WO2022096899A1 - Viral spike proteins and fusion thereof - Google Patents

Viral spike proteins and fusion thereof Download PDF

Info

Publication number
WO2022096899A1
WO2022096899A1 PCT/GB2021/052893 GB2021052893W WO2022096899A1 WO 2022096899 A1 WO2022096899 A1 WO 2022096899A1 GB 2021052893 W GB2021052893 W GB 2021052893W WO 2022096899 A1 WO2022096899 A1 WO 2022096899A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
polypeptide
cov
cell
rbd
Prior art date
Application number
PCT/GB2021/052893
Other languages
French (fr)
Inventor
James SILLIBOURNE
Martin PULÉ
Leila MEKKAOUI
Original Assignee
Autolus Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Autolus Limited filed Critical Autolus Limited
Publication of WO2022096899A1 publication Critical patent/WO2022096899A1/en

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • 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 polypeptides derived from viral spike proteins.
  • SARS-CoV-2 is currently considered a pandemic infection involving 190 countries, with more than 37 million confirmed cases and more than 1 million confirmed deaths worldwide, as of 13 th October 2020. This highlights the need for rapid vaccine development to curb the spread of disease and prevent mortalities.
  • Vaccines to pathogenic viruses or bacteria can be generated through the generation of an attenuated form of the virus or bacterium, the purification of viral (viral-like particles) or bacterial derived membranes (outer membrane vesicles), the production of recombinant protein, or the synthesis of DNA/RNA encoding a potential immunogen.
  • Vaccines to viral or bacterial proteins can consist of attenuated forms of the pathogen, purified membrane (viral-like particles or outer membrane vesicles prepared from viruses or bacteria, respectively), recombinantly produced protein, RNA or DNA encoding the immunogen.
  • purified membrane viral-like particles or outer membrane vesicles prepared from viruses or bacteria, respectively
  • RNA or DNA encoding the immunogen.
  • the yield of viral-like particles or outer membrane vesicles can be variable and the abundance of immunogens within these preparations may not be consistent, complicating the manufacturing process.
  • Recombinant protein production requires substantial optimisation to yield protein that is soluble, folded in its native conformational state and, depending on the type of protein, post-translationally modified to recapitulate the immunogenic viral or bacterial protein. Consequently, recombinant protein production is both time-consuming and expensive.
  • the synthesis of DNA/RNA is relatively inexpensive and the delivery of nucleic acids in lipid nanoparticles (LNP) is usually effective and results in the expression of the viral/bacterial polypeptide and the elicitation of an immune response.
  • LNP lipid nanoparticles
  • problems associated with these approaches include: (i) insufficient secretion of soluble protein; (ii) and, in the case of outer membrane vesicles, exuberant activation of the innate immune system due to the presence of lipopolysaccharides present in the preparation, which activate Toll-like receptors present on the surface of monocytes and macrophages.
  • General approaches adopted to overcome manufacturing issues and improve the expression level of immunogens include codon optimisation, substitution of signal peptide sequences of secreted and membrane bound antigens to improve their expression, and the deletion or mutations of domains known to cause cytotoxicity or have inhibitory effects.
  • the present invention describes a method for reliably expressing immunogenic peptides at the cell surface in a manner that they are accessible to the immune system. This method may be used as a rapid means of generating novel vaccines to raise immunity to pathogenic viruses. It overcomes the disadvantages and limitations of expressing soluble fragments of viral proteins, by attaching them to endogenously or exogenously derived transmembrane anchors.
  • the present inventors have expressed viral proteins as membrane-bound fusions to direct them to the secretory pathway and recapitulate the glycosylation observed on the native proteins.
  • expression of the RBD alone on the surface of the cell may lead to the generation of antibodies recognising this domain, which could potentially be neutralising and block viral infection.
  • transmembrane anchors to display viral or bacterial immunogens will result in a more prolonged expression compared to soluble protein and engage the immune system to produce a profound response leading to long-term memory.
  • membrane-bound immunogens There are two advantages associated with the expression of membrane-bound immunogens: first, they are more likely to be post-translationally modified in the same way as the native protein and serve as relevant immunogens for the generation of neutralising antibodies; and second, they may be retained better than soluble antigens and persist for longer in the host, increasing the likelihood of a profound immune response.
  • the present invention provides a polypeptide comprising an amino acid sequence of at least one receptor-binding domain (RBD) of a viral Spike protein, a spacer and a transmembrane domain, wherein the spacer is a spacer from a mammalian protein.
  • RBD receptor-binding domain
  • the spacer from a mammalian protein may be a CD8a stalk.
  • the viral Spike protein may be a coronavirus Spike protein.
  • the Coronavirus may be SARS-CoV-2.
  • the RBD may comprise the amino acid sequence shown as SEQ ID NO: 4.
  • the polypeptide of the first aspect of the invention may comprise the amino acid sequence shown as SEQ ID NO: 35.
  • the transmembrane domain may be a CD8a transmembrane domain.
  • the polypeptide of the first aspect of the invention may further comprise an endodomain.
  • the endodomain may be a CD8a endodomain.
  • the present invention provides a nucleic acid encoding a polypeptide according to the first aspect of the invention.
  • the present invention provides a nucleic acid construct encoding two or more polypeptides according to the first aspect of the invention.
  • the present invention provides a vector comprising a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention.
  • the present invention provides a cell comprising a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention.
  • the present invention provides a method for making a cell according to the fifth aspect of the invention, which comprises the step of transducing or transfecting a cell with a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a cell or plurality of cells according to the fifth aspect of the invention, and at least one pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.
  • the present invention provides a method for presenting a viral Spike protein-derived peptide, comprising expressing a polypeptide according to the first aspect of the invention in a cell.
  • the viral Spike protein may be a coronavirus Spike protein.
  • the present invention provides a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention for use in preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus.
  • this aspect of the invention may be formulated as a use of a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention in the manufacturing of a medicament for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus.
  • this aspect of the invention may be formulated as a method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, in a subject in need thereof, comprising the step of administrating to the subject a therapeutically effective amount of a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.
  • the virus may be a coronavirus.
  • the coronavirus may be SARS-CoV-2.
  • FIG. 1 Staining of SARS CoV-2 spike SI and S2 fragments expressed on the surface of 293T cells with anti-SARS CoV-2 CR3022 antibody.
  • FIG. 1 Staining of SARS CoV-2 spike SI and S2 fragments expressed on the surface of 293T cells with soluble ACE2-Fc and PE-C7-conjugated anti-HA antibody to detect the fragments of spike protein on the cell surface.
  • FIG. 3 Flow cytometry plots of SupTl cells transduced with y-retrovirus encoding SI subunit and RBD transmembrane fusions and stained with soluble ACE2-Fc and Alexa 647- conjugated anti-human IgG.
  • Rats were immunised with plasmid DNA encoding the RBD-CD8a spacer and transmembrane fusion and sera collected from them. Sera obtained pre- and postimmunisation were used to stain non-transduced SupTl cells or RBD-CD8a spacer and transmembrane expressing SupTl cells and flow cytometric analysis carried out. Positive staining of SupTl RBD-CD8a spacer and transmembrane expressing cells was observed using the post-immunisation sera (top histogram in each panel) but not with the preimmunisation sera.
  • SARS-CoV-2 spike protein showing its structural domains.
  • S signal peptide
  • NTD N-terminal domain
  • RBD receptor binding domain
  • FP fusion peptide
  • HR2, heptad repeat 2 TM
  • CT cytoplasmic domain
  • ER endoplasmic reticulum retention domain.
  • B Diagram of SARS-CoV-2 spike and CD8a stalk fusion constructs.
  • HA haemagglutinin epitope tag
  • STK CD8a stalk
  • TM transmembrane domain
  • CT cytoplasmic domain.
  • FIG. 1 Multi RBD constructs for the expression of SARS-CoV-2 variants.
  • Figure 9 Screening of SARS-CoV-2 patient sera using SupTl-spike and RBD expressing cell lines.
  • the present invention describes a method of anchoring and presenting fragments of viral glycoproteins on the cell surface to elicit an immune response and the generation of neutralising antibodies capable of inhibiting viral entry and preventing viral infection.
  • the present invention provides a polypeptide, hereinafter “the polypeptide of the invention”, comprising an amino acid sequence of at least one receptor-binding domain (RBD) of a viral Spike protein, a spacer and a transmembrane domain.
  • RBD receptor-binding domain
  • polypeptide refers to natural, synthetic, and recombinant proteins or peptides generally having more than 10 amino acids.
  • the polypeptide of the invention comprises an amino acid sequence of at least one RBD of a viral Spike protein.
  • viral spike protein or “S protein”, as used herein, refers to a glycoprotein spike on a viral capsid or viral envelope. It is also termed peplomer. These protrusions bind only to certain receptors on the host cell. Their function is essential for both host specificity and viral infectivity.
  • the viral Spike protein or S protein may be a coronavirus Spike protein.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 SARS- CoV-2.
  • SARS-CoV a lineage B beta-CoV, emerged from bat and palm civet, and infected over 8,000 people and caused about 800 deaths.
  • MERS-CoV a lineage C beta-CoV
  • SARS-CoV-2 a novel coronavirus
  • HCoV-OC43 In addition to the highly pathogenic zoonotic pathogens SARS-CoV, MERS-CoV, and SARS-CoV-2, all belonging to the P-coronavirus genus, four low-pathogenicity coronaviruses are endemic in humans: HCoV-OC43, HCoV-HKUl, HCoV-NL63, and HCoV-229E.
  • coronaviruses are a diverse group of large RNA viruses that cause varieties of diseases in humans and other animals, including respiratory, enteric, renal, and neurological diseases.
  • Coronaviruses are enveloped viruses that contain a large single-stranded RNA genome of positive polarity. At ⁇ 30,000 nucleotides (nt), their genome is the largest found in any of the RNA viruses.
  • Their envelope accommodates three or four membrane proteins of which the membrane (M), envelope (E), and spike (S) proteins are common to all.
  • the S protein is a relatively large, about 180kDa type I glycoprotein, trimers of which form the petal-shaped projections on the surface of the virion that give rise to the characteristic corona solis-like appearance. It has been suggested that the SI subunit constitutes the globular head, while the S2 subunit forms the stalk-like region of the spike.
  • the two functions of the coronavirus S protein appear to be spatially separated.
  • the SI subunit (or the equivalent part in viruses with uncleaved S protein) is responsible for receptor binding, and the S2 subunit is responsible for membrane fusion.
  • NTD N-terminal domain
  • C-domain C-terminal domain
  • RBD receptor-binding domain
  • MHV uses mouse carcinoembryonic antigen related cell adhesion molecule la (mCEACAMla) as the receptor, and the receptor for SARS-CoV and SARS-CoV-2 is human angiotensin-converting enzyme 2 (hACE2) and that of MERS-CoV is dipeptidyl peptidase 4 (DPP4).
  • hACE2 human angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • S proteins of SARS-CoV-2 share about 76% and 97% of amino acid identity with SARS-CoV and RaTG13, respectively, and the amino acid sequence of potential RBD of SARS-CoV-2 is about 74% and 90.1% homologous to that of SARS-CoV and RaTG13, respectively.
  • the ectodomain of the S2 subunit contains two heptad repeat (HR) regions, HR1 and HR2, characteristic of coiled coils, while the fusion peptide (FP) is predicted to be located amino terminally of the first HR region (HR1).
  • HR heptad repeat
  • FP fusion peptide
  • Binding of the SI subunit to the (soluble) receptor has been shown to trigger conformational changes that supposedly facilitate virus entry by activation of the fusion function of the S2 subunit.
  • the conformational changes are thought to expose the fusion peptide and to lead to the formation of a heterotrimeric six-helix bundle by the two HR regions, a characteristic of class I viral fusion proteins, resulting in the close locations of the fusion peptide and the transmembrane domain in the process of membrane fusion.
  • the S2 subunit additionally contains a transmembrane domain required for anchoring of the spike protein and a cytoplasmic endoplasmic reticulum (ER) retention motif.
  • Coronavirus S proteins are typical class I viral fusion proteins, and protease cleavage is required for activation of the fusion potential of S protein.
  • a two-step sequential protease cleavage model has been proposed for activation of S proteins of SARS-CoV and MERS- CoV, priming cleavage between SI and S2 and activating cleavage on S2’ site.
  • CoV S proteins may be cleaved by one or several host proteases, including furin, trypsin, cathepsins, transmembrane protease serine protease-2 (TMPRSS-2), TMPRSS-4, or human airway trypsin-like protease (HAT). Availability of these proteases on target cells largely determines whether coronaviruses enter cells through plasma membrane or endocytosis.
  • the coronavirus S protein may be the S protein of one of the following coronavirus: SARS- CoV-2, SARS-CoV, SARS-like CoV RaTG13, MERS-CoV, HCoV-OC43, HCoV-HKUl, HCoV-NL63, and HCoV-229E.
  • the coronavirus S protein may be the S protein of SARS-CoV-2, SARS-CoV, or SARS-like CoV RaTG13.
  • the coronavirus S protein may be the S protein of SARS-CoV-2.
  • the spike protein of SARS-CoV-2 is depicted under Accession No. P0DTC2 in the Uniprot database on 8 th October 2020.
  • S protein SEQ ID NO: 1; signal sequence underlined
  • subunit SI SEQ ID NO: 2
  • subunit S2 ectodomain SEQ ID NO: 3; HR1 region is underlined and HR2 region is in bold
  • SARS-CoV-2 S protein (SEQ ID NO: 1):
  • SARS-CoV-2 Spike protein SI subunit (SEQ ID NO: 2):
  • SARS-CoV-2 Spike protein S2 subunit (SEQ ID NO: 3):
  • the polypeptide of the invention comprises an amino acid sequence of at least one RBD of a viral Spike protein.
  • receptor binding domain or “RBD”, as used herein, refers to a short immunogenic fragment from a virus that binds to a specific endogenous receptor sequence to gain entry into host cells.
  • the RBD is a part of the Spike glycoprotein (S- protein) which is needed to interact with endogenous receptors to facilitate membrane fusion and delivery to the cytoplasm.
  • S- protein Spike glycoprotein
  • the RBD is also the binding site of neutralizing antibodies.
  • the RBD contains the receptor-binding motif (RBM) that interacts directly with the endogenous receptor.
  • the SARS-CoV-2 RBD has a twisted five-stranded antiparallel P sheet (P 1, P2, P3, P4 and P7) with short connecting helices and loops that form the core. Between the P4 and P7 strands in the core, there is an extended insertion containing the short P5 and P6 strands, a4 and a5 helices and loops. This extended insertion is the RBM, which contains most of the contacting residues of SARS-CoV-2 that bind to ACE2.
  • the SARS-CoV-2 RBD spans residues Arg319 to Phe541 of the sequence shown as SEQ ID NO: 1 (SARS-CoV-2 Spike protein). This is the RBD of the SARS-CoV-2 variant first identified in Wuhan in December 2019.
  • SARS-CoV 2 Other variants of SARS-CoV 2 that have been identified include the delta variant (B.1.617.2), the epsilon variant (B.1.429), the Mu variant (B.1.621), the lambda variant (C.37), the eta variant (B.1.525), the theta variant (P.3), and the variant kappa (B. J .617.1 ) Further variants are expected to emerge.
  • the mutation numbering refers to the SARS-CoV-2 S protein as shown in SEQ ID NO: 1.
  • the RBD may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 44-53.
  • SEQ ID NO: 4 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; Wuhan): RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN CYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
  • SEQ ID NO: 44 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; N501Y; alpha): RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN CYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
  • SEQ ID NO: 45 amino acids 319 to 541 of SARS-CoV-2 Spike protein; K417N_E484K_N501Y; beta
  • SEQ ID NO: 46 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; K417T_E484K_N501Y; gamma): RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
  • SEQ ID NO: 47 amino acids 319 to 541 of SARS-CoV-2 Spike protein; L452R T478K; delta
  • SEQ ID NO: 48 amino acids 319 to 541 of SARS-CoV-2 Spike protein; L452R; epsilon:
  • SEQ ID NO: 49 amino acids 319 to 541 of SARS-CoV-2 Spike protein
  • SEQ ID NO: 50 amino acids 319 to 541 of SARS-CoV-2 Spike protein; L452Q F490S; lambda
  • NCYSPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF SEQ ID NO: 51 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; E484K; eta):
  • SEQ ID NO: 52 amino acids 319 to 541 of SARS-CoV-2 Spike protein; E484K_N501Y; theta):
  • SEQ ID NO: 53 amino acids 319 to 541 of SARS-CoV-2 Spike protein; L452R E484Q; kappa
  • the present invention is not intended to be limited to the above spike protein and RBD variants. Therefore, Spike proteins and RBDs of variants that have not been yet identified and future variants are also within the scope of the present invention.
  • the present invention also contemplates a polypeptide of the invention comprising two or more RBDs.
  • the polypeptide of the invention may comprise three or more, four or more, five or more, six or more RBDs.
  • the two or more RBDs may have the same sequence or different sequences.
  • the two or more RBDs may have different sequences. This is particularly advantageous when the polypeptide of the invention is used to elicit immunological responses against different viruses, viral strains or Spike protein variants.
  • the sequences of the two or more RBDs may be selected from the group consisting of shown as SEQ ID NO: 4 and 44-53.
  • the two or more RBDs may be joined by a linker.
  • the RBD may be joined to the spacer via a linker.
  • linker refers to an oligopeptide which provides spatial separation between domain each of the two or more RBDs that may be present in the polypeptide of the invention.
  • the linker may be a flexible linker. This type of linkers allows for torsion of domain A respective of domain O, which may be beneficial when domain A interacts with the S protein of a coronavirus.
  • Non-limiting examples of flexible linkers that may be used in the first polypeptide of the invention include:
  • the linker may be the (Gly 4 Ser) 3 linker (SEQ ID NO: 6) or Ser(Gly 4 Ser) (SEQ ID NO: 43: SGGGGS).
  • the polypeptide of the invention comprises a spacer.
  • the spacer spatially separates the RBD, or two or more RBDs, from the cell membrane where the polypeptide of the invention is expressed. Together with the transmembrane domain, the spacer acts as an anchor and allows the polypeptide of the invention to present fragments of the viral spike protein on the cell surface.
  • the spacer may be a spacer from a mammalian protein or a viral protein, such as a transmembrane protein.
  • Non-limiting examples of spacers from mammalian proteins include the stalk of alpha chain of CD8 (CD8a; CD8a stalk), the CD4 ectodomain, the CD45 ectodomain, the CD2, and the CD34 ectodomain.
  • Non-limiting examples of spacers from viral proteins include the stalk of the SARS CoV-2 spike glycoprotein (i.e. the S2 subunit stalk of SARS-CoV-2; SEQ ID NO: 36).
  • the spacer may be a spacer from a mammalian protein.
  • the spacer may alternatively comprise a coiled-coil domain, for example as described in WO2016/151315.
  • SEQ ID NO: 12 human CD8 stalk
  • SEQ ID NO: 41 S2 subunit stalk of SARS-CoV-2)
  • SEQ ID NO: 42 (Heptad repeat 2 [HR2] of S2 subunit of SARS-CoV-2) TSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL
  • SEQ ID NO: 13 (human CD4 ectodomain):
  • SEQ ID NO: 14 (human CD45 ectodomain): QSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFERENDFSETTTSLSPDNTST QVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPT PGSNAISDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAY LNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNV NENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHD CTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYK CDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTL CYIKETEKDCLNLDKNLIKYDLQNLKPYTK
  • SEQ ID NO: 16 (CD34 ectodomain)
  • the spacer may comprise a sequence selected from the sequences shown as SEQ ID NOs: 12, 41, 42, or 13 to 17 or a variant thereof having at least 80% sequence identity.
  • a variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 12, 3, or 13 to 17, provided that the sequence provides an effective spacer, i.e. the variant provides sufficient spatial separation between the RBD, or two or more RBDs, and the cell membrane where the polypeptide of the invention is expressed.
  • the polypeptide of the invention may comprise a human CD8a stalk spacer.
  • the polypeptide of the invention comprises a transmembrane domain (TM).
  • TM transmembrane domain
  • the transmembrane domain sequence may, for example, comprise the transmembrane domain of alpha chain of CD8 (CD8a; CD8a TM), the transmembrane domain of the SARS CoV-2 spike glycoprotein (i.e. the S2 subunit of SARS-CoV-2), the CD4 transmembrane domain, the CD45 transmembrane domain, the CD2 transmembrane domain and the and the CD34 ectodomain.
  • SEQ ID NO: 18 (human CD8a transmembrane domain):
  • SEQ ID NO: 19 SARS CoV-2 S2 subunit transmembrane domain: WYIWLGFIAGLIAIVMVTIML
  • SEQ ID NO: 20 human CD4 transmembrane domain
  • SEQ ID NO: 21 (human CD45 transmembrane domain):
  • SEQ ID NO: 22 human CD2 transmembrane domain
  • the transmembrane domain may comprise a sequence selected from the sequences shown as SEQ ID NOs: 18 to 23 or a variant thereof having at least 80% sequence identity.
  • a variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 18 to 23, provided that the sequence provides an effective anchor of the RBD, or two or more RBDs, to the cell membrane where the polypeptide of the invention is expressed.
  • the transmembrane domain may be paired to its respective spacer domain, for example, a human CD8a stalk and transmembrane domain may be comprised in the same polypeptide of the invention.
  • the polypeptide of the invention may comprise a human CD8a transmembrane domain.
  • polypeptide of the invention may further comprise an endodomain (endo) or cytoplasmic domain.
  • the endodomain sequence may, for example, comprise the endodomain of alpha chain of CD8 (CD8a; CD8a endo), the endodomain of the SARS CoV-2 spike glycoprotein (i.e. the S2 subunit of SARS-CoV-2), the endodomain of the SARS CoV-2 spike glycoprotein lacking the ER retention signal, the CD4 endodomain, the CD45 endodomain, the CD2 endodomain, and the CD34 endodomain.
  • SEQ ID NO: 24 (human CD8a endodomain):
  • SEQ ID NO: 25 SARS CoV-2 S2 subunit endodomain
  • SEQ ID NO: 26 SARS CoV-2 S2 subunit endodomain without ER retention signal: CCMTSCCSCLKGCCSCGSCC
  • SEQ ID NO: 27 (human CD4 endodomain):
  • SEQ ID NO: 28 (human CD45 endodomain):
  • SEQ ID NO: 29 (human CD2 endodomain):
  • SEQ ID NO: 30 human CD34 endodomain
  • the endodomain may comprise a sequence selected from the sequences shown as SEQ ID NOs: 24 to 30 or a variant thereof having at least 80% sequence identity.
  • a variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 24 to 30.
  • the endodomain may be paired to its respective spacer domain and transmembrane domain, for example, a human CD8a stalk, CD8a transmembrane domain and CD8a endodomain may be comprised in the same polypeptide of the invention.
  • the polypeptide of the invention may comprise a human CD8a endodomain.
  • the polypeptide of the invention may comprise one of the following membrane anchors derived from SARS-CoV-2 (SEQ ID NO: 54-57):
  • SEQ ID NO: 54 SARS-CoV-2 transmembrane and endodomains
  • SEQ ID NO: 55 SARS-CoV-2 HR2, transmembrane and endodomains
  • SEQ ID NO: 56 (SARS-CoV-2 HR1+HR2, transmembrane and endodomains) YENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFG AISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYD PLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNES LIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGS CC
  • SEQ ID NO: 57 (SARS-CoV-2 HR1, transmembrane and endodomains) YENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFG AISGGGGSGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCG SCO
  • the polypeptide of the present invention may comprise a signal peptide so that when the polypeptide is expressed inside a cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
  • At the end of the signal peptide there is typically a stretch of amino acids that is recognised and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the free signal peptides are then digested by specific proteases.
  • the signal peptide may be at the amino terminus of the molecule.
  • the signal peptide may comprise the SEQ ID NO: 31 to 34 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the protein.
  • the signal peptide of SEQ ID NO: 31 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.
  • SEQ ID NO: 32 MSLPVTALLLPLALLLHAARP
  • the signal peptide of SEQ ID NO: 32 is derived from IgGl.
  • the signal peptide of SEQ ID NO: 33 is derived from CD8.
  • SEQ ID NO: 34 MGWSCIILFLVATATGVHS
  • the signal peptide of SEQ ID NO: 34 is derived from murine Ig heavy chain.
  • the polypeptide of the invention may comprise a TCRBV signal peptide.
  • the polypeptide of the invention may further comprise a detectable marker sequence.
  • Any detectable marker sequence may be used for the purposes of the present invention.
  • detectable markers include the HA epitope tag derived from influenza A Hemagglutinin (Uniprot Q03909; residues 119-127; HA-tag); V5 tag; FLAG tag; C-myc tag; histidine tag (HIS tag); Avi tag; Strep tag; Strep tag II; S-peptide tag (S tag); ALFA tag; AU1 tag; AU5 tag; Glu-Glu tag; HSV tag; KT3 tag; E tag; C tag.
  • polypeptide of the invention may comprise or consist of the sequence shown as SEQ ID NO: 35-40 and 58-70.
  • SEQ ID NO: 35 SARS CoV-2 spike glycoprotein, RBD-CD8a stalk
  • SEQ ID NO: 36 SARS CoV-2 spike glycoprotein RBD, S2 stalk
  • SEQ ID NO: 37 SARS CoV-2 spike glycoprotein RBD, HR2-S2 stalk
  • SEQ ID NO: 38 TRCBV signal peptide, HA tag, SARS CoV-2 spike glycoprotein, RBD- CD8a stalk
  • SEQ ID NO: 39 TRCBV signal peptide, HA tag, SARS CoV-2 spike glycoprotein RBD, S2 stalk
  • SEQ ID NO: 40 TRCBV signal peptide, HA tag, SARS CoV-2 spike glycoprotein RBD, HR2-S2 stalk
  • SEQ ID NO: 58 (signal peptide, HA tag, SI domain, CD8a stalk, TM, endo)
  • SEQ ID NO: 59 (signal peptide, HA tag, RBD, CD8a stalk, TM, endo)
  • SEQ ID NO: 60 (signal peptide, HA tag, SI domain, SARS-CoV-2 TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYT N SFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT
  • SEQ ID NO: 61 (signal peptide, HA tag, SI domain, SARS-CoV-2 HR2, TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYT NSFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGE
  • SEQ ID NO: 62 (signal peptide, HA tag, SI domain, SARS-CoV-2 HR1+HR2, TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYT NSFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNL
  • SEQ ID NO: 63 (signal peptide, HA tag, SI domain, SARS-CoV-2 HR1, TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYT NSFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGE
  • SEQ ID NO: 65 (signal peptide, HA tag, RBD, SARS-CoV-2 HR1+HR2, TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCP FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF
  • SEQ ID NO: 66 (signal peptide, HA tag, RBD, SARS-CoV-2 HR1, TM, endo)
  • RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV SEQ ID NO: 69 ((RBD-CD8STK)x3_tri-cistronic_(RBD_319-541_CD8STK-T2A- RBD 319-54 l_N501 Y CD8 STK-P2 A-RBD 319-41_K417N_E484K_N501 Y CD8 STK)) MGWSCIILFLVATATGVHSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA TVCGPK
  • the invention is also applicable to techniques involving nucleic acid (RNA, DNA) and vector-mediated transgene expression in a context where expression of a transgene leads to therapeutic benefits, for instance vaccination.
  • Nucleic acid vaccination is a technique whereby a nucleic acid encoding a target antigen is introduced into a host either alone or mediated by a vector (for instance viral or bacterial) for the induction of an immune response.
  • the target antigen is expressed by the host cells and can result in the generation of an immunological response and residual immunological memory.
  • the encoded transgene can be expressed and presented directly by antigen presenting cells (APCs), or the target antigen can be expressed by non-APC host cells and then acquired by APCs for presentation, for instance by engulfment of apoptotic target antigen expressing cells.
  • APCs antigen presenting cells
  • An example of a host cell suitable for inoculation with a nucleic acid vaccine is muscle cells.
  • the present invention also provides a nucleic acid sequence encoding a polypeptide of the invention, hereinafter “the nucleic acid of the invention”.
  • polypeptide of the invention has been described in detail in the context of previous aspects of the invention and its definitions and embodiments apply equally to the second aspect of the invention.
  • polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.
  • nucleic acid sequences and constructs of the invention may contain alternative codons in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination.
  • Nucleic acids according to the invention may comprise DNA or RNA. Nucleic acids may be single-stranded or double-stranded. Nucleic acids may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • the nucleic acid may be single stranded or double stranded.
  • the nucleic acid may be DNA or RNA.
  • the RNA may be messenger RNA (mRNA) or circular RNA.
  • Circular RNA is typically generated by chemical or enzymatic ligation of the 5’ and 3’ ends of the RNA molecule, back splicing of intron containing RNA, use of self-splicing sequences or ligation using splint oligonucleotides.
  • Circular RNAs encoding translatable sequences require an internal ribosome entry site (IRES) or a functionally equivalent sequence to recruit the ribosome to the circular RNA and initiate its translation.
  • IRS internal ribosome entry site
  • nucleic acid is RNA
  • nucleotide analogues and other chemical structures with the ability to promote stability and/or translation efficiency may also be used.
  • the RNA preferably has 5’ and 3’ UTRs.
  • the 5’ UTR and/or the 3’ UTR may be between zero and 3000 nucleotides in length.
  • the length of 5’ and 3’ UTR sequences may be modified to achieve optimal translation efficiency.
  • Various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
  • RNAs produced by the methods to include a 5' cap structure can be generated using Vaccinia capping enzyme and 2’-O-methyltransferase enzymes.
  • 5’ cap is provided using techniques known in the art and described herein (Cougot et al., 2001, Trends Biochem Sci 29:436-44; Stepinski et al., 2001, RNA 7: 1468-95; Elango et al., 2005, Biochim Biophys Res Commun 330:958-66).
  • the RNA may be nucleoside-modified RNA.
  • Nucleoside-modified mRNA have particular advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation.
  • Nucleoside-modified mRNA useful in the present invention is further described in patent application W02007024708.
  • the nucleoside-modified RNA may comprise the naturally occurring modified-nucleoside pseudouridine.
  • variant in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
  • the present invention also provides a nucleic acid construct encoding two or more polypeptides of the invention, hereinafter “the nucleic acid construct of the invention”.
  • nucleic acids encoding each of the two or more polypeptides of the invention may be in any order in the construct.
  • Nucleic acids encoding two or more polypeptides may be separated by a co-expression site enabling co-expression of two polypeptides as separate entities. It may be a sequence encoding a cleavage site, such that the nucleic acid construct produces both polypeptides, joined by a cleavage site(s). The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.
  • the cleavage site may be any sequence which enables the two polypeptides to become separated.
  • cleavage is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage.
  • FMDV Foot-and-Mouth disease virus
  • various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82: 1027-1041).
  • cleavage is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.
  • the cleavage site may, for example be a furin cleavage site, a Tobacco Etch Virus (TEV) cleavage site or encode a self-cleaving peptide.
  • TSV Tobacco Etch Virus
  • a ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.
  • the self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus.
  • the primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus.
  • apthoviruses such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus
  • the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C- terminus (Donelly et al (2001) as above).
  • 2A-like sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al (2001) as above).
  • the cleavage site may comprise the 2A-like sequence shown as SEQ ID NO: 71 (RAEGRGSLLTCGDVEENPGP).
  • the nucleic acid of the invention can contain a regulatory sequence operatively linked for the expression of the nucleotide sequence encoding the polypeptide of the invention.
  • operatively linked means that the antibody encoded by the nucleic acid sequence of the invention is expressed in the correct reading frame under control of the expression control or regulating sequences. Therefore, in another aspect, the invention provides an expression cassette, hereinafter “the expression cassette of the invention”, comprising the nucleic acid of the invention or the nucleic acid construct of the invention operatively linked to an expression control sequence.
  • the expression cassette of the invention can be obtained by techniques that are widely known in the art.
  • the expression cassette may comprise one or more control sequences.
  • Control sequences are sequences that control and regulate transcription and, where appropriate, the translation of said immunogen, and include promoter sequences, transcriptional regulators encoding sequences, internal ribosome binding site (IRES), ribosome binding sequences (RBS) and/or transcription terminating sequences.
  • the expression cassette of the present invention may additionally include an enhancer, which may be adjacent to or distant from the promoter sequence and can function to increase transcription from the same.
  • the expression control sequence may be functional in prokaryotic cells or in eukaryotic cells and organisms, such as mammalian cells.
  • the expression cassette may comprise a promoter. Any promoter may be used in this methodology.
  • the present invention also provides a vector, hereinafter “the vector of the invention”, which comprises a nucleic acid of the invention or a nucleic acid construct or an expression cassette of the invention.
  • the vector of the invention may be used to introduce the nucleic acid or expression cassette into a host cell so that it expresses the polypeptide of the invention.
  • polypeptide of the invention “nucleic acid of the invention”, “nucleic acid construct of the invention” and “expression cassette or the invention” have been described in detail in the context of previous aspects of the invention and their definitions and embodiments apply equally to this aspect of the invention.
  • the vector may, for example, be a plasmid, replicating viral vector, non-replicating viral vector, self-replicating RNA viral vector (alphavirus), viral-like particle or synthetic mRNA.
  • the present invention relates to a cell, hereinafter “the cell of the invention”, comprising a polypeptide of the invention, a nucleic acid of the invention, a nucleic acid construct of the invention, an expression cassette of the invention, or a vector of the invention.
  • the cell may comprise a polypeptide, a nucleic acid, a nucleic acid construct, or an expression cassette, or a vector according to the present invention.
  • polypeptide of the invention “nucleic acid of the invention”, “nucleic acid construct of the invention”, “expression cassette or the invention”, and “vector of the invention” have been described in detail in the context of previous aspects of the invention and their definitions and embodiments apply equally to this aspect of the invention.
  • the cell may be prokaryotic or eukaryotic.
  • Cells suitable for performing the invention include, without limitation, mammalian and bacterial cells.
  • Mammalian cells suitable for the present invention include epithelial cell lines, muscle cells, antigen presenting cells (APCs), osteosarcoma cell lines, neuroblastoma cell lines, epithelial carcinomas, glial cells, hepatic cell lines, CHO (Chinese Hamster Ovary) cells, COS, BHK cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293 and 293T cells, SupTl cells, PER.C6 cells, NTERA-2 human ECCs cells, D3 cells of the mESCs line, SHEF1, SHEF2 and HS181, NIH3T3 cells, REH and MCF-7 cells.
  • APCs antigen presenting cells
  • osteosarcoma cell lines include neuroblastoma cell lines, epithelial carcinomas, glial cells, hepatic cell lines, CHO (Chinese Hamster Ovary
  • Bacterial cells include, without limitation, cells from Gram positive bacteria such as species of the genus Bacillus, Streptomyces and Staphylococcus and Gram-negative bacterial cells such as cells of the genus Escherichia and Pseudomonas.
  • the present invention also relates to a method for making a cell of the invention, which comprises a step of introducing a nucleic acid of the invention, a nucleic acid construct of the invention, an expression cassette of the invention, or a vector of the invention into the cell. This may be done by transducing or transfecting a cell with the nucleic acid of the invention, the nucleic acid construct of the invention, the expression cassette of the invention, or the vector of the invention.
  • nucleic acid is RNA
  • it can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation, cationic liposome mediated transfection using lipofection, lipid nanoparticle mediated transfection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns”. 5.
  • electroporation cationic liposome mediated transfection using lipofection
  • lipid nanoparticle mediated transfection lipid nanoparticle mediated transfection
  • polymer encapsulation polymer encapsulation
  • peptide mediated transfection or biolistic particle delivery systems such as “gene guns”.
  • the present invention also relates to a pharmaceutical composition containing the polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the first pharmaceutical composition of the invention”.
  • the present invention also relates to a pharmaceutical composition containing the nucleic acid of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the second pharmaceutical composition of the invention”.
  • the present invention also relates to a pharmaceutical composition containing the nucleic acid construct of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the third pharmaceutical composition of the invention”.
  • the present invention also relates to a pharmaceutical composition containing the vector of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the fourth pharmaceutical composition of the invention”.
  • the present invention also relates to a pharmaceutical composition containing the cell of the invention or a plurality of cells according to the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the fifth pharmaceutical composition of the invention” .
  • the first, second, third, fourth and fifth pharmaceutical compositions of the invention may additionally comprise a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.
  • the first, second, third and fourth pharmaceutical compositions may optionally comprise one or more polypeptides of the invention.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • polypeptide of the invention “nucleic acid of the invention”, “vector of the invention”, and “cell of the invention” have been described in detail in the context of previous aspects of the invention and their definitions and particular embodiments apply equally to this aspect of the invention.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable diluent” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide of the invention.
  • pharmaceutically acceptable excipient refers to an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.
  • excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • Excipients include carriers, diluents, preservatives, colouring agents, and fillers.
  • the term “adjuvant”, as used herein, is defined as any molecule to enhance an antigenspecific adaptive immune response, i.e. a pharmacological or immunological agent that improves the immune response of a vaccine.
  • the first, second, third, fourth and fifth pharmaceutical compositions of the invention may comprise an adjuvant, such as a microparticulate adjuvant, for example liposomes, or an immune stimulating complex (ISCOMS), virus-like particles or nanoparticles, an emulsion, a microparticle, a virosome, a micellar delivery system, a dendrimer delivery system, a plant vaccine, a melt-in-mouth strip (under the tongue) or an immunostimulatory adjuvant (such as cholera toxin, chitosan).
  • an adjuvant such as a microparticulate adjuvant, for example liposomes, or an immune stimulating complex (ISCOMS), virus-like particles or nanoparticles, an emulsion, a microparticle
  • the first, second, third, fourth and fifth pharmaceutical compositions of the invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g. injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, lipid nanoparticles (LNP).
  • liquid solutions e.g. injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes, lipid nanoparticles (LNP).
  • LNP lipid nanoparticles
  • the pharmaceutical composition may comprise a (LNP).
  • LNP LNP
  • Methods for making LNPs are well-known in the art. Typical LNP formation procedures involve the controlled mixing of hydrophobic lipid components dissolved in an organic solvent such as ethanol with an aqueous buffer solution containing the oligonucleotide to be loaded into the resulting particle.
  • the administration of the first, second, third, fourth and fifth pharmaceutical compositions of the invention can be accomplished using any of a variety of routes that make the active ingredient bioavailable.
  • the agent can be administered via an oral, mucosal, buccal, intranasal, intraocular, inhalable, intravenous, subcutaneous, transcutaneous, intramuscular, intraperitoneal, parenteral or topical route.
  • Oral administration may be by inhalation, by nebulisation or nasally.
  • the first, second, third and fourth pharmaceutical compositions of the invention may be administered locally, for example by catheter or stent, or systemically, for example by intravenous injection.
  • the route of administration may be intramuscular.
  • compositions according to the invention may be administered to the subject in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art.
  • Pharmaceutical compositions according to the invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and. then, if necessary, shaping the product.
  • the compositions may be formulated into any of many possible dosage forms
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilisers.
  • the first, second, third, fourth and fifth pharmaceutical compositions of the invention may be administered via the nasal route using a nasal insufflator device.
  • a nasal insufflator device examples of these are already employed for commercial powder systems intended for nasal application (e.g. Fisons Lomudal System). Details of other devices are well-known in the art.
  • compositions of the invention include via the pulmonary route using a powder inhaler or metered dose inhaler, via the buccal route formulated into a tablet or a buccal patch, and via the oral route in the form of a tablet, a capsule or a pellet (which compositions may administer agent via the stomach, the small intestine or the colon), all of which may be formulated in accordance with techniques which are well known to those skilled in the art.
  • the first, second, third, fourth and fifth pharmaceutical compositions of the invention may also be incorporated within a slow- or delayed-release device.
  • a slow- or delayed-release device Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
  • the device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment is required, and which would normally require frequent administration (e.g. at least daily injection).
  • a person skilled in the art would be able to determine the appropriate timing, sequence and dosages of administration for particular pharmaceutical compositions of the present invention. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular subject.
  • the presentation of coronavirus spike protein-derived peptides by the polypeptide of the invention makes this molecule capable of eliciting an immune reaction in a subject. This can be exploited for therapeutic and prophylactic purposes.
  • the invention further provides the use of a polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell or pharmaceutical composition as defined by the invention in the preparation of a coronavirus vaccine for inducing an immune response against a coronavirus in a subject in need thereof.
  • the present invention provides a method for presenting a viral Spike protein-derived peptide, hereinafter “the method of antigen presentation of the invention”, comprising expressing a polypeptide of the invention in a cell.
  • polypeptide of the invention and “cell” have been described in detail in previous aspects of the invention and their definitions and embodiments apply equally to this aspect of the invention.
  • the method of presentation of the invention comprises the step of expressing a polypeptide of the invention in a cell. This may be carried out by transducing or transfecting a cell with a nucleic acid, a nucleic acid construct, an expression cassette or a vector of the invention as has been described in previous aspects of the invention.
  • the resulting cell is a cell according to the invention.
  • the method of presentation of the invention may also comprise a step of contacting the transduced or transfected cell with an immune cell or an antigen-presenting cell (APC).
  • the immune cell may be a population of immune cells.
  • the APC may be a population of APCs.
  • the method of presentation of the invention may involve a step of administering the nucleic acid of the invention, nucleic acid construct of the invention, expression cassette of the invention, vector of the invention, or cell of the invention to a subject.
  • the administration may be by any of the ways described in previous aspects of the invention. 6.2. Method of screening therapeutics
  • the present invention provides a method of testing cross-reactivity of a viral therapeutic, comprising a step of contacting the viral therapeutic with a cell or a plurality of cells of the invention.
  • the specificity of a viral therapeutic that binds to a coronavirus Spike protein can be tested by contacting the viral therapeutic with a cell or a plurality of cells of the invention.
  • the skilled person will immediately appreciate that, by using a cell or plurality of cells which express a polypeptide of the invention comprising the spike protein or RBD of the same virus to which the viral therapeutic is specific, and a cell or plurality of cells which express a polypeptide of the invention comprising the spike protein or RBD of a different virus, it is possible to determine whether the viral therapeutic cross-reacts with other viruses.
  • the specificity against a viral strain or variant can be tested by using a cell or plurality of cells which express a polypeptide of the invention comprising the spike protein or RBD of the same viral variant to which the viral therapeutic is specific, and a cell or plurality of cells which express a polypeptide of the invention comprising the spike protein or RBD of a different viral variant.
  • This method may be carried out by methods that are well-known in the art, such as by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunocytochemistry, or by immunofluorescent techniques such as fluorescence microscopy or flow cytometry.
  • immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunocytochemistry, or by immunofluorescent techniques such as fluorescence microscopy or flow cytometry.
  • an in vitro binding assay such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunocytochemistry, or by immunofluorescent techniques such as fluorescence microscopy or flow cytometry.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the viral therapeutic may be an antibody specific to the spike protein or RBD of a coronavirus.
  • the coronavirus may be SARS-CoV-2.
  • the cell of the invention is particularly useful for screening patients producing high titers of neutralising antibodies. Serum of these patients may be used to treat other patients suffering from the same viral infection.
  • the present invention relates to a method to identify patients suffering from an infection or infectious clinical condition caused by a virus, such as a coronavirus, which have high titers of neutralising antibodies, comprising a step of contacting a blood, serum or plasma sample from the patient with a cell or a plurality of cells of the invention.
  • This method may be carried out by methods that are well-known in the art, such as by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunocytochemistry, or by immunofluorescent techniques such as fluorescence microscopy or flow cytometry.
  • immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunocytochemistry, or by immunofluorescent techniques such as fluorescence microscopy or flow cytometry.
  • an in vitro binding assay such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunocytochemistry, or by immunofluorescent techniques such as fluorescence microscopy or flow cytometry.
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the viral therapeutic may be an antibody specific to the spike protein or RBD of a coronavirus.
  • the coronavirus may be SARS-CoV-2.
  • the present invention provides a polypeptide of the invention, or a nucleic acid of the invention, or a nucleic acid construct of the invention, or an expression cassette of the invention, or a vector of the invention, or a cell of the invention, or a pharmaceutical composition of the invention for use in medicine.
  • the present invention provides a polypeptide of the invention, or a nucleic acid of the invention, or a nucleic acid construct of the invention, or an expression cassette of the invention, or a vector of the invention, or a cell of the invention, or a pharmaceutical composition of the invention for use in preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus, hereinafter “the medical use of the invention”.
  • this aspect of the invention may be formulated as a use of a polypeptide of the invention, or a nucleic acid of the invention, or a nucleic acid construct of the invention, or an expression cassette of the invention, or a vector of the invention, or a cell of the invention, or a pharmaceutical composition of the invention in the manufacture of a medicament for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus.
  • this aspect of the invention may be formulated as a method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus, in a subject in need thereof, hereinafter “the therapeutic method of the invention”, the method comprising the step of administrating to the subject a therapeutically effective amount of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention.
  • the therapeutic method of the invention the method comprising the step of administrating to the subject a therapeutically effective amount of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention.
  • polypeptide of the invention “nucleic acid of the invention”, “nucleic acid construct of the invention”, “expression cassette of the invention”, “vector of the invention”, “cell of the invention”, “pharmaceutical composition of the invention”, and “coronavirus” have been described in detail in previous aspects of the invention and their definitions and embodiments apply equally to this aspect of the invention.
  • a method for method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus, in a subject in need thereof relates to the prophylactic use of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention, which may be administered to a subject who has not been infected with a virus, such as a coronavirus, in order to prevent or lessen their infection with the virus.
  • the virus may be a coronavirus.
  • polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition may be administered to a subject who has not yet contracted the coronavirus infection or condition or disorder resulting from this infection and/or who is not showing any symptoms of the coronavirus infection or condition or disorder resulting from this infection to prevent or impair the coronavirus from infecting the cells of the subject or to reduce or prevent development of at least one symptom associated with the coronavirus infection or condition or disorder resulting from this infection.
  • the subject may have a predisposition for or be thought to be at risk of contracting a coronavirus infection or a condition or disorder resulting from this infection.
  • a method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus infection, or a condition or disorder resulting from this infection also relates to the therapeutic use of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention, which may be administered to a subject who has been infected with the virus, or is suspected to have been infected with the virus, or has tested positive for the virus in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
  • the virus may be a coronavirus.
  • the coronavirus may be SARS-CoV-2
  • These therapeutic applications will comprise the administration of a therapeutically effective amount of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention.
  • the treatment of a coronavirus disease in a subject may comprise the step of administrating the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention to the subject, to trigger an immune reaction against the coronavirus Spike protein which may result in a complete or partial neutralisation of the coronaviruses.
  • subject refers to members of mammalian species.
  • the subject may be a human patient of any gender, age or race.
  • the subject may be a non-human mammal infected with coronavirus.
  • the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be administered to a non-human mammal infected with coronavirus for veterinary purposes or as an animal model of human disease.
  • animal models may be useful for evaluating the therapeutic efficacy of the polypeptides of this invention.
  • Non-limiting examples of non-human mammal that may be subject to treatment according to the invention include a cat or any other feline, a dog or any other canid, a mouse, a rat, a capybara or any other rodent, a pig, a primate, and a bat.
  • terapéuticaally effective amount refers to the amount of the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention which is required to achieve an appreciable prevention, neutralisation, cure, delay, reduction of the severity of, or amelioration of one or more symptoms of a coronavirus disease.
  • a coronavirus infection or a condition or disorder resulting from this infection refers to an infection, condition or disorder caused by a coronavirus.
  • the coronaviruses can cause varieties of diseases in humans and other animals, including respiratory, enteric, renal, and neurological diseases. Particularly important are the diseases caused by coronaviruses SARS-CoV-2 and SARS-CoV because of the severe acute respiratory syndrome that they cause.
  • the coronavirus condition or disorder may be coronavirus disease 2019 (COVID-19).
  • the disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and has since spread globally, resulting in the ongoing 2019-20 coronavirus pandemic.
  • Common symptoms include fever, cough and shortness of breath.
  • Other symptoms may include fatigue, muscle pain, diarrhoea, nausea, sore throat, loss of smell and abdominal pain. While the majority of cases result in mild symptoms, some progress to viral pneumonia and multi-organ failure.
  • COVID-19 manifests as a clotting disorder, which may cause pulmonary embolism and hypoxia. Pulmonary vasculature affected by pulmonary embolism is not fully restored and can cause permanent fibrosis of the lining of blood vessels. Pulmonary fibrosis may also be the result of prolonged mechanical ventilation; even prolonged use of high concentration oxygen can lead to lung injury and result in fibrosis. Permanent fibrosis may lead to chronic thromboembolic pulmonary hypertension (CTEPH). Additionally, the clotting disorder causes end organ damage, primarily kidney. Kidney injury does not fully recover and may lead to chronic kidney disease (CKD) in post- COVID19 patients.
  • CKD chronic kidney disease
  • Severe COVID-19 presents with a cytokine storm or cytokine release syndrome (CRS), which is an immediate and intense response of the immune system to viral infection.
  • CRS cytokine storm or cytokine release syndrome
  • Kawasaki disease is an autoimmune disease in which blood vessels throughout the body become inflamed. It is considered a “post-viral” autoimmune disease.
  • Guillain-Barre syndrome is a neurological disorder where the immune system responds to an infection and ends up mistakenly attacking nerve cells, resulting in muscle weakness and eventually paralysis. Thus, severe COVID-19 may also cause an incidence of other more prevalent autoimmune diseases in recovered patients.
  • the loss of the sense of smell is a direct result of the virus infecting the olfactory neurons. It has been suggested that this may enable the virus to spread from the respiratory tract to the brain.
  • Cells in the human brain express the ACE2 protein on their surface.
  • ACE2 is also found on endothelial cells that line blood vessels. Infection of endothelial cells may allow the virus to pass from the respiratory tract to the blood and then across the blood-brain barrier into the brain. Once in the brain, replication of the virus may cause neurological disorders. Larger studies from China and France have also investigated the prevalence of neurological disorders in COVID-19 patients. These studies have shown that 36% of patients have neurological symptoms. Many of these symptoms were mild and include headache or dizziness that could be caused by a robust immune response.
  • the virus is mainly spread during close contact and by small droplets produced when those infected cough, sneeze or talk. These small droplets may also be produced during breathing, but rapidly fall to the ground or surfaces and are not generally spread through the air over large distances. People may also become infected by touching a contaminated surface and then their face. The virus can survive on surfaces for up to 72 hours. It is most contagious during the first three days after onset of symptoms, although spread may be possible before symptoms appear and in later stages of the disease. The time from exposure to onset of symptoms is typically around five days, but may range from two to 14 days.
  • the standard method of diagnosis is by real-time reverse transcription polymerase chain reaction (RT- PCR) from a nasopharyngeal swab.
  • the infection can also be diagnosed from a combination of symptoms, risk factors and a chest CT scan showing features of pneumonia.
  • the method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by coronavirus in a subject in need thereof may comprise a step of administering the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention to the subject.
  • the skilled person will be able to determine by conventional methods the amount of such polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention that are able to exert a prophylactic or therapeutic effect on the patient.
  • the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be administered once, or it may be administered multiple times.
  • the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be administered from once a day to once every six months, once every year or longer.
  • the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be administered in two doses, 28 days apart.
  • the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention of the invention may also be administered continuously via a minipump.
  • polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention of the invention may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral, or topical route.
  • the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention polypeptide of the invention may be administered locally or systemically.
  • the skilled person will be able to determine the effective dosage of the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention by methods that are well-known in the art.
  • the dosage of polypeptide of the invention will generally be in the range of 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg, and even more preferably 1-10 mg/kg.
  • the immune response triggered by the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be measured by any method known in the art.
  • Example 1 Generation of plasmid DNA constructs encoding fragments of the SARS CoV-2 spike protein fused to transmembrane anchors
  • Fragments of the spike SI subunit were selected for expression on the cell surface as it is this subunit that mediates attachment to the host cell, and it is likely that antibodies recognising this domain would be neutralising and prevent viral infection. Fusion of the SI fragments to a fragment of the S2 subunit could potentially broaden the antibody response and increase the likelihood of developing an immune response that is protective and prevents viral infection. Adopting such an approach would mitigate the risk of a sub-optimal immune response that could lead to individuals becoming more susceptible to viral infection due to antibody-dependent enhancement.
  • the stalk, transmembrane and truncated cytoplasmic domain lacking the ER retention motif (residues 1204-1251); the second heptad repeat (residues 1160-1251); the first and second heptad repeats (917-1251); and the first heptad repeat fused the S2 stalk (residues 917-97 fused to 1204-1254) were used for fusion to the SI fragments.
  • the S2 fragments alone were also cloned to enable for testing of cell surface expression.
  • plasmid DNA constructs were generated and transfected into 293T cells to determine whether the fusion proteins were expressed on the cell surface. All SARS CoV-2 SI and S2 fragments possessed an N-terminal HA epitope tag to facilitate detection at the cell surface using a fluorophore conjugated anti-HA antibody and analysing by flow cytometry. Staining was also carried out with soluble ACE2 tagged to a human IgG2 Fc (fragment crystallisable) to determine if the SI fragments were correctly folded and capable of interacting with their target receptor. Table 1. List of plasmid DNA constructs encoding fragments of the SARS CoV-2 spike protein fused to transmembrane anchors.
  • 293T cells were transiently transfected with the retroviral constructs (Table 1), stained 48 hours later with antibodies to the RBD ( Figure 1) or soluble ACE2-Fc protein and HA epitope tag ( Figure 2) and analysed by flow cytometry.
  • SupTl cells were transduced with y-retroviral supernatant encoding the spike fragments and stained with soluble ACE2-Fc and detected with an Alexa-647 conjugated anti-human IgG Fc antibody (Error! Reference source not found.).
  • the results of this experiment indicated that the RBD can be stably expressed at the cell surface using the CD8a or S2 -derived transmembrane anchors (S2 stalk or the second heptad repeat of S2).
  • the SI subunit was not stably expressed on the surface of SupTl cells, and this could be due to the stability of the protein fragment or differences between the SupTl and 293T cell lines.
  • the SARS CoV-2 RBD can be stably expressed at the cell surface and this fragment of the protein is the most promising target immunogen.
  • three rats were immunised with a plasmid encoding the SARS CoV-2 RBD-CD8a stalk transmembrane fusion and sera collected. These sera were used to stain SupTl cells expressing the RBD-CD8a stalk transmembrane fusion or non-transduced SupTl cells ( Figure 4).
  • the seroconverting capacity of membrane-bound RBD in rats was compared with that of soluble trimeric RBD (RBD fused to the C-terminal domain of T4 bacteriophage fibritin, referred to as foldon domain).
  • soluble trimeric RBD RBD fused to the C-terminal domain of T4 bacteriophage fibritin, referred to as foldon domain.
  • Experiments were carried out as in Example 4. Briefly, three rats were immunised with a plasmid encoding the SARS CoV-2 RBD-CD8a stalk transmembrane fusion or a plasmid encoding the soluble trimeric RBD, and sera were collected.
  • Example 6 Multi RBD constructs for the expression of SARS-CoV-2 variants.
  • Rats immunised with the (RBD)3-CD8STK construct produced titres of antibody comparable to those immunised with the (RBD-CD8STK)3 construct, despite the results obtained from the transient transfection of 293T cells, showing that the cell surface expression of the (RBD)3-CD8STK fusion was lower than that of the (RBD-CD8STK)3 fusions.
  • Example 7 Neutralising capacity of sera from RBD immunised rats.
  • Example 8 Screening of patients using SupTl-RBD-expressing cells
  • Sera from 16 convalescing SARS-CoV-2 patients were used to stain SupTl cells expressing either full-length SARS-CoV-2 spike or the RBD.
  • the SupTl cell lines were stained with pre-SARS-CoV-2 serum to determine the background level of antibody binding ( Figure 9A).
  • the patient sera obtained had been previously screened for anti-spike antibodies using Fortress ELISA (see Table 2) and the titre of antibody determined.

Abstract

The present invention provides a polypeptide comprising an amino acid sequence of at least one receptor-binding domain (RBD) of a viral Spike protein, a spacer and a transmembrane domain. It further provides nucleic acids, nucleic acid constructs, expression cassettes, vectors, cells, pharmaceutical compositions and medical uses that exploit the polypeptides of the invention.

Description

VIRAL SPIKE PROTEINS AND FUSION THEREOF
FIELD OF THE INVENTION
The present invention relates to polypeptides derived from viral spike proteins.
BACKGROUND TO THE INVENTION
In December 2019, a novel coronavirus (SARS-CoV-2, COVID-19 or 2019-nCoV) crossed species barriers to infect humans and was effectively transmitted from person to person, leading to a pneumonia outbreak first reported in Wuhan, China. This virus causes coronavirus disease-19 (COVID-19) with influenza like symptoms ranging from mild disease to severe lung injury and multi-organ failure, eventually leading to death, especially in older patients with other co-morbidities. The WHO has declared that COVID-19 is a public health emergency of pandemic proportions. The SARS-CoV-2 pandemic is not only an enormous burden to public health but has already markedly affected civil societies and the global economy.
SARS-CoV-2 is currently considered a pandemic infection involving 190 countries, with more than 37 million confirmed cases and more than 1 million confirmed deaths worldwide, as of 13th October 2020. This highlights the need for rapid vaccine development to curb the spread of disease and prevent mortalities. Vaccines to pathogenic viruses or bacteria can be generated through the generation of an attenuated form of the virus or bacterium, the purification of viral (viral-like particles) or bacterial derived membranes (outer membrane vesicles), the production of recombinant protein, or the synthesis of DNA/RNA encoding a potential immunogen.
Thus, there is a clear need to be able to develop potential vaccines rapidly and to meet this need reliable methods of vaccine production are required.
Vaccines to viral or bacterial proteins can consist of attenuated forms of the pathogen, purified membrane (viral-like particles or outer membrane vesicles prepared from viruses or bacteria, respectively), recombinantly produced protein, RNA or DNA encoding the immunogen. Each of these approaches has its advantages and disadvantages. The production of attenuated viruses or bacteria is expensive and must be diligently controlled to ensure that the pathogen is no longer viable and capable of replicating in the recipient. Purification of viral-like particles or outer membrane vesicles from viruses or bacteria, respectively, is a way of mitigating the risk associated with the production of attenuated forms of pathogens. However, the yield of viral-like particles or outer membrane vesicles can be variable and the abundance of immunogens within these preparations may not be consistent, complicating the manufacturing process. Recombinant protein production requires substantial optimisation to yield protein that is soluble, folded in its native conformational state and, depending on the type of protein, post-translationally modified to recapitulate the immunogenic viral or bacterial protein. Consequently, recombinant protein production is both time-consuming and expensive. The synthesis of DNA/RNA is relatively inexpensive and the delivery of nucleic acids in lipid nanoparticles (LNP) is usually effective and results in the expression of the viral/bacterial polypeptide and the elicitation of an immune response.
Problems associated with these approaches include: (i) insufficient secretion of soluble protein; (ii) and, in the case of outer membrane vesicles, exuberant activation of the innate immune system due to the presence of lipopolysaccharides present in the preparation, which activate Toll-like receptors present on the surface of monocytes and macrophages. General approaches adopted to overcome manufacturing issues and improve the expression level of immunogens include codon optimisation, substitution of signal peptide sequences of secreted and membrane bound antigens to improve their expression, and the deletion or mutations of domains known to cause cytotoxicity or have inhibitory effects.
In the case of the recent severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) outbreak, the majority of the vaccines being developed utilise the spike glycoprotein as an immunogen, because it mediates attachment and entry of the virus to the host cell and antibodies against the spike protein could potentially be neutralising and immunoprotective. However, use of the full-length spike protein might not lead to the generation of neutralising antibodies because of the spike protein’s conformation that masks the receptor-binding domain. To date, no vaccines are approved against any human-infecting coronaviruses, including SARS-CoV-2. Therefore, there is a need in the art to provide effective vaccines for the immunisation against human-infecting coronaviruses and, especially, SARS-CoV-2.
SUMMARY OF ASPECTS OF THE INVENTION
The present invention describes a method for reliably expressing immunogenic peptides at the cell surface in a manner that they are accessible to the immune system. This method may be used as a rapid means of generating novel vaccines to raise immunity to pathogenic viruses. It overcomes the disadvantages and limitations of expressing soluble fragments of viral proteins, by attaching them to endogenously or exogenously derived transmembrane anchors.
The present inventors have expressed viral proteins as membrane-bound fusions to direct them to the secretory pathway and recapitulate the glycosylation observed on the native proteins. In the case of SARS-CoV-2, expression of the RBD alone on the surface of the cell may lead to the generation of antibodies recognising this domain, which could potentially be neutralising and block viral infection. Moreover, it is anticipated that the use of transmembrane anchors to display viral or bacterial immunogens will result in a more prolonged expression compared to soluble protein and engage the immune system to produce a profound response leading to long-term memory.
There are two advantages associated with the expression of membrane-bound immunogens: first, they are more likely to be post-translationally modified in the same way as the native protein and serve as relevant immunogens for the generation of neutralising antibodies; and second, they may be retained better than soluble antigens and persist for longer in the host, increasing the likelihood of a profound immune response.
Thus, in a first aspect, the present invention provides a polypeptide comprising an amino acid sequence of at least one receptor-binding domain (RBD) of a viral Spike protein, a spacer and a transmembrane domain, wherein the spacer is a spacer from a mammalian protein. The spacer from a mammalian protein may be a CD8a stalk.
The viral Spike protein may be a coronavirus Spike protein.
The Coronavirus may be SARS-CoV-2.
The RBD may comprise the amino acid sequence shown as SEQ ID NO: 4.
The polypeptide of the first aspect of the invention may comprise the amino acid sequence shown as SEQ ID NO: 35.
The transmembrane domain may be a CD8a transmembrane domain.
The polypeptide of the first aspect of the invention may further comprise an endodomain.
The endodomain may be a CD8a endodomain.
In a second aspect, the present invention provides a nucleic acid encoding a polypeptide according to the first aspect of the invention.
In a third aspect, the present invention provides a nucleic acid construct encoding two or more polypeptides according to the first aspect of the invention.
In a fourth aspect, the present invention provides a vector comprising a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention.
In a fifth aspect, the present invention provides a cell comprising a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention. In a sixth aspect, the present invention provides a method for making a cell according to the fifth aspect of the invention, which comprises the step of transducing or transfecting a cell with a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention.
In a seventh aspect, the present invention provides a pharmaceutical composition comprising a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a cell or plurality of cells according to the fifth aspect of the invention, and at least one pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.
In an eighth aspect, the present invention provides a method for presenting a viral Spike protein-derived peptide, comprising expressing a polypeptide according to the first aspect of the invention in a cell.
The viral Spike protein may be a coronavirus Spike protein.
In a ninth aspect, the present invention provides a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention for use in preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus.
Alternatively, this aspect of the invention may be formulated as a use of a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention in the manufacturing of a medicament for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus.
Alternatively, this aspect of the invention may be formulated as a method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, in a subject in need thereof, comprising the step of administrating to the subject a therapeutically effective amount of a polypeptide according to the first aspect of the invention, or a nucleic acid according to the second aspect of the invention, or a nucleic acid construct according to the third aspect of the invention, or a vector according to the fourth aspect of the invention, or a cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.
The virus may be a coronavirus.
The coronavirus may be SARS-CoV-2.
DESCRIPTION OF THE FIGURES
Figure 1. Staining of SARS CoV-2 spike SI and S2 fragments expressed on the surface of 293T cells with anti-SARS CoV-2 CR3022 antibody.
Figure 2. Staining of SARS CoV-2 spike SI and S2 fragments expressed on the surface of 293T cells with soluble ACE2-Fc and PE-C7-conjugated anti-HA antibody to detect the fragments of spike protein on the cell surface.
Figure 3. Flow cytometry plots of SupTl cells transduced with y-retrovirus encoding SI subunit and RBD transmembrane fusions and stained with soluble ACE2-Fc and Alexa 647- conjugated anti-human IgG.
Figure 4. Rats were immunised with plasmid DNA encoding the RBD-CD8a spacer and transmembrane fusion and sera collected from them. Sera obtained pre- and postimmunisation were used to stain non-transduced SupTl cells or RBD-CD8a spacer and transmembrane expressing SupTl cells and flow cytometric analysis carried out. Positive staining of SupTl RBD-CD8a spacer and transmembrane expressing cells was observed using the post-immunisation sera (top histogram in each panel) but not with the preimmunisation sera. Staining was also carried out using soluble ACE2-Fc to ensure that the SupTl cells were expressing the RBD-CD8a spacer and transmembrane domain fusion. These data indicate that the RBD-CD8a spacer and transmembrane domain is a suitable immunogen for use in vaccination, because it elicited a robust immune response in an animal model and the production of antibodies to the SARS CoV-2 spike protein.
Figure 5. Illustration of SARS-CoV-2/CD8a stalk fusion constructs.
Diagram of SARS-CoV-2 spike protein showing its structural domains. S, signal peptide; NTD, N-terminal domain; RBD, receptor binding domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic domain; and ER, endoplasmic reticulum retention domain. B) Diagram of SARS-CoV-2 spike and CD8a stalk fusion constructs. HA, haemagglutinin epitope tag; STK, CD8a stalk; TM, transmembrane domain; and CT, cytoplasmic domain.
Figure 6. Seroconversion of rats immunised with soluble or membrane bound RBD.
Flow cytometry plots of wild-type SupTl, SupTl -RBD or SupTl -spike expressing cells stained with sera from rats immunised with soluble (soluble trimeric RBD-foldon domain) or membrane bound (RBD fused to CD8a stalk). B) Quantification of median fluorescent intensity of rat anti-RBD sera.
Figure 7. Multi RBD constructs for the expression of SARS-CoV-2 variants.
A) Diagram of RBD constructs, showing the (RBD)3-CD8STK mono-cistronic construct (top) where three RBD variants are fused to each other via 16 amino acid serine glycine linkers and are presented on a single CD8a stalk; and the (RBD-CD8STK)3 tri-cistronic construct where each RBD variant is fused to its own CD8a stalk and expression of each polypeptide is facilitated by intervening T2A self-cleaving peptide sequences. B) Flow cytometry plots of transfected 293T cells stained with soluble ACE2-Fc to detect the spike protein or RBDs and quantification of ACE2-Fc median fluorescence intensity. C) Sera from rats immunised with the (RBD)3-CD8STK and (RBD-CD8STK)3 constructs. D) Quantification of rat anti-RBD sera MFI. Figure 8. Neutralising capacity of sera from RBD immunised rats.
Neutralisations assays were conducted with Wuhan (A), D614G (B), alpha (C) and beta (D) SARS-CoV-2 spike protein.
Figure 9. Screening of SARS-CoV-2 patient sera using SupTl-spike and RBD expressing cell lines.
A) The assay’s background was determined by staining SupTl non-transduced, SupTl-spike and SupTl -RBD cells with human pre-SARS-CoV-2 serum. Note that the SARS-CoV-2 spike and RBD expressing cell lines express eBFP as a transduction marker. B) Sera obtained from convalescing SARS-CoV-2 patients was used to the stain SupTl non-transduced, SupTl-spike and SupTl-RBD cells at a 1 : 100 dilution to test for the presence of SARS- CoV-2 spike-specific antibodies. Patients 13 to 16 had the highest titres of antibodies in the group tested.
Figure 10. Neutralisation assays using SARS-CoV-2 patient sera.
Patient sera were diluted and used in neutralisation assays with SARS-CoV-2 spike pseudotyped lentiviral vector. In general, the results of the neutralisation assays mirrored those of the SupTl-spike and SupTl -RBD cell staining assay, with patients having the highest titre of antibodies to spike/RBD exhibiting the highest level of protection to infection by the SARS-CoV-2 spike pseudotyped lentivirus.
Figure 11. Correlation between anti-RBD MFI and neutralisation capacity.
Plotting of the median fluorescence intensities obtained from staining SupTl-spike and SupTl -RBD cells with patient sera against IC50 values from neutralisation assays demonstrated that there was a strong correlation between anti-RBD MFI and neutralisation capacity. These data indicate that RBD MFI can be used as a predictive measure of neutralising antibodies.
DETAILED DESCRIPTION OF THE INVENTION The present invention describes a method of anchoring and presenting fragments of viral glycoproteins on the cell surface to elicit an immune response and the generation of neutralising antibodies capable of inhibiting viral entry and preventing viral infection.
1. Polypeptide
In a first aspect, the present invention provides a polypeptide, hereinafter “the polypeptide of the invention”, comprising an amino acid sequence of at least one receptor-binding domain (RBD) of a viral Spike protein, a spacer and a transmembrane domain.
The term polypeptide, as used herein, refers to natural, synthetic, and recombinant proteins or peptides generally having more than 10 amino acids.
The polypeptide of the invention comprises an amino acid sequence of at least one RBD of a viral Spike protein.
1.1. Viral spike protein (S protein)
The term “viral spike protein” or “S protein”, as used herein, refers to a glycoprotein spike on a viral capsid or viral envelope. It is also termed peplomer. These protrusions bind only to certain receptors on the host cell. Their function is essential for both host specificity and viral infectivity.
The viral Spike protein or S protein may be a coronavirus Spike protein.
Three coronaviruses have crossed the species barrier to cause deadly pneumonia in humans since the beginning of the 21st century: severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS- CoV-2. In 2002-2003, SARS-CoV, a lineage B beta-CoV, emerged from bat and palm civet, and infected over 8,000 people and caused about 800 deaths. In 2012, MERS-CoV, a lineage C beta-CoV, was discovered as the causative agent of a severe respiratory syndrome in Saudi Arabia, currently with 2,494 confirmed cases and 858 deaths, it remains endemic in Middle East, and dromedary camel is considered as the zoonotic reservoir host of MERS-CoV. At the end of 2019, a novel coronavirus, named SARS-CoV-2, was found in patients with severe pneumonia in Wuhan, China. Viruses were isolated from patients and sequenced. Phylogenetical analysis revealed that it is a lineage B beta-CoV and closely related to a SARS-like (SL) CoV, RaTG13, discovered in a cave of Yunnan, China, in 2013. They share about 96% nucleotide sequence identities, suggesting that SARS-CoV-2 might have emerged from a Bat SL-CoV. However, the intermediate host or whether there is an intermediate host remains to be determined.
In addition to the highly pathogenic zoonotic pathogens SARS-CoV, MERS-CoV, and SARS-CoV-2, all belonging to the P-coronavirus genus, four low-pathogenicity coronaviruses are endemic in humans: HCoV-OC43, HCoV-HKUl, HCoV-NL63, and HCoV-229E.
The coronaviruses (order Nidovirales, family Coronaviridae, genus Coronavirus) are a diverse group of large RNA viruses that cause varieties of diseases in humans and other animals, including respiratory, enteric, renal, and neurological diseases. Coronaviruses are enveloped viruses that contain a large single-stranded RNA genome of positive polarity. At ~30,000 nucleotides (nt), their genome is the largest found in any of the RNA viruses. Their envelope accommodates three or four membrane proteins of which the membrane (M), envelope (E), and spike (S) proteins are common to all. The S protein is a relatively large, about 180kDa type I glycoprotein, trimers of which form the petal-shaped projections on the surface of the virion that give rise to the characteristic corona solis-like appearance. It has been suggested that the SI subunit constitutes the globular head, while the S2 subunit forms the stalk-like region of the spike.
The two functions of the coronavirus S protein appear to be spatially separated. The SI subunit (or the equivalent part in viruses with uncleaved S protein) is responsible for receptor binding, and the S2 subunit is responsible for membrane fusion. In the structure, N- and C- terminal portions of SI fold as two independent domains, N-terminal domain (NTD) and C- terminal domain (C-domain). Depending on the virus, either NTD or C-domain can serve as the receptor-binding domain (RBD). While RBD of mouse hepatitis virus (MHV) is located at the NTD, most of other CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV use C-domain to bind their receptors. MHV uses mouse carcinoembryonic antigen related cell adhesion molecule la (mCEACAMla) as the receptor, and the receptor for SARS-CoV and SARS-CoV-2 is human angiotensin-converting enzyme 2 (hACE2) and that of MERS-CoV is dipeptidyl peptidase 4 (DPP4). In terms of sequence identity, S proteins of SARS-CoV-2 share about 76% and 97% of amino acid identity with SARS-CoV and RaTG13, respectively, and the amino acid sequence of potential RBD of SARS-CoV-2 is about 74% and 90.1% homologous to that of SARS-CoV and RaTG13, respectively.
The ectodomain of the S2 subunit contains two heptad repeat (HR) regions, HR1 and HR2, characteristic of coiled coils, while the fusion peptide (FP) is predicted to be located amino terminally of the first HR region (HR1). Binding of the SI subunit to the (soluble) receptor has been shown to trigger conformational changes that supposedly facilitate virus entry by activation of the fusion function of the S2 subunit. The conformational changes are thought to expose the fusion peptide and to lead to the formation of a heterotrimeric six-helix bundle by the two HR regions, a characteristic of class I viral fusion proteins, resulting in the close locations of the fusion peptide and the transmembrane domain in the process of membrane fusion. The S2 subunit additionally contains a transmembrane domain required for anchoring of the spike protein and a cytoplasmic endoplasmic reticulum (ER) retention motif.
Coronavirus S proteins are typical class I viral fusion proteins, and protease cleavage is required for activation of the fusion potential of S protein. A two-step sequential protease cleavage model has been proposed for activation of S proteins of SARS-CoV and MERS- CoV, priming cleavage between SI and S2 and activating cleavage on S2’ site. Depending on virus strains and cell types, CoV S proteins may be cleaved by one or several host proteases, including furin, trypsin, cathepsins, transmembrane protease serine protease-2 (TMPRSS-2), TMPRSS-4, or human airway trypsin-like protease (HAT). Availability of these proteases on target cells largely determines whether coronaviruses enter cells through plasma membrane or endocytosis.
The coronavirus S protein may be the S protein of one of the following coronavirus: SARS- CoV-2, SARS-CoV, SARS-like CoV RaTG13, MERS-CoV, HCoV-OC43, HCoV-HKUl, HCoV-NL63, and HCoV-229E. The coronavirus S protein may be the S protein of SARS-CoV-2, SARS-CoV, or SARS-like CoV RaTG13.
The coronavirus S protein may be the S protein of SARS-CoV-2.
The spike protein of SARS-CoV-2 is depicted under Accession No. P0DTC2 in the Uniprot database on 8th October 2020.
The sequences of S protein (SEQ ID NO: 1; signal sequence underlined), subunit SI (SEQ ID NO: 2) and subunit S2 ectodomain (SEQ ID NO: 3; HR1 region is underlined and HR2 region is in bold) of coronavirus SARS-CoV-2 are shown below.
SARS-CoV-2 S protein (SEQ ID NO: 1):
MFVFLVLLPLVSSOCVNLTTRTOLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFL PFFSNVTWFHAmVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSK TQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTF EYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALE PLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPF GEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT
NVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKF LPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICA SYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVS MTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQV KQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFA MQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQ NAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQ
LIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVT YVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFV SGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNI QKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLC CMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
SARS-CoV-2 Spike protein, SI subunit (SEQ ID NO: 2):
QCVNLTTRTQLPP AYTNSFTRGVYYPDKVFRS SVLHSTQDLFLPFF SNVTWFHAIH VSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNV VIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEG KQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQ TLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALD PLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYA WNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEV RQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLK PFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL HAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTT DAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQL TPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP
SARS-CoV-2 Spike protein, S2 subunit (SEQ ID NO: 3):
RRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCT MYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIK DFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICA QKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFN GIGVTQNVLYENOKLIANOFNS AIGKIQD SLS STAS ALGKLQD VVNONAQALNTL VKOLSSNFGAISSVLNDILSRLDKVEAEVOIDRLITGRLOSLOTYVTOOLIRAAEIRA SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKN FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVI GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRL NEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSC CSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
The polypeptide of the invention comprises an amino acid sequence of at least one RBD of a viral Spike protein. The term “receptor binding domain” or “RBD”, as used herein, refers to a short immunogenic fragment from a virus that binds to a specific endogenous receptor sequence to gain entry into host cells. Specifically, the RBD is a part of the Spike glycoprotein (S- protein) which is needed to interact with endogenous receptors to facilitate membrane fusion and delivery to the cytoplasm. Typically, the RBD is also the binding site of neutralizing antibodies. The RBD contains the receptor-binding motif (RBM) that interacts directly with the endogenous receptor.
The SARS-CoV-2 RBD has a twisted five-stranded antiparallel P sheet (P 1, P2, P3, P4 and P7) with short connecting helices and loops that form the core. Between the P4 and P7 strands in the core, there is an extended insertion containing the short P5 and P6 strands, a4 and a5 helices and loops. This extended insertion is the RBM, which contains most of the contacting residues of SARS-CoV-2 that bind to ACE2. The SARS-CoV-2 RBD spans residues Arg319 to Phe541 of the sequence shown as SEQ ID NO: 1 (SARS-CoV-2 Spike protein). This is the RBD of the SARS-CoV-2 variant first identified in Wuhan in December 2019.
Several variants carrying mutations in S-protein, including in its receptor-binding domain (RBD), have emerged, likely due to the rapid dissemination of the virus coupled with pressure from the patients’ immune response. Of note is the identification of the D614G (Nextstrain clade 20A) in early March 2020 that has rapidly become the dominant strain globally. Additional variants have also gained partial dominance in different regions of the globe. The variants A222V (Nextstrain clade 20A.EU1) and S477N (Nextstrain clade 20A.EU2) have emerged in the summer of 2020 in Spain and have rapidly shown diffusion within Europe. Another variant (clade 20B/501Y.V1, B. l.1.7) characterised by multiple mutations in S-protein (deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, DI 118H) has been associated with a rapid surge in COVID-19 cases in the UK between December 2020 and January 2021. This variant was termed the alpha variant. In the same period, a new variant in South Africa (clade 20C/501Y.V2, B.1.351), also carrying the N501Y mutation in the RBD (L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G and A701V), has been associated with increased transmissibility and reduction of serum neutralisation capacity. This variant was termed the beta variant. Another variant that emerged in Brazil (B.1.1.28) contains mutational hallmarks of both the UK and South Africa variants (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501 Y, H655Y, T1027I), suggesting convergent evolution in SARS-CoV-2 due to similar selective pressures. This variant has been termed the gamma variant. Other variants of SARS-CoV 2 that have been identified include the delta variant (B.1.617.2), the epsilon variant (B.1.429), the Mu variant (B.1.621), the lambda variant (C.37), the eta variant (B.1.525), the theta variant (P.3), and the variant kappa (B. J .617.1 ) Further variants are expected to emerge. The mutation numbering refers to the SARS-CoV-2 S protein as shown in SEQ ID NO: 1.
The RBD may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 44-53.
SEQ ID NO: 4 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; Wuhan): RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN CYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 44 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; N501Y; alpha): RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN CYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 45 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; K417N_E484K_N501Y; beta):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGF NCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 46 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; K417T_E484K_N501Y; gamma): RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGF NCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 47 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; L452R T478K; delta):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 48 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; L452R; epsilon):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 49 (amino acids 319 to 541 of SARS-CoV-2 Spike protein;
R346K_E484K_N501Y; Mu):
RVQPTESIVRFPNITNLCPFGEVFNATKFASVYAWNRKRISNCVADYSVLYNSASF
STFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFT
GCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKG FNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN F
SEQ ID NO: 50 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; L452Q F490S; lambda):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG
CVIAWNSNNLDSKVGGNYNYQYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF
NCYSPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF SEQ ID NO: 51 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; E484K; eta):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 52 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; E484K_N501Y; theta):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG
CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGF NCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 53 (amino acids 319 to 541 of SARS-CoV-2 Spike protein; L452R E484Q; kappa):
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG
CVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVQGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
The present invention is not intended to be limited to the above spike protein and RBD variants. Therefore, Spike proteins and RBDs of variants that have not been yet identified and future variants are also within the scope of the present invention.
The present invention also contemplates a polypeptide of the invention comprising two or more RBDs. The polypeptide of the invention may comprise three or more, four or more, five or more, six or more RBDs.
The two or more RBDs may have the same sequence or different sequences.
Suitably, the two or more RBDs may have different sequences. This is particularly advantageous when the polypeptide of the invention is used to elicit immunological responses against different viruses, viral strains or Spike protein variants. The sequences of the two or more RBDs may be selected from the group consisting of shown as SEQ ID NO: 4 and 44-53.
The two or more RBDs may be joined by a linker.
The RBD, or two or more RBDs, may be joined to the spacer via a linker.
The terms “linker” refers to an oligopeptide which provides spatial separation between domain each of the two or more RBDs that may be present in the polypeptide of the invention. The linker may be a flexible linker. This type of linkers allows for torsion of domain A respective of domain O, which may be beneficial when domain A interacts with the S protein of a coronavirus. Non-limiting examples of flexible linkers that may be used in the first polypeptide of the invention include:
- Ser(Gly4Ser) (SEQ ID NO: 43 : SGGGGS);
- (Gly4Ser)2 (SEQ ID NO: 5 : GGGGSGGGGS);
- (Gly4Ser)3 (SEQ ID NO: 6: GGGGSGGGGSGGGGS);
- (Gly4Ser)4 (SEQ ID NO: 7: GGGGSGGGGSGGGGSGGGGS);
- (Gly4Ser)5 (SEQ ID NO: 8: GGGGSGGGGSGGGGSGGGGSGGGGS);
- SGGGGSGGGGSGGGGS (SEQ ID NO : 9);
- GGGGSGGGGSGGGGS (SEQ ID NO: 10); and
- GGGGSGGGGSGGGAS (SEQ ID NO: 11).
The linker may be the (Gly4Ser)3 linker (SEQ ID NO: 6) or Ser(Gly4Ser) (SEQ ID NO: 43: SGGGGS).
1.2. Spacer
The polypeptide of the invention comprises a spacer. The spacer spatially separates the RBD, or two or more RBDs, from the cell membrane where the polypeptide of the invention is expressed. Together with the transmembrane domain, the spacer acts as an anchor and allows the polypeptide of the invention to present fragments of the viral spike protein on the cell surface. The spacer may be a spacer from a mammalian protein or a viral protein, such as a transmembrane protein. Non-limiting examples of spacers from mammalian proteins include the stalk of alpha chain of CD8 (CD8a; CD8a stalk), the CD4 ectodomain, the CD45 ectodomain, the CD2, and the CD34 ectodomain. Non-limiting examples of spacers from viral proteins include the stalk of the SARS CoV-2 spike glycoprotein (i.e. the S2 subunit stalk of SARS-CoV-2; SEQ ID NO: 36).
The spacer may be a spacer from a mammalian protein.
The spacer may alternatively comprise a coiled-coil domain, for example as described in WO2016/151315.
SEQ ID NO: 12 (human CD8 stalk):
PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
SEQ ID NO: 41 (S2 subunit stalk of SARS-CoV-2)
GKYEQYIKWP
SEQ ID NO: 42 (Heptad repeat 2 [HR2] of S2 subunit of SARS-CoV-2) TSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL
SEQ ID NO: 13 (human CD4 ectodomain):
KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRA DSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQG QSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKK VEFKIDIVVLAFQKAS SIVYKKEGEQVEF SFPLAFTVEKLTGSGELWWQ AERASS S KSWITFDLKNKEVSVKWVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALE AKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKRE KAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQP
SEQ ID NO: 14 (human CD45 ectodomain): QSPTPSPTGLTTAKMPSVPLSSDPLPTHTTAFSPASTFERENDFSETTTSLSPDNTST QVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPT PGSNAISDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAY LNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNV NENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHD CTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYK CDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTL CYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFT TKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNES HKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSK
SEQ ID NO: 15 (CD2 ectodomain)
KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKE KDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKIS WTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNK VSKESSVEPVSCPEKGLD
SEQ ID NO: 16 (CD34 ectodomain)
SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITE TTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDL STTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKD RGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLM KKHQSDLKKLGILDFTEQDVASHQSYSQKT
SEQ ID NO: 17 (COMP spacer)
DLGPQMLRELQETNAALQDVRELLRQQVREITFLKNTVMECDACG
The spacer may comprise a sequence selected from the sequences shown as SEQ ID NOs: 12, 41, 42, or 13 to 17 or a variant thereof having at least 80% sequence identity. A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 12, 3, or 13 to 17, provided that the sequence provides an effective spacer, i.e. the variant provides sufficient spatial separation between the RBD, or two or more RBDs, and the cell membrane where the polypeptide of the invention is expressed. The polypeptide of the invention may comprise a human CD8a stalk spacer.
1.3. Transmembrane domain
The polypeptide of the invention comprises a transmembrane domain (TM). The transmembrane domain, together with the spacer, anchors the RBD, or two or more RBDs, to the cell membrane where the polypeptide of the invention is expressed. This allows the polypeptide of the invention to present fragments of the viral spike protein on the cell surface.
In the polypeptide of the present invention, the transmembrane domain sequence may, for example, comprise the transmembrane domain of alpha chain of CD8 (CD8a; CD8a TM), the transmembrane domain of the SARS CoV-2 spike glycoprotein (i.e. the S2 subunit of SARS-CoV-2), the CD4 transmembrane domain, the CD45 transmembrane domain, the CD2 transmembrane domain and the and the CD34 ectodomain.
SEQ ID NO: 18 (human CD8a transmembrane domain):
IYIWAPLAGTCGVLLLSLVIT
SEQ ID NO: 19 (SARS CoV-2 S2 subunit transmembrane domain): WYIWLGFIAGLIAIVMVTIML
SEQ ID NO: 20 (human CD4 transmembrane domain):
MALIVLGGVAGLLLFIGLGIFF
SEQ ID NO: 21 (human CD45 transmembrane domain):
ALIAFLAFLIIVTSIALLVVL
SEQ ID NO: 22 (human CD2 transmembrane domain)
IYLIIGICGGGSLLMVFVALLVFYIT
SEQ ID NO: 23 (human CD34 transmembrane domain) LIALVTSGALLAVLGITGYFL
The transmembrane domain may comprise a sequence selected from the sequences shown as SEQ ID NOs: 18 to 23 or a variant thereof having at least 80% sequence identity. A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 18 to 23, provided that the sequence provides an effective anchor of the RBD, or two or more RBDs, to the cell membrane where the polypeptide of the invention is expressed.
The transmembrane domain may be paired to its respective spacer domain, for example, a human CD8a stalk and transmembrane domain may be comprised in the same polypeptide of the invention.
The polypeptide of the invention may comprise a human CD8a transmembrane domain.
1.4. Endodomain
The polypeptide of the invention may further comprise an endodomain (endo) or cytoplasmic domain.
In the polypeptide of the present invention, the endodomain sequence may, for example, comprise the endodomain of alpha chain of CD8 (CD8a; CD8a endo), the endodomain of the SARS CoV-2 spike glycoprotein (i.e. the S2 subunit of SARS-CoV-2), the endodomain of the SARS CoV-2 spike glycoprotein lacking the ER retention signal, the CD4 endodomain, the CD45 endodomain, the CD2 endodomain, and the CD34 endodomain.
SEQ ID NO: 24 (human CD8a endodomain):
LYCNHRNRRRVCKCPRPVV
SEQ ID NO: 25 (SARS CoV-2 S2 subunit endodomain):
CCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT
SEQ ID NO: 26 (SARS CoV-2 S2 subunit endodomain without ER retention signal): CCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 27 (human CD4 endodomain):
CVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI
SEQ ID NO: 28 (human CD45 endodomain):
YKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRPFL AEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINAS YIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEY WPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWP DHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAE
NKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLH NMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVP LKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETI GDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSS TYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKN SSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARL GMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVN
PLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGS
SEQ ID NO: 29 (human CD2 endodomain):
KRKKQRSRRNDEELETRAHRVATEERGRKPQQIPASTPQNPATSQHPPPPPGHRSQ APSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLS PSSN
SEQ ID NO: 30 (human CD34 endodomain):
MNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTG QATSRNGHSARQHVVADTEL
The endodomain may comprise a sequence selected from the sequences shown as SEQ ID NOs: 24 to 30 or a variant thereof having at least 80% sequence identity. A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 24 to 30. The endodomain may be paired to its respective spacer domain and transmembrane domain, for example, a human CD8a stalk, CD8a transmembrane domain and CD8a endodomain may be comprised in the same polypeptide of the invention.
The polypeptide of the invention may comprise a human CD8a endodomain.
The polypeptide of the invention may comprise one of the following membrane anchors derived from SARS-CoV-2 (SEQ ID NO: 54-57):
SEQ ID NO: 54 (SARS-CoV-2 transmembrane and endodomains)
GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 55 (SARS-CoV-2 HR2, transmembrane and endodomains) TSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYI WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 56 (SARS-CoV-2 HR1+HR2, transmembrane and endodomains) YENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFG AISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATK MSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYD PLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNES LIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGS CC
SEQ ID NO: 57 (SARS-CoV-2 HR1, transmembrane and endodomains) YENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFG AISGGGGSGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCG SCO
1.5. Signal peptide The polypeptide of the present invention may comprise a signal peptide so that when the polypeptide is expressed inside a cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognised and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.
The signal peptide may be at the amino terminus of the molecule.
The signal peptide may comprise the SEQ ID NO: 31 to 34 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the protein.
SEQ ID NO: 31 (TCRBV signal peptide):
MGTSLLCWMALCLLGADHAD
The signal peptide of SEQ ID NO: 31 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.
SEQ ID NO: 32: MSLPVTALLLPLALLLHAARP
The signal peptide of SEQ ID NO: 32 is derived from IgGl.
SEQ ID NO: 33: MAVPTQVLGLLLLWLTDARC
The signal peptide of SEQ ID NO: 33 is derived from CD8.
SEQ ID NO: 34: MGWSCIILFLVATATGVHS The signal peptide of SEQ ID NO: 34 is derived from murine Ig heavy chain.
The polypeptide of the invention may comprise a TCRBV signal peptide.
The polypeptide of the invention may further comprise a detectable marker sequence. Any detectable marker sequence may be used for the purposes of the present invention. Nonlimiting examples of detectable markers include the HA epitope tag derived from influenza A Hemagglutinin (Uniprot Q03909; residues 119-127; HA-tag); V5 tag; FLAG tag; C-myc tag; histidine tag (HIS tag); Avi tag; Strep tag; Strep tag II; S-peptide tag (S tag); ALFA tag; AU1 tag; AU5 tag; Glu-Glu tag; HSV tag; KT3 tag; E tag; C tag.
The polypeptide of the invention may comprise or consist of the sequence shown as SEQ ID NO: 35-40 and 58-70.
SEQ ID NO: 35 (SARS CoV-2 spike glycoprotein, RBD-CD8a stalk)
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN CYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFS GGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT CGVLLLSLVITLYCNHRNRRRVCKCPRPVV
SEQ ID NO: 36 (SARS CoV-2 spike glycoprotein RBD, S2 stalk)
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN CYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFS GGGGSGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSC C
SEQ ID NO: 37 (SARS CoV-2 spike glycoprotein RBD, HR2-S2 stalk) RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFS
TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG
CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN
CYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFS GGGGSTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK WPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 38 (TRCBV signal peptide, HA tag, SARS CoV-2 spike glycoprotein, RBD- CD8a stalk)
MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCP
FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF
TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG
NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV
GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH RNRRRVCKCPRPVV
SEQ ID NO: 39 (TRCBV signal peptide, HA tag, SARS CoV-2 spike glycoprotein RBD, S2 stalk)
MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCP
FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF
TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG
NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSGKYEQYIKWP WYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 40 (TRCBV signal peptide, HA tag, SARS CoV-2 spike glycoprotein RBD, HR2-S2 stalk)
MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCP
FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF
TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV
GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSTSPDVDLGDIS GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAI VMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 58 (signal peptide, HA tag, SI domain, CD8a stalk, TM, endo)
MGWSCIILFLVATATGVHSDSSYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYTN SFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLPF NDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGV YYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI DGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV KNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS FGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTR AGCLIGAEHVNNSYECDIPIGAGICASYQTQSGGGGSPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCK CPRPVV
SEQ ID NO: 59 (signal peptide, HA tag, RBD, CD8a stalk, TM, endo)
MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCP FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSPAPRPPTPAPTI ASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH RNRRRVCKCPRPVV
SEQ ID NO: 60 (signal peptide, HA tag, SI domain, SARS-CoV-2 TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYT N SFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV KNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS FGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTR AGCLIGAEHVNNSYECDIPIGAGICASYQTQSGGGGSGKYEQYIKWPWYIWLGFIA GLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 61 (signal peptide, HA tag, SI domain, SARS-CoV-2 HR2, TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYT NSFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV KNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS FGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTR AGCLIGAEHVNNSYECDIPIGAGICASYQTQSGGGGSTSPDVDLGDISGINASVVNI QKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLC CMTSCCSCLKGCCSCGSCC
SEQ ID NO: 62 (signal peptide, HA tag, SI domain, SARS-CoV-2 HR1+HR2, TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYT NSFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV KNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS FGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTR AGCLIGAEHVNNSYECDIPIGAGICASYQTQSGGGGSYENQKLIANQFNSAIGKIQD SLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQ IDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYH LMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWF VTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHT SPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYI WLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 63 (signal peptide, HA tag, SI domain, SARS-CoV-2 HR1, TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSSQCVNLTTRTQLPPAYT NSFTRGVYYPDKVFRS S VLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLP FNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLG VYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKN IDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTP CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV KNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS FGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTR AGCLIGAEHVNNSYECDIPIGAGICASYQTQSGGGGSYENQKLIANQFNSAIGKIQD SLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISGGGGSGKYEQYIKWPW YIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC SEQ ID NO: 64 (SARS CoV-2 RBD, transmembrane)
MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCP FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF
TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSGKYEQYIKWP
WYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 65 (signal peptide, HA tag, RBD, SARS-CoV-2 HR1+HR2, TM, endo) MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCP FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF
TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV
GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSYENQKLIANQF
NSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRL
DKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRV DFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF VSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEEL
DKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ YIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 66 (signal peptide, HA tag, RBD, SARS-CoV-2 HR1, TM, endo)
MGTSLLCWMALCLLGADHADAYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCP FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF
TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGG NYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV
GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSYENQKLIANQF NSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISGGGGSGKYE QYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC
SEQ ID NO: 67 (RBDx3-CD8STK_mono-cistronic_(RBD_319-541_linker_ RBD 319- 541_N501 Y_linker_ RBD 319-541_K417N_E484K_N501 Y)) MGWSCIILFLVATATGVHSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR
KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA
PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER
DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA
TVCGPKKSTNLVKNKCVNFSGGGGSGGGGSGGGGSRVQPTESIVRFPNITNLCPFG
EVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTN
VYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNY
NYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGY
QPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSGGGGSGGGGSRV
QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTF
KCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGC
VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFN
CYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFS
GGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT
CGVLLLSLVITLYCNHRNRRRVCKCPRPVV
SEQ ID NO: 68 (HA, RBDx3-CD8STK_mono-cistronic_(HA-RBD_319-541_linker_
RBD 319-541_N501 Y_linker_ RBD 319-541_K417N_E484K_N501 Y))
MGWSCIILFLVATATGVHSDSSYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCPF
GEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT
NVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN
YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG
YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSGGGGSGGGGSR
VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST
FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG
CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFN
CYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFS
GGGGSGGGGSGGGGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRIS
NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQT
GNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST
EIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVC
GPKKSTNLVKNKCVNFSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHT
RGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV SEQ ID NO: 69 ((RBD-CD8STK)x3_tri-cistronic_(RBD_319-541_CD8STK-T2A- RBD 319-54 l_N501 Y CD8 STK-P2 A-RBD 319-41_K417N_E484K_N501 Y CD8 STK)) MGWSCIILFLVATATGVHSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPA TVCGPKKSTNLVKNKCVNFSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVRAEG RGSLLTCGDVEENPGPMGWSCIILFLVATATGVHSRVQPTESIVRFPNITNLCPFGE VFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV YADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQ PYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSPAPRPPTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR RRVCKCPRPVVEGRGSLLTCGDVEENPGPMGWSCIILFLVATATGVHSRVQPTESI VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGV SPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNS NNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQS YGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSP APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL SLVITLYCNHRNRRRVCKCPRPVV
SEQ ID NO: 70 ((RBD-CD8STK)x3_tri-cistronic_(HA-RBD_319-541_CD8STK-T2A-V5- RBD 319-541 N501 Y CD8STK-P2A-FLAG-RBD 319-
541_K417N_E484K_N501 Y CD8 STK))
MGWSCIILFLVATATGVHSDSSYPYDVPDYASGGGGSRVQPTESIVRFPNITNLCPF GEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFT NVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGN YNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG YQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGGGSPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHR NRRRVCKCPRPVVRAEGRGSLLTCGDVEENPGPMGWSCIILFLVATATGVHSEEP GKPIPNPLLGLDSTSGGGGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPA TVCGPKKSTNLVKNKCVNFSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVEGRG SLLTCGDVEENPGPMGWSCIILFLVATATGVHSDSSDYKDDDDKSGGGGSRVQPT ESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCY GVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIA WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYF PLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFSGGG GSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV LLLSLVITLYCNHRNRRRVCKCPRPVV
The above sequences are depicted in Figure 5. These sequences are intended to be exemplary polypeptides according to the invention. Following the teachings described herein, the skilled person will be able to use different components, including different SI and RBD sequences, to generate polypeptides according to the invention.
2. Nucleic acid
The invention is also applicable to techniques involving nucleic acid (RNA, DNA) and vector-mediated transgene expression in a context where expression of a transgene leads to therapeutic benefits, for instance vaccination. Nucleic acid vaccination is a technique whereby a nucleic acid encoding a target antigen is introduced into a host either alone or mediated by a vector (for instance viral or bacterial) for the induction of an immune response. The target antigen is expressed by the host cells and can result in the generation of an immunological response and residual immunological memory. The encoded transgene can be expressed and presented directly by antigen presenting cells (APCs), or the target antigen can be expressed by non-APC host cells and then acquired by APCs for presentation, for instance by engulfment of apoptotic target antigen expressing cells. An example of a host cell suitable for inoculation with a nucleic acid vaccine is muscle cells. Thus, in a second aspect, the present invention also provides a nucleic acid sequence encoding a polypeptide of the invention, hereinafter “the nucleic acid of the invention”.
The term “polypeptide of the invention” has been described in detail in the context of previous aspects of the invention and its definitions and embodiments apply equally to the second aspect of the invention.
As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.
It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
The nucleic acid sequences and constructs of the invention may contain alternative codons in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination.
Nucleic acids according to the invention may comprise DNA or RNA. Nucleic acids may be single-stranded or double-stranded. Nucleic acids may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
The nucleic acid may be single stranded or double stranded. The nucleic acid may be DNA or RNA.
The RNA may be messenger RNA (mRNA) or circular RNA.
Circular RNA is typically generated by chemical or enzymatic ligation of the 5’ and 3’ ends of the RNA molecule, back splicing of intron containing RNA, use of self-splicing sequences or ligation using splint oligonucleotides. Circular RNAs encoding translatable sequences require an internal ribosome entry site (IRES) or a functionally equivalent sequence to recruit the ribosome to the circular RNA and initiate its translation.
Where the nucleic acid is RNA, nucleotide analogues and other chemical structures with the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has 5’ and 3’ UTRs. The 5’ UTR and/or the 3’ UTR may be between zero and 3000 nucleotides in length. The length of 5’ and 3’ UTR sequences may be modified to achieve optimal translation efficiency. Various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
Additionally, the attachment of different chemical groups to the 3 ’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA. 5’ caps on also provide stability to mRNA molecules. In a preferred embodiment, RNAs produced by the methods to include a 5' cap structure. Such cap structure can be generated using Vaccinia capping enzyme and 2’-O-methyltransferase enzymes. Alternatively, 5’ cap is provided using techniques known in the art and described herein (Cougot et al., 2001, Trends Biochem Sci 29:436-44; Stepinski et al., 2001, RNA 7: 1468-95; Elango et al., 2005, Biochim Biophys Res Commun 330:958-66).
The RNA may be nucleoside-modified RNA. Nucleoside-modified mRNA have particular advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in patent application W02007024708. The nucleoside-modified RNA may comprise the naturally occurring modified-nucleoside pseudouridine.
The terms “variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
The present invention also provides a nucleic acid construct encoding two or more polypeptides of the invention, hereinafter “the nucleic acid construct of the invention”.
The nucleic acids encoding each of the two or more polypeptides of the invention may be in any order in the construct.
Nucleic acids encoding two or more polypeptides may be separated by a co-expression site enabling co-expression of two polypeptides as separate entities. It may be a sequence encoding a cleavage site, such that the nucleic acid construct produces both polypeptides, joined by a cleavage site(s). The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.
The cleavage site may be any sequence which enables the two polypeptides to become separated.
The term “cleavage” is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage. For example, for the Foot-and-Mouth disease virus (FMDV) 2A self-cleaving peptide (see below), various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82: 1027-1041). The exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities. The cleavage site may, for example be a furin cleavage site, a Tobacco Etch Virus (TEV) cleavage site or encode a self-cleaving peptide.
A ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self-cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.
The self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus. The primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus. In apthoviruses, such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C- terminus (Donelly et al (2001) as above).
“2A-like” sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al (2001) as above).
The cleavage site may comprise the 2A-like sequence shown as SEQ ID NO: 71 (RAEGRGSLLTCGDVEENPGP).
The nucleic acid of the invention can contain a regulatory sequence operatively linked for the expression of the nucleotide sequence encoding the polypeptide of the invention. As used herein, the term “operatively linked” means that the antibody encoded by the nucleic acid sequence of the invention is expressed in the correct reading frame under control of the expression control or regulating sequences. Therefore, in another aspect, the invention provides an expression cassette, hereinafter “the expression cassette of the invention”, comprising the nucleic acid of the invention or the nucleic acid construct of the invention operatively linked to an expression control sequence. The expression cassette of the invention can be obtained by techniques that are widely known in the art. The expression cassette may comprise one or more control sequences. Control sequences are sequences that control and regulate transcription and, where appropriate, the translation of said immunogen, and include promoter sequences, transcriptional regulators encoding sequences, internal ribosome binding site (IRES), ribosome binding sequences (RBS) and/or transcription terminating sequences. The expression cassette of the present invention may additionally include an enhancer, which may be adjacent to or distant from the promoter sequence and can function to increase transcription from the same. The expression control sequence may be functional in prokaryotic cells or in eukaryotic cells and organisms, such as mammalian cells. The expression cassette may comprise a promoter. Any promoter may be used in this methodology.
3. Vector
In a third aspect, the present invention also provides a vector, hereinafter “the vector of the invention”, which comprises a nucleic acid of the invention or a nucleic acid construct or an expression cassette of the invention. Such a vector may be used to introduce the nucleic acid or expression cassette into a host cell so that it expresses the polypeptide of the invention.
The terms “polypeptide of the invention”, “nucleic acid of the invention”, “nucleic acid construct of the invention” and “expression cassette or the invention” have been described in detail in the context of previous aspects of the invention and their definitions and embodiments apply equally to this aspect of the invention.
The vector may, for example, be a plasmid, replicating viral vector, non-replicating viral vector, self-replicating RNA viral vector (alphavirus), viral-like particle or synthetic mRNA.
4. Cell
In a fourth aspect, the present invention relates to a cell, hereinafter “the cell of the invention”, comprising a polypeptide of the invention, a nucleic acid of the invention, a nucleic acid construct of the invention, an expression cassette of the invention, or a vector of the invention. The cell may comprise a polypeptide, a nucleic acid, a nucleic acid construct, or an expression cassette, or a vector according to the present invention.
The term “polypeptide of the invention”, “nucleic acid of the invention”, “nucleic acid construct of the invention”, “expression cassette or the invention”, and “vector of the invention” have been described in detail in the context of previous aspects of the invention and their definitions and embodiments apply equally to this aspect of the invention.
The cell may be prokaryotic or eukaryotic.
Cells suitable for performing the invention include, without limitation, mammalian and bacterial cells. Mammalian cells suitable for the present invention include epithelial cell lines, muscle cells, antigen presenting cells (APCs), osteosarcoma cell lines, neuroblastoma cell lines, epithelial carcinomas, glial cells, hepatic cell lines, CHO (Chinese Hamster Ovary) cells, COS, BHK cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293 and 293T cells, SupTl cells, PER.C6 cells, NTERA-2 human ECCs cells, D3 cells of the mESCs line, SHEF1, SHEF2 and HS181, NIH3T3 cells, REH and MCF-7 cells. Bacterial cells include, without limitation, cells from Gram positive bacteria such as species of the genus Bacillus, Streptomyces and Staphylococcus and Gram-negative bacterial cells such as cells of the genus Escherichia and Pseudomonas.
The present invention also relates to a method for making a cell of the invention, which comprises a step of introducing a nucleic acid of the invention, a nucleic acid construct of the invention, an expression cassette of the invention, or a vector of the invention into the cell. This may be done by transducing or transfecting a cell with the nucleic acid of the invention, the nucleic acid construct of the invention, the expression cassette of the invention, or the vector of the invention.
Where the nucleic acid is RNA, it can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation, cationic liposome mediated transfection using lipofection, lipid nanoparticle mediated transfection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns”. 5. Pharmaceutical composition
In a fifth aspect, the present invention also relates to a pharmaceutical composition containing the polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the first pharmaceutical composition of the invention”.
In another aspect, the present invention also relates to a pharmaceutical composition containing the nucleic acid of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the second pharmaceutical composition of the invention”.
In another aspect, the present invention also relates to a pharmaceutical composition containing the nucleic acid construct of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the third pharmaceutical composition of the invention”.
In another aspect, the present invention also relates to a pharmaceutical composition containing the vector of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the fourth pharmaceutical composition of the invention”.
In another aspect, the present invention also relates to a pharmaceutical composition containing the cell of the invention or a plurality of cells according to the invention and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant, hereinafter “the fifth pharmaceutical composition of the invention” .
The first, second, third, fourth and fifth pharmaceutical compositions of the invention may additionally comprise a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The first, second, third and fourth pharmaceutical compositions may optionally comprise one or more polypeptides of the invention. Such a formulation may, for example, be in a form suitable for intravenous infusion. The terms “polypeptide of the invention”, “nucleic acid of the invention”, “vector of the invention”, and “cell of the invention” have been described in detail in the context of previous aspects of the invention and their definitions and particular embodiments apply equally to this aspect of the invention.
As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable diluent” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide of the invention.
The term “pharmaceutically acceptable excipient”, as used herein, refers to an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Excipients include carriers, diluents, preservatives, colouring agents, and fillers.
The term “adjuvant”, as used herein, is defined as any molecule to enhance an antigenspecific adaptive immune response, i.e. a pharmacological or immunological agent that improves the immune response of a vaccine. The first, second, third, fourth and fifth pharmaceutical compositions of the invention may comprise an adjuvant, such as a microparticulate adjuvant, for example liposomes, or an immune stimulating complex (ISCOMS), virus-like particles or nanoparticles, an emulsion, a microparticle, a virosome, a micellar delivery system, a dendrimer delivery system, a plant vaccine, a melt-in-mouth strip (under the tongue) or an immunostimulatory adjuvant (such as cholera toxin, chitosan). The first, second, third, fourth and fifth pharmaceutical compositions of the invention may be in a variety of forms, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g. injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, lipid nanoparticles (LNP). The preferred form depends on the intended mode of administration and therapeutic application.
The pharmaceutical composition may comprise a (LNP). Methods for making LNPs are well-known in the art. Typical LNP formation procedures involve the controlled mixing of hydrophobic lipid components dissolved in an organic solvent such as ethanol with an aqueous buffer solution containing the oligonucleotide to be loaded into the resulting particle.
ADMINISTRATION
The administration of the first, second, third, fourth and fifth pharmaceutical compositions of the invention can be accomplished using any of a variety of routes that make the active ingredient bioavailable. For example, the agent can be administered via an oral, mucosal, buccal, intranasal, intraocular, inhalable, intravenous, subcutaneous, transcutaneous, intramuscular, intraperitoneal, parenteral or topical route. Oral administration may be by inhalation, by nebulisation or nasally. The first, second, third and fourth pharmaceutical compositions of the invention may be administered locally, for example by catheter or stent, or systemically, for example by intravenous injection.
The route of administration may be intramuscular.
The first, second, third, fourth and fifth pharmaceutical compositions of the invention may be administered to the subject in a variety of pharmaceutically acceptable dosing forms, which will be familiar to those skilled in the art. Pharmaceutical compositions according to the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and. then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilisers.
For example, the first, second, third, fourth and fifth pharmaceutical compositions of the invention may be administered via the nasal route using a nasal insufflator device. Examples of these are already employed for commercial powder systems intended for nasal application (e.g. Fisons Lomudal System). Details of other devices are well-known in the art.
Other delivery routes for the first, second, third and fourth pharmaceutical compositions of the invention include via the pulmonary route using a powder inhaler or metered dose inhaler, via the buccal route formulated into a tablet or a buccal patch, and via the oral route in the form of a tablet, a capsule or a pellet (which compositions may administer agent via the stomach, the small intestine or the colon), all of which may be formulated in accordance with techniques which are well known to those skilled in the art.
The first, second, third, fourth and fifth pharmaceutical compositions of the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment is required, and which would normally require frequent administration (e.g. at least daily injection).
A person skilled in the art would be able to determine the appropriate timing, sequence and dosages of administration for particular pharmaceutical compositions of the present invention. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular subject.
6. Methods The presentation of coronavirus spike protein-derived peptides by the polypeptide of the invention makes this molecule capable of eliciting an immune reaction in a subject. This can be exploited for therapeutic and prophylactic purposes. The invention further provides the use of a polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell or pharmaceutical composition as defined by the invention in the preparation of a coronavirus vaccine for inducing an immune response against a coronavirus in a subject in need thereof.
6.1. Method of antigen presentation
In another aspect, the present invention provides a method for presenting a viral Spike protein-derived peptide, hereinafter “the method of antigen presentation of the invention”, comprising expressing a polypeptide of the invention in a cell.
The terms “polypeptide of the invention” and “cell” have been described in detail in previous aspects of the invention and their definitions and embodiments apply equally to this aspect of the invention.
The method of presentation of the invention comprises the step of expressing a polypeptide of the invention in a cell. This may be carried out by transducing or transfecting a cell with a nucleic acid, a nucleic acid construct, an expression cassette or a vector of the invention as has been described in previous aspects of the invention. The resulting cell is a cell according to the invention.
The method of presentation of the invention may also comprise a step of contacting the transduced or transfected cell with an immune cell or an antigen-presenting cell (APC). The immune cell may be a population of immune cells. The APC may be a population of APCs.
The method of presentation of the invention may involve a step of administering the nucleic acid of the invention, nucleic acid construct of the invention, expression cassette of the invention, vector of the invention, or cell of the invention to a subject. The administration may be by any of the ways described in previous aspects of the invention. 6.2. Method of screening therapeutics
In another aspect, the present invention provides a method of testing cross-reactivity of a viral therapeutic, comprising a step of contacting the viral therapeutic with a cell or a plurality of cells of the invention.
The term “cell” has been described in detail in previous aspects of the invention and its definition and embodiments apply equally to this aspect of the invention.
In this aspect, the specificity of a viral therapeutic that binds to a coronavirus Spike protein can be tested by contacting the viral therapeutic with a cell or a plurality of cells of the invention. The skilled person will immediately appreciate that, by using a cell or plurality of cells which express a polypeptide of the invention comprising the spike protein or RBD of the same virus to which the viral therapeutic is specific, and a cell or plurality of cells which express a polypeptide of the invention comprising the spike protein or RBD of a different virus, it is possible to determine whether the viral therapeutic cross-reacts with other viruses. Similarly, the specificity against a viral strain or variant can be tested by using a cell or plurality of cells which express a polypeptide of the invention comprising the spike protein or RBD of the same viral variant to which the viral therapeutic is specific, and a cell or plurality of cells which express a polypeptide of the invention comprising the spike protein or RBD of a different viral variant.
This method may be carried out by methods that are well-known in the art, such as by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunocytochemistry, or by immunofluorescent techniques such as fluorescence microscopy or flow cytometry.
The viral therapeutic may be an antibody specific to the spike protein or RBD of a coronavirus. The coronavirus may be SARS-CoV-2.
The cell of the invention is particularly useful for screening patients producing high titers of neutralising antibodies. Serum of these patients may be used to treat other patients suffering from the same viral infection. Thus, in another aspect, the present invention relates to a method to identify patients suffering from an infection or infectious clinical condition caused by a virus, such as a coronavirus, which have high titers of neutralising antibodies, comprising a step of contacting a blood, serum or plasma sample from the patient with a cell or a plurality of cells of the invention.
The term “cell” has been described in detail in previous aspects of the invention and its definition and embodiments apply equally to this aspect of the invention.
This method may be carried out by methods that are well-known in the art, such as by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), competitive ELISA, immunocytochemistry, or by immunofluorescent techniques such as fluorescence microscopy or flow cytometry.
The viral therapeutic may be an antibody specific to the spike protein or RBD of a coronavirus. The coronavirus may be SARS-CoV-2.
6.3. Therapeutic method
In another aspect, the present invention provides a polypeptide of the invention, or a nucleic acid of the invention, or a nucleic acid construct of the invention, or an expression cassette of the invention, or a vector of the invention, or a cell of the invention, or a pharmaceutical composition of the invention for use in medicine.
In another aspect, the present invention provides a polypeptide of the invention, or a nucleic acid of the invention, or a nucleic acid construct of the invention, or an expression cassette of the invention, or a vector of the invention, or a cell of the invention, or a pharmaceutical composition of the invention for use in preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus, hereinafter “the medical use of the invention”.
Alternatively, this aspect of the invention may be formulated as a use of a polypeptide of the invention, or a nucleic acid of the invention, or a nucleic acid construct of the invention, or an expression cassette of the invention, or a vector of the invention, or a cell of the invention, or a pharmaceutical composition of the invention in the manufacture of a medicament for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus.
Alternatively, this aspect of the invention may be formulated as a method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus, in a subject in need thereof, hereinafter “the therapeutic method of the invention”, the method comprising the step of administrating to the subject a therapeutically effective amount of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention.
The terms “polypeptide of the invention”, “nucleic acid of the invention”, “nucleic acid construct of the invention”, “expression cassette of the invention”, “vector of the invention”, “cell of the invention”, “pharmaceutical composition of the invention”, and “coronavirus” have been described in detail in previous aspects of the invention and their definitions and embodiments apply equally to this aspect of the invention.
A method for method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus, in a subject in need thereof relates to the prophylactic use of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention, which may be administered to a subject who has not been infected with a virus, such as a coronavirus, in order to prevent or lessen their infection with the virus. The virus may be a coronavirus. Herein such polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition may be administered to a subject who has not yet contracted the coronavirus infection or condition or disorder resulting from this infection and/or who is not showing any symptoms of the coronavirus infection or condition or disorder resulting from this infection to prevent or impair the coronavirus from infecting the cells of the subject or to reduce or prevent development of at least one symptom associated with the coronavirus infection or condition or disorder resulting from this infection. The subject may have a predisposition for or be thought to be at risk of contracting a coronavirus infection or a condition or disorder resulting from this infection.
A method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, such as a coronavirus infection, or a condition or disorder resulting from this infection also relates to the therapeutic use of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention, which may be administered to a subject who has been infected with the virus, or is suspected to have been infected with the virus, or has tested positive for the virus in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
The virus may be a coronavirus. The coronavirus may be SARS-CoV-2
These therapeutic applications will comprise the administration of a therapeutically effective amount of the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention.
The treatment of a coronavirus disease in a subject may comprise the step of administrating the polypeptide of the invention, or the nucleic acid of the invention, or a nucleic acid construct of the invention, or the expression cassette of the invention, or the vector of the invention, or the cell of the invention, or the pharmaceutical composition of the invention to the subject, to trigger an immune reaction against the coronavirus Spike protein which may result in a complete or partial neutralisation of the coronaviruses.
The term “subject” or “individual”, as used in the context of the present invention, refers to members of mammalian species. The subject may be a human patient of any gender, age or race. Alternatively, the subject may be a non-human mammal infected with coronavirus. The polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be administered to a non-human mammal infected with coronavirus for veterinary purposes or as an animal model of human disease. Such animal models may be useful for evaluating the therapeutic efficacy of the polypeptides of this invention. Non-limiting examples of non-human mammal that may be subject to treatment according to the invention include a cat or any other feline, a dog or any other canid, a mouse, a rat, a capybara or any other rodent, a pig, a primate, and a bat.
The term “therapeutically effective amount”, as used herein, refers to the amount of the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention which is required to achieve an appreciable prevention, neutralisation, cure, delay, reduction of the severity of, or amelioration of one or more symptoms of a coronavirus disease.
The term “a coronavirus infection or a condition or disorder resulting from this infection”, as used herein, refers to an infection, condition or disorder caused by a coronavirus. The coronaviruses can cause varieties of diseases in humans and other animals, including respiratory, enteric, renal, and neurological diseases. Particularly important are the diseases caused by coronaviruses SARS-CoV-2 and SARS-CoV because of the severe acute respiratory syndrome that they cause.
The coronavirus condition or disorder may be coronavirus disease 2019 (COVID-19). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and has since spread globally, resulting in the ongoing 2019-20 coronavirus pandemic. Common symptoms include fever, cough and shortness of breath. Other symptoms may include fatigue, muscle pain, diarrhoea, nausea, sore throat, loss of smell and abdominal pain. While the majority of cases result in mild symptoms, some progress to viral pneumonia and multi-organ failure.
Reports have shown that COVID-19 manifests as a clotting disorder, which may cause pulmonary embolism and hypoxia. Pulmonary vasculature affected by pulmonary embolism is not fully restored and can cause permanent fibrosis of the lining of blood vessels. Pulmonary fibrosis may also be the result of prolonged mechanical ventilation; even prolonged use of high concentration oxygen can lead to lung injury and result in fibrosis. Permanent fibrosis may lead to chronic thromboembolic pulmonary hypertension (CTEPH). Additionally, the clotting disorder causes end organ damage, primarily kidney. Kidney injury does not fully recover and may lead to chronic kidney disease (CKD) in post- COVID19 patients.
Direct infection of SARS-CoV-2 of ACE2-expressing cells has a number of consequences. Infection of the heart muscle cells leads to myocarditis. Patients who have no or minimal pulmonary symptoms but presented with fatigue may experience myocarditis as the primary disease. Myocardial injury may also explain the increase incidence of cardiac arrest in COVID-19 patients. Because ACE2 receptors play a key role in the renin-angiotensin system, which is a primary regulatory mechanism for blood pressure, viral infection of ACE2-expressing cells may lead to malfunction of the system and increased blood pressure.
Severe COVID-19 presents with a cytokine storm or cytokine release syndrome (CRS), which is an immediate and intense response of the immune system to viral infection. However, there are indications that the immune response may not just be temporary. One example is the case reports of Kawasaki disease symptoms in children infected with SARS- CoV-2. Kawasaki disease is an autoimmune disease in which blood vessels throughout the body become inflamed. It is considered a “post-viral” autoimmune disease. Several reports have described COVID-19 patients suffering from Guillain-Barre syndrome Guillain-Barre syndrome is a neurological disorder where the immune system responds to an infection and ends up mistakenly attacking nerve cells, resulting in muscle weakness and eventually paralysis. Thus, severe COVID-19 may also cause an incidence of other more prevalent autoimmune diseases in recovered patients.
The loss of the sense of smell is a direct result of the virus infecting the olfactory neurons. It has been suggested that this may enable the virus to spread from the respiratory tract to the brain. Cells in the human brain express the ACE2 protein on their surface. ACE2 is also found on endothelial cells that line blood vessels. Infection of endothelial cells may allow the virus to pass from the respiratory tract to the blood and then across the blood-brain barrier into the brain. Once in the brain, replication of the virus may cause neurological disorders. Larger studies from China and France have also investigated the prevalence of neurological disorders in COVID-19 patients. These studies have shown that 36% of patients have neurological symptoms. Many of these symptoms were mild and include headache or dizziness that could be caused by a robust immune response. Other more specific and severe symptoms were also seen and include loss of smell or taste, muscle weakness, stroke, seizure and hallucinations. Case studies have described severe COVID-19 encephalitis and stroke in healthy young people with otherwise mild COVID-19 symptoms. These symptoms are seen more often in severe cases, with estimates ranging from 46% to 84% of severe cases showing neurological symptoms. Changes in consciousness, such as disorientation, inattention and movement disorders, were also seen in severe cases and found to persist after recovery. Therefore, brain inflammation in severe COVID-19 might also indirectly cause neurological damage, such as through brain swelling, which is associated with neurodegenerative diseases.
The virus is mainly spread during close contact and by small droplets produced when those infected cough, sneeze or talk. These small droplets may also be produced during breathing, but rapidly fall to the ground or surfaces and are not generally spread through the air over large distances. People may also become infected by touching a contaminated surface and then their face. The virus can survive on surfaces for up to 72 hours. It is most contagious during the first three days after onset of symptoms, although spread may be possible before symptoms appear and in later stages of the disease. The time from exposure to onset of symptoms is typically around five days, but may range from two to 14 days. The standard method of diagnosis is by real-time reverse transcription polymerase chain reaction (RT- PCR) from a nasopharyngeal swab. The infection can also be diagnosed from a combination of symptoms, risk factors and a chest CT scan showing features of pneumonia.
The method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by coronavirus in a subject in need thereof may comprise a step of administering the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention to the subject. The skilled person will be able to determine by conventional methods the amount of such polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention that are able to exert a prophylactic or therapeutic effect on the patient.
The polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be administered once, or it may be administered multiple times. The polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be administered from once a day to once every six months, once every year or longer. Typically, the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be administered in two doses, 28 days apart. The polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention of the invention may also be administered continuously via a minipump. The polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention of the invention may be administered via an oral, mucosal, buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular, parenteral, or topical route. The polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention polypeptide of the invention may be administered locally or systemically.
The skilled person will be able to determine the effective dosage of the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention by methods that are well-known in the art. For example, the dosage of polypeptide of the invention will generally be in the range of 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg, and even more preferably 1-10 mg/kg. The immune response triggered by the polypeptide, nucleic acid, nucleic acid construct, expression cassette, vector, cell, or pharmaceutical composition of the invention may be measured by any method known in the art.
The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES
Example 1: Generation of plasmid DNA constructs encoding fragments of the SARS CoV-2 spike protein fused to transmembrane anchors
Fragments of the spike SI subunit were selected for expression on the cell surface as it is this subunit that mediates attachment to the host cell, and it is likely that antibodies recognising this domain would be neutralising and prevent viral infection. Fusion of the SI fragments to a fragment of the S2 subunit could potentially broaden the antibody response and increase the likelihood of developing an immune response that is protective and prevents viral infection. Adopting such an approach would mitigate the risk of a sub-optimal immune response that could lead to individuals becoming more susceptible to viral infection due to antibody-dependent enhancement.
Three fragments of the SARS CoV-2 spike SI subunit, the SI subunit without its signal peptide (residues 13-677), the ACE2 receptor-binding domain (RBD; residues 319-541) and the ACE2 -interacting face of the RBD (residues 437-508) were expressed on the CD8a stalk or fragments of the SARS CoV-2 S2 subunit. Four fragments of the S2 subunit were used in the study: the stalk, transmembrane and truncated cytoplasmic domain lacking the ER retention motif (residues 1204-1251); the second heptad repeat (residues 1160-1251); the first and second heptad repeats (917-1251); and the first heptad repeat fused the S2 stalk (residues 917-97 fused to 1204-1254) were used for fusion to the SI fragments. The S2 fragments alone were also cloned to enable for testing of cell surface expression.
For the initial testing of the approach, plasmid DNA constructs were generated and transfected into 293T cells to determine whether the fusion proteins were expressed on the cell surface. All SARS CoV-2 SI and S2 fragments possessed an N-terminal HA epitope tag to facilitate detection at the cell surface using a fluorophore conjugated anti-HA antibody and analysing by flow cytometry. Staining was also carried out with soluble ACE2 tagged to a human IgG2 Fc (fragment crystallisable) to determine if the SI fragments were correctly folded and capable of interacting with their target receptor. Table 1. List of plasmid DNA constructs encoding fragments of the SARS CoV-2 spike protein fused to transmembrane anchors.
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Example 2: Testing of SARS CoV-2 SI and S2 transmembrane anchor fusion constructs
To determine if the SARS CoV-2 fragments could be expressed at the cell surface, 293T cells were transiently transfected with the retroviral constructs (Table 1), stained 48 hours later with antibodies to the RBD (Figure 1) or soluble ACE2-Fc protein and HA epitope tag (Figure 2) and analysed by flow cytometry.
Expression of the SI (residues 13-677) and the RBD (residues 319-541) was detected on the cell surface using an antibody to the HA epitope tag and soluble ACE2-Fc, when fused the CD8a stalk and the fragment of spike S2. Positive staining with soluble ACE2-Fc suggested that both the SI subunit and RBD were correctly folded and could potentially be immunogenic (Figure 2). Example 3: Stable expression of the SARS CoV-2 SI transmembrane fusions on SupTl cells
To investigate the stability of expression of the SARS CoV-2 at the cell surface, SupTl cells were transduced with y-retroviral supernatant encoding the spike fragments and stained with soluble ACE2-Fc and detected with an Alexa-647 conjugated anti-human IgG Fc antibody (Error! Reference source not found.). The results of this experiment indicated that the RBD can be stably expressed at the cell surface using the CD8a or S2 -derived transmembrane anchors (S2 stalk or the second heptad repeat of S2).
Surprisingly, the SI subunit was not stably expressed on the surface of SupTl cells, and this could be due to the stability of the protein fragment or differences between the SupTl and 293T cell lines.
Example 4: Validation of RBD-CD8a stalk transmembrane fusion by monitoring seroconversion in rats
As demonstrated in Example 3, the SARS CoV-2 RBD can be stably expressed at the cell surface and this fragment of the protein is the most promising target immunogen. To determine if expression of the RBD at the cell surface via a transmembrane anchor leads to seroconversion and the generation of neutralising antibodies, three rats were immunised with a plasmid encoding the SARS CoV-2 RBD-CD8a stalk transmembrane fusion and sera collected. These sera were used to stain SupTl cells expressing the RBD-CD8a stalk transmembrane fusion or non-transduced SupTl cells (Figure 4).
Positive staining of SupTl cells expressing the RBD-CD8oc spacer and transmembrane domain was obtained using the sera diluted 1 : 100 and 1 : 1000, indicating that the RBD-CD8oc spacer and transmembrane fusion is a potent immunogen eliciting an immune response that results in the production of anti-SARS CoV-2 spike antibodies. Importantly no positive staining of non-transduced SupTl cells was observed using the immunised rat sera, indicating a specific immune response to the immunogen. Together these data indicate that the CD8a stalk and transmembrane domain can be used to present viral peptides on the surface of cells to generate immunogens for vaccination.
Example 5: Comparison of the seroconverting capacity of soluble and membranebound RBD in rats
The seroconverting capacity of membrane-bound RBD in rats was compared with that of soluble trimeric RBD (RBD fused to the C-terminal domain of T4 bacteriophage fibritin, referred to as foldon domain). Experiments were carried out as in Example 4. Briefly, three rats were immunised with a plasmid encoding the SARS CoV-2 RBD-CD8a stalk transmembrane fusion or a plasmid encoding the soluble trimeric RBD, and sera were collected. These sera were used to stain SupTl cells expressing the RBD-CD8a stalk transmembrane fusion, SupTl cells expressing the spike protein-CD8a stalk transmembrane fusion or non-transduced SupTl cells (Figure 6).
Results shown in Figure 6B revealed that the titres of antibody obtained from both groups were comparable, indicating that membrane bound RBD elicited a profound immune response equivalent that of immunisation with the soluble trimer.
Example 6: Multi RBD constructs for the expression of SARS-CoV-2 variants.
The potential of membrane-bound fusion proteins to elicit an immune response against different variants of SARS-CoV-2 was investigated using multimeric RBD constructs. A mono-cistronic construct comprising three RBDs from different SARS-CoV-2 variants (Wuhan, alpha or UK, beta or SA) was generated and compared to a tri-cistronic construct of the same three variants (Figure 7A). Transient expression of these transmembrane fusions was tested in 293T cells. Staining with soluble ACE2-Fc, followed by fluorescence intensity measurements, indicated that expression of the (RBD)3-CD8STK fusion on the cell surface was lower than that of the full-length spike protein and (RBD-CD8STK)3 fusion protein (Figure 7B).
Next, the capacity of these constructs to seroconvert rats was tested as in Examples 4 and 5. Sera from rats immunised with the (RBD)3-CD8STK and (RBD-CD8STK)3 constructs (3 rats per cohort) were used to stain wild-type SupTl, SupTl -spike or SupTl-RBD expressing cells (Figure 7C). Rats immunised with the (RBD)3-CD8STK construct produced titres of antibody comparable to those immunised with the (RBD-CD8STK)3 construct, despite the results obtained from the transient transfection of 293T cells, showing that the cell surface expression of the (RBD)3-CD8STK fusion was lower than that of the (RBD-CD8STK)3 fusions. Overall, the results indicated that expression of each RBD variant on its own CD8a stalk elicited a similar response as to combining the RBDs on a single CD8a stalk.
Example 7: Neutralising capacity of sera from RBD immunised rats.
Sera from rats immunised with soluble RBD or membrane bound RBD (RBD-CD8STK, (RBD)3-CD8STK or (RBD-CD8STK)3 were used in neutralisation assays to prevent the infection of ACE2 and TMPRSS2-expressing 293T cells with SARS-CoV-2 spike pseudotyped lentivirus. The results shown in Figure 8 indicated that sera from rats immunised with the different RBD fusions all produced neutralising antibodies, capable of preventing infection by the spike pseuodtyped lentiviral vector. The neutralisation capacities of the (RBD)3-CD8STK sera were almost equivalent those of the soluble RBD sera, indicating that despite the lower levels of expression of the (RBD)3-CD8STK fusion it was still able to elicit a sterilising immune response.
Example 8: Screening of patients using SupTl-RBD-expressing cells
Sera from 16 convalescing SARS-CoV-2 patients were used to stain SupTl cells expressing either full-length SARS-CoV-2 spike or the RBD. As a control, the SupTl cell lines were stained with pre-SARS-CoV-2 serum to determine the background level of antibody binding (Figure 9A). The patient sera obtained had been previously screened for anti-spike antibodies using Fortress ELISA (see Table 2) and the titre of antibody determined.
Table 2. SARS-CoV-2 patient sera.
Figure imgf000061_0001
Figure imgf000062_0001
Staining of the SupTl -spike and SupTl-RBD cells with the sera showed that there was variation in the titres of anti-SARS-CoV-2 spike antibodies between the patients; however, in general those patients with high titre of anti-spike antibodies on the Fortress ELISA also exhibiting positive staining of SupTl -spike and SupTl -RBD cells (Figure 9B).
Testing of the patient sera in a neutralisation assay with SARS-CoV-2 spike pseudotyped lentivirus demonstrated that many of the patients had neutralising antibodies capable of preventing infection (Figure 10; Table 3).
Table 3. Patient sera IC50 values determined from neutralisation assay.
Figure imgf000062_0002
Figure imgf000063_0001
To determine if there was any correlation between the presence of anti-spike and anti-RBD antibodies and neutralisation capacity, MFIs from the staining experiments were plotted against IC50 values obtained from the neutralisation assays. This showed that there was a strong correlation between the presence of anti-RBD and neutralisation capacity (Spearman correlation coefficient r=0.85) and it was a better predictive value than the presence of antispike antibodies (Spearman correlation coefficient r=0.68) (Figure 11). Together these data indicate that the SupTl-RBD cells could be used to screen patient sera for the presence of anti-RBD antibodies and predict sera neutralising capacity, which would be beneficial for improving SARS-CoV-2 convalescing sera immunotherapy.
This application claims the benefit of United Kingdom application No. 2017649.1 filed 9th November 2020. This application is incorporated herein by reference in its entirety.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

64 CLAIMS
1. A polypeptide comprising an amino acid sequence of at least one receptor-binding domain (RBD) of a viral Spike protein, a spacer and a transmembrane domain, wherein the spacer is a spacer from a mammalian protein.
2. The polypeptide according to claim 1, wherein the viral Spike protein is a coronavirus Spike protein.
3. The polypeptide according to any of claims 1 or 2, wherein the Coronavirus is SARS- CoV-2.
4. The polypeptide according to claim 3, wherein the RBD comprises the amino acid sequence shown as SEQ ID NO: 4.
5. The polypeptide according to claim 3, which comprises the amino acid sequence shown as SEQ ID NO: 35.
6. The polypeptide according to any of claims 1 to 5, wherein the wherein the spacer is a CD8a stalk.
7. The polypeptide according to any of claims 1 to 6, wherein the transmembrane domain is a CD8a transmembrane domain.
8. The polypeptide according to any of claims 1 to 7, which further comprises an endodomain.
9. The polypeptide according to any of claims 1 to 7, which comprises two or more RBDs of a viral spike protein.
10. A nucleic acid encoding a polypeptide according to any of claims 1 to 9. 65 A nucleic acid construct encoding two or more polypeptides according to any of claims 1 to 9. A vector comprising a nucleic acid according to claim 10, or a nucleic acid construct according to claim 11. A cell comprising a polypeptide according to any of claims 1 to 9, or a nucleic acid according to claim 10, or a nucleic acid construct according to claim 11, or a vector according to claim 12. A method for making a cell according to claim 13, which comprises the step of transducing or transfecting a cell with a nucleic acid according to claim 10, or a nucleic acid construct according to claim 11, or a vector according to claim 12. A pharmaceutical composition comprising a polypeptide according to any of claims 1 to 9, or a nucleic acid according to claim 10, or a nucleic acid construct according to claim 11, or a vector according to claim 12, or a cell or plurality of cells according to claim 13, and at least one pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. A method for presenting a viral Spike protein-derived peptide, comprising expressing a polypeptide according to any of claims 1 to 9 in a cell. The method according to claim 16, wherein the viral Spike protein is a coronavirus Spike protein. A polypeptide according to any of claims 1 to 9, or a nucleic acid according to claim 10, or a nucleic acid construct according to claim 11, or a vector according to claim 12, or a cell according to claim 13, or a pharmaceutical composition according to claim 14 for use in preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus. 66 Use of a polypeptide according to any of claims 1 to 9, or a nucleic acid according to claim 10, or a nucleic acid construct according to claim 11, or a vector according to claim 12, or a cell according to claim 13, or a pharmaceutical composition according to claim 14 in the manufacturing of a medicament for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus. Method for preventing, inhibiting, treating, reducing, eliminating, protecting or delaying the onset of an infection or infectious clinical condition caused by a virus, in a subject in need thereof, comprising the step of administrating to the subject a therapeutically effective amount of a polypeptide according to any of claims 1 to 9, or a nucleic acid according to claim 10, or a nucleic acid construct according to claim 11, or a vector according to claim 12, or a cell according to claim 13, or a pharmaceutical composition according to claim 14. he polypeptide, nucleic acid, nucleic acid construct, vector, cell, or pharmaceutical composition according to claim 18, or the use according to claim 19, or the method according to claim 20, wherein the virus is a coronavirus. The polypeptide, nucleic acid, nucleic acid construct, vector, cell, or pharmaceutical composition according to claim 21, or the use according to claim 21, or the method according to claim 17 or claim 21, wherein the coronavirus is SARS-CoV-2.
PCT/GB2021/052893 2020-11-09 2021-11-09 Viral spike proteins and fusion thereof WO2022096899A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB2017649.1A GB202017649D0 (en) 2020-11-09 2020-11-09 Polypeptide
GB2017649.1 2020-11-09

Publications (1)

Publication Number Publication Date
WO2022096899A1 true WO2022096899A1 (en) 2022-05-12

Family

ID=74046320

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2021/052893 WO2022096899A1 (en) 2020-11-09 2021-11-09 Viral spike proteins and fusion thereof

Country Status (2)

Country Link
GB (1) GB202017649D0 (en)
WO (1) WO2022096899A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113755421A (en) * 2021-09-28 2021-12-07 萝芊细胞因子有限公司 Oral vaccine and antibody enhancer for COVID-19
WO2023019309A1 (en) * 2021-08-17 2023-02-23 Monash University Vaccine compositions
CN113755421B (en) * 2021-09-28 2024-04-12 梦芊细胞因子有限公司 Oral vaccine and antibody enhancer for COVID-19

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112608945B (en) * 2021-01-17 2022-09-27 昆明医科大学 Modified PPP1R12A circular RNA vector and application thereof

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007024708A2 (en) 2005-08-23 2007-03-01 The Trustees Of The University Of Pennsylvania Rna containing modified nucleosides and methods of use thereof
WO2009012489A1 (en) * 2007-07-19 2009-01-22 Novavax, Inc. Avian influenza chimeric vlps
WO2016151315A1 (en) 2015-03-23 2016-09-29 Ucl Business Plc Chimeric antigen receptor
WO2017144892A1 (en) * 2016-02-26 2017-08-31 Ucl Business Plc Nucleic acid constructs for producing retroviral vectors
WO2017176596A1 (en) * 2016-04-04 2017-10-12 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Multivalent vaccines for rabies virus and coronaviruses
WO2018211245A1 (en) * 2017-05-15 2018-11-22 Autolus Limited Cell
WO2021213924A1 (en) * 2020-04-22 2021-10-28 BioNTech SE Coronavirus vaccine

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007024708A2 (en) 2005-08-23 2007-03-01 The Trustees Of The University Of Pennsylvania Rna containing modified nucleosides and methods of use thereof
WO2009012489A1 (en) * 2007-07-19 2009-01-22 Novavax, Inc. Avian influenza chimeric vlps
WO2016151315A1 (en) 2015-03-23 2016-09-29 Ucl Business Plc Chimeric antigen receptor
WO2017144892A1 (en) * 2016-02-26 2017-08-31 Ucl Business Plc Nucleic acid constructs for producing retroviral vectors
WO2017176596A1 (en) * 2016-04-04 2017-10-12 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Multivalent vaccines for rabies virus and coronaviruses
WO2018211245A1 (en) * 2017-05-15 2018-11-22 Autolus Limited Cell
WO2021213924A1 (en) * 2020-04-22 2021-10-28 BioNTech SE Coronavirus vaccine

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
"Uniprot", Database accession no. PODTC2
AZAD TAHA ET AL: "Implications for SARS-CoV-2 Vaccine Design: Fusion of Spike Glycoprotein Transmembrane Domain to Receptor-Binding Domain Induces Trimerization", MEMBRANES, vol. 10, no. 9, 30 August 2020 (2020-08-30), pages 215, XP055881583, DOI: 10.3390/membranes10090215 *
COUGOT ET AL., TRENDS BIOCHEM SCI, vol. 29, 2001, pages 436 - 44
DONNELLY ET AL., J. GEN. VIROL., vol. 82, 2001, pages 1027 - 1041
ELANGO ET AL., BIOCHIM BIOPHYS RES COMMUN, vol. 330, 2005, pages 958 - 66
HODGSON JOHN: "The pandemic pipeline", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 38, no. 5, 20 March 2020 (2020-03-20), pages 523 - 532, XP037154993, ISSN: 1087-0156, [retrieved on 20200320], DOI: 10.1038/D41587-020-00005-Z *
KRAMMER FLORIAN: "SARS-CoV-2 vaccines in development", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 586, no. 7830, 23 September 2020 (2020-09-23), pages 516 - 527, XP037277122, ISSN: 0028-0836, [retrieved on 20200923], DOI: 10.1038/S41586-020-2798-3 *
LOES ANDREA N. ET AL: "Attenuated influenza virions expressing the SARS-CoV-2 receptor-binding domain induce neutralizing antibodies in mice", BIORXIV, 3 September 2020 (2020-09-03), XP055888741, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC7430565/pdf/nihpp-2020.08.12.248823.pdf> [retrieved on 20220208], DOI: 10.1101/2020.08.12.248823 *
OZHAROVSKAIA T. A. ET AL: "Immunogenicity of Different Forms of Middle East Respiratory Syndrome S Glycoprotein", ACTA NATURAE, vol. 11, no. 1, 1 March 2019 (2019-03-01), RU, pages 38 - 47, XP055858884, ISSN: 2075-8251, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475872/pdf/AN20758251-11-1-038.pdf> DOI: 10.32607/20758251-2019-11-1-38-47 *
STEPINSKI ET AL., RNA, vol. 7, 2001, pages 1468 - 95
TIAN JING-HUI ET AL: "SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 immunogenicity in baboons and protection in mice", NATURE COMMUNICATIONS, vol. 12, no. 1, 14 January 2021 (2021-01-14), pages 372, XP055844876, Retrieved from the Internet <URL:https://www.nature.com/articles/s41467-020-20653-8.pdf> DOI: 10.1038/s41467-020-20653-8 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023019309A1 (en) * 2021-08-17 2023-02-23 Monash University Vaccine compositions
CN113755421A (en) * 2021-09-28 2021-12-07 萝芊细胞因子有限公司 Oral vaccine and antibody enhancer for COVID-19
CN113755421B (en) * 2021-09-28 2024-04-12 梦芊细胞因子有限公司 Oral vaccine and antibody enhancer for COVID-19

Also Published As

Publication number Publication date
GB202017649D0 (en) 2020-12-23

Similar Documents

Publication Publication Date Title
CN111217918B (en) Novel coronavirus S protein double-region subunit nano vaccine based on 2, 4-dioxotetrahydropteridine synthase
CN111217919B (en) Novel coronavirus S protein double-region subunit nano vaccine based on pyrococcus ferritin
CN111560074B (en) Novel coronavirus S protein single-region subunit nano vaccine based on helicobacter pylori ferritin
CN111607002B (en) Novel coronavirus S protein double-region subunit nano vaccine based on helicobacter pylori ferritin
KR101983989B1 (en) Influenza virus vaccines and uses thereof
EP3166963B1 (en) Influenza virus vaccines and uses thereof
USRE47471E1 (en) Heat-stable respiratory syncytial virus F protein oligomers and their use in immunological compositions
US10517941B2 (en) Influenza virus vaccines and uses thereof
WO2021170131A1 (en) Soluble ace2 and fusion protein, and applications thereof
WO2022096899A1 (en) Viral spike proteins and fusion thereof
US20220098242A1 (en) Compositions and methods for mucosal vaccination against sars-cov-2
JP2010514439A (en) Lentivirus pseudotyped with influenza hemagglutinin and method of use thereof
WO2023138334A1 (en) Recombinant novel coronavirus protein vaccine, and preparation method and use thereof
US20230090422A1 (en) Novel coronavirus s protein double-region subunit nano-vaccine based on bacterial complex
WO2021253172A1 (en) Method for inducing anti-novel coronavirus neutralizing antibody using receptor recognition domain
WO2023138333A1 (en) Recombinant sars-cov-2 protein vaccine, and preparation method therefor and use thereof
WO2023020623A1 (en) Fusion protein and spike protein nanoparticle for preventing or treating coronavirus infections, and use thereof
CN115960180A (en) 2019-nCoV S protein mutant and genetically engineered mRNA and vaccine composition thereof
JP2018052953A (en) Influenza vaccines and uses thereof
CN113801206A (en) Method for inducing anti-neocoronavirus neutralizing antibody by using receptor recognition domain
WO2024061188A1 (en) Coronavirus multivalent vaccine and use thereof
WO2023168880A1 (en) Vaccine booster compositions for respiratory viral diseases
JP2023540778A (en) Coronavirus-derived receptor binding domain mutant with reduced ACE2 binding ability and vaccine composition containing the same
MC et al. international Bureau
NZ715583B2 (en) Influenza virus vaccines and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21814846

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 18/10/2023)