WO2023275538A1 - Vaccins à coronavirus bêta - Google Patents

Vaccins à coronavirus bêta Download PDF

Info

Publication number
WO2023275538A1
WO2023275538A1 PCT/GB2022/051663 GB2022051663W WO2023275538A1 WO 2023275538 A1 WO2023275538 A1 WO 2023275538A1 GB 2022051663 W GB2022051663 W GB 2022051663W WO 2023275538 A1 WO2023275538 A1 WO 2023275538A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
vector
acid sequence
polypeptide
Prior art date
Application number
PCT/GB2022/051663
Other languages
English (en)
Inventor
Sneha VISHWANATH
George CARNELL
Benedikt ASBACH
Ralf Wagner
Rebecca KINSLEY
Jonathan Luke Heeney
David Wells
Original Assignee
Diosynvax Ltd
The Chancellor, Masters And Scholars Of The University Of Cambridge
Universität Regensburg
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
Priority claimed from GBGB2109285.3A external-priority patent/GB202109285D0/en
Priority claimed from GBGB2112499.5A external-priority patent/GB202112499D0/en
Priority claimed from GBGB2114321.9A external-priority patent/GB202114321D0/en
Priority claimed from GBGB2201060.7A external-priority patent/GB202201060D0/en
Application filed by Diosynvax Ltd, The Chancellor, Masters And Scholars Of The University Of Cambridge, Universität Regensburg filed Critical Diosynvax Ltd
Publication of WO2023275538A1 publication Critical patent/WO2023275538A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]

Definitions

  • Coronaviruses cause a wide variety of animal and human disease. Notable human diseases caused by CoVs are zoonotic infections, such as severe acute respiratory syndrome (SARS) and Middle-East respiratory syndrome (MERS). Viruses within this family generally cause mild, self-limiting respiratory infections in immunocompetent humans, but can also cause severe, lethal disease characterised by onset of fever, extreme fatigue, breathing difficulties, anoxia, and pneumonia.
  • SARS severe acute respiratory syndrome
  • MERS Middle-East respiratory syndrome
  • CoVs transmit through close contact via respiratory droplets of infected subjects, with varying degrees of infectivity within each strain.
  • CoVs belong to the Coronaviridae family of viruses, all of which are enveloped.
  • CoVs contain a single-stranded positive-sense RNA genome, with a length of between 25 and 31 kilobases (Siddell S.G.1995, The Coronaviridae), the largest genome so far found in RNA viruses.
  • the Coronaviridae family are subtyped into four genera: ⁇ , ⁇ , ⁇ , and ⁇ coronaviruses (as shown in Figure 1A), based on phylogenetic clustering, with each genus subdivided again into different lineages.
  • Lineage A (subgenus Embecovirus) includes HCoV-OC43 and HCoV-HKU1 (various species) .
  • Lineage B (subgenus Sarbecovirus) includes SARSr-CoV (which includes all its strains such as SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1) .
  • Lineage C (subgenus Merbecovirus) includes Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4), Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), and MERS-CoV (various species) .
  • Lineage D (subgenus Nobecovirus) includes Rousettus bat coronavirus HKU9 (BtCoV- HKU9) A fifth subgenus is also officially recognised: Hibecovirus with one species of bat CoV. CoV virions are spherical with characteristic club-shape spike projections emanating from the surface of the virion.
  • the virions contain four main structural proteins: spike (S); membrane (M); envelope (E); and nucleocapsid (N) proteins, all of which are encoded by the viral genome.
  • Some subsets of ⁇ -CoVs also comprise a fifth structural protein, hemagglutinin- esterase (HE), which enhances S protein-mediated cell entry and viral spread through the mucosa via its acetyl-esterase activity.
  • HE hemagglutinin- esterase
  • homo-trimers of the S glycoprotein make up the distinctive spike structure on the surface of the virus. These trimers are a class I fusion protein, mediating virus attachment to the host receptor by interaction of the S protein and its receptor.
  • S is cleaved by host cell protease into two separate polypeptides – S1 and S2 (as shown in Figure 1D).
  • S1 contains the receptor-binding domain (RBD) of the S protein (the exact positioning of the RBD varies depending on the viral strain), while S2 forms the stem of the spike molecule.
  • RBD receptor-binding domain
  • the total length of SARS-CoV-2 S is 1273 amino acids and consists of a signal peptide (amino acids 1–13) located at the N-terminus, the S1 subunit (14–685 residues), and the S2 subunit (686–1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively.
  • S1 subunit there is an N-terminal domain (14–305 residues) and a receptor-binding domain (RBD, 319–541 residues); the fusion peptide (FP) (788–806 residues), heptapeptide repeat sequence 1 (HR1) (912–984 residues), HR2 (1163–1213 residues), TM domain (1213–1237 residues), and cytoplasm domain (1237–1273 residues) comprise the S2 subunit.
  • S protein trimers visually form a characteristic bulbous, crown-like halo surrounding the viral particle. Based on the structure of coronavirus S protein monomers, the S1 and S2 subunits form the bulbous head and stalk region.
  • FIG. 1(C) shows representative structure of the RBD for SARS-CoV-1 and SARS-CoV-2 (SARS-complex) and MERS.
  • SARS1 and SARS-CoV-2 SARS-complex
  • MERS MERS
  • RNA viruses generally have very high mutation rates compared to DNA viruses, because viral RNA polymerases lack the proofreading ability of DNA polymerases (although CoVs are an exception here as they possess a proofreading system: ExoN, nsp14). This is one reason why the virus can transmit from its natural host reservoir to other species, and from human to human, and is why it is difficult to make effective vaccines to prevent diseases caused by RNA viruses.
  • RNA viruses In most cases, current vaccine candidates against RNA viruses are limited by the viral strain used as the vaccine insert, which is often chosen based on availability of a wild-type strain rather than by informed design.
  • Technical challenges for developing vaccines for enveloped RNA viruses include: i) viral variation of wild-type field isolate glycoproteins (GPs) provide limited breadth of protection as vaccine antigens; ii) selection of vaccine antigens expressed by the vaccine inserts is highly empirical; immunogen selection is a slow, trial and error process; iii) in an evolving or unanticipated viral epidemic, developing new vaccine candidates is time-consuming and can delay vaccine deployment.
  • GPs wild-type field isolate glycoproteins
  • CoVs were only thought to cause mild respiratory problems, and were endemic in the human population, causing 15-30% of respiratory tract infections each year.
  • SARS-CoV SARS-CoV
  • SARS1 SARS-CoV
  • group 2b ⁇ -CoV Two novel virus isolates from bats show more similarity to the human SARS1 than any other virus identified to date, and bind to the same cellular receptor as human derived SARS-CoV – ACE2.
  • MERS is the causative agent of a series of highly pathogenic respiratory tract infections in the Middle East, with an initial mortality rate of 50%.
  • An estimate of 2,494 cases and 858 deaths caused by MERS has been reported since its emergence, with a total estimated fatality rate by the World Health Organisation (WHO) of 34.4%.
  • WHO World Health Organization
  • this novel CoV originated from bats, likely with an intermediate host such as dromedary camels contributing to the spread of the outbreak. This virus utilises DPP4 as its receptor, another peptidase receptor.
  • BNT162b2 BioNTech’s vaccine manufactured by Pfizer
  • mRNA-1273 manufactured by Moderna
  • Both of these vaccines demonstrated >94% efficacy at preventing coronavirus disease 2019 (COVID-19) in phase III clinical studies performed in late 2020 in multiple countries (Polack et al., C4591001 Clinical Trial Group (2020).
  • B.1.1.7 also known as VOC-202012/01 or 501Y.V1, or alpha
  • B.1.1.7 includes three amino acid deletions and seven missense mutations in spike, including D614G as well as N501Y in the ACE2 receptor- binding domain (RBD), and has been reported to be more infectious than D614G.
  • SARS-CoV-2 transmission between humans and minks in Denmark with a variant called mink cluster 5 or B.1.1.298 which includes a two-amino acid deletion and four missense mutations including Y453F in RBD.
  • B.1.429 Another variant that recently emerged in California, termed B.1.429, contains four missense mutations in spike, one of which is a single L452R RBD mutation.
  • B.1.1.298 and B.1.429 variants to evade neutralizing humoral immunity from prior infection or vaccination has yet to be determined.
  • B.1.351 lineage also known as Beta variant, or 501Y.V2
  • This lineage contains three RBD mutations, K417N, E484K, and N501Y, in addition to several mutations outside of RBD. More recently, two lineages have emerged which show increased transmissibility. The earlier of these is B.1.617.2 (Delta variant), first detected in India in December 2020, and which contains four mutations in the RBD: L452R, T478K, K417N, and E484K.
  • the second variant is of greatest concern is B.1.1.529 (Omicron variant), comprising 15 mutations on the RBD which have shown to cause significant humoral immune evasion and high transmissibility.
  • Figure 32 is a table providing details of the greatest VOCs in circulation globally as of 20 January 2022. The emergence of novel variants that appear to escape immune responses has spurred vaccine manufacturers to develop boosters for these spike variants. SARS1, SARS2, and MERS all originated in bats, and through an intermediary, gained the ability to infect humans (Latinne et al., NATURE COMMUNICATIONS (2020) 11:4235 https://doi.org/10.1038/s41467-020-17687-3: Origin and cross-species transmission of bat coronaviruses in China).
  • CoVs The propensity of CoVs to recombine is the main driver in allowing the zoonotic viruses to become transmissible in humans.
  • Viral recombination occurs when viruses of two different strains co-infect the same host cell and interact during replication to generate chimeric virus progeny. Large amounts of genetic information can be shared in one event, which may result in completely novel viral variants emerging.
  • Such vaccines would provide an effective strategy to tackle the current epidemic/pandemic viruses, as well as future spill-over from related zoonotic viruses.
  • Such broadly effective vaccines are provided by use of phylogenetically-, and epitope-optimised sarbecovirus and merbecovirus RBD subunit-based vaccines.
  • Such vaccines may be provided, for example, as nucleic acid vaccines, either as separate polynucleotides, each encoding a different RBD subunit (or RBD dimer) (for administration together or separately) or pieced together in a string as a single polynucleotide encoding all of the RBD subunits (or RBD dimers).
  • the separate polynucleotides may be administered as a mixture together (for example, as a pharmaceutical composition comprising the separate polynucleotides), or co-administered or administered sequentially in any order (in which case, the separate polynucleotides may be provided as a combined preparation for co-administration or sequential administration).
  • Nucleic acid vaccines may be provided as DNA, RNA, or mRNA vaccines. Production and application of multicistronic constructs (for example, where the subunits are provided in a string as a single polynucleotide) is reviewed by Shaimardanova et al. (Pharmaceutics 2019, 11, 580; doi:10.3390/pharmaceutics11110580).
  • polynucleotides RBDs of String 1: According to the invention there is provided an isolated polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), MERS_RBD_M7 amino acid sequence (SEQ ID NO:15), and SARS1_RBD_M7 amino acid sequence (SEQ ID NO:2), or the complement thereof: >SARS2_RBD_M7 (SEQ ID NO:9) Amino acid sequence: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTK LNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYR LFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHANA TVCGPKKSTN >MERS
  • Multicistronic vectors based on IRES nucleotide sequence and self-cleaving 2A peptides are reviewed in Shaimardanova et al. (Pharmaceutics 2019, 11, 580; doi:10.3390/pharmaceutics11110580).
  • 2A self-cleaving peptides are 18–22 amino-acid-long viral oligopeptides that mediate “cleavage” of polypeptides during translation in eukaryotic cells (Liu et al., Scientific Reports 7, Article number: 2193 (2017)).
  • the designation “2A” refers to a specific region of the viral genome and different viral 2As have generally been named after the virus they were derived from. The first discovered 2A was F2A (foot-and-mouth disease virus), after which E2A (equine rhinitis A virus), P2A (porcine teschovirus-12A), and T2A (thosea asigna virus 2A) were also identified.
  • the mechanism of 2A-mediated “self-cleavage” is ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A.
  • a highly conserved sequence GDVEXNPGP is shared by different 2As at the C-terminus, and is essential for the creation of steric hindrance and ribosome skipping.
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO:26, or the complement thereof: >String 1 (SEQ ID NO:26) Nucleic acid sequence: ATGGATGCTATGAAGAGGGGCCTGTGCTGCGTGCTGCTTCTGTGTGGCGCTGTGTTTGTGTCTCCTAG CGCCGCTAGAGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCCAACATCACCAATCTGTGCCCTTTCG GCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGC GTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCC TACCAAGCTGAACGACCTGCTTCACCAACGTGTA
  • a combined preparation which comprises: i) an isolated polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), or the complement thereof; ii) an isolated polynucleotide which comprises nucleotide sequence encoding MERS_RBD_M7 amino acid sequence (SEQ ID NO:15), or the complement thereof; and iii) an isolated polynucleotide which comprises nucleotide sequence encoding SARS1_RBD_M7 amino acid sequence (SEQ ID NO:2), or the complement thereof.
  • nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding MERS_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:16, or the complement thereof.
  • nucleotide sequence encoding SARS1_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:3, or the complement thereof.
  • SARS1_RBD_M7 (SEQ ID NO:2), SARS2_RBD_M7 (SEQ ID NO:9), and MERS_RBD_M7 (SEQ ID NO:15) amino acid sequences shown above are designed RBD amino acid sequences in which a glycosylation site has been introduced towards the C-terminal end of the RBD, compared with the corresponding wild-type RBD sequence.
  • SARS1_RBD_M7 design includes a glycosylation site introduced by an amino acid change at residue position 202.
  • SARS2_RBD_M7 design includes a glycosylation site introduced by an amino acid change at residue position 203.
  • the MERS_RBD_M7 design includes a glycosylation site introduced by an amino acid change at residue position 213.
  • Alignment of SARS1_RBD_M7 amino acid sequence with SARS1_RBD amino acid sequence is shown in Figure 7.
  • Alignment of SARS2_RBD_M7 amino acid sequence with SARS2_RBD amino acid sequence is shown in Figure 8.
  • Alignment of MERS_RBD_M7 amino acid sequence with MERS_RBD amino acid sequence is shown in Figure 9.
  • Masking/de-masking of epitopes has been shown to alter the immune response by masking non- neutralising epitopes, or by de-masking important epitopes in MERS (Du L et. al., Nat. Comm, volume 7, Article number: 13473 (2016)).
  • SARS2_RBD_M7 and wild-type SARS2 RBD DNA are superior to non- glycosylated, or sparsely glycosylated recombinant SARS2 RBD protein in inducing neutralising responses to SARS2.
  • Example 2 describes Mass spectrometry data obtained to study glycosylation of SARS-CoV-2 (SARS2) RBD proteins in supernatants derived from HEK cells transfected with pEVAC plasmid encoding SARS-CoV-2 RBD sequences, compared with recombinant SARS-CoV-2 RBD proteins (see Figures 3 and 4). It was concluded from the results that there are two main glycosylated forms of the proteins obtained from the supernatants, in comparison to purified (recombinant) protein. The purified protein is non-glycosylated or sparsely glycosylated.
  • glycosylation sites surround the epitope region and are conserved in most sarbecoviruses. These glycosylation sites are also important for interaction with some of the antibodies.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:3, or the complement thereof.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:9, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:10, or the complement thereof.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:15.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:15, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:16, or the complement thereof.
  • RBDs of String 2 According to the invention there is also provided an isolated polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11) and Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or the complement thereof: >T2_17 (SEQ ID NO:11) Amino acid sequence: RVAPTKEVVR FPNITNLCPF GEVFNATKFP SVYAWERKKI SNCVADYSVL YNSTSFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGVI ADYNYKLPDD FTGCVIAWNT NNIDSTTGGN YNYLYRSLRK SKLKPFERDI SSDIYSPGGK PCSGVEGFNC YYPLRSYGFF PTNGTGYQPY
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO:28, or the complement thereof: >String 2 (SEQ ID NO:28) Nucleic acid sequence: ATGGATGCTATGAAGAGGGGCCTGTGCTGCGTGCTGCTTCTGTGTGGCGCTGTGTTTGTGTCTCCTAG CGCCGCTAGAGTGGCCCCTACCAAAGAAGTCGTGCGGTTCCCCAACATCACCAATCTGTGCCCTTTCG GCGAGGTGTTCAACGCCACCAAGTTTCCCTCTGTGTACGCCTGGGAGCGCAAAAAGATCAGCAACTGC GTGGCCGACTACAGCGTGCTGTACAACAGCACCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCACC CACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAG TGCGGCAGATTGCCCCTGGACAAACAGGCGTGATCGCCGATTACAACTACAAGCTGCCCGACGACTTC ACCGGCTGTGTGATCGT
  • a combined preparation which comprises: i) an isolated polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11), or the complement thereof; and ii) an isolated polynucleotide which comprises nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or the complement thereof.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • Sequence T2_17 (SEQ ID NO:11) amino acid sequence shown above is a phylogenetically optimised design generated using the sequences represented in Figure 2A.
  • Figure 2A shows a phylogenetic tree of sarbeco lineage of beta-coronavirus. The tree was built using the RBD subunit of spike proteins. Two phylogenetically optimised vaccine designs are represented by black filled circles.
  • the vaccine design labelled as 1 in Figure 2A was further optimised for cross-reactive B-cell epitopes to generate adequate cross-protection against viruses belonging to SARS-complex lineage, and is referred to herein as T2_17.
  • Ancestor_MERS_M7 (SEQ ID NO:17) amino acid sequence is a phylogenetically optimised design generated using the sequences represented in Figure 2B.
  • Figure 2B shows a phylogenetic tree of merbeco lineage of beta-coronavirus. The tree was built using the RBD subunit of spike proteins.
  • the phylogenetically optimised vaccine design is represented by a black filled circle.
  • the vaccine design was generated for MERS and related DPP4 using bat viruses (boxed in black in Figure 2B).
  • Ancestor_MERS_M7 This sequence was further modified to include a glycosylation site at one of the epitopes, and is referred to herein as Ancestor_MERS_M7.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:11.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:11, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:12, or the complement thereof.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:17 There is also provided according to the invention an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:17.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:17, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:18, or the complement thereof.
  • Variants of RBDs of String 2 Variants of T2_17: The amino acid sequence of a SARS2 S protein RBD reference sequence (EPI_ISL_402119_RBD) is shown below.
  • an isolated polynucleotide which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof.
  • an isolated polynucleotide which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof.
  • Variants of Ancestor_MERS_M7 The amino acid differences of the Ancestor_MERS_M7 sequence (SEQ ID NO:17) from the MERS RBD reference sequence (SEQ ID NO:13) are shown in Table 2 below: Table 2
  • an isolated polynucleotide which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, or the complement thereof.
  • an isolated polynucleotide which comprises: nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • an isolated polynucleotide which comprises: nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%
  • a pharmaceutical composition which comprises: a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9
  • a pharmaceutical composition which comprises: a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
  • a combined preparation which comprises: a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9
  • a combined preparation which comprises: a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises one or more of the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • RBDs of String 3 According to the invention there is provided an isolated polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), and Ancestor_SARS1_M7 amino acid sequence (SEQ ID NO:4), or the complement thereof: >SARS2_RBD_M7 (SEQ ID NO:9) Amino acid sequence: RVQPTESIVR FPNITNLCPF GEVFNATRFA SVYAWNRKRI SNCVADYSVL YNSASFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGKI ADYNYKLPDD FTGCVIAWNS NN
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO:30, or the complement thereof: >String 3 (SEQ ID NO:30) Nucleic acid sequence: ATGGATGCTATGAAGAGGGGCCTGTGCTGCGTGCTGCTTCTGTGTGGCGCTGTGTTTGTGTCTCCTAG CGCCGCTAGAGTGCAGCCCACAGAGTCTATCGTGCGGTTCCCCAACATCACCAATCTGTGCCCTTTCG GCGAGGTGTTCAACGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGC GTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCC TACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCAGAGGCGACGAAG TGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCCGATTACAACTACAAGCTGCCCGACGACTTC ACCGGCTGTGTGATT
  • a combined preparation which comprises: i) an isolated polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), or the complement thereof; ii) an isolated polynucleotide which comprises nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or the complement thereof; and iii) an isolated polynucleotide which comprises nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence (SEQ ID NO:4), or the complement thereof.
  • nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:5, or the complement thereof.
  • Ancestor_SARS1_M7 (SEQ ID NO:4) amino acid sequence shown above is another phylogenetically optimised design generated using the sequences represented in Figure 2A.
  • Figure 2A shows a phylogenetic tree of sarbeco lineage of beta-coronavirus. The tree was built using the RBD subunit of spike proteins. Two phylogenetically optimised vaccine designs are represented by black filled circles. The vaccine design labelled as 2 in Figure 2A was optimised to generate cross-protection to SARS-1 and SARS-1 related bat viruses. This sequence was further modified to include a glycosylation site at one of the epitopes, and is referred to herein as Ancestor_SARS1_M7 (SEQ ID NO:4).
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:4.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:4, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:5, or the complement thereof.
  • Variants of Ancestor_SARS1_M7 Alignment of the amino acid sequence of Ancestor_SARS1_M7 (also referred to herein as SARS1_Anc_M7) with SARS1_RBD reference sequence (SEQ ID NO:1) is shown in Figure 7.
  • an isolated polynucleotide which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, or the complement thereof.
  • an isolated polynucleotide which comprises: nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:9 (SARS2_RBD_M7), or the complement thereof; nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, or the complement thereof; and nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7)
  • a pharmaceutical composition which comprises: a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:9 (SARS2_RBD_M7), or the complement thereof; a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, or the complement thereof; and a polynucleotide comprising a nucleotide comprising a nucleo
  • a combined preparation which comprises: a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:9 (SARS2_RBD_M7), or the complement thereof; a polynucleotide comprising a nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, or the complement thereof; and a polynucleotide comprising a nucleotide comprising a nucleo
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises one or more of the amino acid residues of SEQ ID NO:4, at positions corresponding to the amino acid residue positions of SEQ ID NO:1, as shown in Table 2.1.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises the amino acid residues of SEQ ID NO:4, at positions corresponding to the amino acid residue positions of SEQ ID NO:1, as shown in Table 2.1.
  • the nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:5, or the complement thereof.
  • an isolated polynucleotide which comprises nucleotide sequence encoding SARS_complex dimer amino acid sequence (SEQ ID NO:19), and MERS_complex dimer amino acid sequence (SEQ ID NO:21), or the complement thereof: >SARS_complex_dimer (SEQ ID NO:19)
  • Amino acid sequence RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATK LNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYR YLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPAT VCGPKLSTDLIKNQRVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLY
  • the polynucleotide comprises the nucleotide sequence of SEQ ID NO:32, or the complement thereof: >String 4 (SEQ ID NO:32) Nucleic acid sequence: ATGGATGCTATGAAGAGGGGCCTGTGCTGCGTGCTGCTTCTGTGTGGCGCTGTGTTTGTGTCTCCTAG CGCCGCTAGAGTGGTGCCTAGCGGAGATGTTGTGCGGTTCCCCAACATCACCAATCTGTGCCCTTTCG GCGAGGTGTTCAACGCCACCAAGTTTCCCTCTGTGTACGCCTGGGAGCGCAAAAAGATCAGCAACTGC GTGGCCGACTACAGCGTGCTGTACAACAGCACCTTCTTCAGCACCTTTAAGTGCTACGGCGTGTCCGC CACAAAGCTGAACGACCTGTGCTTCAGCAACGTGTACGCCGACAGCTTCGTGGTCAAGGGCGACGATG TTCGGCAGATTGCCCCTGGACAGACAGGCGTGATCGCCGATTACAACTACAAGCTGCCCGACGACTTC ATGGGCTGTGTGTG
  • a combined preparation which comprises: i) an isolated polynucleotide which comprises nucleotide sequence encoding SARS_complex dimer amino acid sequence (SEQ ID NO:19), or the complement thereof; and ii) an isolated polynucleotide which comprises nucleotide sequence encoding MERS_complex dimer amino acid sequence (SEQ ID NO:21), or the complement thereof.
  • the nucleotide sequence encoding SARS_complex dimer amino acid sequence comprises the nucleotide sequence of SEQ ID NO:20, or the complement thereof.
  • the nucleotide sequence encoding MERS_complex dimer amino acid sequence comprises the nucleotide sequence of SEQ ID NO:22, or the complement thereof.
  • the SARS_complex dimer (SEQ ID NO:19) and MERS_complex dimer (SEQ ID NO:21) amino acid sequences shown above are designed chimeric dimer sequences. Dimeric form of RBD has been reported to be more immunogenic than monomeric RBD (Dai, et al., Cell Volume 182, Issue 3, 6 August 2020, Pages 722-733.e11: A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS).
  • SARS_complex dimer SEQ ID NO:19
  • MERS_complex dimer SEQ ID NO:21
  • isolated polypeptide comprising the amino acid sequence of SEQ ID NO:19.
  • isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:19, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:20, or the complement thereof.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:21.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:21, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:22, or the complement thereof.
  • String 5 combines coronavirus S protein RBD design T2_17 (amino acid sequence SEQ ID NO: 11) with coronavirus N protein design COV_N_T2_2 (amino acid sequence SEQ ID NO:45).
  • an isolated polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11), and COV_N_T2_2 amino acid sequence (SEQ ID NO:45), or the complement thereof.
  • the T2_17 and COV_N_T2_2 subunits may be in any order in the String 5 sequence.
  • String 5 amino acid sequence may also include a signal sequence (for example, MDAMKRGLCCVLLLCGAVFVSPSAA; SEQ ID NO:38), upstream of the T2_17 and COV_N_T2_2 subunits, and a 2A self-cleaving peptide sequence between the T2_17 and COV_N_T2_2 subunits (for example, (GSGEGRGSLLTCGDVEENPGP; SEQ ID NO:39 or GSGATNFSLLKQAGDVEENPGP; SEQ ID NO:40).
  • GSGEGRGSLLTCGDVEENPGP SEQ ID NO:39 or GSGATNFSLLKQAGDVEENPGP; SEQ ID NO:40.
  • composition which comprises: i) an isolated polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO: 11), or the complement thereof; and ii) an isolated polynucleotide which comprises nucleotide sequence encoding COV_N_T2_2 amino acid sequence (SEQ ID NO:45), or the complement thereof.
  • a combined preparation which comprises: i) an isolated polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO: 11), or the complement thereof; and ii) an isolated polynucleotide which comprises nucleotide sequence encoding COV_N_T2_2 amino acid sequence (SEQ ID NO:45), or the complement thereof.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • nucleotide sequence encoding COV_N_T2_2 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:46, or the complement thereof.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:45.
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:45, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:46, or the complement thereof.
  • an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:43, or the complement thereof.
  • Variants of Subunit Sequences of String 5 Variants of COV_N_T2_2 The amino acid sequence of a SARS2 N protein reference sequence (EPI_ISL_402119_RBD) is shown below.
  • nucleocapsid phosphoprotein [SARS-CoV-2] (reference sequence) (SEQ ID NO:42) MSDNGPQ-NQ RNAPRITFGG PSDSTGSNQN GERSGARSKQ RRPQGLPNNT ASWFTALTQH GKEDLKFPRG QGVPINTNSS PDDQIGYYRR ATRRIRGGDG KMKDLSPRWY FYYLGTGPEA GLPYGANKDG IIWVATEGAL NTPKDHIGTR NPANNAAIVL QLPQGTTLPK GFYAEGSRGG SQASSRSSSR SRNSSRNSTP GSSRGTSPAR MAGNGGDAAL ALLLLDRLNQ LESKMSGKGQ QQQGQTVTKK SAAEASKKPR QKRTATKAYN VTQAFGRRGP EQTQGNFGDQ ELIRQGTDYK HWPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYTGAIKL
  • Positions 415 and 416 of the SARS2 N protein reference residue position column are italicised as they are not residues of the reference sequences, but include insertions in the N_T2_1 and N_T2_2 sequences.
  • the amino acid changes common to both of the designed sequences are summarised in the table below: Table 3.2
  • Optional additional changes are summarised in the table below: Table 3.3
  • an isolated polynucleotide which comprises nucleotide sequence encoding an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, or the complement thereof.
  • Variants of COV_S_T2_17 Variants of COV_S_T2_17 are as described above for variants of RBDs of String 2.
  • an isolated polynucleotide which comprises: nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, or the complement thereof.
  • an isolated polynucleotide which comprises: nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, or the complement thereof.
  • a pharmaceutical composition which comprises: a polynucleotide comprising nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and a polynucleotide comprising nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, or the complement thereof.
  • a pharmaceutical composition which comprises: a polynucleotide comprising nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and a polynucleotide comprising nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, or the complement thereof.
  • a combined preparation which comprises: a polynucleotide comprising nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and a polynucleotide comprising nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, or the complement thereof.
  • a combined preparation which comprises: a polynucleotide comprising nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and a polynucleotide comprising nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, or the complement thereof.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises one or more of the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • a polypeptide encoded by a polynucleotide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.2 above.
  • the nucleotide sequence encoding N_T2_2 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:46, or the complement thereof.
  • an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45.
  • a polypeptide of the invention comprising an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:45, or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, further comprises at least one, or all of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.2 above.
  • a polypeptide of the invention comprising an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:45, or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, further comprises at least one, or all of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.4 above.
  • an isolated polynucleotide which encodes a polypeptide of the invention.
  • a polynucleotide of the invention, each polynucleotide of a pharmaceutical composition of the invention, or each polynucleotide of a combined preparation of the invention may be provided as part of a vector. Accordingly, there is also provided according to the invention a vector comprising a polynucleotide of the invention.
  • a vector of the invention further comprises a promoter operably linked to the encoding nucleotide sequence of the polynucleotide.
  • a vector of the invention further comprises, for each separate encoding nucleotide sequence of the vector, a separate promoter operably linked to that encoding nucleotide sequence.
  • each promoter may have the same sequence, or a different sequence.
  • a vector comprising a polynucleotide of the invention comprises the nucleotide sequence of SEQ ID NO:26, or the complement thereof.
  • a vector comprising a polynucleotide of the invention comprises the nucleotide sequence of SEQ ID NO:28, or the complement thereof.
  • a vector comprising a polynucleotide of the invention comprises the nucleotide sequence of SEQ ID NO:30, or the complement thereof.
  • a vector comprising a polynucleotide of the invention comprises the nucleotide sequence of SEQ ID NO:32, or the complement thereof.
  • a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), or the complement thereof; ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding MERS_RBD_M7 amino acid sequence (SEQ ID NO:15), or the complement thereof; and iii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS1_RBD_M7 amino acid sequence (SEQ ID NO:2), or the complement thereof.
  • nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding MERS_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:16, or the complement thereof.
  • nucleotide sequence encoding SARS1_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:3, or the complement thereof.
  • a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11), or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or the complement thereof.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence comprises a vector of amino acid SEQ ID NO:41.
  • the nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 8
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises one or more of the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence comprises a vector of amino acid SEQ ID NO:41.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • RBDs of String 3 According to the invention there is provided a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), or the complement thereof; ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or the complement thereof; and iii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence (SEQ ID NO:4), or the complement thereof.
  • nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:5, or the complement thereof.
  • a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), or the complement thereof; ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, or the complement thereof; and iii) a vector comprising a polyn
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises one or more of the amino acid residues of SEQ ID NO:4, at positions corresponding to the amino acid residue positions of SEQ ID NO:1, as shown in Table 2.1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises the amino acid residues of SEQ ID NO:4, at positions corresponding to the amino acid residue positions of SEQ ID NO:1, as shown in Table 2.1.
  • the nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9) comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:5, or the complement thereof.
  • a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS_complex dimer amino acid sequence (SEQ ID NO:19), or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding MERS_complex dimer amino acid sequence (SEQ ID NO:21), or the complement thereof.
  • the nucleotide sequence encoding SARS_complex dimer amino acid sequence comprises the nucleotide sequence of SEQ ID NO:20, or the complement thereof.
  • nucleotide sequence encoding MERS_complex dimer amino acid sequence comprises the nucleotide sequence of SEQ ID NO:22, or the complement thereof.
  • RBDs of String 5 According to the invention there is provided a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO: 11), or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding COV_N_T2_2 amino acid sequence (SEQ ID NO:45), or the complement thereof.
  • nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence comprises a vector of nucleic acid SEQ ID NO:41.
  • nucleotide sequence encoding COV_N_T2_2 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:46, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding N_T2_2 amino acid sequence comprises a vector of nucleic acid SEQ ID NO:48.
  • a vector which comprises nucleotide sequence encoding N_T2_1 amino acid sequence comprises a vector of nucleic acid SEQ ID NO:47.
  • a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a pharmaceutical composition which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11 (COV_
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises one or more of the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence comprises a vector of amino acid SEQ ID NO:41.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.2 above.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.4 above.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.2 above.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.4 above.
  • the nucleotide sequence encoding N_T2_2 amino acid sequence (SEQ ID NO:45) comprises the nucleotide sequence of SEQ ID NO:46, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding N_T2_2 amino acid sequence comprises a vector of amino acid SEQ ID NO:48.
  • each vector of a pharmaceutical composition of the invention comprises a promoter operably linked to the encoding nucleotide sequence.
  • RBDs of String1 There is also provided according to the invention a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), or the complement thereof; ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding MERS_RBD_M7 amino acid sequence (SEQ ID NO:15), or the complement thereof; and iii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS1_RBD_M7 amino acid sequence (SEQ ID NO:2), or the complement thereof.
  • nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding MERS_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:16, or the complement thereof.
  • nucleotide sequence encoding SARS1_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:3, or the complement thereof.
  • RBDs of String 2 There is also provided according to the invention a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11), or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or the complement thereof.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence comprises a vector of amino acid SEQ ID NO:41.
  • the nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 8
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 8
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises one or more of the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence comprises a vector of amino acid SEQ ID NO:41.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • RBDs of String 3 There is also provided according to the invention a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), or the complement thereof; ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or the complement thereof; and iii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence (SEQ ID NO:4), or the complement thereof.
  • nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:5, or the complement thereof.
  • a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), or the complement thereof; ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, or the complement thereof; and iii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises one or more of the amino acid residues of SEQ ID NO:4, at positions corresponding to the amino acid residue positions of SEQ ID NO:1, as shown in Table 2.1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises the amino acid residues of SEQ ID NO:4, at positions corresponding to the amino acid residue positions of SEQ ID NO:1, as shown in Table 2.1.
  • the nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9) comprises the nucleotide sequence of SEQ ID NO:10, or the complement thereof.
  • nucleotide sequence encoding Ancestor_MERS_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:18, or the complement thereof.
  • nucleotide sequence encoding Ancestor_SARS1_M7 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:5, or the complement thereof.
  • RBDs of String 4 There is also provided according to the invention a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS_complex dimer amino acid sequence (SEQ ID NO:19), or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding MERS_complex dimer amino acid sequence (SEQ ID NO:21), or the complement thereof.
  • the nucleotide sequence encoding SARS_complex dimer amino acid sequence comprises the nucleotide sequence of SEQ ID NO:20, or the complement thereof.
  • nucleotide sequence encoding MERS_complex dimer amino acid sequence comprises the nucleotide sequence of SEQ ID NO:22, or the complement thereof.
  • RBDs of String 5 There is also provided according to the invention a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11), or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence encoding COV_N_T2_2 amino acid sequence (SEQ ID NO:45), or the complement thereof.
  • nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence comprises a vector of amino acid SEQ ID NO:41.
  • nucleotide sequence encoding COV_N_T2_2 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:46, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding N_T2_2 amino acid sequence comprises a vector of amino acid SEQ ID NO:48.
  • Variants of RBDs of String 5 comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a combined preparation which comprises: i) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, or the complement thereof; and ii) a vector comprising a polynucleotide which comprises nucleotide sequence which encodes a polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises one or more of the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • the nucleotide sequence encoding T2_17 amino acid sequence comprises the nucleotide sequence of SEQ ID NO:12, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence comprises a vector of amino acid SEQ ID NO:41.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.2 above.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.4 above.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.2 above.
  • the encoded polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.4 above.
  • the nucleotide sequence encoding N_T2_2 amino acid sequence (SEQ ID NO:45) comprises the nucleotide sequence of SEQ ID NO:46, or the complement thereof.
  • the vector comprising a polynucleotide which comprises nucleotide sequence encoding N_T2_2 amino acid sequence comprises a vector of amino acid SEQ ID NO:48.
  • each vector of a combined preparation of the invention comprises a promoter operably linked to the encoding nucleotide sequence.
  • the, or each promoter (of a vector of the invention, or of each vector of a pharmaceutical composition or a combined preparation of the invention) is for expression of a polypeptide encoded by the polynucleotide in mammalian cells.
  • each promoter (of a vector of the invention, or of each vector of a pharmaceutical composition or a combined preparation of the invention) is for expression of a polypeptide encoded by the polynucleotide in yeast or insect cells.
  • the, or each vector is a vaccine vector.
  • the, or each vaccine vector is a viral vaccine vector, a bacterial vaccine vector, or a nucleic acid vaccine vector, such as an RNA vaccine vector, an mRNA vaccine vector, or a DNA vaccine vector.
  • a polynucleotide of the invention may comprise a DNA or an RNA molecule.
  • RNA molecule for example, an mRNA
  • nucleic acid sequence of the polynucleotide will be the same as that recited in the respective SEQ ID, or the complement thereof, but with each ‘T’ nucleotide replaced by ‘U’.
  • a polynucleotide of the invention may include one or more modified nucleosides.
  • a polynucleotide of the invention may include one or more nucleotide analogs known to those of skill in the art.
  • Viral vaccine vectors use live viruses to deliver nucleic acid (for example, DNA or RNA) into human or non-human animal cells.
  • the nucleic acid contained in the virus encodes one or more antigens that, once expressed in the infected human or non-human animal cells, elicit an immune response. Both humoral and cell-mediated immune responses can be induced by viral vaccine vectors.
  • Viral vaccine vectors combine many of the positive qualities of nucleic acid vaccines with those of live attenuated vaccines. Like nucleic acid vaccines, viral vaccine vectors carry nucleic acid into a host cell for production of antigenic proteins that can be tailored to stimulate a range of immune responses, including antibody, T helper cell (CD4+ T cell), and cytotoxic T lymphocyte (CTL, CD8+ T cell) mediated immunity.
  • Viral vaccine vectors unlike nucleic acid vaccines, also have the potential to actively invade host cells and replicate, much like a live attenuated vaccine, further activating the immune system like an adjuvant.
  • a viral vaccine vector therefore generally comprises a live attenuated virus that is genetically engineered to carry nucleic acid (for example, DNA or RNA) encoding protein antigens from an unrelated organism.
  • nucleic acid for example, DNA or RNA
  • viral vaccine vectors are generally able to produce stronger immune responses than nucleic acid vaccines, for some diseases viral vectors are used in combination with other vaccine technologies in a strategy called heterologous prime-boost. In this system, one vaccine is given as a priming step, followed by vaccination using an alternative vaccine as a booster.
  • the heterologous prime-boost strategy aims to provide a stronger overall immune response.
  • Viral vaccine vectors may be used as both prime and boost vaccines as part of this strategy.
  • Viral vaccine vectors are reviewed by Ura et al., 2014 (Vaccines 2014, 2, 624-641) and Choi and Chang, 2013 (Clinical and Experimental Vaccine Research 2013;2:97-105).
  • the viral vaccine vector is based on a viral delivery vector, such as a Poxvirus (for example, Modified Vaccinia Ankara (MVA), NYVAC, AVIPOX), herpesvirus (e.g. HSV, CMV, Adenovirus of any host species), Morbillivirus (e.g. measles), Alphavirus (e.g.
  • a viral delivery vector such as a Poxvirus (for example, Modified Vaccinia Ankara (MVA), NYVAC, AVIPOX), herpesvirus (e.g. HSV, CMV, Adenovirus
  • Adenoviruses are by far the most utilised and advanced viral vectors developed for SARS2 vaccines. They are non-enveloped double-stranded DNA (dsDNA) viruses with a packaging capacity of up to 7.5 kb of foreign genes. Almost all SARS2 adenovirus based vaccines have been engineered for the expression of the SARS2 S protein or the RBD subunit.
  • dsDNA non-enveloped double-stranded DNA
  • Adenovirus vectors are widely used because of their high transduction efficiency, high level of transgene expression, and broad range of viral tropism. These vaccines are highly cell specific, highly efficient in gene transduction, and efficient at inducing an immune response. Adenovirus vaccines are effective at triggering and priming T cells, leading to long term and high level of antigenic protein expression and therefore long lasting protection.
  • AZD1222 manufactured by AstraZeneca
  • vaccine construct comprises a recombinant adenoviral vector vaccine encoding the SARS2 S protein.
  • the recombinant adenovirus genome comprises SARS2 S gene at the E1 locus.
  • each designed RBD subunit i.e. each RBD subunit of String 1, 2, 3, or 4
  • each designed RBD subunit is encoded as part of the same viral vaccine vector.
  • a viral vaccine vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), MERS_RBD_M7 amino acid sequence (SEQ ID NO:15), and SARS1_RBD_M7 amino acid sequence (SEQ ID NO:2), or the complement thereof.
  • the viral vaccine vector comprises the nucleotide sequence of SEQ ID NO:26, or the complement thereof.
  • a viral vaccine vector comprising a polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11) and Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), or the complement thereof.
  • the viral vaccine vector comprises the nucleotide sequence of SEQ ID NO:28, or the complement thereof.
  • a viral vaccine vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), and Ancestor_SARS1_M7 amino acid sequence (SEQ ID NO:4), or the complement thereof.
  • the viral vaccine vector comprises the nucleotide sequence of SEQ ID NO:30, or the complement thereof.
  • a viral vaccine vector comprising a polynucleotide which comprises nucleotide sequence encoding SARS_complex dimer amino acid sequence (SEQ ID NO:19) and MERS_complex dimer amino acid sequence (SEQ ID NO:21), or the complement thereof.
  • the viral vaccine vector comprises the nucleotide sequence of SEQ ID NO:32, or the complement thereof.
  • the nucleic acid expression vector is a nucleic acid expression vector, and a viral pseudotype vector.
  • the nucleic acid expression vector is a vaccine vector.
  • the nucleic acid expression vector comprises, from a 5’ to 3’ direction: a promoter; a splice donor site (SD); a splice acceptor site (SA); and a terminator signal, wherein a multiple cloning site is located between the splice acceptor site and the terminator signal.
  • the promoter comprises a CMV immediate early 1 enhancer/promoter (CMV-IE- E/P) and/or the terminator signal comprises a terminator signal of a bovine growth hormone gene (Tbgh) that lacks a KpnI restriction endonuclease site.
  • the nucleic acid expression vector further comprises an origin of replication, and nucleic acid encoding resistance to an antibiotic.
  • the origin of replication comprises a pUC-plasmid origin of replication and/or the nucleic acid encodes resistance to kanamycin.
  • the vector is a pEVAC-based expression vector.
  • the nucleic acid expression vector comprises a nucleic acid sequence of SEQ ID NO:34 (pEVAC).
  • the pEVAC vector has proven to be a highly versatile expression vector for generating viral pseudotypes as well as direct DNA vaccination of animals and humans.
  • the pEVAC expression vector is described in more detail in Example 11 below.
  • Figure 6 shows a plasmid map for pEVAC.
  • the, or each vaccine vector is an RNA vaccine vector.
  • the, or each vaccine vector is an mRNA vaccine vector.
  • a polynucleotide of the invention may comprise a DNA molecule.
  • the or each polynucleotide of a pharmaceutical composition, a combined preparation, or a vector, of the invention may comprise a DNA molecule.
  • a vector of the invention may be a DNA vector.
  • the or each vector of a pharmaceutical composition or a combined preparation of the invention may be a DNA vector.
  • a polynucleotide of the invention, or a polynucleotide of a pharmaceutical composition, a combined preparation, or a vector, of the invention may be provided as part of a DNA vaccine.
  • a DNA vaccine which comprises a polynucleotide of the invention, a vector of the invention, or a pharmaceutical composition or a combined preparation of the invention which comprises one or more polynucleotides, wherein the or each polynucleotide is a DNA molecule.
  • a polynucleotide of the invention may comprise an RNA molecule.
  • the or each polynucleotide of a pharmaceutical composition, a combined preparation, or a vector, of the invention may comprise an RNA molecule.
  • a vector of the invention may be an RNA vector.
  • the or each vector of a pharmaceutical composition or a combined preparation of the invention may be an RNA vector.
  • a polynucleotide of the invention, or a polynucleotide of a pharmaceutical composition, a combined preparation, or a vector, of the invention may be provided as part of an RNA vaccine.
  • an RNA vaccine which comprises a polynucleotide of the invention, a vector of the invention, or a pharmaceutical composition or a combined preparation of the invention which comprises one or more polynucleotides, wherein the or each polynucleotide is an RNA molecule.
  • a polynucleotide of the invention may comprise an mRNA molecule.
  • the or each polynucleotide of a pharmaceutical composition, a combined preparation, or a vector, of the invention may comprise an mRNA molecule.
  • a vector of the invention may be an mRNA vector.
  • the or each vector of a pharmaceutical composition or a combined preparation of the invention may be an mRNA vector.
  • a polynucleotide of the invention, or a polynucleotide of a pharmaceutical composition, a combined preparation, or a vector, of the invention may be provided as part of an mRNA vaccine.
  • an mRNA vaccine which comprises a polynucleotide of the invention, a vector of the invention, or a pharmaceutical composition or a combined preparation of the invention which comprises one or more polynucleotides, wherein the or each polynucleotide comprises an mRNA molecule.
  • mRNA vaccines are a new form of vaccine (recently reviewed in Pardi et al., Nature Reviews Drug Discovery Volume 17, pages 261–279(2018); Wang et al., Molecular Cancer (2021) 20:33: mRNA vaccine: a potential therapeutic strategy).
  • the first mRNA vaccines to be approved for use were BNT162b2 (BioNTech’s vaccine manufactured by Pfizer) and mRNA-1273 (manufactured by Moderna) during the COVID-19 pandemic.
  • mRNA vaccines have a unique feature of temporarily promoting the expression of antigen (typically days). The expression of the exogenous antigen is controlled by the lifetime of encoding mRNA, which is regulated by cellular degradation pathways.
  • mRNA based vaccines trigger an immune response after the synthetic mRNA which encodes viral antigens transfects human cells.
  • the cytosolic mRNA molecules are then translated by the host’s own cellular machinery into specific viral antigens. These antigens may then be presented on the cell surface where they can be recognised by immune cells, triggering an immune response.
  • the structural elements of a vaccine vector mRNA molecule are similar to those of natural mRNA, comprising a 5’ cap, 5’ untranslated region (UTR), coding region (for exampole, comprising an open reading frame encoding a polypeptide of the invention), 3’ UTR, and a poly(A) tail.
  • the 5′ UTR also known as a leader sequence, transcript leader, or leader RNA
  • the 5′ UTR is the region of an mRNA that is directly upstream from the initiation codon. This region is important for the regulation of translation of a transcript. In many organisms, the 5′ UTR forms complex secondary structure to regulate translation.
  • the 5′ UTR begins at the transcription start site and ends one nucleotide (nt) before the initiation sequence (usually AUG) of the coding region.
  • nt nucleotide
  • AUG initiation sequence
  • the length of the 5′ UTR tends to be anywhere from 100 to several thousand nucleotides long. The differing sizes are likely due to the complexity of the eukaryotic regulation which the 5′ UTR holds as well as the larger pre-initiation complex that must form to begin translation.
  • the eukaryotic 5′ UTR contains the Kozak consensus sequence (ACCAUG (initiation codon underlined) (SEQ ID NO:36), which contains the initiation codon AUG.
  • RNA vaccine constructs described herein contain an elongated Kozak sequence: GCCACCAUG (initiation codon underlined) (SEQ ID NO:37).
  • Two major types of RNA are currently studied as vaccines: non-replicating mRNA and virally derived, self-amplifying RNA. While both types of vaccines share a common structure in mRNA constructs, self-amplifying RNA vaccines contain additional sequences in the coding region for RNA replication, including RNA-dependent RNA polymerases.
  • BNT162b2 vaccine construct comprises a lipid nanoparticle (LNP) encapsulated mRNA molecule encoding trimerised full-length SARS2 S protein with a PP mutation (at residue positions 986-987).
  • LNP lipid nanoparticle
  • mRNA-1273 vaccine construct is also based on an LNP vector, but the synthetic mRNA encapsulated within the lipid construct encodes the full-length SARS2 S protein.
  • US Patent No. 10,702,600 B1 (ModernaTX) describes betacoronavirus mRNA vaccines, including suitable LNPs for use in such vaccines.
  • a nucleic acid vaccine (for example, a mRNA) of the invention may be formulated in a lipid nanoparticle.
  • mRNA vaccines have several advantages in comparison with conventional vaccines containing inactivated (or live attenuated) disease-causing organisms.
  • mRNA-based vaccines can be rapidly developed due to design flexibility and the ability of the constructs to mimic antigen structure and expression as seen in the course of a natural infection.
  • mRNA vaccines can be developed within days or months based on sequencing information from a target virus, while conventional vaccines often take years and require a deep understanding of the target virus to make the vaccine effective and safe.
  • these novel vaccines can be rapidly produced. Due to high yields from in vitro transcription reactions, mRNA production can be rapid, inexpensive and scalable (due to chemical synthesis rather than biological growth of cells or bacteria). Thirdly, vaccine risks are low. mRNA does not contain infectious viral elements or cell debris that pose risks for infection and insertional mutagenesis (as the mRNA is generated synthetically).
  • mRNA is the minimally immunogenic genetic vector, allowing repeated administration of the vaccine.
  • the challenge for effective application of mRNA vaccines lies in cytosolic delivery.
  • mRNA isolates are rapidly degraded by extracellular RNases and cannot penetrate cell membranes to be transcribed in the cytosol.
  • efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm.
  • numerous delivery methods have been developed including lipid-, polymer-, or peptide-based delivery, virus-like replicon particle, cationic nanoemulsion, naked mRNAs, and dendritic cell- based delivery (each reviewed in Wang et al., supra).
  • Decationic lipid nanoparticle (LNP) delivery is the most appealing and commonly used mRNA vaccine delivery tool.
  • Exogenous mRNA may be highly immunostimulatory.
  • Single-stranded RNA (ssRNA) molecules are considered a pathogen associated molecular pattern (PAMP), and are recognised by various Toll-like receptors (TLR) which elicit a pro-inflammatory reaction.
  • TLR Toll-like receptors
  • ssRNA Single-stranded RNA
  • TLR Toll-like receptors
  • a strong cellular and humoral immune response is desirable in response to vaccination, the innate immune reaction elicited by exogenous mRNA may cause undesirable side-effects in the subject.
  • the U-rich sequence of mRNA is a key element to activate TLR (Wang et al., supra).
  • dsRNA double stranded RNA
  • IVT in vitro transcription
  • dsRNA double stranded RNA
  • IVT in vitro transcription
  • dsRNA is a potent PAMP, and elicits downstream reactions resulting in the inhibition of translation and the degradation of cellular mRNA and ribosomal RNA (Pardi et al., supra).
  • the mRNA may suppress antigen expression and thus reduce vaccine efficacy.
  • nucleoside modification also suppresses recognition of dsRNA species (Pardi et al., supra) and can reduce innate immune sensing of exogenous mRNA translation (Hou et al. Nature Reviews Materials, 2021, https://doi.org/10.1038/s41578-021- 00358-0).
  • Other nucleoside chemical modifications include, but are not limited to, 5-methylcytidine (m5C), 5-methyluridine (m5U), N1-methyladenosine (m1A), N6- methyladenosine (m6A), 2- thiouridine (s2U), and 5-methoxyuridine (5moU) (Wang et al., supra).
  • the IVT mRNA molecules used in the mRNA-1273 and BNT162b2 COVID-19 vaccines were prepared by replacing uridine with m1 ⁇ , and their sequences were optimized to encode a stabilized pre- fusion spike protein with two pivotal proline substitutions (Hou et al., supra).
  • CureVac s mRNA vaccine candidate, CVnCoV, uses unmodified nucleosides and relies on a combination of mRNA sequence alterations to allow immune evasion without affecting the expressed protein. Firstly, CVnCoV has a higher GC content (63%) than rival vaccines (BNT162b2 has 56%) and the original SARS-CoV-2 virus itself (37%).
  • the vaccine comprises C-rich motifs which bind to poly(C)-binding protein, enhancing both the stability and expression of the mRNA.
  • CVnCoV contains a histone stem-loop sequence as well as a poly(A) tail, to enhance the longevity and translation of the mRNA (Hubert, B., 2021.
  • the CureVac Vaccine and a brief tour through some of the wonders of nature. URL https://berthub.eu/articles/posts/curevac-vaccine-and-wonders-of- biology/.(accessed 15.09.21).
  • the vaccine had disappointing results from phase III clinical trials, which experts assert are down to the decision not to incorporate chemically modified nucleosides into the mRNA sequence.
  • a polynucleotide of the invention may comprise an mRNA molecule.
  • the or each polynucleotide of a pharmaceutical composition, a combined preparation, or a vector, of the invention may comprise an mRNA molecule.
  • a vector of the invention may be an mRNA vector.
  • the or each vector of a pharmaceutical composition or a combined preparation of the invention may be an mRNA vector.
  • a polynucleotide of the invention, or a polynucleotide of a pharmaceutical composition, a combined preparation, or a vector, of the invention may be provided as part of an mRNA vaccine.
  • an mRNA vaccine which comprises a polynucleotide of the invention, a vector of the invention, or a pharmaceutical composition or a combined preparation of the invention which comprises one or more polynucleotides, wherein the or each polynucleotide comprises an mRNA molecule.
  • RNA or mRNA of a polynucleotide of the invention, or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention may be produced by in vitro transcription (IVT).
  • IVT in vitro transcription
  • a polynucleotide of the invention, or a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention may comprise one or more modified nucleosides.
  • the one or more modified nucleosides may be present in DNA or RNA of a polynucleotide of the invention, or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention.
  • At least one chemical modification is selected from pseudouridine, N1- methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine.
  • the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is a N1-methylpseudouridine. In some embodiments, the chemical modification is a N1-ethylpseudouridine.
  • an RNA or an mRNA of a polynucleotide of the invention, or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention may comprise one or more of the following modified nucleosides: pseudouridine ( ⁇ ); N1- methylpseudouridine (m1 ⁇ ) 5-methylcytidine (m5C) 5-methyluridine (m5U) N1-methyladenosine (m1A) N6- methyladenosine (m6A) 2-thiouridine (s2U) 5- methoxyuridine (5moU) In some embodiments, 100% of the uracil in the open reading frame have a chemical modification.
  • a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is a N1-methyl pseudouridine. In some embodiments, 100% of the uracil in the open reading frame have a N1-methyl pseudouridine in the 5-position of the uracil.
  • the polynucleotide may contain from about 1% to about 100% modified nucleotides (or nucleosides) (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 20% to 9
  • a polynucleotide of the invention or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the polynucleotide is the same as that recited in the respective SEQ ID, or the complement thereof, but with each ‘U’ replaced by m1 ⁇ .
  • a polynucleotide of the invention or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention, comprises an mRNA molecule in which the nucleic acid sequence of the polynucleotide is the same as that recited in the respective SEQ ID, or the complement thereof, but with each ‘U’ replaced by m1 ⁇ .
  • a polynucleotide of the invention or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the polynucleotide is the same as that recited in the respective SEQ ID, or the complement thereof, but with at least 50% of the ‘U’s replaced by m1 ⁇ .
  • the remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
  • a polynucleotide of the invention or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention, comprises an mRNA molecule in which the nucleic acid sequence of the polynucleotide is the same as that recited in the respective SEQ ID, or the complement thereof, but with at least 50% of the ‘U’s replaced by m1 ⁇ .
  • the remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
  • a polynucleotide of the invention or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the polynucleotide is the same as that recited in the respective SEQ ID, or the complement thereof, but with at least 90% of the ‘U’s replaced by m1 ⁇ .
  • the remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
  • a polynucleotide of the invention or of a polynucleotide of a pharmaceutical composition, a combined preparation, a vector, or a vaccine, of the invention, comprises an mRNA molecule in which the nucleic acid sequence of the polynucleotide is the same as that recited in the respective SEQ ID, or the complement thereof, but with at least 90% of the ‘U’s replaced by m1 ⁇ .
  • the remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides.
  • mRNA vaccines of the invention may be co-administered with an immunological adjuvant, for example MF59 (Novartis), TriMix, RNActive (CureVac AG), RNAdjuvant (again reviewed in Wang et al., supra).
  • an immunological adjuvant for example MF59 (Novartis), TriMix, RNActive (CureVac AG), RNAdjuvant (again reviewed in Wang et al., supra).
  • each designed RBD subunit i.e. each RBD subunit of String 1, 2, 3, or 4, or variant thereof of String 2
  • each designed subunit sequence of String 5 or variant thereof
  • each vector of a pharmaceutical composition, or combined preparation, of the invention is an mRNA vaccine vector.
  • an isolated cell comprising or transfected with a vector of the invention.
  • an isolated cell comprising a vector of the invention.
  • Polypeptides Vaccine constructs of the invention may also be provided, for example, either as separate polypeptides, each comprising a different designed RBD subunit (or RBD dimer) or pieced together in a string as a single polypeptide comprising all of the RBD subunits (or RBD dimers).
  • the separate polypeptides may be administered as a mixture together (for example, as a pharmaceutical composition comprising the separate polypeptides), or co-administered or administered sequentially in any order (in which case, the separate polypeptides may be provided as a combined preparation for co-administration or sequential administration).
  • RBDs of String 1 There is also provided according to the invention an isolated polypeptide which comprises SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), MERS_RBD_M7 amino acid sequence (SEQ ID NO:15), and SARS1_RBD_M7 amino acid sequence (SEQ ID NO:2).
  • polypeptide comprises the amino acid sequence of SEQ ID NO:25: >String 1 (SEQ ID NO:25) Amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPSAARVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNC VADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGF QPTNGVGYQPYRVVVLSFELLHANATVCGPKKSTNGSGEGRGSLLTCGDVEENPGPMDAMKRGLCCVL LLCGAVFVSPSAAQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPA AIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLI
  • a combined preparation which comprises: i) an isolated polypeptide which comprises SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9); ii) an isolated polypeptide which comprises MERS_RBD_M7 amino acid sequence (SEQ ID NO:15); and iii) an isolated polypeptide which comprises SARS1_RBD_M7 amino acid sequence (SEQ ID NO:2).
  • RBDs of String 2 There is also provided according to the invention an isolated polypeptide which comprises T2_17 amino acid sequence (SEQ ID NO:11) and Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17).
  • polypeptide comprises the amino acid sequence of SEQ ID NO:27: >String 2 (SEQ ID NO:27)
  • Amino acid sequence MDAMKRGLCCVLLLCGAVFVSPSAARVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNC VADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDF TGCVIAWNTNNIDSTTGGNYNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGF FPTNGTGYQPYRVVLSFELLNAPATVCGPKLSTDGSGEGRGSLLTCGDVEENPGPMDAMKRGLCCVL LLCGAVFVSPSAAQPNSVECDFSPMLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFMVNEFSCNGISPD AIARGCYSSLTVDYFAYPLSMKSYMQPGSAGVISQYNYKQSFANPT
  • a combined preparation which comprises: i) an isolated polypeptide which comprises T2_17 amino acid sequence (SEQ ID NO:11); and ii) an isolated polypeptide which comprises Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17).
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:11.
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:17.
  • variants of RDBs of String 2 There is also provided according to the invention an isolated polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8.
  • an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11.
  • an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17.
  • an isolated polypeptide which comprises: an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8; and an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17.
  • an isolated polypeptide which comprises: an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11; and an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17.
  • composition which comprises: i) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8; and ii) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11 (COV_S_
  • composition which comprises: i) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11; and ii) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11; and ii)
  • a combined preparation which comprises: i) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8; and ii) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11 (COV
  • a combined preparation which comprises: i) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11; and ii) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11; and
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises one or more of the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid sequence of SEQ ID NO:11.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid sequence of SEQ ID NO:17.
  • RBDs of String 3 There is also provided according to the invention an isolated polypeptide which comprises SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9), Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17), and Ancestor_SARS1_M7 amino acid sequence (SEQ ID NO:4).
  • polypeptide comprises the amino acid sequence of SEQ ID NO:29: >String 3 (SEQ ID NO:29) Amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPSAARVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNC VADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDF TGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGF QPTNGVGYQPYRVVVLSFELLHANATVCGPKKSTNGSGEGRGSLLTCGDVEENPGPMDAMKRGLCCVL LLCGAVFVSPSAAQPNSVECDFSPMLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFMVNEFSCNGISPD AIARGCYSSLTVDYFAYPLSMKSYMQPGSAGVISQYNYKQSFANPT
  • a combined preparation which comprises: i) an isolated polypeptide which comprises SARS2_RBD_M7 amino acid sequence (SEQ ID NO:9); ii) an isolated polypeptide which comprises Ancestor_MERS_M7 amino acid sequence (SEQ ID NO:17); and iii) an isolated polypeptide which comprises Ancestor_SARS1_M7 amino acid sequence (SEQ ID NO:4).
  • variants of RBDs of String 3 There is also provided according to the invention an isolated polypeptide, which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, or the complement thereof.
  • an isolated polypeptide which comprises: an amino acid sequence of SEQ ID NO:9 (SARS2_RBD_M7); an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17; and an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4.
  • a pharmaceutical composition which comprises: i) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:9 (SARS2_RBD_M7); ii) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17; and iii) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ
  • a combined preparation which comprises: i) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:9 (SARS2_RBD_M7); ii) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17; and iii) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises one or more of the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid residues of SEQ ID NO:17, at positions corresponding to the amino acid residue positions of SEQ ID NO:13, as shown in Table 2.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises one or more of the amino acid residues of SEQ ID NO:4, at positions corresponding to the amino acid residue positions of SEQ ID NO:1, as shown in Table 2.1.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises the amino acid residues of SEQ ID NO:4, at positions corresponding to the amino acid residue positions of SEQ ID NO:1, as shown in Table 2.1.
  • a polypeptide of the invention, or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:17 (Ancestor_MERS_M7), or an amino acid sequence which has at least 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:17, comprises the amino acid sequence of SEQ ID NO:17.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:4 (Ancestor_SARS1_M7), or an amino acid sequence which has at least 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:4, comprises the amino acid SEQ ID NO:4.
  • RBDs of String 4 There is also provided according to the invention an isolated polypeptide which comprises SARS_complex dimer amino acid sequence (SEQ ID NO:19) and MERS_complex dimer amino acid sequence (SEQ ID NO:21).
  • polypeptide comprises the amino acid sequence of SEQ ID NO:31: >String 4 (SEQ ID NO:31) Amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPSAARVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNC VADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDF MGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFY TTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQRVVPSGDVVRFPNITNLCPFGEVFNATKF PSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQT GVIADYNYKLPDDFMGCVLAWNTRNIDSKVGGNYNYLY
  • a combined preparation which comprises: i) an isolated polypeptide which comprises SARS_complex dimer amino acid sequence (SEQ ID NO:19); and ii) an isolated polypeptide which comprises MERS_complex dimer amino acid sequence (SEQ ID NO:21).
  • Subunit Sequences of String 5 There is also provided according to the invention an isolated polypeptide which comprises T2_17 amino acid sequence (SEQ ID NO:11), and COV_N_T2_2 amino acid sequence (SEQ ID NO:45).
  • a pharmaceutical composition which comprises: i) an isolated polypeptide which comprises T2_17 amino acid sequence (SEQ ID NO:11); and ii) an isolated polypeptide which comprises COV_N_T2_2 amino acid sequence (SEQ ID NO:45).
  • a combined preparation which comprises: i) an isolated polypeptide which comprises T2_17 amino acid sequence (SEQ ID NO:11); and ii) an isolated polypeptide which comprises COV_N_T2_2 amino acid sequence (SEQ ID NO:45).
  • an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:45.
  • a fusion protein comprising a polypeptide of the invention.
  • a pseudotyped virus particle also known as a pseudovirus
  • Variants of Subunit Sequences of String 5 According to the invention there is provided an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45.
  • an isolated polypeptide which comprises: an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8; and an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45.
  • an isolated polypeptide which comprises: an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11; and an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45.
  • composition which comprises: i) isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8; and ii) an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45.
  • isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9
  • composition which comprises: i) isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11; and ii) an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45.
  • isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9
  • a combined preparation which comprises: i) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8; and ii) an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45.
  • a combined preparation which comprises: i) an isolated polypeptide which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11; and ii) an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:8, comprises the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises one or more of the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid residues of SEQ ID NO:11, at positions corresponding to the amino acid residue positions of SEQ ID NO:8, as shown in Table 1.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), or an amino acid sequence which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:11, comprises the amino acid sequence of SEQ ID NO:11.
  • the polypeptide comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17).
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.2 above.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises one or more of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.4 above.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.2 above.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 3.4 above.
  • a polypeptide of the invention or a pharmaceutical composition or combined preparation of the invention which comprises a polypeptide, which comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2), or an amino acid sequence which has at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO:45, comprises the amino acid sequence of SEQ ID NO:45.
  • the polypeptide comprises an amino acid sequence of SEQ ID NO:45 (COV_N_T2_2).
  • Vaccines of the invention in combination with a licensed vaccine may be administered before, with, or after a licensed beta coronavirus vaccine (such as the AZD1222, BNT162b2, or mRNA-1273 vaccine).
  • Vaccines comprising a single designed RBD subunit
  • a phylogenetically-, and epitope-optimised sarbecovirus or merbecovirus RBD subunit-based vaccine of the invention may be administered alone, without another RBD subunit-based vaccine of the invention.
  • an isolated polynucleotide which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11), or an isolated polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), may be administered alone, without another RBD subunit-based vaccine of the invention.
  • a designed RBD subunit-based vaccine of the invention may be administered before, with, or after a licensed beta coronavirus vaccine (such as the AZD1222, BNT162b2, or mRNA-1273 vaccine).
  • an isolated polynucleotide (DNA, RNA, or mRNA) which comprises nucleotide sequence encoding T2_17 amino acid sequence (SEQ ID NO:11), or an isolated polypeptide, which comprises an amino acid sequence of SEQ ID NO:11 (COV_S_T2_17), may be administered before, with, or after the AZD1222 vaccine.
  • Pharmaceutical compositions According to the invention there is also provided a pharmaceutical composition which comprises a polynucleotide of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. According to the invention there is also provided a pharmaceutical composition which comprises a vector of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • a pharmaceutical composition which comprises a polypeptide of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent.
  • a pharmaceutical composition of the invention further comprises an adjuvant for enhancing an immune response in a subject to the polypeptide, or to a polypeptide encoded by the polynucleotide, of the composition.
  • a pharmaceutical composition of the invention further comprises an adjuvant for enhancing an immune response in a subject to the polypeptides, or to polypeptides encoded by the polynucleotides, of the composition.
  • combined preparation refers to a "kit of parts" in the sense that the combination components (i) and (ii), or (i), (ii) and (iii), as defined above, can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination components (i) and (ii), or (i), (ii) and (iii).
  • the components can be administered simultaneously or one after the other. If the components are administered one after the other, preferably the time interval between administration is chosen such that the therapeutic effect of the combined use of the components is greater than the effect which would be obtained by use of only any one of the combination components (i) and (ii), or (i), (ii) and (iii).
  • the components of the combined preparation may be present in one combined unit dosage form, or as a first unit dosage form of component (i) and a separate, second unit dosage form of component (ii), or as a first unit dosage form of component (i), a separate, second unit dosage form of component (ii), and a separate, third unit dosage form of component (iii).
  • the ratio of the total amounts of the combination component (i) to the combination component (ii), or of the combination component (i) to the combination component (ii) and to the combination component (iii), to be administered in the combined preparation can be varied, for example in order to cope with the needs of a patient sub-population to be treated, or the needs of the single patient, which can be due, for example, to the particular disease, age, sex, or body weight of the patient.
  • there is at least one beneficial effect for example an enhancing of the effect of the component (i), or an enhancing of the effect of the component (ii), or a mutual enhancing of the effect of the combination components (i) and (ii), or an enhancing of the effect of the component (i), or an enhancing of the effect of the component (ii), or an enhancing of the effect of the component (iii), or a mutual enhancing of the effect of the combination components (i), (ii), and (iii), for example a more than additive effect, additional advantageous effects, fewer side effects, less toxicity, or a combined therapeutic effect compared with an effective dosage of one or both of the combination components (i) and (ii), or (i), (ii), and (iii), and very preferably a synergism of the combination components (i) and (ii), or (i), (ii), and (iii).
  • a combined preparation of the invention may be provided as a pharmaceutical combined preparation for administration to a mammal, preferably a human.
  • the component (i) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, and/or the component (ii) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, or the component (i) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, and/or the component (ii) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent and/or the component (iii) may optionally be provided together with a pharmaceutically acceptable carrier, excipient, or diluent.
  • Embodiments of the invention in which the designed RBD subunits (or other subunits) are encoded as part of the same polynucleotide, or are provided in the same polypeptide (i.e. as “strings” of different subunits), are particularly advantageous since use of such a “string” as part of a vaccine requires testing only of the single product containing the “string” for safety and efficacy, rather than testing each different subunit individually. This dramatically reduces the time and cost of developing the vaccine compared with individual subunits.
  • a combination of different strings polynucleotide and/ or polypeptide
  • a combination of one or more strings and one or more single subunits may be used.
  • Methods of treatment and medical uses there is also provided according to the invention a method of inducing an immune response to a beta-coronavirus in a subject, which comprises administering to the subject an effective amount of: a polynucleotide of the invention; a pharmaceutical composition of the invention; a combined preparation of the invention; a vector of the invention; or a polypeptide of the invention.
  • a method of immunising a subject against a beta-coronavirus which comprises administering to the subject an effective amount of: a polynucleotide of the invention; a pharmaceutical composition of the invention; a combined preparation of the invention; a vector of the invention; or a polypeptide of the invention.
  • An effective amount is an amount to produce an antigen-specific immune response in a subject.
  • a polynucleotide of the invention a pharmaceutical composition of the invention, a combined preparation of the invention, a vector of the invention, or a polypeptide of the invention, for use as a medicament.
  • a polynucleotide of the invention a pharmaceutical composition of the invention, a combined preparation of the invention, a vector of the invention, or a polypeptide of the invention, for use in the prevention, treatment, or amelioration of a beta-coronavirus infection.
  • beta-coronavirus is a lineage B or C beta-coronavirus.
  • beta-coronavirus is a lineage B beta-coronavirus.
  • the lineage B beta-coronavirus is SARS-CoV or SARS-CoV-2.
  • the lineage C beta-coronavirus is MERS-CoV.
  • an immune response is induced against more than one lineage B beta- coronavirus.
  • an immune response is induced against SARS-1 and SARS-2 beta-coronavirus.
  • an immune response is induced against SARS-1 and MERS beta-coronavirus.
  • an immune response is induced against SARS-2 and MERS beta-coronavirus.
  • an immune response is induced against SARS-1, SARS-2, and MERS beta- coronavirus.
  • the beta-coronavirus is a variant of concern (VOC).
  • the beta-coronavirus is a SARS-CoV-2 VOC.
  • the beta-coronavirus is a SARS-CoV-2 lineage B1.248 (Brazil P1 lineage) VOC.
  • the beta-coronavirus is a SARS-CoV-2 lineage B1.351 (South Africa) VOC (also termed a beta VOC).
  • the beta-coronavirus is a SARS-CoV-2 delta VOC.
  • the beta-coronavirus is a SARS-CoV-2 omicron VOC.
  • the beta-coronavirus is a SARS-CoV-2 delta or omicron VOC.
  • the beta-coronavirus is a SARS-CoV-2 beta, gamma, or delta VOC.
  • the beta-coronavirus is a SARS-CoV-2 beta, gamma, delta, or omicron VOC.
  • the beta-coronavirus is a SARS-CoV-2 alpha, beta, gamma, or delta VOC.
  • the beta-coronavirus is a SARS-CoV-2 alpha, beta, gamma, delta, or omicron VOC.
  • an immune response is induced against two or more SARS-CoV-2 VOCs, for example beta and gamma, gamma and delta, beta and delta, beta and omicron, gamma and omicron, or delta and omicron.
  • an immune response is induced against three or more SARS-CoV-2 VOCs, for example: beta, gamma, and delta; beta, gamma, and omicron; gamma, delta, and omicron.
  • beta-CoV-2 VOCs for example: beta, gamma, and delta; beta, gamma, and omicron; gamma, delta, and omicron.
  • a pseudotype neutralisation assay as described in any of the examples below (see Examples 3-5, 8-10, 12) may be used.
  • Administration Any suitable route of administration may be used. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal, intranasal, inhalation or oral.
  • Parenteral administration such as subcutaneous, intravenous or intramuscular administration, is generally achieved by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Administration can be systemic or local. Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes.
  • Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections.
  • Compositions may be administered in any suitable manner, such as with pharmaceutically acceptable carriers.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • An aqueous carrier for parenteral administration may comprise phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid
  • Administration can be accomplished by single or multiple doses.
  • the dose administered to a subject in the context of the present disclosure should be sufficient to induce a beneficial therapeutic response in a subject over time, or to inhibit or prevent infection.
  • the dose required will vary from subject to subject depending on the species, age, weight and general condition of the subject, the severity of the infection being treated, the particular composition being used and its mode of administration. An appropriate dose can be determined by one of ordinary skill in the art using only routine experimentation. Multiple doses may be administered to a subject with a period of several days or a few weeks between consecutive administrations.
  • a polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention may be administered as a first dose, and a second dose of the polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation administered 3 to 12 weeks after the first dose.
  • a polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention may be administered at weeks 0, 4, and 12.
  • a vaccine, polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention may be administered as a booster.
  • a vaccine, polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention may be administered as part of a heterologous prime- boost protocol.
  • a first vaccine may be administered as a priming step, followed by vaccination using a vaccine, polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention vaccine as a booster.
  • the first vaccine is a different vaccine to the booster.
  • the first vaccine may be any suitable coronavirus vaccine, such as the AstraZeneca/Oxford COVID-19 vaccine (AZD1222) BNT162b2 (BioNTech’s vaccine manufactured by Pfizer), or mRNA-1273 (manufactured by Moderna).
  • the booster may be administered at any suitable time after the first vaccine, for example within 4 weeks, 6 weeks, 8 weeks, 12 weeks, 6 months or more after the first vaccine.
  • two or more doses of the first vaccine may be administered before administration of the booster.
  • the booster may be administered at any suitable time after the second, third (or further) dose of the first vaccine, for example after 6 weeks, 8 weeks, 12 weeks, 6 months or more after the second (or further) dose of the first vaccine.
  • Each dose of the first vaccine may be separate by at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 6 months or a year.
  • the present disclosure includes methods comprising administering an RNA vaccine or a DNA vaccine to a subject in need thereof.
  • RNA or DNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the RNA may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • An effective amount of the RNA or DNA, as provided herein, may be as low as 20 pg, administered for example as a single dose or as two 10 pg doses.
  • the effective amount is a total dose of 20 ⁇ g-300 ⁇ g or 25 ⁇ g-300 ⁇ g.
  • an effective amount may be a total dose of 20 ⁇ g, 25 ⁇ g, 30 ⁇ g, 35 ⁇ g, 40 ⁇ g, 45 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g, 180 ⁇ g, 190 ⁇ g, 200 ⁇ g, 250 ⁇ g, or 300 ⁇ g.
  • an effective amount is a total dose of 20 ⁇ g. In some embodiments, an effective amount is a total dose of 25 pg. In some embodiments, an effective amount is a total dose of 50 ⁇ g. In some embodiments, an effective amount is a total dose of 75 ⁇ g. In some embodiments, an effective amount is a total dose of 100 ⁇ g. In some embodiments, an effective amount is a total dose of 150 ⁇ g. In some embodiments, an effective amount is a total dose of 200 ⁇ g. In some embodiments, an effective amount is a total dose of 250 pg. In some embodiments, an effective amount is a total dose of 300 ⁇ g.
  • RNA or DNA described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).
  • an RNA (e.g., mRNA) or DNA vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.
  • an effective amount is a total dose of 25 ⁇ g to 1,500 ⁇ g, 25 ⁇ g to 1,200 ⁇ g, 25 ⁇ g to 1000 ⁇ g, or 50 ⁇ g to 1000 ⁇ g.
  • an effective amount is a total dose of 100 ⁇ g. In some embodiments, an effective amount is a dose of 25 ⁇ g administered to the subject a total of two times. In some embodiments, an effective amount is a dose of 100 ⁇ g administered to the subject a total of two times. In some embodiments, an effective amount is a dose of 400 ⁇ g administered to the subject a total of two times. In some embodiments, an effective amount is a dose of 500 ⁇ g administered to the subject a total of two times. Optionally, an effective amount is a dose of 100-300 ⁇ g administered to the subject at least two times. Optionally, an effective amount is a dose of 300-500 ⁇ g administered to the subject at least two times.
  • an effective amount is a dose of 500-700 ⁇ g administered to the subject at least two times.
  • an effective amount is a dose of 700-900 ⁇ g administered to the subject at least two times.
  • an effective amount is a dose of 900-1100 ⁇ g administered to the subject at least two times.
  • an effective amount is a dose of 1100-1300 ⁇ g administered to the subject at least two times.
  • a first dose is administered to the subject at least two weeks before a second dose.
  • a first dose is administered to the subject at least three weeks before a second dose.
  • a dosage of between 10 ⁇ g/kg and 400 ⁇ g/kg of the nucleic acid vaccine is administered to the subject.
  • a dosage of between 10 ⁇ g/kg and 1,000 ⁇ g/kg of the nucleic acid vaccine is administered to the subject.
  • the dosage of the RNA or DNA polynucleotide is 1-5 ⁇ g, 5-10 ⁇ g, 10-15 ⁇ g, 15-20 ⁇ g, 10-25 ⁇ g, 20-25 ⁇ g, 20- 50 ⁇ g, 30-50 ⁇ g, 40-50 ⁇ g, 40-60 ⁇ g, 60-80 ⁇ g, 60-100 ⁇ g, 50-100 ⁇ g, 80-120 ⁇ g, 40-120 ⁇ g, 40-150 ⁇ g, 50-150 ⁇ g, 50-200 ⁇ g, 80-200 ⁇ g, 100-200 ⁇ g, 120-250 ⁇ g, 150-250 ⁇ g, 180-280 ⁇ g, 200-300 ⁇ g, 50-300 ⁇ g, 80-300 ⁇ g, 100-300 ⁇ g, 40-300 ⁇ g, 50-350 ⁇ g, 100-350 ⁇ g, 200- 350
  • the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.
  • Polynucleotides, polypeptides, pharmaceutical compositions, combined preparations, and vaccines of the invention may induce a broadly neutralising immune response to protect against disease caused by viruses of the sarbecovirus and merbecovirus lineages.
  • the term “broadly neutralising immune response” is used herein to mean an immune response elicited in a subject that is sufficient to inhibit (i.e.
  • a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of more than one type of ⁇ -coronavirus (for example, SARS-CoV, and SARS-CoV-2).
  • a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of more than one type of ⁇ -coronavirus within the same ⁇ -coronavirus lineage (for example, more than one type of ⁇ -coronavirus within the subgenus Sarbecovirus, such as SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1).
  • a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of coronaviruses of different ⁇ -coronavirus lineages, such as lineage B (for example, SARS-CoV, and SARS-CoV-2) and lineage C (for example, MERS-CoV).
  • a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of most or all different ⁇ -coronaviruses.
  • the immune response may be humoral and/or a cellular immune response.
  • a cellular immune response is a response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen or vaccine.
  • An immune response can include any cell of the body involved in a host defence response, including for example, an epithelial cell that secretes an interferon or a cytokine.
  • An immune response includes, but is not limited to, an innate immune response or inflammation.
  • a polynucleotide, polypeptide, pharmaceutical composition, combined preparation, or vaccine of the invention of the invention induces a protective immune response.
  • a protective immune response refers to an immune response that protects a subject from infection or disease (i.e. prevents infection or prevents the development of disease associated with infection).
  • Methods of measuring immune responses include, for example, measuring proliferation and/or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, or antibody production.
  • a polynucleotide, polypeptide, pharmaceutical composition, combined preparation, or vaccine of the invention is able to induce the production of antibodies and/or a T-cell response in a human or non-human animal to which the polypeptide has been administered (either as a polypeptide or, for example, expressed from an administered nucleic acid expression vector).
  • Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile, and the formulation suits the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used.
  • the medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like.
  • compositions comprise a pharmaceutically acceptable carrier and/or an adjuvant.
  • the adjuvant can be alum, Freund’s complete adjuvant, a biological adjuvant or immunostimulatory oligonucleotides (such as CpG oligonucleotides).
  • the pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA , 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, such as one or more influenza vaccines, and additional pharmaceutical agents.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions for example, powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • a polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention is administered intramuscularly.
  • a polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention is administered intramuscularly, intradermally, or subcutaneously, for example by needle or by gene gun, or electroporation, or by needle-free injection (for example, using a needle-free injection device of Pharmajet).
  • a vaccine, polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention is administered by needle-free injection.
  • a vaccine, polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention is administered intradermally by needle-free injection.
  • a vaccine of the invention is administered intradermally by needle-free injection.
  • a vector of the invention is administered intradermally by needle-free injection.
  • a pharmaceutical composition of the invention comprising a vector or a polynucleotide is administered intradermally by needle-free injection.
  • a polynucleotide of the invention is administered intradermally by needle-free injection.
  • a vaccine of the invention may comprise any polypeptide, polynucleotide, vector, pharmaceutical composition, or combined preparation of the invention.
  • Conservative Amino Acid Substitutions A polypeptide of the invention may include one or more conservative amino acid substitutions. Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original polypeptide, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • substitutions which in general are expected to produce the greatest changes in protein properties will be non-conservative, for instance changes in which (a) a hydrophilic residue, for example, serine or threonine, is substituted for (or by) a hydrophobic residue, for example, leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, for example, lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, for example, glutamate or aspartate; or (d) a residue having a bulky side chain, for example, phenylalanine, is substituted for (or by) one not having a side chain, for example, glycine.
  • a hydrophilic residue for example, serine or threonine
  • a hydrophobic residue for example, leucine,
  • Sequence identity The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a given gene or protein will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.2:482, 1981; Needleman and Wunsch, J. Mol. Biol.48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.
  • Biol.215:403-410, 1990 is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
  • Sequence identity between nucleic acid sequences, or between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same nucleotide, or amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at positions shared by the compared sequences.
  • optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences.
  • Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties. Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector.
  • Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4: 29; program available from http://bitincka.com/ledion/matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol.48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol.
  • sequence comparisons may be undertaken using the “needle” method of the EMBOSS Pairwise Alignment Algorithms, which determines an optimum alignment (including gaps) of two sequences when considered over their entire length and provides a percentage identity score.
  • Default parameters for amino acid sequence comparisons (“Protein Molecule” option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum 62.
  • the sequence comparison may be performed over the full length of the reference sequence.
  • Corresponding Positions Sequences described herein include reference to an amino acid sequence comprising an amino acid residue “at a position corresponding to an amino acid residue position” of another sequence.
  • Figure 1A shows the phylogenetic tree of the coronavirus family of viruses
  • Figure 1B shows the gross morphology of the viral S-protein across the CoV family
  • Figure 1C shows a cartoon representation of the structure of the receptor binding domain of SARS-complex (SARS1, SARS2), and MERS.
  • Figure 1D shows a schematic of the domain structure of the CoV S protein
  • Figure 2A shows a phylogenetic tree of the sarbeco lineage of ⁇ -coronavirus built using S protein RBD sequences.
  • the black filled circles represent two phylogenetically optimised vaccine designs using the RBD sequences
  • Figure 2B shows a phylogenetic tree of the merbeco lineage of ⁇ -coronavirus built using the S protein RBD sequences.
  • the black circle represents a phylogenetically optimised vaccine design using the RBD sequences; and
  • Figure 2C shows designed S protein RBD string constructs 1-4 which have pan ⁇ - coronavirus coverage;
  • Figure 3 shows the spectra overlap (MALDI MS) of supernatants derived from HEK cells transfected with pEVAC plasmid encoding S protein RBD sequences;
  • Figure 4 shows spectra for recombinant RBD proteins;
  • Figure 5 provides a reference for glycosylation of the S protein;
  • Figure 6 shows a plasmid map for pEVAC DNA vector;
  • Figure 7 shows a multiple sequence alignment of the S-protein RBD for SARS-1 and related vaccine constructs (String 1 and String3).
  • Figure 8 shows a multiple sequence alignment of the S-protein RBD for SARS-2 and related vaccine constructs (String 1, String 2 and String 3). The position at which the glycosylation is introduced is highlighted in grey;
  • Figure 9 shows a multiple sequence alignment of the S-protein RBD for MERS and related vaccine constructs (String 1, String 2, and String 3);
  • Figure 10 shows a multiple sequence alignment of the S-protein RBD for SARS-CoV-1 and SARS-CoV-2 dimer (SARS-complex dimer);
  • Figure 11 shows a multiple sequence alignment of the S-protein RBD for MERS and HKU5 dimer (MERS-complex dimer);
  • Figure 12 In-silico design of a vaccine according to an embodiment of the invention: A.
  • Phylogenetic tree generated for sarbecoviruses using protein sequence of receptor binding domain (RBD) of the spike protein.
  • the tree was generated using IQ-Tree.
  • Human viruses are represented in green, palm civet viruses in pink and bat viruses in dark grey.
  • B Structural model of the antibody-RBD complex.
  • the antibodies are represented as cartoon and coloured green and orange and the RBD is represented as both cartoon and surface and coloured pink.
  • the different epitope regions are labelled as A, B and C.
  • C Sequence alignment of SARS-1 and SARS-2 RBDs. Only the non-conserved amino acids are shown. The epitope C is boxed in black.
  • Figure 13(A) shows a Western Blot of sera from mice immunised with vaccine designs T2_13-T2_20, including T2_17 of Example 3.
  • Figure 13(B) shows antibody binding responses of Cell Surface expression bleed 2.
  • Figure 14A shows a multiple sequence alignment of constructs CoV_S_T2_13-18, including construct CoV_S_T2_13 of Example 3.
  • the coloured boxes show the residues of discontinuous epitopes present in sequences COV_S_T2_14-18 shown in different colour.
  • the changes made relative to the COV_S_T2_13 sequence to provide discontinuous epitopes that elicit a broader or more potent immune response are shown by the boxed regions.
  • Figure 14B Neutralisation data: Neutralisation curves of vaccine designs, SARS-1 RBD and SARS-2 RBD against SARS1 pseudotype (upper panel) and SARS2 pseudotype (lower panel).
  • the X- axis represents the dilution of the sera and the Y-axis represent the percentage of neutralisation observed.
  • Each curve in the plots represents an individual mouse.
  • Figure 15 represents the study protocol of a dose finding study of DNA encoding COV_S_T2_17 (SEQ ID NO:12).
  • Figure 16 shows the results of ELISA to determine the level of antibodies to the RBD of SARS- CoV-2, and SARS. Panel A (left) Plates coated with SARS-CoV-2 RBD.
  • Panel B (right) Plates coated with SARS RBD; Figure 17 shows virus neutralisation at day 28 after 1 immunisation (Pseudotype MicroNeutralisation or pMN assay).
  • Panel A (left) Antibody neutralisation of SARS-CoV-228 days after 1 dose.
  • Panel B (right) Antibody neutralisation of SARS 28 days after 1 dose.
  • Figure 18 shows (for Groups 1, 2, and 3) comparison of virus neutralisation responses after first to second immunisation.
  • Panel A left SARS-CoV-2) Comparing bleeds 2 (pre) and 3 (post) second immunisation (boost).
  • Panel B (right SARS) Comparing bleeds 2 (pre) and 3 (post) second immunisation (boost).
  • Figure 19 shows (for groups 4, 5 and 6) comparison of virus neutralisation responses after first to second immunisation.
  • Panel A left SARS-CoV-2 Comparing bleeds 2 (pre) and 3 (post) second immunisation (boost).
  • Panel B right SARS
  • Figure 20 shows neutralisation of variants of concern (B1.351(SA) & B1.248(P1 BZ) is superior with T2_17 vs T2_8).
  • Figure 21 shows SARS CoV-1, SARS-CoV-2, and WIV-16 pseudotype neutralisation for vaccine constructs of Example 5;
  • Figure 22 shows a Western Blot of sera from mice immunised with the vaccine designs of Example 6;
  • Figure 23 shows binding (by ELISA) of mouse sera, collected following immunisation of mice with DNA encoding MERS RBD (MERS RBD), and DNA encoding ancestor MERS M7 RBD (panMERS), to MERS S protein, as described in Example 7;
  • Figures 24-26 show neutralisation of SARS-CoV-1, SARS-CoV-2, and WIV-16 pseudoviruses with sera collected after immunisation of mice with the vaccine constructs listed in Examples 8-10;
  • Figure 27 shows in silico design and in-vivo selection of vaccine antigen candidate;
  • Figure 28 shows immunogenicity studies in Guinea pigs and rabbits;
  • Figure 29 shows multiple sequence alignment of the known sarbecoviruses;
  • Figure 30A shows ELISA binding data of K18
  • spike protein amino acid changes are included – this is not a full reference for assignment of the variants. It includes changes to spike protein residues 319- 541 (receptor binding domain) and 613-705 (the S1 part of the S1/S2 junction and a small stretch on the S2 side, and any additional unusual changes specific to the variant.
  • DOI https://www.ecdc.europa.eu/en/covid-19/variants-concern, accessed on 26 Jan 2022.
  • Figure 32B is a multiple sequence alignment of the VOCs with the ancestral sequence (Wuhan-Hu- 1 isolate).
  • Figure 33 compares the neutralisation responses of String 1, String 4, and a T2_23 Sarbeco dimer: left panel shows the results of neutralisation against SARS-CoV-1; right panel shows the results of neutralisation against SARS-CoV-2.
  • SARS1_RBD amino acid sequence (AY274119_RBD) 2 SARS1_RBD_M7 amino acid sequence 3 SARS1_RBD_M7 encoding nucleotide sequence 4 Ancestor_SARS1_M7 amino acid sequence 5 Ancestor_SARS1_M7 encoding nucleotide sequence 6 WIV16 amino acid sequence 7 WIV16 encoding nucleotide sequence 8 SARS2_RBD amino acid sequence (EPI_ISL_402119_RBD) 9 SARS2_RBD_M7 amino acid sequence 10 SARS2_RBD_M7 encoding nucleotide sequence 11 T2_17 amino acid sequence 12 T2_17 encoding nucleotide sequence 13 MERS_RBD amino acid sequence 14 MERS_RBD encoding nucleotide sequence (encoding amino acid residues 1- 235 of SEQ ID NO:13) 15 MERS_RBD_M7 amino acid sequence 16 MERS_RBD_M
  • Example 1 Broad coverage beta-coronavirus vaccine constructs
  • Four different broad coverage beta-coronavirus string-based vaccine constructs (Strings 1-4) have been designed using phylogenetically optimised vaccine constructs for sarbecovirus, phylogenetically optimised vaccine constructs for merbecovirus, and dimeric RBD subunit- based vaccine constructs.
  • Phylogenetically optimised vaccine construct for sarbecoviruses Two phylogenetically optimised vaccine designs were generated using the sequences represented in Figure 2A.
  • One vaccine design (labelled as 1 in Figure 2A) was further optimised for cross-reactive B-cell epitopes to generate adequate cross-protection against viruses belonging to SARS-complex lineage, referred to herein as “T2_17” (amino acid sequence: SEQ ID NO:11; encoding nucleotide sequence: SEQ ID NO:12).
  • Another vaccine design (labelled as 2 in Figure 2A) was optimised to generate cross-protection to SARS-1 and SARS-1 related bat viruses.
  • sequence was further modified to provide a glycosylation site at one of the epitopes, referred to herein as “Ancestor_SARS1_M7”, or “SARS1_Anc_M7” (amino acid sequence: SEQ ID NO:4; encoding nucleotide sequence: SEQ ID NO:5).
  • a modified SARS-2 RBD, and SARS-1 RBD were generated by introducing a glycosylation site at one of the epitopes in the wild-type sequence, referred to herein as “SARS2_M7” or “SARS2_RBD_M7” (amino acid sequence: SEQ ID NO:9; encoding nucleotide sequence: SEQ ID NO:10), and “SARS1_M7” (amino acid sequence: SEQ ID NO:2; encoding nucleotide sequence: SEQ ID NO:3), respectively.
  • a phylogenetically optimised vaccine design (black filled circle in Figure 2B) was generated for MERS and related DPP4 using bat viruses (boxed in black in Figure 2B). This sequence was further modified to provide a glycosylation site at one of the epitopes, referred to herein as “Ancestor_MERS_M7” (amino acid sequence: SEQ ID NO:17; encoding nucleotide sequence: SEQ ID NO:18).
  • a modified MERS RBD was generated by introducing a glycosylation site at one of the epitopes in the wild-type sequence, referred to herein as “MERS_M7” or “MERS_RBD_M7” (amino acid sequence: SEQ ID NO:15; encoding nucleotide sequence: SEQ ID NO:16) (L. Du et. al., 2016, Nat. Comm).
  • Dimeric RBD subunit-based vaccine constructs Dimeric form of RBD has been reported to be more immunogenic than monomeric RBD (L. Dai, 2020, Cell).
  • String 1 comprises an isolated polynucleotide comprising nucleotide sequence encoding designed SARS2_RBD_M7, MERS_RBD_M7, and SARS1_RBD_M7 monomers, covalently linked.
  • String 2 comprises an isolated polynucleotide comprising nucleotide sequence encoding designed T2_17 (SARS2 ancestral designed sequence) and Ancestor_MERS_M7 monomers, covalently linked.
  • String 3 comprises an isolated polynucleotide comprising nucleotide sequence encoding designed SARS2_RBD_M7, Ancestor_MERS_M7, and Ancestor_SARS1_M7, covalently linked.
  • String 4 comprises an isolated polynucleotide comprising nucleotide sequence encoding designed SARS-Complex (SARS-CoV-1 and SARS-CoV-2 RBD) dimer and MERS-Complex (MERS and HKU5 RBD) dimer, covalently linked.
  • T2_17 SARS2 ancestral designed sequence
  • Ancestor_MERS_M7 monomers, covalently linked.
  • the amino acid sequence of this design is shown below.
  • the sequence of T2_17 is designed using a the sequences represented in Figure 2A, and is optimised to generate adequate cross-protection against viruses belonging to SARS-CoV-1 and SARS-CoV-2 lineages.
  • the sarbeco lineage construct comprises an isolated polynucleotide comprising nucleotide sequence encoding designed SARS-Complex (SARS-CoV-1 and SARS-CoV-2 RBD) dimer.
  • the merbeco lineage construct comprises an isolated polynucleotide comprising nucleotide sequence encoding designed MERS-Complex (MERS and HKU5 RBD) dimer.
  • String4 a vaccine construct, comprising an isolated polynucleotide comprising nucleotide sequence encoding designed SARS-Complex dimer and a designed MERS-complex dimer, covalently linked.
  • the amino acid sequences of the designed SARS-Complex and MERS-Complex dimers are shown below, along with the String4 designed construct.
  • Figure 7 shows a multiple sequence alignment of the S-protein RBD for SARS1 and related vaccine constructs (String1 and String3). The position at which the glycosylation is introduced is highlighted in grey.
  • Figure 8 shows a multiple sequence alignment of the S-protein RBD for SARS2 and related vaccine constructs (String1, String2 and String3). The position at which the glycosylation is introduced is highlighted in grey.
  • Figure 9 shows a multiple sequence alignment of the S-protein RBD for MERS and related vaccine constructs (String1, String2, and String3).
  • Figure 10 shows a multiple sequence alignment of the S-protein RBD for SARS-CoV-1 and SARS-CoV-2 dimer (SARS-complex dimer).
  • Figure 11 shows a multiple sequence alignment of the S-protein RBD for MERS and HKU5 dimer (MERS-complex dimer).
  • Example 2 Glycosylation of S protein RBD proteins
  • Figure 3 shows the spectra overlap (MALDI MS) of supernatants derived from HEK cells transfected with pEVAC plasmid encoding various S protein RBD sequences, including the following: . COV_S_T2_5 (wild-type SARS1 RBD) . COV_S_T2_6 (wild-type SARS2 RBD) . T2_17 . M7 RBD . TM RBD The results show that the RBD is peaking at 25-26 kDa, and a second peak appears at 29 kDa.
  • FIG. 4 shows the spectra for the following examples of recombinant RBD proteins: .
  • RBD one sample labelled “RBDr-1”
  • RBDr-2 His-tagged RBD
  • RBDr-3 Another RBD protein sample labelled “RBDr-3”.
  • RBD protein (Leader - RBD – Tag) is below: MKRGLCCVLLLCGAVFVSPSAARVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCN GVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNGGSGLNDIF EAQKIEWHEGSHHHHHH (SEQ ID NO:35)
  • Figure 4 shows that the RBDr-1 and RBDr-2 proteins peak at ⁇ 26 KDa (RBDr-1 is the higher peak in the figure), and that the RBDr-3 sample peaks at ⁇ 31-32 KDa.
  • Figure 5 provides a reference for glycosylation of the “S” Spike protein.
  • the glycosylation pattern of the spike protein is mixed.
  • the mass for each glycan is ⁇ 2 kDa.
  • the mass of “RBDr-3” protein is 29.2 kDa.
  • the mass of the designed RBD proteins, and wild- type RBD is ⁇ 24kDa.
  • Example 3 Epitope optimised broad coverage vaccine design for Sarbecoviruses Overview: To increase the coverage of our receptor binding domain (RBD) based vaccine designs to all the extant sarbecovirus sub-genus of Beta-coronaviruses, a phylogenetically optimised vaccine design was constructed. This design is further used as backbone for designing both epitope optimised and immune re-focussed designs. The epitope information is derived largely from the known high-resolution structural data of spike protein-antibody complex. Few of these epitopes are reported to cross protect SARS-1 and SARS-2 and were included in the designs to increase the coverage of the vaccine designs.
  • RBD receptor binding domain
  • each epitope was modified to match the epitope sequences from SARS-1 and SARS-2.
  • Three conformational epitopes are identified through structural analyses of RBD-antibody complex (see Figure 12B). Two of these epitopes (henceforth termed as A and B) are reported to bind antibodies that neutralise both SARS-1 and SARS-2. The third epitope (henceforth termed as C) is in and around the receptor binding region. This epitope shows maximum divergence (Figure 12C) and is expected to generate a virus specific antibody response.
  • FIG. 13(A) shows a Western Blot of sera from mice immunised with the vaccine designs, including T2_17.
  • Figure 13(B) shows antibody binding responses of Cell Surface expression bleed 2.
  • Neutralisation data Sera from mice injected with T2_17, SARS-1 RBD and SARS-2 RBD are checked for neutralisation of SARS-1 and SARS-2 pseudotypes.
  • human sera from an infected individual are used.
  • the neutralisation curves are shown in Figure 14B.
  • Each amino acid position in the sequence is chosen based on the phylogenetic relatedness of the input sequences.
  • the novel sequence generated neutralising response against SARS-2 but not much against SARS-1.
  • SARS-1 and SARS-2 On comparison of the epitopes in the novel sequence and SARS-1 and SARS-2, it was observed that the epitopes were more biased towards SARS-2 compared to SARS-1.
  • T2_17 To focus the immune response towards the conserved epitopes, a glycosylation site was introduced at the more diverged third epitope (T2_17). The introduction of the glycosylation site indeed broadened the immune response to both SARS-1 and SARS-2, with cross-neutralisation observed for the design.
  • Epitope identification Available structural data for Spike protein-antibody complexes for SARS-1 and SARS-2 were downloaded from the Protein Databank (PDB). These structural data were further pruned for antigen-antibody complexes where the epitope region is in the RBD. Amino acid residues of antigen that have at least one atom within 5 ⁇ radii of at least one atom of amino acid of antibody are defined as epitope residues. An epitope region is defined as contiguous stretch of at least 5 amino acids. . Molecular modelling Structural models were generated for COV_S_T2_13 using MODELLER algorithm. The structural model with the highest DOPE score was chosen as the working model for the further molecular modelling.
  • T2_17 SEQ ID NO:12
  • BUILD pan-Sarbeco Coronavirus Vaccine DNA candidate
  • Figure 15 To determine the optimal dose of DNA, a pre-clinical vaccine study was undertaken in mature Hartley Guinea pigs. Animals were randomised into six groups of eight animals and pre-bled to determine the absence of anti-SARS-CoV-2 antibodies.
  • Group 1 (control) group received the high dose of 400ug (2mg/ml) of the modified SARS-CoV- 2 RBD COV_S_T2_8 DNA subcutaneously, to compare to a second group (Group 6) the same control DNA of COV_S_T2_8 at 400ug administered intradermally (ID) by the PharmaJet Tropis device.
  • the remaining four groups received the pan-Sarbeco vaccine candidate, T2_17 at 100ug (0.5mg/ml) (two groups, one receiving 2, the other 3 doses), 200ug (1mg/ml), or 400ug (2mg/ml) intradermally at day 0 and 28. Animals were bled at days 14, 28, 42, 56 and 70.
  • ELISA to determine the level of antibodies to the RBD of SARS-CoV-2, and SARS
  • Figure 16 Panel A (left) Plates coated with SARS-CoV-2 RBD. 28 days following the first immunisation, an ELISA assay was performed to determine the titre of anti-SARS-CoV-2 RBD, or anti-SARS RBD antibodies induced 28 days after one DNA immunisation.
  • the top left panel (T2_8 at 400ug sc) demonstrates the antibody responses to SARS-CoV-2 in 5 out of 8 animals, compared to the bottom right hand panel (T2_8 at 400ug DNA administered ID by the Tropis Pharmajet) where 7 of 8 animals respond strongly to SARS-CoV-2 RBD.
  • the 4 remaining groups receiving T2_17 ID by PharmaJet delivery showed similar anti-SARS-CoV-2 responses to 400ug of the SARS-CoV-2 RBD DNA administered at the maximal dose.
  • Panel B (right) Plates coated with SARS RBD. The same 28 day serum samples at serial dilutions were tested for binding to the SARS RBD.
  • the top left panel (T2_8 at 400ug sc) demonstrates low titre antibodies, with only 2 of 8 animals reaching an OD of 0.5.
  • the same dose of the SARS-CoV-2 RBD vaccine given by the PharmaJet device bottom right hand panel
  • pan-Sarbeco T2_17 groups respond strongly to the SARS RBD in a dose-dependent manner, with all animals in the high (400ug) (bottom row left in panel B) and medium doses (200ug) groups (middle row panel B) responding strongly, and a more variable but distinct response in all 8 animals in the lowest (100ug) T2_17 group (top right, panel B).
  • Virus Neutralisation at day 28 after 1 immunisation Pseudotype MicroNeutralisation or pMN assay
  • Figure 17 Panel A (left) Antibody neutralisation of SARS-CoV-228 days after 1 dose.
  • T2_8 at 400ug sc had low level responses compared to the same vaccine candidate (T2_8 at 400ug DNA) administered ID by the Tropis Pharmajet device, which was the strongest of all the groups.
  • T2_17 ID by PharmaJet delivery showed lower but significant responses to SARS-CoV-2.
  • Panel B (right) Antibody neutralisation of SARS 28 days after 1 dose. The same 28 day serum samples at serial dilutions were tested for neutralising to SARS pseudotyped viruses. At this time point, after 1 administration, responses were absent in the T2_8 groups (top left and bottom right of panel B (right)).
  • pan-Sarbeco T2_17 groups respond at low and variable levels after 1 dose of vaccine, again with the best but weak response in the highest dose group (400ug) (bottom row left in panel B) Groups 1 to 3, Comparison of Virus Neutralisation responses after first to second immunisation (Figure 18): Panel A (left, SARS-CoV-2) Comparing bleeds 2 (pre) and 3 (post) second immunisation (boost) There was significant boost effect with increased neutralising responses to SARS-CoV-2 in all groups, though not all animals in group 1 (T2_8 at 400ug) administered subcutaneously. Groups 2 and 3, middle and lower rows of panel B, right, were more uniform and comparably boosted neutralising titres to SARS-CoV-2.
  • Panel B (right, SARS) Comparing bleeds 2 (pre) and 3 (post) second immunisation (boost).
  • boost effect 5 of 8 animals to SARS in group 1 (T2_8 at 400ug).
  • Example 5 Neutralisation of SARS-CoV-1, SARS-CoV-2, and WIV-16 pseudotype induced by String 3 RBD DNA vaccine Mice were immunised with different RBD DNA vaccines listed below, then sera was collected and tested for SARS-CoV-1, SARS-CoV-2, and SARS-CoV-1 closely related bat coronavirus WIV-16 pseudotype neutralisation.
  • the DNA vaccines used were: . PBS control .
  • String 3 (DNA encoding SARS-CoV-2 M7 RBD, ancestor MERS M7 RBD, ancestor SARS-CoV-1 M7 RBD); .
  • Mix String 3 mixture of individual plasmids encoding the constituents of the String 3 construct, injected at 3 different sites); .
  • Figure 21 shows SARS CoV-1, SARS-CoV-2, and WIV-16 (SARS-CoV-1 closely related bat coronavirus) pseudotype neutralisation log(IC 50 ) values for sera collected (bleed 5) from the mice immunised with the vaccines listed above. The dots in Figure 21 show log(IC 50 ) values for individual mice.
  • FIG 21 also shows that the PanSARS (ancestor_SARS1_M7) vaccine is effective against SARS-CoV-1 pseudovirus and WIV-16 pseudovirus, and the M7 (T2_8) vaccine is effective against SARS-CoV-2 pseudovirus.
  • the panMERS (ancestor_MERS_M7) vaccine construct elicited a weaker neutralising response to SARS-CoV-2.
  • Example 6 Western Blot showing expression of MERS RBD vaccine construct
  • Figure 22 shows a Western Blot of sera from mice immunised with the vaccine designs listed below. Mice were immunised with different CoV RBD DNA vaccines, then sera were collected and tested for binding to SARS-2 S1 Subunit (His-tagged), or SARS-1 Spike (His- tagged). The DNA vaccines used were: .
  • RBD String 3 (DNA encoding SARS-CoV-2 M7 RBD, ancestor MERS M7 RBD, ancestor SARS-CoV-1 M7 RBD) .
  • Mixed mixture of individual plasmids encoding the constituents of the String 3 construct, injected at 3 different sites
  • Single individual plasmids encoding the constituents of the String 3 construct, injected at 3 different sites simultaneously.
  • Pan MERS Pan SARS1 . SARS-2 RBD The results are shown in Figure 22.
  • Figure 22 shows that mice vaccinated with the String 3, Mixed, and Single constructs separately elicit antibodies that bind to SARS-2 S1 Subunit, and to SARS-1 Spike protein, confirming that the constructs have expressed their encoded polypeptides.
  • Example 7 Ability of MERS RBD and panMERS vaccine constructs to induce antibodies to MERS S protein Mice were immunised with DNA encoding MERS RBD (MERS RBD), and DNA encoding ancestor MERS M7 RBD (panMERS), and the sera were collected and tested at different dilutions for binding (by ELISA) to MERS S protein RBD.
  • the sera were heat inactivated (HI) to check for non-specific interactions in the ELISA.
  • the results are shown in Figure 23.
  • the binding of the sera to MERS S protein RBD was tested using ELISA.
  • the ELISA protocol is as follows: Materials and Reagents: . F96 Nunc Maxisorp flat-bottom plates (Cat #: 44-2404-21, Thermo Scientific) . Plate sealers (Cat #: 676001, Greiner Bio-one) . Shaker (Cat #: 544-11200-00, Heidolph Instruments Titramax 100) . 50mL and 100mL reservoirs (Cat #4870 Corning and #B3110-100 Argos) .
  • HRP-conjugated secondary antibodies o Anti-mouse IgG-horseradish peroxidase (HRP) conjugated secondary antibody (Cat #:715-035-150, Jackson ImmunoResearch) o Anti-human IgG/IgM/IgA-horseradish peroxidase (HRP) conjugated secondary antibody (Cat #: 109-035-064, Jackson ImmunoResearch) .
  • 1-StepTM Ultra TMB (Cat # 34029, Thermo Scientific) . Stop solution of H 2 SO 4 (add 28mL of 1.84kg/L H 2 SO 4 to 472mL milliQ water) .
  • Serum samples (about 4ul is needed to run a duplicate, starting at 1:50 dilution with 10-fold serial dilutions; about 5.5ul is needed to run a duplicated, starting at 1:50 dilution with 2-fold serial dilutions) .
  • Positive control anti-histidine .
  • Figure 23 shows that the DNA encoding MERS RBD (MERS RBD), and DNA encoding ancestor MERS M7 RBD (panMERS) induced a relatively strong binding response to MERS S protein RBD.
  • MERS RBD DNA encoding MERS RBD
  • panMERS DNA encoding ancestor MERS M7 RBD
  • Figure 24 shows antibody neutralisation of SARS-CoV-1 pseudoviruses after immunisation with the vaccine constructs listed below. Mice were immunised with the different RBD DNA vaccines, then sera were collected and tested for SARS-CoV-1 pseudovirus neutralisation.
  • the DNA vaccines used were: . PBS control .
  • String 3 (DNA encoding SARS-CoV-2 M7 RBD, ancestor MERS M7 RBD, ancestor SARS-CoV-1 M7 RBD); .
  • Mix String 3 mixture of individual plasmids encoding the constituents of the String3 construct, injected at 3 different sites); .
  • Individual String 3 (individual plasmids encoding the constituents of the String3 construct, injected at 3 different sites simultaneously); .
  • PanMERS DNA encoding ancestor MERS M7 RBD
  • PanSARS1 DNA encoding ancestor SARS1 M7 RBD
  • PanSARS2 DNA encoding SARS2 M7 RBD. The results are shown in Figure 24.
  • Figure 24 shows bleed 5 neutralisation of SARS CoV-1 PV for sera collected from the mice immunised with the vaccines listed above.
  • the individual lines of Figure 24 represent each mouse tested, and each dot shows neutralisation values for the mouse at the respective serum dilution.
  • the results show that the Mix String 3 vaccine construct elicited a very strong and consistent neutralisation response.
  • a strong neutralisation response was also seen for the individual RBD constituents of String 3 (Individual String 3), and for the String 3 vaccine construct.
  • mice were immunised with the different RBD DNA vaccines, then sera was collected and tested for SARS-CoV-2 pseudovirus neutralisation.
  • the DNA vaccines used were: . PBS control .
  • String 3 (DNA encoding SARS-CoV-2 M7 RBD, ancestor MERS M7 RBD, ancestor SARS-CoV-1 M7 RBD); .
  • Mix String 3 (mixture of individual plasmids encoding the constituents of the String3 construct, injected at 3 different sites); .
  • Individual String 3 individual plasmids encoding the constituents of the String3 construct, injected at 3 different sites simultaneously); .
  • PanMERS (DNA encoding ancestor MERS M7 RBD); .
  • PanSARS1 (DNA encoding ancestor SARS1 M7 RBD); .
  • PanSARS2 (DNA encoding SARS2 M7 RBD).
  • Figure 25 shows bleed 5 neutralisation assay of original Wuhan SARS CoV-2 strain using sera collected from the mice immunised with the vaccines listed above.
  • the individual lines of Figure 25 represent each mouse tested, and each dot shows neutralisation values for the mouse at the respective serum dilution.
  • the results show that the String 3 vaccine construct generates a neutralisation response against SARS-CoV-2 PV.
  • the Mix String 3 and Individual String 3 vaccine constructs show strong neutralisation of SARS-CoV-2 PV.
  • Example 10 WIV-16 (SARS-CoV-1 like ⁇ -coronavirus) pseudovirus neutralisation after immunisation with vaccine constructs
  • Figure 26 shows antibody neutralisation of WIV-16 pseudovirus after immunisation with the vaccine constructs listed below. Mice were immunised with the different RBD DNA vaccines, then sera were collected and tested for WIV-16 pseudovirus neutralisation. The DNA vaccines used were: . PBS control .
  • String 3 (DNA encoding SARS-CoV-2 M7 RBD, ancestor MERS M7 RBD, ancestor SARS-CoV-1 M7 RBD); .
  • Mix String 3 mixture of individual plasmids encoding the constituents of the String 3 construct, injected at 3 different sites); .
  • Individual String 3 (individual plasmids encoding the constituents of the String 3 construct, injected at 3 different sites simultaneously); .
  • PanMERS DNA encoding ancestor MERS M7 RBD
  • PanSARS DNA encoding ancestor SARS1 M7 RBD
  • PanSARS2 DNA encoding SARS2 M7 RBD. The results are shown in Figure 26.
  • Figure 26 shows WIV-16 neutralisation assay using sera collected from the mice immunised with the vaccines listed above at bleed 5.
  • the individual lines of Figure 26 represent each mouse tested, and each dot shows neutralisation values for the mouse at the respective serum dilution.
  • the results show that the String 3, Mix String 3, and Individual String 3 vaccine constructs all elicited a strong WIV16 PV neutralising antibody response.
  • the results show that the neutralising antibody response elicited by the String 3 vaccine construct was comparable to the responses elicited by the Mix String 3, and Individual String 3 vaccine constructs, although the Mix String 3 response was superior.
  • panSARS1 vaccine also elicited a strong neutralising effect, and the SARS2 M7 RBD (panSARS2) and MERS M7 RBD (panMERS) vaccine constructs showed no effect.
  • SARS2 M7 RBD panSARS2 M7 RBD
  • MERS M7 RBD panMERS
  • Example 11 pEVAC Expression Vector DNA sequences encoding the antigens are optimised for expression in mammalian cells before inserting into a DNA plasmid expression vector, such as pEVAC.
  • the pEVAC vector is a flexible vaccine platform and any combination of antigens can be inserted to produce a different vaccine.
  • a previous version was used in a SARS-1 clinical trial (Martin et al, Vaccine 200825:633). This platform is clinically proven and GMP compliant allowing rapid scale-up.
  • the DNA vaccine may be administered using pain-free needle-free technology causing patients’ cells to produce the antigens, which are recognised by the immune system to induce durable protection against beta-coronavirus.
  • Figure 6 shows a map of the pEVAC expression vector. The sequence of the multiple cloning site of the vector is given below, followed by its entire nucleotide sequence.
  • ACE-2 angiotensin-converting enzyme 2
  • Bats are the reservoir of a large number of SARS-CoV-like ACE-2 binding sarbecoviruses which pose a constant threat for future spill-over into human population, and potentially new epidemics 3,4 .
  • VOCs variants of concerns
  • RBD receptor binding domain
  • the delta VOC 11 was at one point the most contagious variant reported to date, with L452R and T478K mutations, while the Omicron has multiple mutations (Fig.32A and 32B) in the RBD.
  • the majority of these mutations reported in VOCs are in or around the region in RBD that interacts with ACE-2 as well as the regions that induce highly potent neutralising antibodies 12,13 .
  • This design was further used as a backbone for designing both epitope optimised and immune re-focussed designs using available structural data from a number of high-quality structural data available for spike protein in complex with monoclonal antibodies, specifically those targeting the ACE-2 receptor binding domain (RBD), such as S309 14 and CR3022 15 that bind both SARS-CoV-1 and SARS-CoV-2.
  • RBD ACE-2 receptor binding domain
  • the nucleic acid sequence of these in silico designed vaccine antigens were optimised for expression in humans and synthetic genes expressing each unique antigen structure was shuttled in an expression cassette for consecutive in vitro and in vivo screens in different species to select the best-in-class immunologically optimal antigen as the vaccine candidate for nucleic acid vaccine delivery.
  • T2_13 To further understand the importance of amino-acid composition of epitopes in generating antibody responses, we further modified T2_13 to display the epitopes of SARS- CoV-1 for monoclonal antibodies - S309 14 (T2_14), and CR3022 15 (T2_15) and of SARS- CoV-2 for monoclonal antibody - B38 12 (T2_16).
  • the sequence of epitopes for monoclonal antibodies - S309 14 , and CR3022 15 are highly conserved across the sequences considered in this study while the sequence of epitopes for monoclonal antibody - B38 12 is highly divergent (Fig.27B).
  • T2_13 and T2_17 membrane bound forms of T2_13 and T2_17 (T2_19 (also denoted T2_13_TM) and T2_20 (also denoted T2_17_TM) respectively) were generated.
  • T2_19 also denoted T2_13_TM
  • T2_20 also denoted T2_17_TM
  • Fig.27C Structural models of these vaccine antigens.
  • In vivo screening in BALB/c mice was performed by immunising different the lead antigen designs and assaying for cross reactive antibodies against different sarbeco Spike proteins by FACS cell-surface display (Fig.27D).
  • Sera taken two weeks following the second immunisation with antigen designs (T2_13 through to T2_18 and the membrane bound designs T2_13_TM and T2_17_TM), demonstrated the binding profile of the vaccine candidates for different spike proteins (Fig.27E).
  • sera from SARS-CoV-1 RBD immunised mice bound strongly to both homologous SARS-CoV-1 spike protein and closely related WIV16 spike in comparison to other vaccine designs, while sera from SARS-CoV-2 RBD immunised mice binds homologous SARS-CoV-2 spike protein and closely related RaTG13 spike protein in the similar range of other vaccine designs.
  • T2_16 design in which the epitope region for mAb B38 was matched identical to SARS- CoV-2, binding to SARS-CoV-1, WIV16, and RaTG13 declined in comparison to T2_13 (Mann-Whitney U test, p-value ⁇ 0.05) without statistical changes in binding to SARS-CoV-2. This observation is suggestive of immunodominance of this region in comparison to other sites. Matching of the epitopes of S309 and CR3022 to SARS-CoV-1 (T2_14 and T2_15), enhanced the binding to SARS-CoV-1 (Mann-Whitney U test, p-value ⁇ 0.05) but not to other spike proteins.
  • T2_17 significantly enhanced the binding of SARS-CoV-1 and RaTG13 (Mann-Whitney U test, p-value ⁇ 0.01) in comparison to T2_14, but no difference is observed in T2_18 in comparison to T2_15. There was no difference between trans-membrane and non- trans-membrane bound designs. As T2_17 has either the best (or second best) median binding to the four spike proteins apart from the homologous RBD vaccine antigen, we chose T2_17 as the lead candidate for further immunological assays.
  • T2_17 Elicitation of cross-binding antibodies by T2_17 was further confirmed by ELISA with SARS- CoV-1 RBD and SARS-CoV-2 RBD (Fig.27F) revealing robust antibody responses to both SARS-CoV-1 and SARS-CoV-2 within two weeks of the second immunisation. While the T2_17 antigen elicited stronger responses against SARS-CoV-1, it was lower than those induced by the homologous SARS-CoV-1 antigen, but significantly higher than SARS-CoV- 2. against SARS-CoV-2, all the three antigens – SARS-CoV-1 RBD, SARS-CoV-2 RBD, and T2_17 generated similar binding antibody responses.
  • T2_17 and SARS2_RBD_P521N generated binding antibodies against both SARS-CoV-1 and SARS-CoV-2 (Fig.28C) after one immunisation
  • T2_17 elicited significantly higher antibodies than SARS2_RBD_P521N to SARS-CoV-1 and comparable antibodies against SARS-CoV-2.
  • Higher binding antibodies were detected for T2_17 to SARS-CoV-1 in comparison to SARS2_RBD_P521N after two immunisations while the responses were comparable for SARS-CoV-2.
  • SARS2_RBD_P521N had developed a bias response to SARS-CoV-2, while T2_17 had higher responses to SARS-CoV.
  • a GMP lot of pEVAC T2_17 was manufactured and evaluated for safety and immunogenicity in rabbits using the same gene delivery device to ensure uniform intradermal administration (Fig.28G). After one immunisation, binding antibodies to SARS-CoV-1 and SARS-CoV-2 were elicited (Fig.28H), increasing on subsequent immunisations until a plateau was reached by the fourth immunisation.
  • pan-sarbecovirus vaccine antigen candidates were immune selected to elicit a broad neutralisation profile demonstrated in 3 species against SARS-CoV-1, WIV16, RaTG13, SARS-CoV-2 and it’s VOCs.
  • the success of this vaccine antigen strategy for broader vaccine protection was further corroborated by the efficacy of the rabbit sera to neutralise all the variants of concern, which emerged much later in the pandemic and were not used as input in our design pipeline.
  • the epitopes that were modified to match the wild-type SARS-CoV-1 (coloured orange) and wild-type SARS-CoV-2 (coloured grey) are represented in spheres. Further glycosylation site modification is represented in green sphere.
  • the X-axis represents the mean fluorescence intensity (MFI), and the Y-axis represents all the vaccine designs considered for screening.
  • mice were challenged after 8 weeks with either Victoria strain of SARS-CoV-2 or the Delta variant.
  • I Elicitation of binding antibodies against SARS-CoV-1 and SARS-CoV-2 before challenge was confirmed using ELISA using K18 hACE2 mice sera 4 weeks post boost (bleed4).
  • Boost by T2_17, and SARS2_RBD (not shown) significantly increased the binding antibody titres in comparison to boost by AZD1222.
  • the X-axis represents the vaccine designs, and the Y-axis represents the area under the curve (AUC) for ELISA binding curves.
  • J Neutralisation of SARS-CoV-1, SARS-CoV-2, and Delta variant of SARS-CoV-2 by K18 hACE2 mice sera 4 weeks post boost (bleed4).
  • mice boosted with T2_17 significantly neutralised the Delta variant (B.1.617.2) in comparison to those boosted by AZD1222.
  • the X-axis represents the bleed number, and the Y-axis represents the log 10 IC 50 values for neutralisation curves.
  • K Weight loss profile of K18 hACE2 mice following challenge by the Victoria strain and the Delta variant. All the mice, except na ⁇ ve were protected. Mann-Whitney U test is used as statistical significance test in all the plots (p-value: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001).
  • Figure 28 - Immunogenicity studies in Guinea pigs and Rabbits A. Immunisation and bleed schedule of Guinea pigs.
  • Guinea pigs were immunised using DNA delivered intradermally (i.d) by the Tropis ParmaJet device at 28 day intervals and bled every 14 days (although Figure 28A indicates immunisation at 30 day intervals and bleeding every 15 days).
  • B Structure models of the vaccine designs used for the study in Guinea pigs. The glycosylation site and the modified epitope are represented as green and orange spheres respectively.
  • C Elicitation of binding antibodies against SARS-CoV-1 and SARS- CoV-2 by T2_17 and SARS2_RBD_P521N was confirmed using ELISA. T2_17 and SARS2_RBD_P521N generated cross-binding antibodies after one immunisation.
  • the pre- bleed (Bleed 0) is considered as the control for non-specific binding.
  • the X-axis represents the bleed number, and the Y-axis represents the area under the curve (AUC) for ELISA binding curve.
  • D. Neutralisation by Guinea pig sera immunised with T2_17 and SARS2_RBD_P521N. Both T2_17 and SARS2_RBD_P521N generated neutralising antibodies against SARS-CoV-1 and SARS-CoV-2.
  • the X-axis represents the bleed number, and the Y-axis represents the log10IC50 values for neutralisation curves.
  • T2_17 generated cross-binding antibodies after one immunisation.
  • the X-axis represents the bleed number, and the Y-axis represents the area under the curve (AUC) for ELISA binding curve.
  • AUC area under the curve
  • the X-axis represents the bleed number
  • the Y-axis represents the log 10 IC 50 values for neutralisation curves.
  • IQ-TREE A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol. Biol. Evol.32, 268–274 (2015). 23. Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res.44, W344–W350 (2016). 24. Schrodinger, L. & DeLano, W. PyMOL. (2020). 25. Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res.28, 235–242 (2000). 26. Martinez, D. R. et al. Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice.
  • BioRxiv Prepr. Serv. Biol.2021.03.11.434872 (2021) doi:10.1101/2021.03.11.434872. 27. Walls, A. C. et al. Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines. Cell (2021) doi:10.1016/j.cell.2021.09.015.
  • Methods Phylogenetic analysis Protein sequences of spike proteins were downloaded from the NCBI virus database for the sarbecoviruses. Multiple sequence alignment (MSA) was generated using the MUSCLE algorithm 1 . The resulting MSA was pruned to the RBD region and used as input for phylogenetic tree reconstruction.
  • MSA Multiple sequence alignment
  • the phylogenetic tree was generated using IQTREE algorithm 2 using protein model with best AIC score. The resultant tree was used for generation of phylogenetically optimised design using HyPhy algorithm 3 .
  • Epitope identification Available structural data for Spike protein-antibody complexes for SARS-CoV-1 and SARS- CoV-2 were downloaded from the Protein Databank (PDB) 4 . Structural data were then pruned for antigen-antibody complexes where the epitopes are on the RBD. Amino acid residues of antigen that have at least one atom within 5 ⁇ radii of at least one atom of amino acid of antibody are defined as epitope residues. And epitope regions are defined as contiguous stretches of at least 5 amino acids.
  • Fluorescence assisted cell sorting (FACS) assay HEK293T cells were transfected with an expression plasmid expressing wild-type Spike glycoprotein of each of the four ACE-2 binding sarbecoviruses including SARS-CoV-1, SARS--CoV-2, RaTG13, and WIV16.48 hours after transfection, cells were transferred into V-bottom 96-well plates (50,000 cells/well).
  • FACS Fluorescence assisted cell sorting
  • ELISA Enzyme-linked immunosorbent assay
  • mice 11 groups of female 8–10-week-old BALB/c mice were purchased from Charles River Laboratories (Kent, United Kingdom). Mice were immunised a total of four times with 30 days intervals. A total volume of 50 ⁇ l of PBS containing 50 ⁇ g of plasmid DNA was administered via subcutaneous route in the rear flank. Blood was sampled from the saphenous vein at 15 days intervals, and animals were terminally bled by cardiac puncture under non-recovery anaesthesia at day 150. Vaccine boost efficacy studies in K18 hACE2 mice.
  • mice Ten groups of six female 8–15-week-old homozygous K18-hACE2 mice (Jax) were primed with 1.4x10 9 viral particles of AZD1222 or PBS by intramuscular route, in a total volume of 100 ⁇ l split over the two rear legs. After 28 days, two groups of six mice were boosted with PBS, AZD1222, or T2_17 DNA. Mice were bled at two-week intervals and challenged at day 84 with either Victoria/1/2020 (B-type) or Delta SARS-CoV-2 by intranasal route, in a total volume of 40 ⁇ l over both nares. Mice were weighed daily and monitored for clinical signs for a period of 10 days before being culled by terminal bleed.
  • B-type Victoria/1/2020
  • Delta SARS-CoV-2 Delta SARS-CoV-2
  • Intradermal nucleic acid immunisation with Tropis PharmaJet delivery in Guinea pigs Two groups of female 7-week-old Dunkin Hartley Guinea pigs ( Envigo RMS, Blackthorn, United Kingdom) were immunised a total of three times with 30 or 28 day intervals. A total volume of 200 ⁇ l of PBS containing 400 ⁇ g of plasmid DNA was administered by PharmaJet Tropis intradermal device.100 ⁇ l was administered to each hind leg. Blood was sampled from the saphenous vein at 15 or 14 day intervals. Intradermal nucleic acid immunisation with Tropis PharmaJet delivery in in Rabbits.
  • serum from bleed 6 guinea pigs were diluted in PBS across an 8 point 1:2 dilution series from a starting concentration of 1:50.
  • Samples were further diluted in the provided sample buffer at a 1:9 ratio, and then mixed with HRP conjugated to SARS-CoV-2 RBD protein, incubated at 37°C for 30 min and added to human ACE-2 protein coated wells in 96-well plate format.
  • the reaction was incubated at 37°C for 15 min and then washed four times with provided wash buffer.
  • TMB solution was then added, incubated for 15 minutes in the dark at R.T to allow the reaction to develop.
  • the reaction was then quenched using the provided stop solution, and then absorbance read at 450 nm.
  • FIG. 30A ELISA binding data of K18 hACE2 sera Binding antibodies were observed 4 weeks post immunisation with AZD1222 and 4 weeks post boosting with different AZD1222/T2_17/SARS2_RBD.
  • Figure 30B Neutralisation data of K18 hACE2 sera Neutralising antibodies against SARS-CoV-1 and delta variant of SARS-CoV-1 were observed two-week post boost (bleed 3) and the levels were maintained 6 weeks post boost (bleed 5).
  • Figure 32B Multiple sequence alignment of the variants of concern Multiple sequence alignment of the variants of concern with the ancestral sequence (Wuhan- Hu-1 isolate). The conserved amino acids in reference to the ancestral sequence is represented as dots.
  • T2_17 + pEVAC Expression Vector (SEQ ID NO:41) This example provides the nucleic acid sequence encoding the T2_17 vaccine construct (amino acid sequence SEQ ID NO:11) within the pEVAC expression vector.
  • Sequence COV_N_T2_2 was designed using a methodology and algorithm which selected predicted epitopes to include based on their conservation across the sarbecoviruses (whilst minimising redundancy), the frequency and number of MHC alleles the epitope is restricted by the predicted epitope quality, and a handful of user specified weightings.
  • nucleocapsid phosphoprotein [SARS-CoV-2] (reference sequence) (SEQ ID NO:42) MSDNGPQ-NQ RNAPRITFGG PSDSTGSNQN GERSGARSKQ RRPQGLPNNT ASWFTALTQH GKEDLKFPRG QGVPINTNSS PDDQIGYYRR ATRRIRGGDG KMKDLSPRWY FYYLGTGPEA GLPYGANKDG IIWVATEGAL NTPKDHIGTR NPANNAAIVL QLPQGTTLPK GFYAEGSRGG SQASSRSSSR SRNSSRNSTP GSSRGTSPAR MAGNGGDAAL ALLLLDRLNQ LESKMSGKGQ QQQGQTVTKK SAAEASKKPR QKRTATKAYN VTQAFGRRGP EQTQGNFGDQ ELIRQGTDYK HWPQIAQFAP SASAFFGMSR IGMEVTPSGT WLTYTGAIKL
  • Positions 415 and 416 are italicised as they are not residues of the reference sequences, but include insertions in the N_T2_1 and N_T2_2 sequences.
  • the amino acid changes common to both of the designed sequences are summarised in the table below: Table 3.2
  • Example 15 Clinical Trial Design The following clinical trial design protocol is directed to immunisation with pEVAC_T2_17 (SEQ ID NO:41).
  • the study will consist of thirty six volunteers that are SARS-CoV-2 PCR and/or, Nucleocapsid antibody negative healthy human volunteers enrolled for this trial, who agree to self-isolate and report back during the two immunisations, in order to demonstrate safety and immunogenicity.
  • the first of 3 study Groups will consist of: .
  • Group 1 9 volunteers receiving 2 administrations of 0.2mg with 28 days between administration 1 and 2 .
  • Group 2 9 volunteers receiving 2 administrations of 0.4mg with 28 days between administration 1 and 2
  • Group 3 9 volunteers receiving 2 administrations of 0.8mg with 28 days between administration 1 and 2
  • Group 4 9 volunteers receiving 2 administrations of 1.2mg with 28 days between administration 1 and 2 All will receive the vaccine administered by the PharmaJet needless delivery system, which minimises the barriers to people taking the vaccine. Power calculations are based on an estimated standard deviation of 0.27 log10 units, using the ELISA data from the SARS clinical Trial (Martin et al, Vaccine, 2008). The primary endpoint will be safety immunisation (complete safety data for 28 days), while the secondary endpoints will be analysed when the last volunteer has completed 3 months following primary, and immunogenicity primary and key secondary endpoints to 3 months).
  • Example 16 Clinical Trial Design The following clinical trial design protocol is directed to immunisation with GMP lot pEVAC_T2_17 (pEVAC_PS) (SEQ ID NO:41). Study design overview This is an adaptive, open label phase I dose escalation study of the safety, tolerability, immunogenicity and intra-dermal administration dose finding for the pEVAC_PS vaccine candidate against SARS-CoV and SARS-CoV-2. This study will recruit up to 36 healthy volunteers in 4 groups.
  • All volunteers recruited will be healthy, male or non-pregnant female adults aged between 18 and 50 years who have been vaccinated with 2 or 3 doses of a SARS- CoV-2 vaccine, optimally with the most recent dose at least 84 days prior to day 0, and have no history of infection and are seronegative for Nucleocapsid IgG (determined at screening).
  • Group 1 9 volunteers receiving 2 administrations of 0.2mg with 28 days between administration 1 and 2
  • Group 2 9 volunteers receiving 2 administrations of 0.4mg with 28 days between administration 1 and 2
  • Group 3 9 volunteers receiving 2 administrations of 0.8mg with 28 days between administration 1 and 2
  • Group 4 9 volunteers receiving 2 administrations of 1.2mg with 28 days between administration 1 and 2

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pulmonology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des vaccins qui induisent une réponse immunitaire largement neutralisante pour protéger contre une maladie provoquée par des bêta-coronavirus, plus particulièrement des bêta-coronavirus des lignées sarbecovirus et merbecovirus. Les vaccins sont fournis par l'utilisation de sous-unités RBD de sarbecovirus et de merbecovirus optimisées sur le plan phylogénétique et épitopique. Les vaccins sont fournis en tant que vaccins à acides nucléiques, soit en tant que polynucléotides séparés, chacun codant pour une sous-unité RBD différente (ou dimère RBD), ou rattachés ensemble dans une chaîne en tant que polynucléotide unique codant pour la totalité des sous-unités RBD (ou dimères RBD). Les polynucléotides séparés peuvent être administrés en mélange, co-administrés ou administrés séquentiellement dans n'importe quel ordre. L'invention concerne également des polynucléotides, des polypeptides, des vecteurs, des cellules, des protéines de fusion, des compositions pharmaceutiques, des préparations combinées et leur utilisation en tant que vaccins contre des virus de la famille des coronavirus. L'invention concerne également des procédés d'induction d'une réponse immunitaire à, ou l'immunisation d'un sujet contre, un bêta-coronavirus chez un sujet par l'administration d'une quantité efficace d'un vaccin au sujet.
PCT/GB2022/051663 2021-06-28 2022-06-28 Vaccins à coronavirus bêta WO2023275538A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
GB2109285.3 2021-06-28
GBGB2109285.3A GB202109285D0 (en) 2021-06-28 2021-06-28 Beta-coronavirus vaccines
GBGB2112499.5A GB202112499D0 (en) 2021-09-02 2021-09-02 Beta-coronavirus vaccines
GB2112499.5 2021-09-02
GBGB2114321.9A GB202114321D0 (en) 2021-10-06 2021-10-06 Beta-coronavirus Caccines
GB2114321.9 2021-10-06
GBGB2201060.7A GB202201060D0 (en) 2022-01-27 2022-01-27 Beta-coronavirus vaccines
GB2201060.7 2022-01-27

Publications (1)

Publication Number Publication Date
WO2023275538A1 true WO2023275538A1 (fr) 2023-01-05

Family

ID=82547322

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2022/051663 WO2023275538A1 (fr) 2021-06-28 2022-06-28 Vaccins à coronavirus bêta

Country Status (1)

Country Link
WO (1) WO2023275538A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014045254A2 (fr) * 2012-09-23 2014-03-27 Erasmus University Medical Center Rotterdam Lignée c de coronavirus bêta humains et identification de la peptidase dipeptidylique n-terminale en tant que récepteur viral
US20180244756A1 (en) * 2015-02-24 2018-08-30 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services Middle east respiratory syndrome coronavirus immunogens, antibodies, and their use
WO2020065349A2 (fr) * 2018-09-28 2020-04-02 The Chancellor, Masters And Scholars Of The University Of Cambridge Vaccins et méthodes
US10702600B1 (en) 2015-10-22 2020-07-07 Modernatx, Inc. Betacoronavirus mRNA vaccine
WO2021045836A1 (fr) * 2020-04-02 2021-03-11 Regeneron Pharmaceuticals, Inc. Anticorps anti-glycoprotéine spike du sars-cov 2 et fragments de liaison à l'antigène de ceux-ci
WO2021072501A1 (fr) * 2019-10-17 2021-04-22 Monash University Procédés de détection de réponse immunitaire

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014045254A2 (fr) * 2012-09-23 2014-03-27 Erasmus University Medical Center Rotterdam Lignée c de coronavirus bêta humains et identification de la peptidase dipeptidylique n-terminale en tant que récepteur viral
US20180244756A1 (en) * 2015-02-24 2018-08-30 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services Middle east respiratory syndrome coronavirus immunogens, antibodies, and their use
US10702600B1 (en) 2015-10-22 2020-07-07 Modernatx, Inc. Betacoronavirus mRNA vaccine
WO2020065349A2 (fr) * 2018-09-28 2020-04-02 The Chancellor, Masters And Scholars Of The University Of Cambridge Vaccins et méthodes
WO2021072501A1 (fr) * 2019-10-17 2021-04-22 Monash University Procédés de détection de réponse immunitaire
WO2021045836A1 (fr) * 2020-04-02 2021-03-11 Regeneron Pharmaceuticals, Inc. Anticorps anti-glycoprotéine spike du sars-cov 2 et fragments de liaison à l'antigène de ceux-ci

Non-Patent Citations (71)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410, Retrieved from the Internet <URL:http://www.ebi.ac.uk/fasta>
ALTSCHUL ET AL., NATURE GENET, vol. 6, 1994, pages 119 - 129
AMANAT, F. ET AL.: "A serological assay to detect SARS-CoV-2 seroconversion in humans", NAT. MED., vol. 26, 2020, pages 1033 - 1036, XP037191537, DOI: 10.1038/s41591-020-0913-5
ASHKENAZY, H. ET AL.: "ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules", NUCLEIC ACIDS RES, vol. 44, 2016, pages W344 - W350
BADEN ET AL.: "Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine", N. ENGL. J. MED., vol. 384, 2021, pages 403 - 416
BERMAN, H. M. ET AL.: "The Protein Data Bank", NUCLEIC ACIDS RES, vol. 28, 2000, pages 235 - 242
CAMPANELLA ET AL., BMC BIOINFORMATICS, vol. 4, 2003, pages 29, Retrieved from the Internet <URL:http://bitincka.com/ledion/matgat>
CAMPBELL, F. ET AL.: "Increased transmissibility and global spread of SARS-CoV-2 variants of concern as at June 2021", EUROSURVEILLANCE, vol. 26, 2021, pages 2100509
CARNELL, G.GREHAN, K.FERRARA, F.MOLESTI, E.TEMPERTON, N. J.: "An Optimised Method for the Production using PEI, Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes", BIO-PROTOC, vol. 7, 2017
CHOICHANG, CLINICAL AND EXPERIMENTAL VACCINE RESEARCH, vol. 2, 2013, pages 97 - 105
CORPET ET AL., NUCLEIC ACIDS' RESEARCH, vol. 16, 1988, pages 10881 - 10890
CRAVEN: "COVID-19 Vaccine Tracker", NEWS ARTICLES, 2020, pages 3, Retrieved from the Internet <URL:https://www.raps.org/news-and-articles/news-articles/2020/3/covid-19-vaccine-tracker>
DAI ET AL.: "A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS", CELL, vol. 182, 6 August 2020 (2020-08-06), pages 722 - 733
DAI LIANPAN ET AL: "A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS", CELL, ELSEVIER, AMSTERDAM NL, vol. 182, no. 3, 28 June 2020 (2020-06-28), pages 722, XP086239965, ISSN: 0092-8674, [retrieved on 20200628], DOI: 10.1016/J.CELL.2020.06.035 *
DICKEY THAYNE H. ET AL: "Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response", BIORXIV, 10 May 2021 (2021-05-10), XP055915754, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2021.05.09.443238v1.full.pdf> [retrieved on 20220426], DOI: 10.1101/2021.05.09.443238 *
E. W. MARTIN: "Remington's Pharmaceutical Sciences", 1975, MACK PUBLISHING CO.
EDGAR, R. C.: "MUSCLE: a multiple sequence alignment method with reduced time and space complexity", BMC BIOINFORMATICS, vol. 5, 2004, pages 113, XP021000496, DOI: 10.1186/1471-2105-5-113
EGGINK, D.GOFF, P. H.PALESE, P.: "Guiding the immune response against influenza virus hemagglutinin toward the conserved stalk domain by hyperglycosylation of the globular head domain", J. VIROL., vol. 88, 2014, pages 699 - 704, XP055480641, DOI: 10.1128/JVI.02608-13
ESWAR, N. ET AL.: "Curr. Protoc. Protein Sci.", 2007, article "Comparative protein structure modeling using MODELLER"
GARCIA-BELTRAN ET AL., CELL, vol. 184, 2021, pages 2372 - 2383
HATCHER, E. L. ET AL.: "Virus Variation Resource - improved response to emergent viral outbreaks", NUCLEIC ACIDS RES, vol. 45, 2017, pages D482 - D490
HIGGINSSHARP, CABIOS, vol. 5, 1989, pages 151 - 153
HIGGINSSHARP, GENE, vol. 73, 1988, pages 237 - 244
HORSPOOL, A. M. ET AL.: "SARS-CoV-2 B.1.1.7 and B. 1.351 variants of concern induce lethal disease in K18-hACE2 transgenic mice despite convalescent plasma therapy", BIORXIV PREPR. SERVO BIOL., 2021
HOU ET AL., NATURE REVIEWS MATERIALS, 2021, Retrieved from the Internet <URL:https://doi.org/10,1Q38/s41578-Q21-00358-0>
HU, B. ET AL.: "Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus", PLOS PATHOG, vol. 13, 2017, pages e1006698
HUBERT, B., THE CUREVAC VACCINE, AND A BRIEF TOUR THROUGH SOME OF THE WONDERS OF NATURE, 2021, Retrieved from the Internet <URL:https://berthub.eu/articles/posts/curevac-vaccine-and-wonders-of-biology>
HUNTER, J. D.: "Matplotlib: A 2D Graphics Environment", COMPUT. SCI. ENG., vol. 9, 2007, pages 90 - 95
HWANG, W. C. ET AL.: "Structural Basis of Neutralization by a Human Anti-severe Acute Respiratory Syndrome Spike Protein Antibody", J. BIOL. CHEM., vol. 281, 2006, pages 34610 - 34616, XP055598159, DOI: 10.1074/jbc.M603275200
KRIVOV, G. G.SHAPOVALOV, M. V.DUNBRACK, R. L.: "Improved prediction of protein side-chain conformations with SCWRL4: Side-Chain Prediction with SCWRL4", PROTEINS STRUCT. FUNCT. BIOINFORMA., vol. 77, 2009, pages 778 - 795, XP055643151, DOI: 10.1002/prot.22488
KRUSKALADDISON WESLEY, TIME WARPS, STRING EDITS AND MACROMOLECULES: THE THEORY AND PRACTICE OF SEQUENCE COMPARISON, 1983, pages 1 - 44, Retrieved from the Internet <URL:http://www.ebi.ac.uk/tools/emboss/align>
L. DAI, CELL, 2020
L. DU, NAT. COMM, vol. 7, 2016
LARKIN ET AL., BIOINFORMATICS, vol. 23, 2007, pages 2947 - 2948, Retrieved from the Internet <URL:http://www.ebi.ac.uk/tools/clustalw2>
LATINNE: "Origin and cross-species transmission of bat coronaviruses in China", NATURE COMMUNICATIONS, vol. 11, 2020, pages 4235, Retrieved from the Internet <URL:https://doi.orq/10.1038/s41467-020-17687-3>
LAVIE, M.HANOULLE, X.DUBUISSON, J.: "Glycan Shielding and Modulation of Hepatitis C Virus Neutralizing Antibodies", FRONT. IMMUNOL., vol. 9, 2018, pages 910
LEUNG, K.SHUM, M. H.LEUNG, G. M.LAM, T. T.WU, J. T.: "Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom", EURO SURVEILL. BULL. EUR. SUR MAL. TRANSM. EUR. COMMUN. DIS. BULL., vol. 26, no. 2021, October 2020 (2020-10-01)
LI, W.: " Animal Origins of the Severe Acute Respiratory Syndrome Coronavirus: Insight from ACE2-S-Protein Interactions", J. VIROL., vol. 80, 2006, pages 4211 - 4219
LIU ET AL., SCIENTIFIC REPORTS, vol. 7, 2017
LIU, K. ET AL.: "Cross-species recognition of SARS-CoV-2 to bat ACE2", PROC. NATL. ACAD. SCI. U. S. A., 2021, pages 118
MARTIN ET AL., VACCINE, vol. 25, 2008, pages 633
MARTINEZ, D. R. ET AL.: "Chimeric spike mRNA vaccines protect against Sarbecovirus challenge in mice", BIORXIV PREPR. SERV. BIOL., 2021
MENACHERY, V. D. ET AL.: "A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence", NAT. MED., vol. 21, 2015, pages 1508 - 1513, XP037143869, DOI: 10.1038/nm.3985
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
NGUYEN, L.-T.SCHMIDT, H. A.VON HAESELER, A.MINH, B. Q.: "IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies", MOL. BIOL. EVOL., vol. 32, 2015, pages 268 - 274
OLIVAL, K. J. ET AL.: "Possibility for reverse zoonotic transmission of SARS-CoV-2 to free-ranging wildlife: A case study of bats", PLOS PATHOG, vol. 16, 2020, pages e1008758
PARDI ET AL., NATURE REVIEWS DRUG DISCOVERY, vol. 17, 2018, pages 261 - 279
PEARSONLIPMAN, PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 2444
PEDREGOSA, F. ET AL.: "Scikit-learn: Machine Learning in Python", J. MACH. LEARN. RES., vol. 12, 2011, pages 2825 - 2830
PINTO, D. ET AL.: "Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody", NATURE, vol. 583, 2020, pages 290 - 295, XP037289888, DOI: 10.1038/s41586-020-2349-y
PLANAS, D. ET AL.: "Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B. 1.351 variants to neutralizing antibodies", NAT. MED., vol. 27, 2021, pages 917 - 924, XP037452990, DOI: 10.1038/s41591-021-01318-5
POLACK ET AL.: "Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine", N. ENGL. J. MED., vol. 383, 2020, pages 2603 - 2615, XP055820495, DOI: 10.1056/NEJMoa2034577
POND, S. L. K.FROST, S. D. W.MUSE, S. V.: "HyPhy: hypothesis testing using phylogenies", BIOINFORMATICS, vol. 21, 2005, pages 676 - 679
RAAB, D.GRAF, M.NOTKA, F.SCHODL, T.WAGNER, R.: "The GeneOptimizer 518 Algorithm: using a sliding window approach to cope with the vast sequence space in 519 multiparameter DNA sequence optimization", SYST. SYNTH. BIOL., vol. 4, 2010, pages 215 - 225, XP055065511, DOI: 10.1007/s11693-010-9062-3
SALI, A.BLUNDELL, T. L.: "Comparative protein modelling by satisfaction of spatial restraints", J. MOL. BIOL., vol. 234, 1993, pages 779 - 815, XP024008717, DOI: 10.1006/jmbi.1993.1626
SCHRODINGER, L.DELANO, W., PYMOL, 2020
SCHYMKOWITZ, J. ET AL.: "The FoldX web server: an online force field", NUCLEIC ACIDS RES, vol. 33, 2005, pages W382 - W388, XP055064918, DOI: 10.1093/nar/gki387
SHAIMARDANOVA ET AL., PHARMACEUTICS, vol. 11, 2019, pages 580
SHEN, M.SALI, A.: "Statistical potential for assessment and prediction of protein structures", PROTEIN SCI. PUBL. PROTEIN SOC., vol. 15, 2006, pages 2507 - 2524
SMITHWATERMAN, ADV. APPL. MATH., vol. 2, 1981, pages 482
TIAN, F. ET AL.: "Mutation N501Y in RBD of Spike Protein Strengthens the Interaction between COVID-19 and its Receptor ACE2", BIORXIV 2021.02.14.431117, 2021
URA ET AL., VACCINES, vol. 2, 2014, pages 624 - 641
VAN DER SPOEL, D. ET AL.: "GROMACS: fast, flexible, and free", J. COMPUT. CHEM., vol. 26, 2005, pages 1701 - 1718, XP055028875, DOI: 10.1002/jcc.20291
VILAR, S.ISOM, D. G.: "One Year of SARS-CoV-2: How Much Has the Virus Changed?", BIOLOGY, 2021, pages 10
WALLS, A. C. ET AL.: "Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines", CE, 2021
WANG, P. ET AL.: "Increased resistance of SARS-CoV-2 variant P.1 to antibody neutralization", CELL HOST MICROBE, vol. 29, 2021, pages 747 - 751
WANG: "mRNA vaccine: a potential therapeutic strategy", MOLECULAR CANCER, vol. 20, 2021, pages 33, XP055907500, DOI: 10.1186/s12943-021-01311-z
WATANABE, Y. ET AL.: "Structure of the Lassa virus glycan shield provides a model for immunological resistance", PROC. NATL. ACAD. SCI. U. S. A., vol. 115, 2018, pages 7320 - 7325
WU, Y. ET AL.: "A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2", SCIENCE, vol. 368, 2020, pages 1274 - 1278, XP055799109, DOI: 10.1126/science.abc2241
YANG JINGYUN ET AL: "A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity", NATURE, vol. 586, no. 7830, 22 October 2020 (2020-10-22), pages 572 - 577, XP037341143, ISSN: 0028-0836, DOI: 10.1038/S41586-020-2599-8 *
YUAN, M. ET AL.: "A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV", SCIENCE, vol. 368, 2020, pages 630 - 633

Similar Documents

Publication Publication Date Title
WO2021254327A1 (fr) Vaccin à vecteur viral de type à remplacement d&#39;enveloppe et procédé de construction associé
CN116113431A (zh) 冠状病毒疫苗
KR100571479B1 (ko) Hiv에 대한 폴리엔브백신으로서 재조합 백시니아벡터의 혼합물
CN113185613A (zh) 新型冠状病毒s蛋白及其亚单位疫苗
JP2023524054A (ja) ベータコロナウイルスの予防と治療
PL220281B1 (pl) Szczepionka DNA, sposób indukowania odpowiedzi immunologicznej, przeciwciała specyficznie rozpoznające białko hemaglutyniny H5 wirusa grypy i zastosowanie szczepionki DNA
CN113174392B (zh) 一种狂犬病毒的重组基因、重组假病毒及其构建方法和应用
CN107847579A (zh) 人和禽h5n1流感的计算优化的广泛反应性抗原的协同共同给药
JP2021536228A (ja) ヘマグルチニン（ｈａ）タンパク質内の非ドミナントエピトープに対する免疫応答を誘起するためのベクター
CN111556896A (zh) 交叉免疫抗原疫苗及其制备方法
Sun et al. Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein
US10792357B2 (en) Optimized HIV envelope gene and expression thereof
US20220040284A1 (en) Vaccines and methods
CN107530417A (zh) H1n1流感的计算优化的广泛反应性抗原的协同共同给药
US20230149530A1 (en) Influenza vaccines
WO2023275538A1 (fr) Vaccins à coronavirus bêta
Wang et al. A novel multi-variant epitope ensemble vaccine against avian leukosis virus subgroup J
WO2023057769A1 (fr) Vaccins contre le coronavirus
JP6622326B2 (ja) インフルエンザａ／上海／２／２０１３ ｈ７配列の改変ｈ７赤血球凝集素糖タンパク質
Tan et al. Efficacy of seasonal pandemic influenza hemagglutinin DNA vaccines delivered by electroporation against aseasonal H1N1 virus challenge in mice
CN107841513A (zh) 基于M2e表位的广谱型流感疫苗
KR102401682B1 (ko) SARS-CoV-2 항원을 발현하는 재조합 마이코박테리움 균주 및 이를 포함하는 백신 조성물
CN114591406B (zh) 一种传染性法氏囊病病毒重组vp2蛋白及其在疫苗中的用途
CA3234653A1 (fr) Vaccins contre la grippe
EP3888677A1 (fr) Virus de la vaccine recombiné dérivé d&#39;une souche dis ayant un nouveau gène de protéine hémagglutinine dérivé du virus de la grippe

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: 22741553

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22741553

Country of ref document: EP

Kind code of ref document: A1