the RBD. In any embodiment, the receptor binding domain may be N334- P527 of a WT (“Wuhan”), alpha, beta, gamma, kappa, delta, lambda, mu, iota or omicron strains and others that may emerge such as delta plus, for example Y.1 , AY.2, AY.3 and AY.4.2 and omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5. The alpha variant carries 1 mutation in the RBD compared to the original WT (“Wuhan”) strain: N501 Y. The beta variant carries three mutations in the RBD compared to the original WT (“Wuhan”) strain: N501Y, E484K and K417N. The gamma variant carries three mutations in the RBD compared to the original WT (“Wuhan”) strain: K417T, E484K and N501 Y. The kappa variant carries two mutations in the RBD compared to the original WT (“Wuhan”) strain: L452R and E484Q. The delta variant carries two mutations in the RBD compared to the original WT (“Wuhan”) strain: L452R and T478K. The lambda variant carries two mutations in the RBD compared to the original WT (“Wuhan”) strain: L452Q and F490S. The iota variant carries 1 mutation in the RBD compared to the original WT (“Wuhan”) strain: E484K. The mu variant carries two mutations in the RBD compared to the original WT (“Wuhan”) strain: E484K and N501 Y. The omicron subvariant BA.1 carries fifteen mutations in the RBD compared to the original WT (“Wuhan”) strain: G339D, S371 L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501 Y and Y505H. [0104] Without being bound to theory, a person skilled in the art would recognise that vaccine cross-strain efficacy is, in part, linked to the mutations shared across the strains. For example, the beta RBD (with mutations (N501Y, K417N and E484K) is closer to omicron than a wildtype (“Wuhan”) RBD because it shares with omicron the RBD mutations N501Y and K417N (two important mutations known to influence binding to ACE2 (N501Y) and immune evasion (K417N)). For similar reasons, the beta RBD is closer to gamma (N501 Y, E484K (immune evasion) and K417T), mu (N501 Y and E484K) and iota (E484K). A beta RBD should therefore drive superior responses against gamma, mu, iota and omicron.
[0105] Table 3: Summary of SARS-CoV-2 variants or strains and RBD mutations
Figure imgf000042_0001
Figure imgf000043_0001
[0106] As such, peptides, polypeptides or polynucleotides encoding peptides or polypeptides as described herein may contain substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this invention. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation.
Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminus and/or N-terminus residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like may be substituted with alternative sequences that achieve the same or a similar function. In some embodiments, cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids. In other embodiments, buried hydrogen bond networks may be replaced with hydrophobic resides to improve stability. In yet other embodiments, glycosylation sites may be removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminus and/or C-terminus ends) that may be deleted, for example, prior to use in the preparation of a vaccine composition.
[0107] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of coronavirus RBD antigens of interest. For example, unless the context provides otherwise, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference antigen sequence but otherwise identical) of a reference protein, provided that the fragment is immunogenic and confers a protective immune response to the coronavirus. In addition to variants that are identical to the reference protein but are truncated or mutated, in some embodiments, an antigen includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of the sequences provided or referenced herein.
[0108] In any aspect, a receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 12 or an SARS-CoV-2 variant that has evolved from the original WT (“Wuhan”) strain of the virus (e.g. genome reference sequence: GenBank accession NC_045512.2), including those described in Table 3, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 12 or a SARS-CoV-2 variant that has evolved from the original WT (“Wuhan”) strain of the virus (e.g. genome reference sequence: GenBank accession NC_045512.2), including those described in Table 3. In one embodiment, the amino acid sequence may include one or more of the mutations as shown in Figure 9, Table 3 or described herein in relation to a SARS-CoV-2 variant, such as an alpha, beta, gamma, kappa, delta, delta plus, lambda, mu, iota and omicron strain (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5).
[0109] In any aspect, a receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 12 or a SARS-CoV-2 variant that has evolved from the original WT (“Wuhan”) strain of the virus (e.g genome reference sequence: GenBank accession NC_045512.2), including those described in Table 3, having 0 to 16 amino acid insertions, deletions, substitutions or additions (or a combination thereof). In some embodiments, the relevant amino acid sequence may have from 0 to 16, preferably from 0 to 15, preferably from 0 to 14, preferably from 0 to 13, preferably from 0 to 12, preferably from 0 to 11 , preferably from 0 to 10, preferably from 0 to 9, preferably from 0 to 8, preferably from 0 to 7, preferably from 0 to 6, preferably from 0 to 5, preferably from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1 amino acid insertions, deletions, substitutions or additions (or a combination thereof), wherein the amino acid insertions, deletions, substitutions or additions (or a combination thereof) are located at the N- and/or C-terminus.
Chimeric or fusion proteins
[0110] A particularly preferred coronavirus Spike receptor binding domain (RBD) based vaccine comprises, consists essentially of or consists of a chimeric or fusion protein as described herein, particularly this section.
[0111] In one embodiment, the coronavirus Spike receptor binding domain (RBD) antigen may be a chimeric or fusion protein as described herein. In one embodiment, the nucleic acid encoding a coronavirus Spike RBD antigen may encode a chimeric or fusion protein as described herein.
[0112] In one embodiment, the chimeric or fusion protein comprises 2 or more polypeptides comprising or consisting of an amino acid sequence of a receptor binding domain (RBD) from a Spike protein of a coronavirus linked to an Fc region of an antibody.
[0113] In another embodiment, the chimeric or fusion protein comprises 2 or more polypeptides each comprising or consisting of an amino acid sequence of a receptor binding domain from a Spike protein of a coronavirus linked to a polypeptide comprising an Fc receptor binding domain.
[0114] In another embodiment, the chimeric or fusion protein comprises a dimer of receptor binding domains from a Spike protein of a coronavirus linked to an Fc region of an antibody. [0115] In another embodiment, the chimeric or fusion protein comprises a dimer of receptor binding domains from a Spike protein of a coronavirus linked to a polypeptide comprising an Fc receptor binding domain. In one embodiment, the chimeric or fusion protein is a single chain dimer wherein a contiguous polypeptide chain comprises two RBD chain sequences that are covalently linked.
[0116] In any embodiment the chimeric or fusion protein comprises comprises, consists essentially of or consists of a sequence as set forth in any one of SEQ ID NO: 2, 4, 5, 7, 8, 9, 12, 13 or 18.
Fc region of an antibody and Fc receptor binding domains
[0117] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. In other words, the Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. In the context of the present invention, the Fc region comprises two heavy chain fragments, preferably the CH2 and CH3 domains of said heavy chain. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, s, Y, and p.
[0118] In some embodiments, the RBD-Fc dimer does not exhibit any effect function or any detectable effector function. “Effector functions” or “effector activities” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity). The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991 ). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821 ,337 (see Bruggemann, M. et aL, J. Exp. Med. 166:1351 - 1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wl). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et aL, J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101 :1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929 Al).
[0119] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581 ). For example, an antibody variant may comprise an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, the substitutions are L234A and L235A (LALA) (See, e.g., WO 2012/130831 ). Further, alterations may be made in the Fc region that result in altered (i.e., diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551 , WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0120] In some aspects, the Fc region includes mutations to the complement (C1q) and/or to Fc gamma receptor (FcyR) binding sites. In some aspects, such mutations can render the fusion protein incapable of antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
[0121] The term “Fc region” also includes native sequence Fc regions and variant Fc regions. The Fc region may include the carboxyl-terminus of the heavy chain.
Antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Amino acid sequence variants of the Fc region of an antibody may be contemplated. Amino acid sequence variants of an Fc region of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the Fc region of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., inducing or supporting an anti-inflammatory response.
[0122] The Fc region of the antibody may be an Fc region of any of the classes of antibody, such as IgA, IgD, IgE, IgG, and IgM. The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. Accordingly, as used in the context of the present invention, the antibody may be an Fc region of an IgG. For example, the Fc region of the antibody may be an Fc region of an IgG 1 , an lgG2, an lgG3 or an lgG4. In some aspects, the fusion protein of the present invention comprises an IgG of an Fc region of an antibody. In the context of the present invention, the Fc region of the antibody is an Fc region of an IgG, preferably lgG1. [0123] The Fc region is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen.
[0124] An Fc receptor binding domain is any protein or polypeptide that binds to the Fc receptor on the surface of a cell. The Fc receptor binding domain may be an antigen binding domain of an antibody. The Fc receptor binding domain also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.
Linkers
[0125] Moreover, the herein provided fusion proteins may comprise a linker (or “spacer”). In the context of the present invention, the 2 or more polypeptides comprising or consisting of an amino acid sequence of a receptor binding domain, or the dimer of receptor binding domains from a spike protein of a coronavirus, is fused via a linker at the C-terminus to the Fc region or Fc receptor binding domain.
[0126] A linker is usually a peptide having a length of up to 20 amino acids. The term “linked”, “linked to” or “fused to” refers to a covalent bond, e.g., a peptide bond, formed between two moieties. Accordingly, in the context of the present invention the linker may have a length of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. For example, the herein provided fusion protein may comprise a linker between the 2 or more polypeptides comprising or consisting of an amino acid sequence of a receptor binding domain, or the dimer of receptor binding domains from a spike protein of a coronavirus, and the Fc region of the antibody, such as between the N-terminus of the Fc regions/FcR binding domains and the C-terminus of the receptor binding domain polypeptide. Such linkers have the advantage that they can make it more likely that the different polypeptides of the fusion protein fold independently and behave as expected.
[0127] Thus, in the context of the present invention the 2 or more polypeptides comprising or consisting of an amino acid sequence of a receptor binding domain, or the dimer of receptor binding domains from a spike protein of a coronavirus, and the Fc region of an antibody or Fc receptor binding domain may be comprised in a single covalently associated multi-functional polypeptide.
[0128] In some aspects, the fusion protein of the present invention includes a peptide linker. In some aspects, the peptide linker can include the amino acid sequence Gly- Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS) or Gly-Gly-Gly-Gly-Ser (GGGGS). In some aspects, the peptide linker can include the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer. The linker may a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS)n where n is any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS) or longer (GS)11 or longer. It will be appreciated that n can be any number including 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. Fusion proteins having linkers of such length are included within the scope of the present invention. Additionally, fusion proteins that have no linker are included within the scope of the present invention.
Adjuvants
[0129] In some embodiments, the present invention provides a vaccine compositions that may also include a pharmaceutically acceptable adjuvant in addition to the peptides as defined herein. Adjuvants are added in order to enhance the immunogenicity of the vaccine composition. A single vaccine may include two or more of said adjuvants.
[0130] The adjuvants for use in the present invention may be modulators and/or agonists of Toll-Like Receptors (TLR). For example, they may be agonists of one or more of the human TLR1 , TLR2, TLR3, TLR4, TLR7, TLR8, and/or TLR9 proteins. Preferred agents are agonists of TLR7 (e.g. imidazoquinolines) and/or TLR9 (e.g. CpG oligonucleotides). These agents are useful for activating innate immunity pathways.
[0131] Suitable adjuvants are known in the art and include any one described herein, preferably a TLR2-agonist, more preferably a Pam-2-Cys containing molecule such as PEG-R4-Pam-2-Cys, or preferably a stimulator of NKT cells, more preferably alpha- Galactosylceramide (also referred to herein as “a-GalCer”), alpha-glucosylceramide, alpha-glucosyldiacylglycerol, alpha-galactosyldiacylglycerol, beta-mannosylceramide, or other NKT cell-stimulatory lipid molecules and analogues thereof comprising variations in acyl and sphingosine chain lengths, saturation, and variations in polar head group composition. [0132] In some embodiments, the TLR2-agonist can be selected from the group consisting of Pam3CSK4, PEG-R4-Pam-2-Cys, MALP-2, lipoteichoic acid, OspA, Porin, LcrV, lipomannan, Lysophosphatidylserine, Lipophosphoglycan (LPG), Glycophosphatidylinositol (GPI) and Zymosan.
[0133] In some embodiments, the adjuvant can be selected from the group consisting of is selected from the group consisting of poly-l:C, CpG, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, B(C, CP-870,893, CpG7909, ASO3, ASO4, MatrixM, CyaA, dSLIM, GM-CSF, IC30, IC31 , Imiquimod, 3dMPL, ImuFact IMP321 , IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59®, AddaVax™, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA V, Montanide ISA-51 , OK- 432, OM-174, OM-197-MP-EC, ONTAK. PEPTEL, vector system, PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21 stimulon.
Oil-in-Water Emulsion Adjuvants
[0134] Oil-in-water emulsions have been found to be particularly suitable for use in adjuvanting viral vaccines. Various such emulsions are known, and they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible. The oil droplets in the emulsion are generally less than 5 pm in diameter, and may even have a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions. Droplets with a size less than 220 nm are preferred as they can be subjected to filter sterilization.
[0135] The invention can be used with oils such as those from an animal (such as fish) or vegetable source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5- carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoids known as squalene, 2,6,10,15,19,23- hexamethyl-2,6,10,14,18,22-tetracosahexaene, which is particularly preferred herein. Squalane, the saturated analog to squalene, is also a preferred oil. Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art. Other preferred oils are the tocopherols (see below). Mixtures of oils can be used.
[0136] Surfactants can be classified by their ‘HLB’ (hydrophile/lipophile balance). Preferred surfactants for vaccine composition have a HLB of at least 10, preferably at least 15, and more preferably at least 16. The invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-1 ,2-ethanediyl) groups, with octoxynol- 9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Non-ionic surfactants are preferred. Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100.
[0137] Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
[0138] Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.
[0139] In some embodiments, the adjuvant comprises a metabolizable oil and an emulsifying agent (such as a detergent or surfactant). Preferably, the oil and the emulsifying agent are present in the form of an oil-in-water emulsion having oil droplets substantially all of which are less than 1 micron in diameter. Exemplary metabolizable oils and emulsifying agents are described in US6,299,884 and US6,086,901 . In one embodiment, the adjuvant comprises an oil-in-water emulsion. Preferably, the oil is squalene. Preferably, the aqueous phase is a citrate buffer (for example 10mM at pH 6.5).
[0140] In one embodiment, the adjuvant comprises squalene in an oil-in-water emulsion. Preferably, the adjuvant further comprises TWEEN® 80 (polyoxyethylenesorbitan monooleate) and Span® 85 (sorbitan trioleate). The adjuvant may comprise 4.3% squalene, 0.5% TWEEN® 80 (polyoxyethylenesorbitan monooleate), 0.5% Span® 85 (sorbitan trioleate), optionally with 400 pig/ml MTP-PE (N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1 ,2-dipalmitoyl-sn-glycero-3- 3(hydroxyphosphoryl-oxy)]ethylamide)
[0141] In one embodiment, the composition comprises 50%vol/vol adjuvant, preferably the adjuvant is MF59®. MF59® is an oil-in-water emulsion of squalene oil. Squalene, a naturally occurring substance found in humans, animals and plants, is highly purified for the vaccine manufacturing process. MF59 adjuvant (MF59C.1 ) is an oil-in-water emulsion with a squalene internal oil phase and a citrate buffer external aqueous phase. See, e.g., U.S. Pat. Nos. 6,299,884 and 6,086,901 ; Ott et al. “MF59 — Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines,” Vaccine Design: The Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, pp. 277-296 (1995). Two nonionic surfactants, sorbitan trioleate and polysorbate 80, serve to stabililize the emulsion. The safety of the MF59 adjuvant has been demonstrated in animals and in humans in combination with a number of antigens. See, e.g., Higgins et al., “MF59 Adjuvant Enhances the Immunogenicity of Influenza Vaccine in Both Young and Old Mice,” Vaccine 14(6):478- 484 (1996). MF59 is 4.3% squalene, 0.5% TWEEN® 80 (polyoxyethylenesorbitan monooleate), 0.5% Span® 85 (sorbitan trioleate), optionally with 400 pg/ml MTP-PE (N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1 ,2-dipalmitoyl-sn-glycero-3- 3(hydroxyphosphoryl-oxy)]ethylamide). An exemplary composition of MF59 comprises citrate buffer pH 6.5 (10mM citrate, 140mM NaCI, 0.02% PS80, pH 6.5) and 39mg/ml Squalene, 4.7mg/ml Polysorbate 80, 4.7mg/ml Sorbitan Trioleate, 2.65mg/ml Sodium Citrate, 0.17mg/ml Citric Acid monohydrate.
[0142] In some embodiments the adjuvant is an emulsion of squalene, a tocopherol, and Tween 80. The emulsion may include phosphate buffered saline. It may also include Span 85 (e.g. at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene:tocopherol is preferably ^1 as this provides a more stable emulsion. Squalene and Tween 80 may be present volume ratio of about 5:2. One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-a-tocopherol and 5 ml squalene), then microfluidising the mixture. The resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250 nm, preferably about 180 nm.
[0143] An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X- 100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer.
[0144] An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an a-tocopherol succinate). The emulsion may include these three components at a mass ratio of about 75:11 :10 (e.g. 750 pg/ml polysorbate 80, 110 pg/ml Triton X-100 and 100 pg/ml a-tocopherol succinate), and these concentrations should include any contribution of these components from antigens. The emulsion may also include squalene. The emulsion may also include a 3d-MPL (see below). The aqueous phase may contain a phosphate buffer.
[0145] An emulsion of squalane, polysorbate 80 and poloxamer 401 (“Pluronic™ L121 ”). The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the “SAF-1” adjuvant (Allison & Byars, Res Immuno , 1992, 143:519- 25) (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the “AF” adjuvant (Hariharan et al., Cancer Re, 1995, 55:3486-9 (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
[0146] An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or ‘Span 80’). The emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm (US2007/014805) The emulsion may also include one or more of alditol; a cryoprotective agent (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or an alkylpolyglycoside. Such emulsions may be lyophilized.
[0147] An emulsion having from 0.5-50% of an oil, 0.1 -10% of a phospholipid, and 0.05-5% of a non-ionic surfactant. As described in WO95/11700, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.
[0148] A submicron oil-in-water emulsion of a non-metabolisable oil (such as light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such as Qui1 A saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in US6,080,725, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyldioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis(2- hydroxyethyl)propanediamine.
[0149] An emulsion in which a saponin (e.g. Qu i 1 A or QS21 ) and a sterol (e.g. a cholesterol) are associated as helical micelles (W02005/097181 ).
[0150] An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer) (W02006/113373). [0151] An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer) (W02006/113373).
[0152] The emulsions may be mixed with antigen extemporaneously, at the time of delivery. Thus the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use. The antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids. The volume ratio of the two liquids for mixing can vary (e.g. between 5:1 and 1 :5) but is generally about 1 :1 .
[0153] Where a composition includes a tocopherol, any of the a, |3, y, 5,
Figure imgf000056_0001
tocopherols can be used, but a-tocopherols are preferred. The tocopherol can take several forms e.g. different salts and/or isomers. Salts include organic salts, such as succinate, acetate, nicotinate, etc. D-a-tocopherol and DL-a-tocopherol can both be used. Tocopherols are advantageously included in vaccines for use in elderly patients (e.g. aged 60 years or older) because vitamin E has been reported to have a positive effect on the immune response in this patient group. They also have antioxidant properties that may help to stabilize the emulsions. A preferred a-tocopherol is DL-a- tocopherol, and the preferred salt of this tocopherol is the succinate. The succinate salt has been found to cooperate with TNF-related ligands in vivo. Moreover, a-tocopherol succinate is known to be compatible with influenza vaccines and to be a useful preservative as an alternative to mercurial compounds.
Aluminium Salt Adjuvants
[0154] The adjuvants known as aluminium hydroxide and aluminium phosphate may be used. These names are conventional, but are used for convenience only, as neither is a precise description of the actual chemical compound which is present (e.g. see chapter 9 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)). The invention can use any of the “hydroxide” or “phosphate” adjuvants that are in general use as adjuvants.
[0155] The adjuvants known as “aluminium hydroxide” are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AIO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide AI(OH)3, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070 cm-1 and a strong shoulder at 3090-3100 cm-1 (chapter 9 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867- X)). The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology (e.g. as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants. The pl of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1 .8-2.6 mg protein per mg Al+++ at pH 7.4 have been reported for aluminium hydroxide adjuvants.
[0156] The adjuvants known as “aluminium phosphate” are typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. Hydroxyphosphates generally have a PCU/AI molar ratio between 0.3 and 1 .2. Hydroxyphosphates can be distinguished from strict AlPC by the presence of hydroxyl groups. For example, an IR spectrum band at 3164 cm-1 (e.g. when heated to 200° C.) indicates the presence of structural hydroxyls.
[0157] The PC /AI3+ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1 .2, preferably between 0.8 and 1 .2, and more preferably 0.95+0.1 . The aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts. A typical adjuvant is amorphous aluminium hydroxyphosphate with PO4/AI molar ratio between 0.84 and 0.92, included at 0.6 mg Al3+/ml. The aluminium phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5-20 pm (e.g. about 5-10m) after any antigen adsorption. Adsorptive capacities of between 0.7-1 .5 mg protein per mg Al+++ at pH 7.4 have been reported for aluminium phosphate adjuvants. [0158] The point of zero charge (PZC) of aluminium phosphate is inversely related to the degree of substitution of phosphate for hydroxyl, and this degree of substitution can vary depending on reaction conditions and concentration of reactants used for preparing the salt by precipitation. PZC is also altered by changing the concentration of free phosphate ions in solution (more phosphate=more acidic PZC) or by adding a buffer such as a histidine buffer (makes PZC more basic). Aluminium phosphates used according to the invention will generally have a PZC of between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.
[0159] Suspensions of aluminium salts used to prepare vaccine compositions may contain a buffer (e.g. a phosphate or a histidine or a Tris buffer), but this is not always necessary. The suspensions are preferably sterile and pyrogen-free. A suspension may include free aqueous phosphate ions e.g. present at a concentration between 1 .0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM. The suspensions may also comprise sodium chloride.
[0160] The invention can use a mixture of both an aluminium hydroxide and an aluminium phosphate. In this case there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2:1 e.g. ^5:1 , ^6:1 , ^7:1 , ^8:1 , ^9:1 , etc. The concentration of Al+++ in a composition for administration to a patient is preferably less than 10 mg/ml e.g. ^5 mg/ml, ^4 mg/ml, ^3 mg/ml, ^2 mg/ml, ^1 mg/ml, etc. A preferred range is between 0.3 and 1 mg/ml. A maximum of 0.85 mg/dose is preferred.
Immunostimulatory Oligonucleotides
[0161] Immunostimulatory oligonucleotides can include nucleotide modifications/analogs such as phosphorothioate modifications and can be doublestranded or (except for RNA) single-stranded. References Kandimalla et al.
(2003) Nucleic Acids Research 31 :2393-2400, WO02/26757 and WO99/62923 disclose possible analog substitutions e.g. replacement of guanosine with T-deoxy-7- deazaguanosine. A CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT. The CpG sequence may be specific for inducing a Th1 immune response, such as a CpG-A ODN (oligodeoxynucleotide), or it may be more specific for inducing a B cell response. Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences may be attached at their 3' ends to form “immunomers”. A useful CpG adjuvant is CpG7909, also known as ProMune™ (Coley Pharmaceutical Group, Inc.).
[0162] As an alternative, or in addition, to using CpG sequences, TpG sequences can be used WO01/22972. These oligonucleotides may be free from unmethylated CpG motifs.
[0163] The immunostimulatory oligonucleotide may be pyrimidine-rich. For example, it may comprise more than one consecutive thymidine nucleotide (e.g. TTTT, as disclosed in ref. 158), and/or it may have a nucleotide composition with >25% thymidine (e.g. >35%, >40%, >50%, >60%, >80%, etc.). For example, it may comprise more than one consecutive cytosine nucleotide (e.g. CCCC), and/or it may have a nucleotide composition with >25% cytosine (e.g. >35%, >40%, >50%, >60%, >80%, etc.). These oligonucleotides may be free from unmethylated CpG motifs.
[0164] Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They may comprise fewer than 100 nucleotides.
3 de-O-acylated monophosphoryl lipid A
[0165] In some embodiments, the adjuvant is 3dMPL (also known as 3 de-O-acylated monophosphoryl lipid A or 3-0-desacyl-4'-monophosphoryl lipid A). 3dMPL is an adjuvant in which position 3 of the reducing end glucosamine in monophosphoryl lipid A has been de-acylated. 3dMPL has been prepared from a heptoseless mutant of Salmonella minnesota, and is chemically similar to lipid A but lacks an acid-labile phosphoryl group and a base-labile acyl group. It activates cells of the monocyte/macrophage lineage and stimulates release of several cytokines, including IL- 1 , IL-12, TNF-a and GM-CSF. Preparation of 3dMPL was originally described in GB-A- 2220211.
[0166] In aqueous conditions, 3dMPL can form micellar aggregates or particles with different sizes e.g. with a diameter <150 nm or >500 nm. Either or both of these can be used with the invention, and the better particles can be selected by routine assay. Smaller particles (e.g. small enough to give a clear aqueous suspension of 3dMPL) are preferred for use according to the invention because of their superior activity. Preferred particles have a mean diameter less than 220 nm, more preferably less than 200 nm or less than 150 nm or less than 120 nm, and can even have a mean diameter less than 100 nm. In most cases, however, the mean diameter will not be lower than 50 nm. These particles are small enough to be suitable for filter sterilization. Particle diameter can be assessed by the routine technique of dynamic light scattering, which reveals a mean particle diameter. Where a particle is said to have a diameter of x nm, there will generally be a distribution of particles about this mean, but at least 50% by number (e.g. 60%, 170%, 180%, 190%, or more) of the particles will have a diameter within the range x±25%.
[0167] 3dMPL can advantageously be used in combination with an oil-in-water emulsion. Substantially all of the 3dMPL may be located in the aqueous phase of the emulsion.
[0168] The 3dMPL can be used on its own, or in combination with one or more further compounds. For example, it is known to use 3dMPL in combination with the QS21 saponin (including in an oil-in-water emulsion), with an immunostimulatory oligonucleotide, with both QS21 and an immunostimulatory oligonucleotide, with aluminium phosphate, with aluminium hydroxide, or with both aluminium phosphate and aluminium hydroxide.
[0169] In any embodiment, the adjuvant is any one described herein, preferably PEG- R4-Pam-2-Cys, alpha-Galactosylceramide (also referred to herein as “a-GalCer”) or MF59.
Vaccine compositions
[0170] In any aspect, the present invention provides the use of a vaccine composition that comprises (or consists essentially of or consists of) a protein that comprises, consists essentially of or consists of an amino acid sequence of a Spike protein RBD of a coronavirus, or a nucleic acid (preferably RNA, more preferably mRNA) that comprises, consists essentially of or consists of a nucleotide sequence encoding a Spike protein RBD of a coronavirus, however neither the protein nor the nucleic acid comprises, or encodes for, a full length coronavirus Spike protein.
[0171] Vaccine compositions as described herein are pharmaceutically acceptable and are typically in aqueous form. They may include components in addition to the antigen or nucleic acid encoding the antigen (and, where applicable, the adjuvant) e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s). A pharmaceutically acceptable excipient, after administered to a subject, does not cause undesirable physiological effects. The excipient in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with mRNA or protein or polypeptide and can be capable of stabilizing it. One or more excipients (e.g., solubilizing agents) can be utilized as pharmaceutical carriers for delivery of the antigen (such as mRNA). Examples of a pharmaceutically acceptable excipients include, but are not limited to, biocompatible vehicles (e.g., LNPs), carriers, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other excipients include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical excipients, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.
[0172] In some embodiments, the vaccine is formulated using one or more excipients to: (1 ) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
[0173] In some embodiments, a composition comprising mRNA or protein does not include an adjuvant (they are adjuvant free).
[0174] Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
[0175] Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the mRNA or protein into association with an excipient (e.g., a mixture of lipids and/or a lipid nanoparticle), and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
[0176] Relative amounts of the mRNA or protein, the pharmaceutically-acceptable excipient, and/or any additional ingredients in a composition in accordance with the disclosure may vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
[0177] Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will typically be included in the 5-20 mM range.
[0178] The pH of a composition will generally be between 5.0 and 8.1 , and more typically between 6.0 and 8.0 e.g. between 6.5 and 7.5, between 7.0 and 7.8. A process of vaccine composition may therefore include a step of adjusting the pH of the bulk vaccine prior to packaging.
[0179] The composition is preferably sterile. The composition is preferably non- pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. The composition is preferably gluten free.
[0180] The composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a ‘multidose’ kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material.
Dosages, timing and route of administration
[0181] The present invention provides methods comprising administering vaccine compositions to a subject in need thereof. The exact amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
[0182] The phrase “therapeutically effective amount” generally refers to an amount of a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen as described herein that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein, when applied in a method or use of the invention. Undesirable effects, e.g., side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount".
[0183] In some such embodiments, a spike based vaccine composition described herein is administered to subjects of age 16 or older (including, e.g., 16-85 years). In some such embodiments, a spike based vaccine composition described herein is administered to subjects of age 18-55. In some such embodiments, a spike based vaccine composition escribed herein is administered to subjects of age 56-85. In some embodiments, a spike based vaccine composition described herein is administered (e.g., by intramuscular injection) as a single dose.
[0184] In one embodiment, the vaccination regimen comprises a RBD based booster vaccination wherein a subject has received at least two doses of a vaccine described herein, e.g., two doses of the whole spike based vaccine as described herein or wherein the subject has been previously infected with coronavirus and optionally has had at least one dose of a vaccine described herein, e.g., one dose of the whole spike based vaccine as described herein.
[0185] In some embodiments, an effective amount of a RBD antigen described herein, for example a chimeric or fusion protein described herein, for a human subject lies in the range of about 0.25 nmoles/kg body weight/dose to 0.0001 nmoles/kg body weight/dose. Preferably, the range is about 0.25 nmoles/kg body weight/dose to 0.0001 nmoles/kg body weight/dose. More preferably, the range is about 0.002 nmoles/kg body weight/dose to 0.001 nmoles/kg body weight/dose. In some embodiments, the body weight/dose range is about 0.25 nmoles/kg, to 0.001 nmoles/kg, about 0.1 nmoles/kg to 0.001 nmoles/kg, about 0.025 nmoles/kg to 0.001 nmol/kg, about 0.01 nmoles/kg to 0.001 nmoles/kg, or about 0.005 nmoles/kg to 0.001 nmoles/kg body weight/dose. In some embodiments, the amount is at, or about, 0.25 nmoles, 0.1 nmoles, 0.05 nmoles, 0.01 nmoles, 0.005 nmoles, 0.002 nmoles, or 0.001 nmoles/kg body weight/dose of the RBD antigen, chimeric or fusion protein as described herein. Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic dose.
[0186] In some embodiments, an effective amount of a RBD antigen described herein, for example a chimeric or fusion protein as described herein for a human subject lies in the range of about 0.01 pg to 100 pg per dose. Preferably, the range is about 0.1 pg to 50 pg per dose. More preferably, the range is about 0.1 pg to 20 pg per dose. In some embodiments, the dose is about 1 pg to 100 pg, about 5 pg to 50 pg, about 10 pg to 45 pg, about 10 pg to 25 pg or about 10 pg to 20 pg per dose. In some embodiments, the dose is at, or about, 0.01 pg. 0.1 pg, 0.2 pg, 0.5 pg, 1 pg, 5 pg, 10 pg, 15 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg. Preferably the dose is 15 pg.
[0187] In some embodiments, an effective amount of nucleic acid (preferably DNA or RNA, more preferably mRNA) encoding an RBD antigen, for example a chimeric or fusion protein as described herein for a human subject is provided. For example, the mRNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the mRNA may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective amount, prophylactically effective, or appropriate imaging dose level for any particular subject may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
[0188] The effective amount of the RNA, as provided herein, may range from about 25 pg - 500 pg, administered as a single dose or as multiple booster doses. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 25 pg mRNA. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 100 pg mRNA. In some embodiments, a single dose of a vaccine composition (e.g., administered once, twice, three times, or more) comprises about 250 pg mRNA.
[0189] In some embodiments, a total amount of mRNA administered to a subject is about 5 pg, 10 pg, 25 pg, 50 pg, about 100 pg, or about 200 pg. In some embodiments, a total amount of mRNA administered to a subject is about 25 pg. In some embodiments, a total amount of mRNA administered to a subject is about 50 pg. In some embodiments, a total amount of mRNA administered to a subject is about 100 pg. In some embodiments, a total amount of mRNA administered to a subject is about 200 pg-
[0190] The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount". However, an appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. In one aspect, the dose administered to a subject is any dose that reduces viral load.
[0191] The RBD antigen (or nucleic acid encoding thereof), chimeric or fusion protein or vaccine compositions thereof can be administered using immunisation schemes known by persons of ordinary skill in the art to induce protective immune responses.
[0192] In any aspect, the present invention provides for single or multiple immunisations in a boosting strategy in subjects who have previously received one or more coronavirus vaccine compositions, and/or previously been infected with coronavirus. Preferably, the coronavirus vaccine composition is a whole spike based vaccine. For example, a single or multiple boosting immunisation is provided to a subject who has received one, two or more doses of a whole spike based vaccine.
[0193] A boosting immunisation or third dose of a coronavirus Spike receptor binding domain (RBD) based vaccine in a vaccination schedule of the invention can be administered at a time after a subject has received a second dose immunisation or has been previously infected with coronavirus that is days, weeks, months or even years after the prime immunisation. In some embodiments, a booster dose of a vaccine composition as described herein is administered following a second dose immunisation. Preferably, the second dose immunisation is provided by a whole (or full length) spike based coronavirus vaccine. A booster dose is a dose that is given at a certain interval after completion of a second dose or after a subject is infected with a coronavirus that is intended to boost immunity to, and therefore prolong protection against, the disease that is to be prevented. The time between administration of a second dose of a vaccine composition, or infection of subject with coronavirus, and a booster dose may be, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 1 year, 2 years, 3 years, 4 years, 5 years or 10 years.
[0194] In some embodiments, the time between administration of a second dose of a vaccine composition, or infection of subject with coronavirus, and a booster dose of a coronavirus Spike receptor binding domain (RBD) based vaccine is 1 to 6 months. In some embodiments, the time between administration of an initial dose of a vaccine and a booster dose is less than 1 month. In certain embodiments, a boosting immunisation is administered 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or 12 months or more after a subject has received a second dose immunisation or has been previously infected with coronavirus.
[0195] Additionally multiple boost immunisations can be administered weekly, every other week, monthly, every other month, every third month, every sixth month, or more. In other embodiments, the boost immunisation is administered every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, or every 24 weeks. In certain embodiments, boosting immunisation can continue until a protective anti-coronavirus antibody titre is seen in the subject’s serum. In certain embodiments, a subject is given one boost immunisation, two boost immunisations, three boost immunisations, or four or more boost immunisations, as needed to obtain and maintain a protective antibody titre.
[0196] In some embodiments, the booster dose or administration of a coronavirus Spike RBD based vaccine (and/or other subsequent dose) may be administered by intramuscular injection. In some embodiments, a booster dose may be administered in the deltoid muscle. [0197] In some embodiments, a provided composition is established to achieve elevated antibody titres, and/or B cell and/or T-cell titres (e.g., specific for a relevant portion of a coronavirus spike protein) for a period of time longer than about 3 weeks; in some such embodiments, a whole Spike based vaccine dosing regimen (the administrations prior to the Spike RBD based vaccine) may involve only a single dose, or may involve two or more doses, which may, in some embodiments, be separated from one another by a period of time that is longer than about 21 days or three weeks. For example, in some such embodiments, such period of time may be about 4 weeks, 5 weeks, 6 weeks 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or more, or about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10, months, 11 months, 12 months or more, or in some embodiments about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years or more. In some embodiments, a first dose and a second dose of a whole Spike based vaccine (and/or other subsequent dose) may be administered by intramuscular injection. In some embodiments, a first dose and a second dose may be administered in the deltoid muscle. In some embodiments, a first dose and a second dose may be administered in the same arm. In some embodiments, a whole spike based vaccine composition described herein is administered (e.g., by intramuscular injection) as a series of two doses (e.g., 0.3 ml each) 21 days part.
[0198] In some embodiments, the composition as described herein can be administered by any convenient route as described herein, such as via the intramuscular, dermal, intranasal, subcutaneous, intravenous, intraperitoneal or oral routes.
[0199] In some embodiments, the composition as described herein is formulated for or adapted for administration by any convenient route as described herein, such as via the intramuscular, intranasal, subcutaneous, dermal, intravenous, intraperitoneal or oral routes.
[0200] As used herein, the upper respiratory tract (URT) may include the following regions: nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords). [0201] Typically, the lower respiratory tract (LRT) includes the following regions: portion of the larynx below the vocal folds, trachea, bronchi and bronchioles. The lungs can be included in the lower respiratory tract and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli. In any aspect of the present invention, administration to the URT may be administration to the nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords). Also contemplated is administration to any one or more regions of the URT provided that the compound is retained in the URT or does not contact a region of the LRT.
[0202] In some embodiments, the composition as described herein may be formulated for administration to the URT only. Limitation to the URT may be achieved by an amount, particularly volume and composition of form i.e. particle size, physical form whether dry powder or solution droplet, of composition that would otherwise be administered to the LRT or TRT. Alternatively, the vaccine composition as described herein may be administered via a device that ensures retention in the URT only.
[0203] In some embodiments, the composition as described herein may be formulated for intranasal administration, including dry powder, sprays, mists, or aerosols. This may be particularly preferred for treatment of coronavirus infection.
[0204] There are a number of options that can be employed to limit delivery to the URT using intranasal delivery: (1) Ensuring the droplet/particle size is sufficiently large to prevent access into the LRT (>10um); (2) For liquids limiting dose volume to minimise run-off/d rain age; (3) Similarly administration with the head in an inverted position also minimises run-off/drainage; (4) Inclusion of a viscosity enhancer/ mucoadhesive to promote retention in the nasal cavity and prevent run-off/drainage; (5) Use a nasal device that entirely eliminates the potential for LRT exposure e.g. the Optinose bidirectional delivery device. One or a combination of these methods can be applied.
[0205] The selection of appropriate carriers depends upon the particular type of administration that is contemplated. For administration via the upper respiratory tract, e.g., the nasal mucosal surfaces, the compound can be formulated into a solution, e.g., water or isotonic saline, buffered or unbuffered, or as a suspension, for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2 (Remington's, Id. at page 1445). Of course, the ordinary artisan can readily determine a suitable saline content and pH for an innocuous aqueous carrier for nasal and/or upper respiratory administration.
[0206] Other ingredients, such as art known preservatives, colorants, lubricating or viscous mineral or vegetable oils, perfumes, natural or synthetic plant extracts such as aromatic oils, and humectants and viscosity enhancers such as, e.g., glycerol, can also be included to provide additional viscosity, moisture retention and a pleasant texture and odour for the formulation. For nasal administration of solutions or suspensions according to the invention, various devices are available in the art for the generation of drops, droplets and sprays. For example, a vaccine composition described herein can be administered into the nasal passages by means of a simple dropper (or pipet) that includes a glass, plastic or metal dispensing tube from which the contents are expelled drop by drop by means of air pressure provided by a manually powered pump, e.g., a flexible rubber bulb, attached to one end.
[0207] The tear secretions of the eye drain from the orbit into the nasal passages, thus, if desirable, a suitable pharmaceutically acceptable ophthalmic solution can be readily provided by the ordinary artisan as a carrier for the vaccine composition described herein to be delivered and can be administered to the orbit of the eye in the form of eye drops to provide for both ophthalmic and intranasal administration.
[0208] In one embodiment, a premeasured unit dosage dispenser that includes a dropper or spray device containing a solution or suspension for delivery as drops or as a spray is prepared containing one or more doses of the drug to be administered. The invention also includes a kit containing one or more unit dehydrated doses of vaccine composition, together with any required salts and/or buffer agents, preservatives, colorants and the like, ready for preparation of a solution or suspension by the addition of a suitable amount of water. The water may be sterile or nonsterile, although sterile water is generally preferred.
[0209] The composition may be administered via a skin patch. Vaccine Efficacy
[0210] In any aspect, the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein are administered in effective amounts to induce an immune response to a coronavirus.
[0211] As used herein, an immune response to a vaccine composition is the development in a subject of a humoral response to a (one or more) coronavirus protein(s) present in the composition. For purposes of the present invention, a "humoral" immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules.
[0212] In some embodiments, an immune response is assessed by determining [protein] antibody titre in the subject. In some embodiments, the ability of serum or antibody from an immunized subject is tested for its ability to neutralise viral uptake or reduce viral transformation of human cells. In some embodiments, the ability to promote a robust T cell response(s) is measured.
[0213] In some embodiments, an antigen-specific immune response is characterized by measuring an anti-antigen antibody titre produced in a subject administered a composition as provided herein, wherein the antigen is a SARS-CoV-2 S protein (e.g., prefusion stabilized S protein). An antibody titre is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen. Antibody titre is typically expressed as the inverse of the greatest dilution that provides a positive result.
[0214] A variety of serological tests can be used to measure antibody against encoded antigen of interest, for example, SARS-CoV-2 virus or SARS-CoV-2 viral antigen, e.g., SARS-CoV-2 spike or S protein, of domain thereof. These tests include the hemagglutination-inhibition test, complement fixation test, fluorescent antibody test, enzyme-linked immunosorbent assay (ELISA), microneutralisation test, pseudovirus neutralisation test, surrogate virus neutralisation tests (RBD-ACE2 binding inhibition assays) and plaque reduction neutralisation test (PRNT). These tests measures antibody activities in a range of different settings. In exemplary embodiments, neutralising assays such as a plaque reduction neutralisation test, or PRNT (e.g., PRNT50 or PRNT80), or microneutralisation assays, areis used as a serological correlates of protection. These assays measure the biological parameter of in vitro virus neutralisation and are the most serologically virus-specific test among certain classes of viruses, correlating well to serum levels of protection from virus infection.
[0215] The basic design of the PRNT allows for virus-antibody interaction to occur in a test tube or microtitre plate, and then measuring antibody effects on viral infectivity by plating the mixture on virus-susceptible cells, preferably cells of mammalian origin. The cells are overlaid with a semi-solid media that restricts spread of progeny virus. Each virus that initiates a productive infection produces a localized area of infection (a plaque), that can be detected in a variety of ways. Plaques are counted and compared back to the starting concentration of virus to determine the percent reduction in total virus infectivity. In PRNT, the serum sample being tested is usually subjected to serial dilutions prior to mixing with a standardized amount of virus. The concentration of virus is held constant such that, when added to susceptible cells and overlaid with semi-solid media, individual plaques can be discerned and counted. In this way, PRNT end-point titres can be calculated for each serum sample at any selected percent reduction of virus activity.
[0216] In functional assays intended to assess vaccinal immunogenicity, the serum sample dilution series for antibody titration should ideally start below the "seroprotective" threshold titre. Regarding SARS-CoV-2 neutralising antibodies, the "seroprotective" threshold titre remains unknown; but a seropositivity threshold of 1 : 10 can be considered a seroprotection threshold in certain embodiments.
[0217] PRNT end-point titres are expressed as the reciprocal of the last serum dilution showing the desired percent reduction in plaque counts. The PRNT titre can be calculated based on a 50% or greater reduction in plaque counts (PRNT50). A PRNT50 titre is preferred over titres using higher cut-offs (e.g., PRNT80) for vaccine sera, providing more accurate results from the linear portion of the titration curve.
[0218] There are several ways to calculate PRNT titres. The simplest and most widely used way to calculate titres is to count plaques and report the titre as the reciprocal of the last serum dilution to show >50% reduction of the input plaque count as based on the back-titration of input plaques. Use of curve fitting methods from several serum dilutions may permit calculation of a more precise result. There are a variety of computer analysis programs available for this (e.g., SPSS or GraphPad Prism).
[0219] In some embodiments, an antibody titre is used to assess whether a subject has had an infection or to determine whether immunisations are required. In some embodiments, an antibody titre is used to determine the strength of an autoimmune response, to determine whether a booster immunisation is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. In accordance with the present invention, an antibody titre may be used to determine the strength of an immune response induced in a subject by a vaccine composition.
[0220] In some embodiments, antibody-mediated immunogenicity in a subject is assessed at one or more time points. Methods of assessing antibody-mediated immunogenicity are known and include geometric mean concentration (GMC) of antibody to antigen, geometric mean fold rise (GMFR) in serum antibody, geometric mean titre (GMT), median, minimum, maximum, 95% confidence interval (Cl), geometric mean ratio (GMR) of post-baseline/ baseline titres, and seroconversion rate.
[0221] The GMC is the average antibody concentration for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data. GMT is the average antibody titre for a group of subjects calculated by multiplying all values and taking the nth root of this number, where n is the number of subjects with available data.
[0222] A control, in some embodiments, is an anti-antigen antibody titre produced in a subject who has not been administered a vaccine composition, or who has been administered a saline placebo (an unvaccinated subject).
Neutralising antibody titre
[0223] In some embodiments, an immune response may comprise generation of a neutralising antibody titre against coronavirus protein (including, e.g., a stabilized prefusion spike trimer in some embodiments) or a fragment thereof. In some embodiments, an immune response may comprise generation of a neutralising antibody titre against the receptor binding domain (RBD) of the coronavirus spike protein. In some embodiments, a provided immunogenic composition has been established to achieve a neutralising antibody titre in an appropriate system (e.g., in a human infected with coronavirus and/or a population thereof, and/or in a model system thereof).
[0224] In some embodiments, a neutralising antibody titre is a titre that is (e.g., that has been established to be) sufficient to reduce viral infection relative to that observed for an appropriate control. In some such embodiments, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.
[0225] In some embodiments, induction of a neutralising antibody titre may be characterized by an elevation in the number of B cells, which in some embodiments may include plasma cells, class-switched lgG1 - and lgG2-positive B cells, and/or germinal center B cells.
[0226] In some embodiments, induction of a neutralising antibody titre may be characterized by a reduction in the number of circulating B cells in blood.
Methods of prevention and treatment
[0227] In any aspect, the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein generates antibodies to, preferably neutralising antibodies, any one or more of the coronavirus strains described herein, particularly those in Table 3. In any aspect, the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein provides a therapeutic or prophylactic treatment for any or more of the coronavirus strains described herein, particularly those in Table 3. In the aspects and embodiments defined below, reference to “a coronavirus” also includes reference to one or more strains of coronavirus as described herein, including but not limited to those in Table 3, for example: WT, alpha and beta strains; WT, alpha, beta and VIC2089 strains; WT, alpha, beta, and delta strains; WT, alpha, beta, delta, delta plus, gamma, lambda, mu, kappa, iota and omicron strains (including omicron subvariants BA.1 , BA.2, BA.3, BA.4 and BA.5). The RBD antigen, chimeric or fusion protein as described herein, or composition as described herein may provide a therapeutic or prophylactic benefit against SARS-CoV-2 strains that are different from which the RBD amino acid sequence in the RBD antigen, chimeric or fusion protein was derived. [0228] Exemplary strains against which the RBD antigen, chimeric or fusion protein as described herein, or composition as described herein provides a therapeutic or prophylactic benefit are shown in the Examples.
[0229] In one aspect, the present invention provides a method of treating and/or preventing a disease associated with a coronavirus, the method comprising administering to the subject in need thereof, a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby treating and/or preventing a disease associated with a coronavirus, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0230] In another aspect, the present invention provides a method of treating and/or preventing a disease associated with, or caused by, a coronavirus, the method comprising administering to a subject in need thereof a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby treating and/or preventing a disease associated with, or caused by, a coronavirus, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0231] In another aspect, the present invention provides a method of treating and/or preventing a respiratory disease or condition associated with a coronavirus infection, the method comprising administering to a subject in need thereof, a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby treating and/or preventing a respiratory disease or condition associated with a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0232] In another aspect, the present invention provides a method for reducing airway inflammation associated with, or caused by, a coronavirus, the method comprising administering to a subject in need thereof, a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby reducing airway inflammation associated with, or caused by, a coronavirus, preferably the subject has received one, two or more doses of a whole spike based vaccine. [0233] In another aspect, the present invention also provides a method of improving the ability of a subject to control a respiratory disease or condition during a coronavirus infection, the method comprising administering to a subject in need thereof, a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby improving the ability of a subject to control a respiratory disease or condition during a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0234] In another aspect, the present invention provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for raising an immune response in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0235] In another aspect, the present invention provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for increasing an immune response in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine
[0236] In another aspect, the present invention provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for treating and/or preventing a disease caused by a coronavirus in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0237] In another aspect, the present invention further provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for treating and/or preventing a respiratory disease or condition associated with a coronavirus infection in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0238] In another aspect, the present invention further provides for use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for treating and/or preventing a coronavirus infection in a subject who has received one, two or more doses of a whole spike based vaccine.
[0239] In another aspect, the present invention further provides use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for reducing airway inflammation in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0240] In another aspect, the present invention further provides use of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, immune stimulating composition as described herein, in the preparation of a medicament or vaccine for improving the ability of a subject to control a respiratory disease or condition during a coronavirus infection preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0241] In one aspect, the present invention provides for a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in raising an innate immune response in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0242] In one aspect, the present invention provides for a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in increasing an innate immune response in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine
[0243] In another aspect, the present invention provides for a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in treating and/or preventing a disease caused by a coronavirus in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0244] In another aspect, the present invention provides for a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in treating and/or preventing a respiratory disease or condition associated with a coronavirus infection in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0245] In another aspect, the invention provides a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in reducing airway inflammation in a subject diagnosed with, or suspected of having, a coronavirus infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0246] In another aspect, the invention provides a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein, for use in controlling a respiratory disease or condition during a coronavirus infection in a subject, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0247] In any aspect of the invention, where prevention or prophylaxis is intended or required, a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein is administered to the subject before any clinically or biochemically detectable symptoms of viral infection, preferably the subject has received one, two or more doses of a whole spike based vaccine.
[0248] In any aspect of the invention, administration of a coronavirus Spike receptor binding domain (RBD) antigen, a nucleic acid encoding a coronavirus Spike RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein to a subject reduces viral load in the subject, preferably the subject has received one, two or more doses of a whole spike based vaccine. Preferably, the viral load is reduced in the respiratory tract, for example the upper and/or lower respiratory tract. Preferably, the viral load is reduced in the nasal cavity and pharynx (i.e. throat). In alternative embodiments, the viral load may be in the gastrointestinal tract, in the peripheral circulation, in the heart, liver, kidney, spleen or other organ known to be susceptible to infection with coronavirus, preferably to infection with SARS-CoV-2.
[0249] The term “coronavirus infection” or “CoV infection” as used herein, refers to infection with a coronavirus such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term includes coronavirus respiratory tract infections, often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhoea, organ failure (kidney failure and renal dysfunction), septic shock, and death in severe cases.
[0250] A reduction in coronavirus infection may be determined using any method known in the art or described herein, including measuring viral load in a sample from the subject after treatment and comparing it to viral load in a sample from the same subject before treatment. The sample may be any biological sample obtained from the subject, and may include blood, saliva, urine, faeces, nasal wash, sputum, and mucous secretions. The sample may be taken from the respiratory tract, preferably the upper respiratory tract, for example the nose or pharynx (i.e. throat). The term 'respiratory disease' or 'respiratory condition' refers to any one of several ailments that involve inflammation and affect a component of the respiratory system including the upper (including the nasal cavity, pharynx and larynx) and lower respiratory tract (including trachea, bronchi and lungs). The inflammation in the upper and lower respiratory tract may be associated with or caused by viral infection. [0251] A symptom of respiratory disease may include cough, excess sputum production, a sense of breathlessness or chest tightness with audible wheeze.
[0252] The existence of, improvement in, treatment of or prevention of a respiratory disease may be determined by any clinically or biochemically relevant method of the subject or a biopsy therefrom. For example, a parameter measured may be the presence or degree of lung function, signs and symptoms of obstruction; exercise tolerance; night time awakenings; days lost to school or work; bronchodilator usage; Inhaled corticosteroid (ICS) dose; oral glucocorticoid (GC) usage; need for other medications; need for medical treatment; hospital admission.
[0253] As used herein, the term respiratory infection means an infection anywhere in the respiratory tract. Examples of respiratory infection include but are not limited to colds, sinusitis, throat infection, tonsillitis, laryngitis, bronchitis, pneumonia, or bronchiolitis. Preferably, in any embodiment of the invention the respiratory infection is a cold. An individual or subject may be identified as having a respiratory tract infection by viral testing and may exhibit systems of itchy watery eyes, nasal discharge, nasal congestion, sneezing, sore throat, cough, headache, fever, malaise, nausea, vomiting, fatigue and weakness. In one aspect, a subject having a respiratory infection may not have any other respiratory condition. Detection of the presence or amount of virus, preferably coronavirus, may be by PCR/sequencing of RNA isolated from clinical samples (nasal wash, sputum, BAL) or serology.
[0254] As used herein, the term respiratory infection means an infection by a coronavirus, preferably by SARS-CoV-2, anywhere in the respiratory tract.
[0255] An individual or subject may be identified as having a respiratory tract infection by viral testing and may exhibit symptoms of itchy watery eyes, nasal discharge, nasal congestion, sneezing, sore throat, cough, headache, fever, malaise, nausea, vomiting, fatigue and weakness. In one aspect, a subject having a respiratory infection may not have any other respiratory condition. Detection of the presence or amount of virus may be by PCR/sequencing of RNA isolated from clinical samples (nasal wash, sputum, BAL) or serology. The terms "treatment" or "treating" of a subject includes the application or administration of an RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein to a subject (or application or administration of an RBD antigen, chimeric or fusion protein, composition, vaccine or immune stimulating composition as described herein to a cell or tissue from a subject) with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.
[0256] Given the breadth of symptoms caused by coronaviruses, including by SARS- CoV-2, it will be appreciated that a positive response to vaccination according to the methods described herein, may include any amelioration or improvement of symptoms experienced by the subject.
[0257] For example, a positive response to vaccination may be a reduction in general levels of fatigue, muscle pain, headache and/or lethargy in the subject. A positive response may also include a reduction in fever, and a return to afebrile state in the subject.
[0258] A positive response to vaccination may also be prevention or attenuation of worsening of respiratory symptoms following a respiratory virus infection. This could be assessed by comparison of the mean change in disease score from baseline to end of study period, for example, based on a questionnaire, and could also assess lower respiratory symptom score (LRSS - symptoms of chest tightness, wheeze, shortness or breath and cough) daily following infection/onset of cold symptoms. Change from baseline lung function (peak expiratory flow PEF) could also be assessed and a positive response to therapy could be a significant attenuation in reduced PEF. For example, a placebo treated group would show a significant reduction in morning PEF of 15% at the peak of exacerbation whilst the treatment group would show a non-significant reduction in PEF less than 15% change from baseline. [0259] A positive response to vaccination may also be a reduction in the presence of ground-glass type opacities in the lung periphery or near the pleura (for example, as determined using chest CT imaging techniques).
[0260] A positive response to vaccination may also include an increase or return to normal levels of blood oxygenation levels.
[0261] A positive response to vaccination may also include an improvement in cardiovascular disorders such as alterations in blood pressure and increased presence of clotting factors.
[0262] Protective immune responses can include humoral immune responses and cellular immune responses. Protection against SARS-CoV-2 is believed to be conferred through serum neutralising antibodies (humoral immune response) directed to the spike protein, with mucosal IgA antibodies and cell-mediated immune responses also playing a role. Cellular immune responses are useful in protection against SARS-CoV-2 infection with CD4+ and CD8+ T cell responses and memory B cell responses being particularly important. CD8+ immunity is of particular importance in killing virally infected cells. Natural killer cells and NKT cells may also be important for killing and/or clearance of virally infected cells.
Examples
[0263] The inventors performed various studies to:
- investigate whether a WT or variant (Beta) focussed RBD vaccine was able to boost immune response to the Beta variant following priming (and boosting) with WT strain whole spike vaccine (to emulate the antigen exposure that most human vaccine recipients have received in the form of WT whole spike-based vaccines)
- assess whether the Beta variant RBD vaccine could perform as well as, or better than, a Beta-specific whole spike protein vaccine in its ability to boost immunity in general, and whether it can overcome imprinting, in particular in the WT strain spike primed mice - compare the immune responses elicited by WT or the beta variant targeted boosts against other variant SARS-CoV-2 coronaviruses including alpha, beta, gamma, delta, kappa and omicron.
Example 1 - Materials and Methods
RBD-human IgG 1 Fc-fusion:
[0264] To produce recombinant RBD-human lgG1 Fc-fusion protein (SEQ ID NO: 5), recombinant DNA fragments encoding the truncated receptor binding domain (RBD) of SARS-CoV-2 (N334-P527; genbank accession NC_045512.2; SEQ ID NO: 2) fused via a GSGSG linker (SEQ ID NO: 10) to the Fc domain of human IgG 1 (SEQ ID NO: 11 ) from the core-hinge region to the C-terminal lysine followed by a stop codon were codon-optimised for mammalian expression (GeneArt Gene Strings, Thermo Scientific) and synthesised between a 5’ Nhel and a 3’ Xhol cloning site (IDT). This was then cloned into the mammalian expression vector pHLSec (SEQ ID NO: 3).
RBD-beta human IgG 1 Fc fusion
[0265] To enable the cloning of the recombinant DNA fragments encoding the truncated RBD of WT isolate of SARS-CoV-2 (N334-P527; GenBank accession NC-045512; SEQ ID NO: 5) fused via a GSGSG linker (SEQ ID NO: 10) to the Fc domain of human IgG 1 (SEQ ID NO: 11 ) from the core-hinge region to the C-terminal lysine followed by a stop codon from the mammalian expression vector pHLSec into the mammalian expression vector pXC-17.4 PCR primers were designed. The resulting PCR product was cloned into the mammalian expression vector pXC-17.4 using the restriction enzymes sites Hindlll and EcoRI to produce the polynucleotide sequence as set forth in SEQ ID NO: 14. To introduce the mutations K417N, E484K and N501 Y PCR mutagenesis was used. This was the construct used for production of the DoCo-Pro- RBD-1 ADP.
Protein Expressions:
[0266] The RBD-human IgG 1 Fc fusion protein, was expressed by transient transfection of Expi293S cells using ExpiFectamine 293 Transfection Kits as per manufacturer’s instruction (ThermoFisher Scientific). RBD-Fc protein was harvested on day six. RBD-Fc protein was purified from supernatants by Protein A Sepharose (CL- 4B, Cytiva). The RBD-Fc protein was further purified by gel filtration size exclusion chromatography using a Superdex-200 column (Cytiva). The RBD-Fc protein was sterile filtered and stored at -80°C prior to use.
[0267] A research cell bank (RCB) for a stable pool expressing the beta RBD-hFc protein was developed at the National Biologies Facility (QLD node). Cells from a single vial were revived and expanded in shake flasks and the protein produced in a fed batch shaker flask production over 12 days according to the Lonza GSv9™ recommendations. The conditioned media was harvested by depth filtration and 0.2 pm filtration. The antigen was captured on a protein A resin, MabSelect PrismA (Cytiva) with elution at pH 4.0. The antigen was subjected to low pH viral inactivation, with acidification to pH 3.5, holding for > 60 min, then neutralisation to pH 5.5. The antigen was buffer exchanged to pH 6.5 citrate buffer and formulated with PS80 to 0.02 % and diluted to 1 .06 mg/mL before being aseptically filled to vials.
Enzyme-linked immunosorbent assay (ELISA) for measurement of RBD-specific antibody responses
[0268] RBD-specific antibody titres were determined by ELISA. Flat bottom 96 well maxisorp plates (ThermoFisher Scientific) were coated with 50 pl/well of RBD monomer at a concentration of 2 pg/ml in Dulbecco’s phosphate buffered saline (DPBS; Gibco Life Technologies). Plates were incubated overnight at 4°C in a humidified atmosphere. Unbound antibody was removed, and wells were blocked with 100 pl/well of 1 % bovine serum albumin (BSA fraction V, Invitrogen Corporation, Gibco) in PBS for 1 -2 hours before washing 3 times with PBS containing 0.05% v/v Tween-20 (PBST). Serial dilutions of mouse sera were added to wells and left to incubate overnight at room temperature. After washing, bound Ab was detected using horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig Abs (Dako, Denmark). The detection antibody was incubated for 1 hour at room temperature in a humidified atmosphere and the plates then washed five times with PBST. 10OpI of tetramethylbenzidine substrate (TMB, BD Biosciences, cat# 555214) was then added to each well and the reaction was stopped after 5-7 minutes by the addition of 100 pl/well of 1 M orthophosphoric acid (BDH Chemicals, Australia). A Labsystems Multiskan microplate reader (Labsystems, Finland) was used to measure the optical density (OD) of each well at wavelengths of 450 nm and 540 nm. The titres of Ab are expressed as the reciprocal of the highest dilution of serum required to achieve an OD of 0.3.
Microneutralisation Assay
[0269] SARS-CoV-2 isolates used in the microneutralisation assay were propagated in Vero cell cultures and stored at -80°C. Flat-bottom 96-well plates were seeded with Vero cells at 2 x 104 cells/well the day before assay. Serial 2-fold dilutions of heat- inactivated sera were incubated with 100 TCIDso (50% tissue culture infectious dose) of SARS-CoV-2 for 1 hour and residual virus infectivity was assessed in quadruplicate wells of Vero cells. Plates were incubated at 37°C and viral cytopathic effect was read on day 5. The neutralising antibody titre was calculated using the Reed/Muench method.
SARS-CoV-2 Surrogate Virus Neutralisation Test
[0270] The detection of circulating antibodies directed against the spike protein receptor binding domain (RBD) based on antibody-mediated blockage of interaction between the ACE2 receptor protein and RBD was measured in the mouse serum using SARS-CoV-2 Surrogate Virus Neutralisation Test (GenScript, USA) according to the manufacturer’s instructions. In brief, 10 pl of mouse serum was diluted with 90 pl of sample dilution buffer and incubated with horseradish peroxidase conjugated SARS- CoV-2 RBD protein (HRP-RBD); the test solution was added to wells coated with fixed ACE2 receptor. The degree to which serum inhibited binding of the HRP-RBD to ACE2 receptors, compared to control serum, was determined by optical density reading, with 20% inhibition and above considered a positive result.
Example 2 - Administering a booster dose of RBD-hFc dimer + MF59® to mice previously vaccinated with WT Spike vaccines to assess serological responses and to evaluate immunological imprinting.
[0271] Groups of 5 C57BL/6 mice were inoculated via the intramuscular route with 4.5pg of WT Spike protein, 10pg of WT RBD-hFc, 10pg of Beta RBD-hFc or 4.5pg of Beta spike protein in the presence of MF59®. Mice were primed on day 0 and boosted on day 21 . Mice were bled prior to the first inoculation (pre-bleed), 21 days after the first immunisation (1° bleeds), and 2 weeks (day 35) and 5 weeks (day 56) following the second immunisation (2° bleeds). [0272] Humoral responses were evaluated after the first and second immunisation and included anti-RBD antibody responses assessed by ELISA and measured against WT RBD monomer, Beta variant RBD monomer and Delta variant RBD monomer.
[0273] Total anti-WT RBD antibody titres in primary (day 21 ) and secondary (day 35) sera were determined using an RBD-specific ELISA. As shown in Figure 1 strong primary responses were observed in the sera of all groups 21 days after priming. However, in this experiment, the Beta RBD-hFc primed and boosted group displayed a 3-fold lower mean titre relative to the other groups which displayed comparable titres. Nonetheless, antibody titres were still very high in these mice. Increased levels of WT RBD-specific antibodies were detected in sera collected 2 weeks after the second dose (day 35) in all groups of mice. It is noteworthy that the antigen being targeted in this ELISA-based experiment was the WT RBD, which may explain why the response following Beta-RBD prime-boost was lower.
[0274] Total antibody titres to WT RBD, Beta variant (0) RBD and Delta variant (5) RBD were determined in secondary sera collected 5 weeks after the second dose (day 56). Sera from all groups displayed strong binding against all three variants of the RBD monomers assessed, indicating that the WT spike protein, WT RBD-hFc, Beta RBD-hFc and the Beta spike protein administered with MF59® in the combinations assessed can all elicit potent antibody responses against the WT RBD, Beta variant RBD and Delta variant RBD monomers (Figure 2). Furthermore, the mean antibody reactivity against the beta variant RBD from mice receiving the beta-RBD prime and boost, was moderately higher than the antibody reactivity against the WT RBD and delta RBD, and comparable to that of mice that received WT spike prime and boost.
Example 3 - IgG antibody responses to WT, Beta and Delta RBD monomers in mice vaccinated with 2 doses of WT Spike and boosted with WT RBD-hFc, Beta RBD-hFc, WT-spike or Beta spike.
[0275] As a continuation of the study described above, 4 groups of 5 C57BL/6 mice vaccinated via the intramuscular route on days 0 and 21 with 4.5|ig of WT-Spike protein in the presence of MF59®, were boosted on day 70 with a third dose of either 4.5|ig of WT Spike protein, 10|ig of WT RBD-hFc, 10|ig of Beta RBD-hFc or 4.5|ig of Beta spike protein with MF59®. Secondary sera collected 5 weeks (day 56) following two doses of the WT-Spike protein (2° bleed) and tertiary sera collected 16 days (day 86) following the third immunisation with a Spike protein or an RBD-hFc vaccine (3° bleed) were assessed using a multiplex bead-based assay for binding to WT RBD, Beta RBD and Delta RBD (Figure 3).
[0276] Comparing the mean levels of RBD binding antibodies between the 2° and 3° bleeds, it is evident that for mice previously vaccinated with 2 doses of WT-Spike protein, boosting with either WT RBD-hFc (Figure 3A) or Beta RBD-hFc (Figure 3B) enhanced the level of RBD specific antibodies against all 3 RBD antigens assessed. Mice previously vaccinated with 2 doses of WT-Spike protein and boosted with WT- spike (Figure 3C) or Beta-spike (Figure 3D) did not display this level of enhanced RBD binding in 3° bleeds collected after boosting with whole spike antigens. For comparison, human Ab binding levels against the 3 RBD antigens were also assessed in day 35 plasma samples from 5 adults vaccinated on day 0 and 21 with Comirnaty (Pfizer mRNA) vaccine (Figure 3E). Relative to all the mouse sera assessed, plasma from these 5 vaccinees displayed the lowest level of binding to all 3 of the RBD antigens assessed.
[0277] The ability of serum antibodies in tertiary (day 86) sera collected from the mice immunised intramuscularly with 2 doses of WT-spike and then boosted with WT RBD- hFc, Beta RBD-hFc, WT-spike or Beta spike were further assessed for their ability to bind to WT RBD and 8 RBD variants (Figure 4, Figure 5). Sera from all tertiary bleeds exhibited very strong binding to all the RBD antigens assessed. However, mice previously vaccinated with 2 doses of WT-Spike protein and boosted with WT RBD-hFc (Figure 4A, Figure 5A) or Beta RBD-hFc (Figure 4B, Figure 5B) developed the highest mean Ab binding levels against all the RBD antigens compared to the levels observed in mice vaccinated with 2 doses of WT-Spike protein and boosted with WT-spike (Figure 4C, Figure 5C) or Beta spike (Figure 4D, Figure 5D). Plasma samples from the 5 adults vaccinated with Comirnaty exhibited the lowest Ab binding levels against all the RBD antigens assessed (Figure 5E). Example 4 - Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or Beta RBD-hFc + MF59®
[0278] Next, neutralising Ab (nAb) against both WT and Beta variant SARS-CoV-2 strain viruses were assessed using a micro-neutralisation assay. Four sets of mouse samples were selected for testing:
1 . Sera from mice that were primed and boosted with WT Spike + MF59®;
2. Sera from the same mice that were subsequently administered a third injection on day 70 with WT spike + MF59®;
3. Sera from mice that were primed and boosted with WT Spike + MF59®;
4. Sera from the same mice that were subsequently administered a third injection on day 70 with Beta-RBD-hFc + MF59®.
[0279] These results (Figure 6) demonstrated that the Beta-RBD-hFc + MF59® boost provided enhanced mean nAb responses not only against the WT virus but even more prominently against the Beta virus strain. Importantly, the boost provided by the Beta- RBD + MF59® was equal to, or better than, the boost provided by the third dose of WT spike + MF59® vaccine.
[0280] As it was not possible to carry out extensive testing against all the key variants of concern using micro-neutralisation assays, nAb responses were assessed by examining the ability of antibodies in 2° and 3° bleeds to inhibit the interaction between RBD antigens and human ACE2 using the RBD-ACE2 multiplex inhibition assay (Figure 7). Strong neutralising activity against the WT RBD, Beta RBD and Delta RBD (indicated as the half-maximal inhibitory dilution (ID50)) was observed in secondary sera from all groups of mice. After the third vaccination, mean nAb levels were highest in mice that had received a third dose of an RBD-hFc vaccine (Figure 7A-B) relative to the mice that were boosted with spike-based vaccines (Figure 7C-D). Furthermore, these boosted neutralising antibody levels were almost an order of magnitude higher than the neutralising activity observed in 2° sera from the human vaccinees (Figure 7E). Interestingly, there was not a clear difference in the boosting potential comparing the WT and the Beta variant RBD-hFc vaccine, except for the Delta response where the mean titre was moderately higher for the Beta variant RBD-hFc vaccine.
[0281] The neutralising capacity of tertiary sera was further assessed by examining their ability to inhibit the interaction between human ACE2 and WT RBD, and an additional 8 RBD antigens (Figure 8). Antibodies in sera from all tertiary bleeds were able inhibit the interaction between ACE2 and all RBD antigens assessed. However, again it was observed that mice given a third dose of WT RBD-hFc + MF59® (Figure 8A) or Beta RBD-hFc + MF59® (Figure 8B) developed more potent nAb levels against all the RBD antigens than mice given a third dose of WT-spike + MF59® (Figure 8C) or Beta spike + MF59® (Figure 8D). These nAb levels were all higher than those observed from the 5 adults vaccinated with Comirnaty (Figure 8E).
Example 5 - Neutralising Ab responses in mice vaccinated intramuscularly with 2 doses of WT-Spike + MF59® and boosted with a 3rd dose of WT-Spike + MF59® or WT RBD-hFc + MF59®’ or Beta RBD-hFc + MF59®, or Beta Spike + MF59®.
[0282] An in vitro micro-neutralisation assay measured the level of SARS-CoV-2- specific nAb in sera of immunised mice. Stocks of SARS-CoV-2 WT VIC01 used in the microneutralisation assay were propagated and assayed in Vero cell cultures. Stocks of the SARS-CoV-2 omicron variant were propagated and assayed in VeroE6/TMPRSS-2 cells (Matsuyama et al. Proc Natl Acad Sci U S A. 2020 Mar 31 ;117(13):7001 -7003) that overexpress the human transmembrane serine protease, TMPRSS2 (Cell Bank Australia JCRB1819 http://www.cellbankaustralia.com/veroe6-tmprss2.html). Viral stocks were stored at -80°C. Flat-bottom 96-well plates were seeded with Vero or VeroE6/TMPRSS2 cells at 2 x 104 cells/well the day before assay. Serial 2-fold dilutions of heat-inactivated sera were incubated with 10 TCID50 (50% tissue culture infectious dose) of SARS-CoV-2 for 1 hour and residual virus infectivity was assessed in quadruplicate wells. Plates were incubated at 37°C and viral cytopathic effect was read on day 5 for VIC01 , and day 2 for the omicron variant. The dilution of serum that completely prevented CPE in 50% of the wells (ID50) was calculated by the Reed- Muench formula (Reed and Muench, 1938; American Journal of Epidemiology.1938; 27(3):493-497).
[0283] Figure 12 shows nAb responses of experimental replicates against infection of
WT VIC01 (open circle) in Vero cells or omicron variant BA.1 (filled diamond) in VeroE6/TMPRSS2 cells with the tertiary sera (3°) collected 50 days following the third immunisation from groups of 5 mice immunised intramuscularly with WT-spike/WT- spike/WT-spike (groupl ), or WT-spike/WT-spike/WT- RBD-hFc (group 2), or WT-spike/ WT-spike/Beta RBD-hFc (group 3), or WT-spike/WT-spike/Beta-spike (group 4) on day 70. The three vaccinations were administered on day 0, 21 and 70 in the presence of MF59®. The half-maximal inhibitory dilution (ID50) was calculated based on the reciprocal dilution of serum that completely prevented cytopathic effect (CPE) in 50% of the wells and was calculated by the Reed-Muench formula. (LOD = limit of detection, MNS = mouse non-immune serum, WT-Spike = wild type spike, WT RBD-hFc = wild type RBD-human Fc dimer, beta-RBD-hFc = beta variant RBD-human Fc dimer and beta-spike = beta variant spike).
[0284] The data in Figure 12 regarding Omicron variant BA.1 shows
Group 1 = WT spike x 3; Average titre 112
Group 2 = WT spike x 2, WT RBD x 1 ; Ave titre 203
Group 3 = WT spike x 2, beta RBD x 1 ; Ave titre 290
Group 4 = WT spike x 2, beta spike x 1 ; Ave titres 51 .
[0285] The data in Figure 12 clearly shows that a dosing regimen of the invention is capable of driving a neutralising response to an omicron variant (in mice) - with a higher average titre than the whole Spike vaccine that was run in parallel as a booster. While both RBD based vaccines (WT and Beta) as a booster were superior in generating nAb responses compared to whole Spike vaccines as a booster, the beta RBD based vaccine resulted in the highest level of neutralisation. Whereas the whole Spike vaccine boosts drove some nAb responses to the ancestral strain (VIC01 ) they, especially the whole beta Spike vaccine, lost a larger amount of nAb activity when tested against the omicron strain, suggesting that the Spike RBD based vaccine is providing broader cross strain immunity.
[0286] The inventors next tested serum samples from these mice for their ability to neutralise the omicron variants-of-concern (using a later bleed collected 50 days after the third dose). The inventors performed the micro-neutralisation assay using 100 TCID50 virus units/well of omicron BA.1 virus. All samples from mice immunised with 3 doses of WT Spike vaccine showed no neutralising activity above the lower limit of detection (Figure 13) noting the 10-fold higher TCID50 dose used in this assay compared to the previous assay (Figure 12). Similarly, all but one sample from beta spike vaccine boosted mice had undetectable nAb. In contrast, 2/5 samples from WT RBD vaccine boosted mice and 4/5 from beta RBD vaccine boosted mice had clearly detectable nAb titres against omicron BA.1 virus.
[0287] As new variants-of-concern are constantly emerging, including omicron sublineages BA.2, BA.3, BA.4 and BA.5, it is increasingly clear that a vaccine that can provide broad immunity against SARS-CoV-2 variants and ideally the broader family of beta-coronaviruses, is required. Therefore, the inventors tested serum samples from the heterologous third dose boost experiments described above, in a different RBD-ACE2 binding inhibition (sVNT) bead assay carrying a broad panel of CoV-derived RBDs of CoV-derived RBDs including some of the SARS-CoV-2 variants-of-concern RBDs described above, as well as omicron BA.2, plus bat CoV BANAL-52 and BANAL-236 and the pangolin CoV GD-1 . Briefly, samples were collected 30 days (day 100) following the third immunisation from groups of 5 mice immunised intramuscularly with WT-spike/WT-spike/WT-spike (group 1 ), or WT-spike/WT-spike/WT- RBD-hFc (group 2), or WT-spike/ WT-spike/Beta RBD-hFc (group 3), or WT-spike/WT-spike/Beta-spike (group 4). Vaccinations were administered on days 0, 21 and 70, all in the presence of MF59®.
[0288] The results highlighted the breadth of nAb induced by the WT and beta RBD- hFc vaccines. Thus, mean ID50 readings were between 3 and 7-fold higher for samples from the WT and beta RBD boosted mice for all of the SARS-CoV-2 RBDs tested, including WT, alpha, beta, gamma, delta, delta+, kappa, lambda, mu, omicron BA.1 , omicron BA.2 (Figure 14). Inhibition of omicron BA.5 RBD-ACE-2 binding was also tested in a similar assay with the WT and beta-RBD vaccine boosts resulting in two to three-fold higher mean titres against the BA.5 variant compared to WT S boost (Data no shown).
[0289] Similarly, increased mean titres against several of the bat CoVs including BANAL-52, BANAL-236 and the pangolin CoV GD-1 (Figure 15).
[0290] Taken together, the results described herein suggest that the RBD-hFc vaccine works well not only as a prime and boost vaccine, but also as a booster vaccine for mice that have previously been primed and boosted with WT spike vaccines. Furthermore, the response to the RBD vaccines (WT and beta) were stronger than those from a third dose of whole spike vaccine (WT and beta), which suggests the third dose RBD vaccine boost is focussing the immune response on the critical part of the spike, being the RBD, for enhancing the nAb response. Interestingly, while there was not clear evidence of selective beta-specific immunity, the beta RBD-Fc vaccine enhanced nAb responses across all variant-RBDs tested, including the original WT, lambda, gamma, mu and importantly, the delta, delta plus and omicron variants (including subvariants BA.1 and BA.2).
[0291] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims

1 . A method for increasing an immune response in a subject who has been exposed to a coronavirus whole Spike protein, the method comprising administering to the subject a therapeutically effective amount of a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby increasing an immune response in the subject.
2. A method according to claim 1 , wherein the subject has been exposed to a whole Spike protein through a naturally acquired infection of a coronavirus, preferably an infection with SARS-CoV-2 or variants thereof.
3. A method according to claim 1 , wherein the subject has been exposed to a whole Spike protein by immunisation with a vaccine comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen.
4. A method according to claim 3, wherein the vaccine comprising a coronavirus whole Spike antigen is a protein based vaccine that comprises a whole Spike protein.
5 A method according to claim 4, wherein the protein based vaccine comprises an inactivated coronavirus, preferably an inactivated SARS-CoV-2.
6. A method according to claim 3, wherein the vaccine comprising a coronavirus whole Spike antigen is nucleic acid based vaccine that encodes a whole Spike antigen.
7. A method according to claim 6, wherein the nucleic acid is DNA.
8. A method according to claim 6, wherein the nucleic acid is RNA.
9. A method according to claim 8, wherein the RNA is mRNA.
10. A method according to claim 6, wherein the nucleic acid is a viral vector comprising a nucleotide sequence that encodes a whole Spike antigen, preferably the viral vector is an adenoviral vector, preferably a chimp or human adenoviral vector.
11. A method according to any one of claims 3 to 10, wherein the subject has received a prime (e.g. first dose) and boost immunisation (e.g. second or further dose).
12. A method according to claim 11 , wherein the prime and boost immunisations are with the same vaccine comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen.
13. A method according to claim 11 , wherein the prime and boost immunisations are with different vaccines comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen.
14. A method according to any one of claims 11 to 13, wherein the prime (or first), boost (or second) are administered by the same route of administration.
15. A method according to claim 14, wherein the route of administration is intramuscular.
16. A method according to any one of claims 3 to 10, wherein the subject has been immunised with at least two doses of the vaccine comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen.
17. A method according to claim 16, wherein the subject was immunised with the same vaccine comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen.
18. A method according to claim 16, wherein the subject was immunised with different vaccines comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen.
19. A method according to any one of claims 1 to 18, further comprising a step of identifying a subject who has been exposed to a whole Spike protein through a naturally acquired infection of a coronavirus or by immunisation with a coronavirus whole Spike based vaccine.
20. A method for immunizing a subject against a coronavirus infection, the method comprising a step of administering to a subject a vaccine comprising a coronavirus Spike receptor binding domain (RBD) antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, wherein the subject has been exposed to a coronavirus whole Spike protein, thereby immunizing a subject against a coronavirus infection.
21 . A method for immunizing a subject against a coronavirus infection, the method comprising the steps of:
(i) administering a first and second dose of a vaccine comprising a coronavirus whole Spike antigen or a nucleic acid encoding a coronavirus whole Spike antigen; and
(ii) subsequently to (i), administering a dose of a vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen, thereby immunizing the subject against a coronavirus infection.
22. A method according any one of claims 1 to 21 , wherein the coronavirus Spike receptor binding domain (RBD) antigen is a chimeric or fusion protein.
23. A method according to any one of claims 1 to 21 , wherein the nucleic acid encoding a coronavirus Spike RBD antigen encodes a chimeric or fusion protein.
24. A method according to any one of claims 22 or 23, wherein the chimeric or fusion protein comprises a dimer of receptor binding domains from a Spike protein of a coronavirus linked to an Fc region of an antibody.
25. A method according to any one of claims 22 to 24, wherein the chimeric or fusion protein comprises a dimer of receptor binding domains from a Spike protein of a coronavirus linked to a polypeptide comprising an Fc receptor binding domain.
26. A method according to any one of claims 1 to 25, wherein the coronavirus Spike RBD antigen is from a coronavirus from any of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus or Deltacoronavirus.
27. A method according to any one of claims 1 to 26, wherein the coronavirus Spike RBD antigen is from a coronavirus from one of the Alphacoronavirus subgroup clusters 1 a and 1 b or one of the Betacoronavirus subgroup clusters 2a, 2b, 2c, and 2d.
28. A method according to any one of claims 1 to 27, wherein the coronavirus Spike RBD antigen is from SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43 or HKU1.
29. A method according to claim 28, wherein the receptor binding domain comprises an amino acid sequence from a WT (“Wuhan”), alpha, beta, gamma, kappa, omicron or delta SARS-CoV-2 strain or any other strain defined in Table 3.
30. A method according to claim 29, wherein the receptor binding domain comprises an amino acid sequence from a WT SARS-CoV-2 strain.
31 . A method according to claim 29, wherein the receptor binding domain comprises an amino acid sequence from a beta SARS-CoV-2 strain.
32. A method according to claim 29, wherein the Spike RBD antigen comprises an amino acid sequence from an omicron SARS-CoV-2 strain.
33. A method according to any one of claims 1 to 29, wherein the receptor binding domain comprises an amino acid sequence from N334 to P527 of SEQ ID NO: 1 .
34. A method according to any one of claims 1 to 29, wherein the receptor binding domain consists of an amino acid sequence from N334 to P527 of SEQ ID NO: 1 .
35. A method according to any one of claims 1 to 29, wherein the receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 2 or 12, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 2 or 12.
36. A method according to claim 35, wherein the amino acid sequence includes one or more of the mutations as shown in Figure 9, or Table 3.
37. A method according to any one of claims 1 to 29, wherein the receptor binding domain from a Spike protein of a coronavirus comprises, consists essentially of or consists of an amino acid sequence of any one or more of SEQ ID NO: 2 or 12 having with 0 to 8 amino acid insertions, deletions, substitutions or additions (or a combination thereof), preferably from 0 to 7, preferably from 0 to 6, preferably from 0 to 5, preferably from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1 amino acid insertions, deletions, substitutions or additions (or a combination thereof), wherein the amino acid insertions, deletions, substitutions or additions (or combination thereof) are located at the N- and/or C-terminus.
38. A composition according to any one of claims 24 to 37, wherein the Fc region of the antibody is an Fc region of an IgG.
39. A method according to claim 38, wherein the IgG is IgG 1 .
40. A method according to claim 39, wherein the IgG is human.
41 . A method according to any one of claims 24 to 40, wherein the Fc region of the chimeric or fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of said heavy chain.
42. A method according to any one of claims 24 to 41 , wherein the Fc region of an antibody comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 16 or 18, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 16 or 18.
43. A method according to any one of claims 24 to 42, wherein the Fc region of an antibody comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 16 or 18 having with 0 to 8 amino acid insertions, deletions, substitutions or additions (or a combination thereof), preferably from 0 to 7, preferably from 0 to 6, preferably from 0 to 5, preferably from 0 to 4, preferably from 0 to 3, preferably from 0 to 2, preferably from 0 to 1 amino acid insertions, deletions, substitutions or additions (or a combination thereof).
44. A method according to any one of claims 22 to 43, wherein the chimeric or fusion protein comprises, consists essentially of or consists of a sequence as set forth in any one of SEQ ID NO: 2, 4, 5, 7, 8, 9, 12, 13 or 18.
45. A method according to any one of claims 22 to 44, wherein the chimeric or fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 13.
46. A method according to any one of claims 22 to 44, wherein the chimeric or fusion protein consists of an amino acid sequence as set forth in SEQ ID NO: 13.
47. A method according to any one of claims 22 to 44, wherein the chimeric or fusion protein comprises an amino acid sequence as set forth in SEQ ID NO: 5.
48. A method according to any one of claims 22 to 44, wherein the chimeric or fusion protein consists of an amino acid sequence as set forth in SEQ ID NO: 5.
49. A method according to any one of claims 1 to 48, wherein the vaccine comprising a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen is administered with an adjuvant.
50. A method according to claim 49, wherein the adjuvant is a TLR2-agonist, more preferably a Pam-2-Cys containing molecule such as PEG-R4-Pam-2-Cys, or a stimulator of NKT cells, more preferably alpha-Galactosylceramide (also referred to herein as “a-GalCer”), alpha-glucosylceramide, beta-mannosylceramide, or other NKT cell-stimulatory lipid molecules and analogues thereof.
51 . A method according to claim 49, wherein the adjuvant is selected from the group consisting of Pam3CSK4, PEG-R4-Pam-2-Cys, MALP-2, lipoteichoic acid, OspA, Porin, LcrV, lipomannan, Lysophosphatidylserine, Lipophosphoglycan (LPG), Glycophosphatidylinositol (GPI) and Zymosan.
52. A method according to claim 49, wherein the adjuvant is selected from the group consisting of poly-l:C, CpG, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, B(C, CP-870,893, CpG7909, CyaA, ASO3, ASO4, MatrixM, dSLIM, GM-CSF, IC30, IC31 , Imiquimod, ImuFact IMP321 , IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59®, AddaVax™, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA V, Montanide ISA-51 , OK-432, OM-174, OM-197-MP-EC, ONTAK. PEPTEL, vector system, PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam-3-Cys, and Aquila's QS21 stimulon.
53. A method according to any one of claims 49 to 52, wherein the adjuvant comprises a metabolizable oil and an emulsifying agent.
54. A method according to claim 53, wherein the oil and the emulsifying agent are present in the form of an oil-in-water emulsion having oil droplets substantially all of which are less than 1 micron in diameter.
55. A method according to claim 54, wherein the oil is squalene.
56. A method according to any one of claims 53 to 55, wherein the adjuvant further comprises polyoxyethylenesorbitan monooleate and sorbitan trioleate.
57. A method according to claim 56, wherein the adjuvant comprises 4.3% squalene, 0.5% polyoxyethylenesorbitan monooleate, 0.5% sorbitan trioleate.
58. A method according to any one of claims 19 to 57, wherein the coronavirus infection is an infection with a coronavirus from any of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus or Deltacoronavirus.
59. A method according to any one of claims 19 to 58, wherein the coronavirus infection is an infection from one of the Alphacoronavirus subgroup clusters 1 a and 1 b or one of the Betacoronavirus subgroup clusters 2a, 2b, 2c, and 2d.
60. A method according to any one of claims 19 to 59, wherein the coronavirus infection is an infection with SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43 or HKU1 .
61 . A method according to claim 60, wherein the coronavirus infection is an infection with SARS-CoV-2 preferably a variant described in Table 3, more preferably the omicron variant.
62. A method according to any one of claims 4 to 19 and 21 to 61 , wherein the coronavirus whole Spike antigen is from a coronavirus from any of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus or Deltacoronavirus.
63. A method according to any one of claims 4 to 19 and 21 to 62, wherein the coronavirus whole Spike antigen is from a coronavirus from one of the Alphacoronavirus subgroup clusters 1 a and 1 b or one of the Betacoronavirus subgroup clusters 2a, 2b, 2c, and 2d.
64. A method according to any one of claims 4 to 19 and 21 to 63 to 63, wherein the coronavirus whole Spike antigen is from SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV- NL63, HCoV-229E, HCoV-OC43 or HKU1.
65. A method according to claim 64, wherein the coronavirus whole Spike antigen is from SARS-CoV-2.
66. A method according to claim 65, wherein the coronavirus whole Spike antigen is from SARS-CoV-2 variant described in Table 3.
67. A method according to claim 66, wherein the coronavirus whole Spike antigen is from the omicron variant.
68. A method according to claim 1 or 20, wherein the coronavirus infection is a SARS-CoV-2 infection, the coronavirus whole Spike antigen is a SARS-CoV-2 whole Spike antigen and the coronavirus Spike RBD antigen is a SARS-CoV-2 WT or beta variant Spike RBD antigen.
69. Use of a coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen in the preparation of a medicament or vaccine for treating and/or preventing (a) a disease associated with, or caused by, a coronavirus, or (b) a coronavirus infection in a subject in need thereof who has been exposed to a coronavirus whole Spike protein.
70. A coronavirus Spike RBD antigen or a nucleic acid encoding a coronavirus Spike RBD antigen for use for treating and/or preventing (a) a disease associated with, or caused by, a coronavirus, or (b) a coronavirus infection in a subject in need thereof who has been exposed to a coronavirus whole Spike protein.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021178971A1 (en) * 2020-03-06 2021-09-10 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Vaccines against sars-cov-2 and other coronaviruses
WO2022115471A1 (en) * 2020-11-24 2022-06-02 Tiba Biotech Llc Vaccination by heterologous boost immunization
WO2022133547A1 (en) * 2020-12-23 2022-06-30 The University Of Melbourne Coronavirus vaccine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021178971A1 (en) * 2020-03-06 2021-09-10 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Vaccines against sars-cov-2 and other coronaviruses
WO2022115471A1 (en) * 2020-11-24 2022-06-02 Tiba Biotech Llc Vaccination by heterologous boost immunization
WO2022133547A1 (en) * 2020-12-23 2022-06-30 The University Of Melbourne Coronavirus vaccine

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CAO Y. ET AL.: "Humoral immunogenicity and reactogenicity of CoronaVac or ZF2001 booster after two doses of inactivated vaccine", CELL RESEARCH, vol. 32, 3 December 2021 (2021-12-03), pages 107 - 109, XP037654386, DOI: 10.1038/s41422-021-00596-5 *
LI WENJUAN, LI XINGXING, ZHAO DANHUA, LIU JINGJING, WANG LING, LI MIAO, LIU XINYU, LI JIA, WU XIAOHONG, LI YUHUA: "Heterologous prime-boost with AdC68- and mRNA-based COVID-19 vaccines elicit potent immune responses in mice", SIGNAL TRANSDUCTION AND TARGETED THERAPY, vol. 6, no. 1, XP093086086, DOI: 10.1038/s41392-021-00843-6 *
SONG SHUO, ZHOU BING, CHENG LIN, LIU WEILONG, FAN QING, GE XIANGYANG, PENG HUA, FU YANG-XIN, JU BIN, ZHANG ZHENG: "Sequential immunization with SARS-CoV-2 RBD vaccine induces potent and broad neutralization against variants in mice", VIROLOGY JOURNAL, vol. 19, no. 1, 1 December 2022 (2022-12-01), XP093046924, DOI: 10.1186/s12985-021-01737-3 *
TAN HYON-XHI, JUNO JENNIFER A., LEE WEN SHI, BARBER-AXTHELM ISAAC, KELLY HANNAH G., WRAGG KATHLEEN M., ESTERBAUER ROBYN, AMARASENA: "Immunogenicity of prime-boost protein subunit vaccine strategies against SARS-CoV-2 in mice and macaques", NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, UK, vol. 12, no. 1, 3 March 2021 (2021-03-03), UK, pages 1403 - 1403, XP055923220, ISSN: 2041-1723, DOI: 10.1038/s41467-021-21665-8 *
WANG XUN, ZHAO XIAOYU, SONG JIEYU, WU JING, ZHU YUQI, LI MINGHUI, CUI YUCHEN, CHEN YANJIA, YANG LULU, LIU JUN, ZHU HUANZHANG, JIAN: "Homologous or heterologous booster of inactivated vaccine reduces SARS-CoV-2 Omicron variant escape from neutralizing antibodies", EMERGING MICROBES & INFECTIONS, vol. 11, no. 1, 31 December 2022 (2022-12-31), pages 477 - 481, XP093086088, DOI: 10.1080/22221751.2022.2030200 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11878055B1 (en) 2022-06-26 2024-01-23 BioNTech SE Coronavirus vaccine

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