WO2024184626A2 - Coronavirus vaccines - Google Patents

Coronavirus vaccines Download PDF

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WO2024184626A2
WO2024184626A2 PCT/GB2024/050568 GB2024050568W WO2024184626A2 WO 2024184626 A2 WO2024184626 A2 WO 2024184626A2 GB 2024050568 W GB2024050568 W GB 2024050568W WO 2024184626 A2 WO2024184626 A2 WO 2024184626A2
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mrna
seq
cov
amino acid
sequence
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PCT/GB2024/050568
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French (fr)
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Jonathan Luke Heeney
Sneha VISHWANATH
Ralf Wagner
George CARNELL
Benedikt ASBACH
Martina BILLMEIER
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Diosynvax Ltd
Universitat Regensburg
Cambridge Enterprise Limited
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Priority claimed from GBGB2303150.3A external-priority patent/GB202303150D0/en
Priority claimed from GBGB2305092.5A external-priority patent/GB202305092D0/en
Priority claimed from GBGB2312403.5A external-priority patent/GB202312403D0/en
Priority claimed from GBGB2312413.4A external-priority patent/GB202312413D0/en
Priority claimed from GBGB2315819.9A external-priority patent/GB202315819D0/en
Priority claimed from GBGB2318080.5A external-priority patent/GB202318080D0/en
Application filed by Diosynvax Ltd, Universitat Regensburg, Cambridge Enterprise Limited filed Critical Diosynvax Ltd
Publication of WO2024184626A2 publication Critical patent/WO2024184626A2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
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  • General Chemical & Material Sciences (AREA)
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  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Designed messenger RNAs (mRNAs) encoding coronavirus polypeptides are described, as well as mRNA vaccine vectors, pharmaceutical compositions comprising the mRNAs or vectors, and mRNA vaccines, and their use to induce an immune response against viruses of the coronavirus family. The designed sequences include mRNA sequences encoding designed coronavirus receptor binding domain (RBD) sequences CoV_S_T2_17, CoV_S_T2_17 comprising a transmembrane domain sequence (CoV_S_T2_20), CoV_S_T3_3 (T2_20v2), and CoV_S_T3_4 (T2_17_T2_20 dimer). Polypeptides, nucleic acid molecules encoding the polypeptides, vectors, fusion proteins, pharmaceutical compositions, and their use as vaccines against viruses of the coronavirus family are also described.

Description

Coronavirus Vaccines This invention relates to messenger RNAs (mRNAs), mRNA vaccine vectors, pharmaceutical compositions comprising the mRNAs or vectors, and mRNA vaccines, and their use to induce an immune response against viruses of the coronavirus family. Coronaviruses (CoVs) 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. 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: α, β, γ, and δ coronaviruses, based on phylogenetic clustering, with each genus subdivided again into clusters depending on the strain of the virus. For example, within the genus β-CoV (Group 2 CoV), four lineages (a, b, c, and d) are commonly recognized: • 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) 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 β-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. 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. In most CoVs, S is cleaved by host cell protease into two separate polypeptides – S1 and S2. 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. Figure 13 shows SARS S-protein architecture. The Studies show that the N-terminal region of the S protein is much more diverse than the C-terminal region, which is highly conserved (Dong et al, Genomic and protein structure modelling analysis depicts the origin and infectivity of 2019-nCoV, a new coronavirus which caused a pneumonia outbreak in Wuhan, China.2020). 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). N-terminal sequence is responsible for relaying extracellular signals intracellularly. The last two regions (S1 and S2 subunits) are responsible for receptor binding and membrane fusion, respectively. In the 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), transmembrane (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. Structure of the SARS-CoV-2 trimeric S protein has been determined by cryo-electron microscopy/x-ray crystallography at the atomic level, revealing different conformations of the S RBD domain in opened and closed states and its corresponding functions. Amongst the coronaviruses, of the greatest pandemic risk are the angiotensin-converting enzyme 2 (ACE-2) binding viruses of β-Coronaviruses genus (1, 2). Over the last two decades, two ACE-2 binding sarbecoviruses (a sub-genus of β-coronaviruses) have spilled over into human population causing the SARS epidemic in 2002/2003 and the current on- going SARS-CoV-2 pandemic. Bats are a reservoir of a large number of SARS-CoV-like ACE-2 binding sarbecoviruses which pose a constant threat for future spill-overs into humans with the potential to cause new epidemics (3, 4). In addition to emergence of new ACE-2 binding viruses from zoonotic reservoirs, another concern is the emergence of mutations in variants of these viruses capable of escaping vaccine-induced immunity, a constant observation and concern in the current on-going pandemic. As human infections increase globally during the current pandemic, the virus has continued to accrue mutations, most significantly in the spike protein (5). An accumulating number of variants of concern (VOCs) have implications for increased transmission and escape from natural and vaccine immunity (6–9). The N501Y, asparagine to tyrosine substitution in the receptor binding domain (RBD) of the spike protein is a common feature of VOCs and is associated with increased affinity of the viral spike protein to the ACE-2 receptor and subsequent increase in transmission (10). Notably, 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 (11, 12). In the key RBD epitopes, the Delta VOC (13) has L452R and T478K mutations, while the Omicron lineage VOCs have multiple mutations. The continued emergence of these VOCs during the on-going COVID-19 pandemic, and the constant threat of new zoonotic spill overs of coronaviruses from animals to humans, highlights the need for next generation vaccines with broader protection from ACE-2 binding sarbecoviruses as well as the emerging VOCs. Two of the SARS-CoV-2 vaccines currently in use worldwide, BNT162b2 (BioNTech’s vaccine manufactured by Pfizer) and mRNA-1273 (manufactured by Moderna), are based on lipid nanoparticle delivery of mRNA encoding a prefusion stabilized form of spike protein derived from SARS-CoV-2 isolated early in the epidemic from Wuhan, China. 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). Safety and Efficacy of the BNT162b2 mRNA Covid- 19 Vaccine. N. Engl. J. Med. 383, 2603–2615; Baden et al., COVE Study Group (2021). Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N. Engl. J. Med.384, 403– 416). However, the emergence of novel circulating variants has raised significant concerns about the effectiveness of the current vaccines, especially in countries where the epidemic is dominated by variant strains (Garcia-Beltran et al., 2021, Cell 184, 2372–2383: Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity). There is a need, therefore, to provide effective vaccines that induce a broadly neutralising immune response to protect against emerging and re-emerging diseases caused by CoVs, especially β-CoVs, such as SARS-CoV and the recent SARS-CoV-2. There is also a need to provide improved coronavirus vaccines that elicit broadly neutralising antibodies against SARS-CoV-2 variants, in particular against current and recent variants of concern. Furthermore, there is a need to provide vaccines that successfully combat vaccine escape of new SARS-CoV-2 variants. WO 2021/198706 (Example 32, Figure 37B) describes evaluation of the ability of a designed receptor binding domain (RBD) sequence (known as “COV_S_T2_17”, or “T2_17”) of SARS- CoV-2 spike protein to induce an immune response. DNA encoding T2_17 induced an immune response in mice to both SARS-CoV and SARS-CoV-2. Similar immunogenic responses in mice were observed for T2_17, and for T2_17 attached to a transmembrane domain (known as “COV_S_T2_17_TM”, “T2_17_TM”, “COV_S_T2_20”, or “T2_20”). It has now been found that mRNA expressing T2_20 surprisingly induced significantly higher SARS-CoV-2 binding antibody titres at relatively low doses, compared with mRNA expressing the corresponding untethered RBD (T2_17). Unexpectedly, compared with mRNA expressing full-length spike antigen, mRNA expressing T2_20 also induced binding antibodies against SARS-CoV more rapidly, induced significantly higher antibody titres against SARS-CoV-2 more rapidly, and induced a more broadly neutralising antibody response (including a neutralising response against SARS-CoV-2 omicron variant, compared with almost negligible neutralising titres for this variant from mRNA expressing the full-length spike). According to the invention there is provided an isolated messenger RNA (mRNA) encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:1 (T2_17), or an amino acid sequence which has at least 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:1, and an amino acid sequence of a transmembrane domain. T2_17 amino acid sequence (SEQ ID NO:1): RVAPTKEVVR FPNITNLCPF GEVFNATKFP SVYAWERKKI SNCVADYSVL YNSTSFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGVI ADYNYKLPDD FTGCVIAWNT NNIDSTTGGN YNYLYRSLRK SKLKPFERDI SSDIYSPGGK PCSGVEGFNC YYPLRSYGFF PTNGTGYQPY RVVVLSFELL NAPATVCGPK LSTD Optionally the amino acid sequence of the encoded transmembrane domain is C-terminal to the amino acid sequence of SEQ ID NO:1, or the amino acid sequence which has at least 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:1. Optionally the amino acid sequence of the encoded transmembrane domain is linked to the amino acid sequence of SEQ ID NO:1, or the amino acid sequence which has at least 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:1 by a linker amino acid sequence of upto 10 amino acid residues. Optionally the amino acid sequence of the encoded transmembrane domain is linked directly (i.e. with no linker amino acid sequence) to the amino acid sequence of SEQ ID NO:1, or the amino acid sequence which has at least 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:1. The encoded transmembrane domain may comprise any suitable transmembrane domain amino acid sequence, including for example, an amino acid sequence of a transmembrane domain of a coronavirus spike protein. Optionally the encoded transmembrane domain comprises an amino acid sequence of SEQ ID NO:2, or an amino acid sequence which has at least 70%, 75%, 80%, 85%, 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:2. Transmembrane domain amino acid sequence (SEQ ID NO:2): GGGGSGGGGS GGGGSGGGGS KSSIASFFFI IGLIIGLFLV LRVGIHLCIK LKHTKKRQIY TDIEMNRLGK Optionally an mRNA of the invention encodes an amino acid sequence of SEQ ID NO:1. Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:8, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:8 and which encodes an amino acid sequence of SEQ ID NO:1. Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:8. RNA sequence encoding COV_S_T2_17 (SEQ ID NO:8): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUCGGCGAGG UGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAGAAAAUCAGCAACUGCGUGGC CGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGUCACCCACC AAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGC GGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAAGCUGCCCGACGACUUCAC CGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUG UACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCC CUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUU CUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUGAAU GCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGAC There is also provided according to the invention an isolated RNA, which comprises an RNA sequence of SEQ ID NO:8, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:8 and which encodes an amino acid sequence of SEQ ID NO:1, or the complement thereof. There is also provided according to the invention an isolated RNA, which comprises an RNA sequence of SEQ ID NO:8, or the complement thereof. There is also provided according to the invention an isolated messenger RNA (mRNA) encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:1 (T2_17), or an amino acid sequence which has at least 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:1. There is also provided according to the invention an isolated mRNA encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:1. Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:8, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:8 and which encodes an amino acid sequence of SEQ ID NO:1. Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:8. Optionally an mRNA of the invention comprises an mRNA sequence of SEQ ID NO:7. mRNA sequence encoding COV_S_T2_17 (SEQ ID NO:7): m GP G- GGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUGU UUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAA UCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAG AAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGU GCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGU GAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUAC AAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCG GCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAU CAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUAC CCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCC UGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGACUGAUGAGA AUU-Poly(~A ) ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5-ÚTR (minimal) =GGAGACGCCACC 3-ÚTR (unspecific) = GAAUU Coding sequence = bold format text Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:4, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:4 and which encodes an amino acid sequence of SEQ ID NO:1. RNA sequence encoding COV_S_T2_17 (SEQ ID NO:4): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUC GGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAAAAGAUC AGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGAC AGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUC GCCGAUUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACC AACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAG UCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCCCUGGCGGCAAG CCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUUCUUC CCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUG AAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACCGAC Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:4. There is also provided according to the invention an isolated RNA comprising a sequence of SEQ ID NO:4, or a sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:4 and which encodes an amino acid sequence of SEQ ID NO:1, or the complement thereof. Optionally an mRNA of the invention encodes an amino acid sequence of SEQ ID NO:2. Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:5, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:5 and which encodes an amino acid sequence of SEQ ID NO:2. RNA sequence encoding a transmembrane domain of amino acid sequence SEQ ID NO:2 (SEQ ID NO:5): GGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUU CUAUCGCCAGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAU CCACCUGUGCAUCAAGCUGAAACACACCAAGAAGCGGCAAAUCUACACCGACAUCGAGAUGAACCGG CUGGGCAAA Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:5. There is also provided according to the invention an isolated RNA comprising a sequence of SEQ ID NO:5, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:5 and which encodes an amino acid sequence of SEQ ID NO:2, or the complement thereof. Optionally an mRNA of the invention encodes an amino acid sequence of SEQ ID NO:3 (T2_20), or an amino acid sequence which has at least 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:3. T2_20 amino acid sequence (SEQ ID NO:3): RVAPTKEVVR FPNITNLCPF GEVFNATKFP SVYAWERKKI SNCVADYSVL YNSTSFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGVI ADYNYKLPDD FTGCVIAWNT NNIDSTTGGN YNYLYRSLRK SKLKPFERDI SSDIYSPGGK PCSGVEGFNC YYPLRSYGFF PTNGTGYQPY RVVVLSFELL NAPATVCGPK LSTDGGGGSG GGGSGGGGSG GGGSKSSIAS FFFIIGLIIG LFLVLRVGIH LCIKLKHTKK RQIYTDIEMN RLGK Optionally an mRNA of the invention encodes an amino acid sequence of SEQ ID NO:3. Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:10, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:10 and which encodes an amino acid sequence of SEQ ID NO:3. RNA sequence encoding T2_20 (SEQ ID NO:10): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUCGGCGAGGU GUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAGAAAAUCAGCAACUGCGUGGCCG ACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGUCACCCACCAAGC UGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAG AUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAAGCUGCCCGACGACUUCACCGGCUGU GUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUGUACAGAAGCC UGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCCCUGGCGGCAAG CCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUUCUUCCCCACAAA UGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAG UGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGG AAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCAGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCC UGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGUGCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUC UACACCGACAUCGAGAUGAACCGGCUGGGCAAG Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:10. Optionally an mRNA of the invention comprises an mRNA sequence of SEQ ID NO:9. mRNA sequence encoding COV_S_T2_20 (SEQ ID NO:9): m27,3´-oGP3 G- GGGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGU GUUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCA AUCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAA GAAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGU GCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUG AUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACA AGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGC GGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCA GCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCC UCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUG AGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAG GAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCA GCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGU GCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCAAG UGAUGAGAAUU-Poly(~A120) ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5ÚTR (minimal) =GGGAGACGCCACC 3ÚTR (unspecific) = GAAUU Coding sequence = bold format text Optionally an mRNA of the invention comprises an mRNA sequence of SEQ ID NO:46. mRNA sequence encoding COV_S_T2_20 (SEQ ID NO:46): m2 7,3´-oGP3 G- GGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUG UUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAA UCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAG AAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUG CUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGA UCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAA GCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCG GCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGC AGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUC UGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAG CUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAGGA UCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCAGC UUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGUGC AUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCAAGU GAUGAGAAUU-Poly(~A120) ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5ÚTR (minimal) = GGAGACGCCACC 3ÚTR (unspecific) = GAAUU Coding sequence = bold format text Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:6, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:6 and which encodes an amino acid sequence of SEQ ID NO:3. RNA sequence encoding T2_20 (SEQ ID NO:6): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUC GGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAAAAGAUC AGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGAC AGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUC GCCGAUUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACC AACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAG UCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCCCUGGCGGCAAG CCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUUCUUC CCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUG AAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAGGAUCUGGC GGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCAGC UUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCAC CUGUGCAUCAAGCUGAAACACACCAAGAAGCGGCAAAUCUACACCGACAUCGAGAUGAAC CGGCUGGGCAAA Optionally an mRNA of the invention comprises an RNA sequence of SEQ ID NO:6. There is also provided according to the invention an isolated RNA comprising a sequence of SEQ ID NO:6, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:6 and which encodes an amino acid sequence of SEQ ID NO:3, or the complement thereof. There is also provided according to the invention an isolated RNA which encodes an amino acid sequence of SEQ ID NO:1. Optionally, the RNA comprises the RNA sequence of SEQ ID NO:8, SEQ ID NO: 27, SEQ ID 28, SEQ ID NO:29, or SEQ ID NO:30 or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:8, SEQ ID NO: 27, SEQ ID 28, SEQ ID NO:29, or SEQ ID NO:30 and which encodes an amino acid sequence of SEQ ID NO:1. There is also provided according to the invention an isolated RNA which encodes an amino acid sequence of SEQ ID NO:3. Optionally, the RNA comprises an RNA sequence of SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34 and which encodes an amino acid sequence of SEQ ID NO:3. A further aspect of the invention is an isolated RNA which encodes an amino acid sequence of SEQ ID NO: 43. Optionally, the RNA comprises an RNA sequence of SEQ ID NO: 42, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:43. Optionally, the mRNA is a modified version of the mRNA comprising modified nucleosides. Optionally the one or more modified nucleosides are 5- iodouridine and 5-iodocytidine.Optionally at least 50% of the uridines in the ORF have been modified. Optionally at least 50% of the uridines in the mRNA have been modified. Optionally at least 50% of the uridines in the ORF have been modified to m1ψ. Optionally, 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5- iodocytidine. Optionally, 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine. We have appreciated that advantageous immunogenic properties (for example, increased antibody response and/or increased breadth of immune response) may also be provided with mRNA immunogens encoding other tethered coronavirus spike protein receptor binding domains. According to the invention there is also provided an isolated mRNA encoding a polypeptide comprising an amino acid sequence of a coronavirus spike protein receptor binding domain (RBD) linked at its C-terminal end directly, or by a linker amino acid sequence of up to 10 amino acid residues, to an amino acid sequence of a transmembrane domain. Optionally the encoded RBD is a prefusion-stabilised RBD. Optionally the encoded RBD is a SARS-CoV-2 RBD, for example a prefusion-stabilised SARS-CoV-2 RBD. The encoded transmembrane domain may be any suitable transmembrane domain, for example as described above. Optionally an mRNA of the invention is a product of in-vitro transcription (IVT). Optionally an IVT mRNA of the invention comprises a polyadenylation (poly(A)) tail downstream of an open reading frame (ORF) encoding the polypeptide. Optionally an mRNA of the invention comprises one or more modified nucleosides. Optionally the or each modified nucleoside is selected from any of the following: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 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, 5-iodo-uridine, 2′-O-methyl uridine, 5-methylcytosine, 5-methylcytidine, 5- iodo-cytidine, N1-methyladenosine, N6-methyladenosine. Optionally the one or more modified nucleosides comprise a 1-methylpseudouridine (m1ψ) modification. Optionally the one or more modified nucleosides comprise at least one N1- methylpseudouridine (N1ψ) modification. Optionally the one or more modified nucleosides are 5-iodouridine and 5-iodocytidine. Optionally at least 50% of the uridines in the ORF have been modified. Optionally at least 50% of the uridines in the ORF have been modified to m1ψ. Optionally, 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine. Optionally, 5% to 50% of the uridine nucleotides are 2- thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine. There is also provided according to the invention an mRNA vaccine vector comprising an mRNA of the invention. There is also provided according to the invention an mRNA vaccine, which comprises an mRNA of the invention, or an mRNA vaccine vector of the invention, encapsulated in a lipid nanoparticle (LNP). There is further provided according to the invention a pharmaceutical composition comprising an mRNA of the invention, an mRNA vaccine vector of the invention, or an mRNA vaccine of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. There is also provided according to the invention an mRNA of the invention, an mRNA vaccine vector of the invention, an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, for use as a medicament. There is further provided according to the invention an mRNA of the invention, an mRNA vaccine vector of the invention, an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, for use in the prevention, treatment, or amelioration of a coronavirus infection. There is also provided according to the invention use of an mRNA of the invention, an mRNA vaccine vector of the invention, an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, in the manufacture of a medicament for the prevention, treatment, or amelioration of a coronavirus infection. There is also provided according to the invention a method of inducing an immune response to a coronavirus in a subject, which comprises administering to the subject an effective amount of an mRNA of the invention, an mRNA vaccine vector of the invention, an mRNA vaccine of the invention, or a pharmaceutical composition of the invention. There is also provided according to the invention a method of immunising a subject against a coronavirus, which comprises administering to the subject an effective amount of an mRNA of the invention, an mRNA vaccine vector of the invention, an mRNA vaccine of the invention, or a pharmaceutical composition of the invention. Optionally a method of the invention comprises administering to the subject an mRNA of the invention, an mRNA vaccine vector of the invention, an mRNA vaccine of the invention, or a pharmaceutical composition of the invention, as part of a prime boost regimen. Optionally the coronavirus is a beta-coronavirus. Optionally the beta-coronavirus is a lineage B or C beta-coronavirus. Optionally the beta-coronavirus is a lineage B beta-coronavirus. Optionally the lineage B beta-coronavirus is SARS-CoV or SARS-CoV-2. Optionally the lineage C beta-coronavirus is MERS-CoV. Optionally the beta-coronavirus is a variant of concern (VOC). Optionally the beta-coronavirus is a SARS-CoV-2 VOC. Optionally the beta-coronavirus is a SARS-CoV-2 beta, gamma, delta, or omicron VOC. Optionally the beta-coronavirus is a SARS-CoV-2 alpha virus. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2.12.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2.75. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2.3.20. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BQ.1.1. Optionally the omicron VOC is XBB. Optionally the omicron VOC is XBB.1.5. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBB.1.5. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBB.1.19.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBC.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BQ.1.12. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBB.1.9.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron CH.1.1.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2.86. Optionally the SARS-CoV-2 is a Wuhan variant. Optionally the subject is a human subject. Optionally the coronavirus infection has resulted in long Covid following an initial infection with SARS-CoV-2. Long COVID is broadly defined as signs, symptoms, and conditions that continue or develop after an initial SARS-CoV-2 infection. mRNA Vaccines An mRNA of the invention may be provided as part of an mRNA vaccine. Messenger RNA (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 (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. While this transient nature of protein expression requires repeated administration for the treatment of genetic diseases and cancers, it is extremely beneficial for vaccines, where prime or prime-boost vaccination is sufficient to develop highly specific adaptive immunity without any exposure to the contagion. 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 example, 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) 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. In eukaryotes, 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 may contain a Kozak consensus sequence (ACCAUG (initiation codon underlined), which contains the initiation codon AUG. An elongated Kozak sequence may be used: GCCACCAUG (initiation codon underlined). The 5′ and 3′ UTR elements flanking the coding sequence profoundly influence the stability and translation of mRNA, both of which are critical concerns for vaccines. These regulatory sequences can be derived from viral or eukaryotic genes and greatly increase the half-life and expression of therapeutic mRNAs. For example, a 5’UTR of an mRNA of the invention may comprise, with an initiation codon of the mRNA, a Kozak consensus sequence, or an elongated Kozak sequence. Optionally a 5’UTR of an mRNA of the invention comprises any one of the following sequences: GGAGACGCCACC (SEQ ID NO:11), GGGAGACGCCACC (SEQ ID NO:47), or GGGAGACUGCCACC (SEQ ID NO:14), immediately upstream of an initiation codon sequence. Optionally, a 5’UTR of an mRNA of the invention comprises immediately upstream of an initiation codon sequence a T7, T3, SP6, or K11 polymerase binding domain, a minimal UTR and a Kozak sequence as follows: GGAGACGCCACC (SEQ ID NO:11), GGGAGACGCCACC (SEQ ID NO:47), GGGACGCCACC (SEQ ID NO:12), GGGACGCCACC (SEQ ID NO:13), GGGAGACUGCCACC (SEQ ID NO:14), GAAGCTGCCACC (SEQ ID NO:15), or GGGACTGCCACC (SEQ ID NO:16). A 5′ cap structure is required for efficient protein production from mRNA. Various versions of 5′ caps can be added during or after the transcription reaction using a vaccinia virus capping enzyme, or by incorporating synthetic cap or anti-reverse cap analogues (see Pardi et al., supra). Anti-Reverse Cap Analog (ARCA) is a cap analog used during in vitro transcription for the generation of capped transcripts. ARCA is modified in a way that ensures incorporation in the forward orientation only. Anti-Reverse Cap Analog (ARCA) is a modified cap analog in which the 3' OH group (closer to m7G) is replaced with –OCH3:
Figure imgf000018_0001
Conventional Cap Analog: R=H, m7G(5’)pppG; ARCA: R=CH3, 3’-0-Me-m7G(5’)pppG Because of this substitution, the RNA polymerase can only initiate transcription with the remaining hydroxyl group thus forcing ARCA incorporation in the forward orientation. As a result, unlike transcripts synthesized with conventional cap analog, 100% of the transcripts synthesized with ARCA at the 5' end are translatable leading to a strong stimulatory effect on translation. The 3’ UTR may comprise a sequence for generation of a restriction site when in a vector, such as GAAUU. Alternatively, a 3’ UTR that may be used is 3’ UTR of CYBA (CCUCGCCCCGGACCUGCCCUCCCGCCAGGUGCACCCACCUGCAAUAAAUGCAGCG AAGCCGGGA, SEQ ID NO:26. The poly(A) tail also plays an important regulatory role in mRNA translation and stability; thus, an optimal length of poly(A) must be added to mRNA either directly from the encoding DNA template, by using poly(A) polymerase (see Pardi et al., supra), or ligation after in-vitro transcription. The poly(A) may have a length of 90 A nucleotides (A90) or more, 100 A nucleotides (A100) or more, 110 A nucleotides (A110) or more, 120 A nucleotides (A120) or more, 130 A nucleotides (A130) or more, 150 A nucleotides (A150) or more, 180 A nucleotides (A180) or more, 190 A nucleotides (A190) or more. An example of a suitable length of poly(A) tail is poly(~A120). The poly(A) tail may be a segmented poly(A) tail, as disclosed in WO 2020074642 A1, which is herein incorporated by reference. Optionally the segmented poly(A) may have the structure A55-65-S-A55-65 wherein S is a single nucleotide selected from C, G, T or U. Optionally the poly(A) have the structure: A55-65-N-S4-N-A55-65, wherein N is a nucleotide that is not adenine, and wherein S4 are four nucleotides selected from A, C, G, T or U..Optionally, the segmented poly(A) is a poly(A) of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40). The codon usage additionally has an impact on protein translation. Replacing rare codons with frequently used synonymous codons that have abundant cognate tRNA in the cytosol is a common practice to increase protein production from mRNA. Enrichment of G:C content constitutes another form of sequence optimization that has been shown to increase steady- state mRNA levels in vitro and protein expression in vivo (see Pardi et al., supra). 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). The mRNA is encapsulated in 80 nm ionizable cationic lipid nanoparticles. 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. An mRNA vaccine 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. Firstly, 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. Secondly, these novel vaccines can be rapidly produced. Due to high yields from in vitro transcription reactions, mRNA production can be rapid, inexpensive and scalable. Thirdly, vaccine risks are low. mRNA does not contain infectious viral elements that pose risks for infection and insertional mutagenesis. Anti-vector immunity is also avoided as 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. However, efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm. To date, 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). Dicationic 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. Although 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). Additionally, enzymatically synthesised mRNA preparations contain double stranded RNA (dsRNA) contaminants as aberrant products of the in vitro transcription (IVT) process. 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). Thus, the mRNA may suppress antigen expression and thus reduce vaccine efficacy. Studies over the past decade have shown that the immunostimulatory effect of mRNA can be shaped by the purification of IVT mRNA, the introduction of modified nucleosides, complexing the mRNA with various carrier molecules (Pardi et al., supra), adding poly(A) tails or optimising mRNA with GC-rich sequence (Wang et al., supra). Chemical modification of uridine is a common approach to minimise the immunogenicity of foreign mRNA. Incorporation of pseudouridine (ψ) and N1- methylpseudouridine (m1ψ) to IVT mRNA prevents TLR activation and other innate immune sensors, thus reducing pro-inflammatory signalling in response to the exogenous mRNA. Such 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 m1ψ, and their sequences were optimized to encode a stabilized pre-fusion spike protein with two pivotal proline substitutions (Hou et al., supra). However, 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%). Secondly, the vaccine comprises C-rich motifs which bind to poly(C)-binding protein, enhancing both the stability and expression of the mRNA. A further modification of CVnCoV is that it 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). However, 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. Nonetheless, CureVac and Acuitas Therapeutics delivered erythropoietin (EPO)-encoding mRNA, which has rich GC codons, to pigs with lipid nanoparticles (LNPs). Their results indicated EPO-related responses were elicited without immunogenicity (Wang et al., supra), suggesting that there is still scope for unmodified mRNA nucleoside-based vaccines. An RNA of the invention may comprise an mRNA. An mRNA of the invention, a pharmaceutical composition, or a vector of the invention, may be provided as part of an mRNA vaccine. A Vector of the invention may comprise the corresponding DNA sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO: 8 and optionally immediately upstream of an initiation codon sequence anyone of the following sequences: TAATACGACTCACTATA GGGAGACGCCACC (SEQ ID NO:17), AATTAACCCTCACTAAA GGGAGACGCCACC (SEQ ID NO:18), ATTTAGGTGACACTATA GAAGCGCCACC (SEQ ID NO:19), AATTAGGGCACACTATA GGGACGCCACC (SEQ ID NO:20), TAATACGACTCACTATA GGGAGA CTGCCACC (SEQ ID NO:21), AATTAACCCTCACTAAAGGGAGA CTGCCACC (SEQ ID NO:22), ATTTAGGTGACACTATAGAAG CTGCCACC (SEQ ID NO:23), AATTAGGGCACACTATAGGGA CTGCCACC (SEQ ID NO:24), or CGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCC (SEQ ID NO:25). The above sequences may be placed upstream of the ATG of any of the mRNA sequences of the invention, including the full-length spike (SEQ ID NO:44), COV_S_T2_17 (SEQ ID NO:4) and/or T2_20 (SEQ ID NO:6). An mRNA, a pharmaceutical composition, 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 an RNA or mRNA of the invention, or in mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention. Optionally, 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, 5-Iodo- uridine, and 2′-O-methyl uridine. In some embodiments, 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. For example, an RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, may comprise one or more of the following modified nucleosides: pseudouridine (ψ); N1- methylpseudouridine (m1ψ) 5-methylcytidine (m5C) 5-methyluridine (m5U) N1-methyladenosine (m1A) N6- methyladenosine (m6A) 2-thiouridine (s2U) 5- methoxyuridine (5moU) 5-iodouridine 5-iodocytidine. In some embodiments, 100% of the uracil of the whole mRNA have a chemical modification. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, 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 of the mRNA have a N1-methyl pseudouridine in the 5- position of the uracil. In some embodiments, 100% of the uracil in the open reading frame have a N1-methyl pseudouridine in the 5-position of the uracil. In some embodiments, 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine. In some embodiments, 5 to 50% of the uridine nucleotides are 5-iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine. In some embodiments, 5 to 50% of the uridine nucleotides are 2-thiouridine and 5 to 50% of the cytidine nucleotides are 5- methylcytidine. RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, 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 (or nucleoside), 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 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). Any remaining percentage is accounted for by the presence of unmodified A, G, U, or C. Optionally RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with each ‘U’ replaced by m1ψ. Optionally RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 50% of the ‘U’s replaced by m1ψ. The remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides. Optionally RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 70% of the ‘U’s replaced by m1ψ. The remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides. Optionally RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with at least 90% of the ‘U’s replaced by m1ψ. The remaining ‘U’s may all be unmodified, or may comprise unmodified and one or more other modified nucleosides. Optionally RNA or mRNA of the invention, or mRNA of a pharmaceutical composition, a vector, or a vaccine, of the invention, comprises an RNA molecule in which the nucleic acid sequence of the molecule is the same as that recited in the respective SEQ ID, but with 100% of the ‘U’s replaced by m1ψ. 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).
Figure imgf000024_0001
According to the invention there is provided an isolated polynucleotide comprising a first nucleotide sequence encoding SEQ ID NO:53 (CoV_S_T2_20 scaffold sequence), or the complement thereof, and a second nucleotide sequence encoding SEQ ID NO:53 (CoV_S_T2_20 scaffold sequence), or the complement thereof. SEQ ID NO:53 below shows a scaffold RBD sequence for CoV_S_T2_20 (SEQ ID NO:3), CoV_S_T3_3 (SEQ ID NO:50), and CoV_S_T3_4 (SEQ ID NO:52) designed structures (without leader sequence), in which the amino acid sequence of the constant regions of the scaffold is provided, with each variable amino acid residue (i.e. amino acid residues which can be varied to provide antigen which induces neutralising immune response against new and/or future SARS-CoV-2 variants) represented with an X (shown underlined in the sequence below) >CoV_S_T2_20 Scaffold Sequence (SEQ ID NO:53): RVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNXXXFXXFK 60 CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNT 120 NNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFF 180 PTNGTGYQPYRVVVLSFELLXAPATVCGPKLSTD 214 Examples of sequences provided herein which are covered by this scaffold sequence are SEQ ID NOs:3 and 48 (CoV_T2_20 without and with leader sequence, respectively), SEQ ID NOs:49 and 50 (CoV_S_T3_3 (T2_20v2) with and without leader sequence, respectively), and SEQ ID NOs:51 and 52 (CoV_S_T3_4 (T2_17_T2_20 dimer) with and without leader sequence). Example 5 below provided detail of the scaffold sequence. According to the invention there is provided an isolated polynucleotide comprising a first nucleotide sequence encoding SEQ ID NO:1 (T2_17), or the complement thereof, and a second nucleotide sequence encoding SEQ ID NO:1 (T2_17), or the complement thereof. Optionally an isolated polynucleotide according to the invention further comprises a nucleotide sequence encoding SEQ ID NO:2 (transmembrane domain amino acid sequence). According to the invention there is provided an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:50 (CoV_S_T3_3), or the complement thereof. According to the invention there is provided an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:52 (CoV_S_T3_4), or the complement thereof. Optionally an isolated polynucleotide according to the invention further comprises a nucleotide sequence encoding a leader amino acid sequence, preferably SEQ ID NO:54 (leader amino acid sequence). According to the invention there is provided an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:48 (T2_20). According to the invention there is provided an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:49 (CoV_S_T3_3). According to the invention there is provided an isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:51 (CoV_S_T3_4). Also provided is a pharmaceutical composition which comprises an isolated polynucleotide of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. Further provided according to the invention is a pharmaceutical composition which comprises an isolated polynucleotide according to the invention, which further comprises an adjuvant for enhancing an immune response in a subject to a polypeptide, or to a polypeptide encoded by a nucleotide, of the composition. Also provided is a vector comprising an isolated polynucleotide according to the invention, and a separate promoter operably linked to each different nucleotide sequence of the polynucleotide. The or each vector of a pharmaceutical composition or a combined preparation of the invention may be an mRNA vector. There is also provided according to the invention an isolated cell comprising a vector of the invention.
Figure imgf000026_0001
According to the invention there is provided an isolated polypeptide comprising first amino acid sequence of SEQ ID NO:53 (CoV_S_T2_20 scaffold sequence), and a second amino acid sequence of SEQ ID NO:53 (CoV_S_T2_20 scaffold sequence). According to the invention there is provided an isolated polypeptide comprising a first amino acid sequence of SEQ ID NO:1 (T2_17), and a second amino acid sequence of SEQ ID NO:1 (T2_17). Optionally, an isolated polypeptide according to the invention further comprises an amino acid sequence of SEQ ID NO:2 (transmembrane domain amino acid sequence). According to the invention there is provided an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:50 (CoV_S_T3_3). According to the invention there is provided an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:52 (CoV_S_T3_4). Optionally, an isolated polypeptide according to the invention further comprises a leader amino acid sequence, preferably SEQ ID NO:54 (leader amino acid sequence). According to the invention there is provided an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:48 (T2_20). According to the invention there is provided an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:49 (CoV_S_T3_3). According to the invention there is provided an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:51 (CoV_S_T3_4). There is also provided a pharmaceutical composition which comprises an isolated polypeptide of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. There is also provided a pharmaceutical composition which comprises an adjuvant for enhancing an immune response in a subject to a polypeptide of the composition. There is further provided according to the invention a fusion protein comprising an isolated polypeptide of the invention. There is also provided according to the invention a pseudotyped virus particle comprising an isolated polypeptide of the invention. There is also provided according to the invention an isolated polynucleotide of the invention, an isolated polypeptide of the invention, a pharmaceutical composition of the invention, or a vector of the invention, for use as a medicament. There is also provided according to the invention an isolated polypeptide of the invention, a pharmaceutical composition of the invention, or a vector of the invention, for use in the prevention, treatment, or amelioration of a coronavirus infection. There is also provided according to the invention use of an isolated polypeptide of the invention, a pharmaceutical composition of the invention, or a vector of the invention, in the manufacture of a medicament for the prevention, treatment, or amelioration of a coronavirus infection. There is also provided according to the invention an isolated polynucleotide of the invention, an isolated polypeptide of the invention, a pharmaceutical composition of the invention, or a vector of the invention, for use in inducing an immune response to a coronavirus in a subject. There is also provided according to the invention use of an isolated polynucleotide of the invention, an isolated polypeptide of the invention, a pharmaceutical composition of the invention, or a vector of the invention, in the manufacture of a medicament for inducing an immune response to a coronavirus in a subject. There is also provided according to the invention an isolated polynucleotide of the invention, an isolated polypeptide of the invention, a pharmaceutical composition of the invention, or a vector of the invention, for use in immunising a subject against a coronavirus. There is also provided according to the invention use of an isolated polynucleotide of the invention, an isolated polypeptide of the invention, a pharmaceutical composition of the invention, or a vector of the invention, in the manufacture of a medicament for immunising a subject against a coronavirus. There is also provided according to the invention a method of inducing an immune response to a coronavirus in a subject, which comprises administering to the subject an effective amount of: isolated polynucleotide of the invention; an isolated polypeptide of the invention; a pharmaceutical composition of the invention; or a vector of the invention. There is also provided according to the invention a method of immunising a subject against a coronavirus, which comprises administering to the subject an effective amount of: isolated polynucleotide of the invention; an isolated polypeptide of the invention; a pharmaceutical composition of the invention; or a vector of the invention. Methods of treatment and uses There is also provided according to the invention a method of inducing an immune response to a coronavirus in a subject, which comprises administering to the subject an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention. There is also provided according to the invention a method of immunising a subject against a coronavirus, which comprises administering to the subject an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention. An effective amount is an amount to produce an antigen-specific immune response in a subject. Optionally, the method comprises administering an effective amount of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention to a subject that has previously been seroconverted with an mRNA, a vector, a pharmaceutical composition or a vaccine, coding or comprising a full-length spike protein of a coronavirus. Optionally, the coronavirus is a Sarbecovirus. Optionally, the mRNA of the invention, the vector of the invention, the pharmaceutical composition of the invention or the vaccine of the invention comprises or consists of SEQ ID NO: 4 or SEQ ID NO: 7. Optionally, said mRNA, vector, pharmaceutical composition or vaccine, coding or comprising a full-length spike protein of a coronavirus is an mRNA comprising or consisting of any one of SEQ ID NOs:41 to 43. There is further provided according to the invention an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, for use as a medicament. There is further provided according to the invention an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, for use in the prevention, treatment, or amelioration of a coronavirus infection. There is also provided according to the invention use of an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention, in the manufacture of a medicament for the prevention, treatment, or amelioration of a coronavirus infection. Optionally the coronavirus is a β-coronavirus. Optionally the β-coronavirus is a lineage B or C β-coronavirus. Optionally the β-coronavirus is a lineage B β-coronavirus. Optionally the lineage B β-coronavirus is SARS-CoV or SARS-CoV-2. Optionally the lineage C β-coronavirus is MERS-CoV. Optionally an immune response is induced against more than one lineage B beta- coronavirus. Optionally an immune response is induced against SARS-1 and SARS-2 beta-coronavirus. Optionally an immune response is induced against SARS-1 and MERS beta-coronavirus. Optionally an immune response is induced against SARS-2 and MERS beta-coronavirus. Optionally an immune response is induced against SARS-1, SARS-2, and MERS beta- coronavirus. Optionally the beta-coronavirus is a variant of concern (VOC). Optionally the beta-coronavirus is a SARS-CoV-2 VOC. Optionally the beta-coronavirus is a SARS-CoV-2 lineage B1.248 (Brazil P1 lineage) VOC. Optionally the beta-coronavirus is a SARS-CoV-2 lineage B1.351 (South Africa) VOC. Optionally the beta-coronavirus is a SARS-CoV-2 beta, gamma, or delta VOC. Optionally the beta-coronavirus is a SARS-CoV-2 alpha virus. Optionally the beta-coronavirus is a SARS-CoV-2 beta VOC. Optionally the beta-coronavirus is a SARS-CoV-2 gamma VOC. Optionally the beta-coronavirus is a SARS-CoV-2 delta VOC. Optionally the beta-coronavirus is a SARS-CoV-2 alpha VOC. Optionally the beta-coronavirus is a SARS-CoV-2 omicron VOC. Optionally the beta-coronavirus is SARS-CoV-2 omicron BA.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2.12.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2.75. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2.3.20. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BQ.1.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBB. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBB.1.5. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBB.1.19.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBC.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BQ.1.12. Optionally the beta-coronavirus is a SARS-CoV-2 omicron XBB.1.9.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron CH.1.1.1. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2. Optionally the beta-coronavirus is a SARS-CoV-2 omicron BA.2.86. It can readily be determined whether an immune response has been induced to a beta- coronavirus using methods well-known to the skilled person. For example, a pseudotype neutralisation assay as described in the example below may be used. Optionally the coronavirus infection has resulted in long Covid following an initial infection with SARS-CoV-2. Long COVID is broadly defined as signs, symptoms, and conditions that continue or develop after an initial SARS-CoV-2 infection. Optionally the subject is a human subject. 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. For lipid nanoparticles, the administration route is often determined by the properties of the nanoparticles and therapeutic indications. After intravenous (i.v.) administration, many lipid nanoparticles can accumulate in the liver. The liver is inherently capable of producing secretory proteins and, therefore, i.v. administration of lipid nanoparticle–mRNA formulations can be used to produce proteins that are missing in inherited metabolic and haematological disorders, or to produce antibodies to neutralize pathogens or target cancer cells. These applications require protein translation without stimulation of an immune response, which may limit the efficiency of repeated dosing. However, i.v. administration may also lead to accumulation of lipid nanoparticles in multiple lymph nodes throughout the body, which could increase immune responses to mRNA vaccines. For example, i.v. administration of mRNA vaccines has been shown to induce stronger antigen-specific cytotoxic T cell responses compared with local injection. Broad distribution of mRNA vaccines in the body may lead to systemic adverse effects, and, thus, it may be necessary to develop lipid nanoparticles that allow targeted delivery of mRNA vaccines into tissues with abundant immune cells. Topical administration routes have also been explored for mRNA therapeutics. Topical administration aims at achieving local therapeutic effects; for example, local injection of lipid nanoparticle–mRNA formulations enables supplementation of therapeutic proteins in specific tissues, such as heart, eyes and brain. Moreover, lipid nanoparticle–mRNA formulations can be administered into the lungs by inhalation. Local administration of mRNA vaccines can also prime systemic responses; for example, intradermal (i.d.), intramuscular (i.m.) and subcutaneous (s.c.) injection are commonly used for vaccination, because resident and recruited antigen-presenting cells (APCs) are present in the skin and muscle, which can internalize and process mRNA-encoded antigens. Furthermore, the vascular and lymphatic vessels of these tissues help APCs and mRNA vaccines to centre the draining lymph nodes to stimulate T cell immunity. Indeed, both i.m. and i.d. administration of lipid nanoparticle–mRNA vaccines produce robust immune responses at a well-tolerated dose in human trials. Vaccination can also be done by intranasal administration, because APCs in the peripheral lymph nodes can readily endocytose administered lipid nanoparticle–mRNA formulations. mRNA vaccines delivered by lipid nanoparticle may comprise cationic lipids and/or ionisable lipids, see review: Lipid Nanoparticles for mRNA Delivery, Nature Reviews Materials, 61078- 1094, 2021. In addition to cationic or ionizable lipids, lipid nanoparticle–mRNA formulations typically contain other lipid components, such as phospholipids (for example, phosphatidylcholine and phosphatidylethanolamine), cholesterol or polyethylene glycol (PEG)-functionalized lipids (PEG-lipids). These lipids can improve nanoparticle properties, such as particle stability, delivery efficacy, tolerability and biodistribution. 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 nonaqueous 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. 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. 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. The present disclosure includes methods comprising administering an mRNA vaccine to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The mRNA vaccine is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the mRNA vaccine 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. The effective amount of the mRNA, as provided herein, may be as low as 20 pg, administered for example as a single dose or as two 10 pg doses. In some embodiments, the effective amount is a total dose of 20 μg-300 μg or 25 μg-300 μg. For example, the effective amount may be a total dose of 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, or 300 μg. In some embodiments, the effective amount is a total dose of 20 μg. In some embodiments, the effective amount is a total dose of 25 pg. In some embodiments, the effective amount is a total dose of 50 μg. In some embodiments, the effective amount is a total dose of 75 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a total dose of 150 μg. In some embodiments, the effective amount is a total dose of 200 μg. In some embodiments, the effective amount is a total dose of 250 pg. In some embodiments, the effective amount is a total dose of 300 μg. An mRNA vaccine 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). Optionally, an mRNA vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject. In some embodiments, the effective amount is a total dose of 1 μg to 1000 μg, 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a dose of 25 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 100 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 400 μg administered to the subject a total of two times. In some embodiments, the effective amount is a dose of 500 μg administered to the subject a total of two times. Optionally a dosage of between 10 μg/kg and 400 μg/kg of the mRNA vaccine is administered to the subject. In some embodiments the dosage of the mRNA is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg, 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40- 300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg per dose. In some embodiments, the mRNA vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the mRNA vaccine is administered to the subject on day zero. In some embodiments, a second dose of the mRNA vaccine is administered to the subject on day twenty one. In a strategy called “prime-boost”, a first dose of the mRNA vaccine is given as a priming step, followed by a second dose as a booster. The prime-boost strategy aims to provide a stronger overall immune response. The boost may be administered at least a day, at least a week, or at least two, three, four, five, six, or seven weeks, or at least two, three, four, five,, or six months after the primer. For example, the boost may be administered at least three weeks after the primer.
Figure imgf000035_0001
vehicles and/or carriers The pharmaceutical composition may comprise a vehicle solution and/or a pharmaceutical acceptable carrier. The vehicle solution and/or the pharmaceutically acceptable carriers may 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. Other media that can be used with the compositions and methods provided herein are normal saline and sesame oil. The vehicle solution and/or the carrier may comprise a triblock copolymer which contains one poly(propylene oxide) block and two poly(ethylene oxide) blocks. Preferably, the triblock copolymer is an A-B-A triblock copolymer which contains one poly(propylene oxide) block B of formula (p-1):
Figure imgf000036_0001
wherein s is an integer of 15 to 67, preferably 20 to 40, and two poly(ethylene oxides) blocks A of formula (p-2):
Figure imgf000036_0002
wherein r is, independently for each block, an integer of 2 to 130, preferably 50 to 100, and more preferably 60 to 90. More preferably, the triblock copolymer has the following structure: wherein r and t are independently of each other integers of 2 to 130, preferably 50 to 100, and more preferably 60 to 90, and s is an integer of 15 to 67, preferably 20 to 40. Most preferably, Poloxamer P188 is used as the triblock copolymer. The vehicle solution and/or carrier may comprise the triblock copolymer dissolved therein. However, as will be appreciated by the skilled reader, this does not exclude the possibility that a certain amount of the copolymer molecules is adsorbed to the lipid or lipidoid nanoparticles which are contained in the composition and will be considered component (p) of the LNPs/LiNPs. Preferably, the composition for intramuscular administration or for aerosol formation comprises the triblock copolymer at a concentration of 0.05 to 5 % w/v (i.e. gram per 100 mL) preferably 0.1 to 2 %, based on the total volume of the composition. In addition to the triblock copolymer, other excipients may be present in the vehicle solution. Preferably, the vehicle solution further comprises at least one of sucrose and NaCl, more preferably sucrose and NaCl. The pharmaceutical formulation in accordance with the invention can be conveniently prepared e.g. by a method including adding the triblock copolymer to a suspension comprising a vehicle solution and the lipid or lipidoid nanoparticles, or including adding the lipid or lipidoid nanoparticles to a vehicle solution comprising the triblock copolymer. Pharmaceutical compositions for RNA delivery As aspect of the invention relates to a pharmaceutical composition comprising an mRNA of the invention, an mRNA vaccine vector of the invention, or an mRNA vaccine of the invention, and a pharmaceutically acceptable carrier, excipient, or diluent. The mRNA or the mRNA vaccine of the invention can advantageously be combined in the pharmaceutical composition with further components and/or compounds which ease delivery of the mRNA to the target cells or the target tissue and/or which increase its stability. One possibility in this regard is the formation of the RNA into liposomes or nanoparticles with suitable substances such as those described herein and, e.g. in EP3013964B1, which is incorporated herein in its entirety. In particular, the mRNA or the mRNA vaccine of the invention might be formulated with liposomes, to generate lipoplexes or with subsequent generations of lipid nanocarriers, such as lipid nanoparticles (LNPs), lipidoid nanoparticles (LiNPs), nanostructured lipid carriers, and/or cationic lipid–nucleic acid complexes. In some embodiments, the nucleic acid of the invention can be delivered to target cells and/or target tissues in vivo, ex-vivo and/or in vitro using LNPs or LiNPs. LNPs and LiNPs can be distinguished from other carriers due to their small size, their homogenous size distribution and their structure and are especially suited for immunization of a subject. The skilled person knows method for the production of LNPs and LiNPs. The production of LNPs or LiNPs involves a combination of lipids or lipidoids, such as phospholipids, cholesterol, and other specialized lipids, which are mixed together in a solvent, such as an alcohol. This mixture is then subjected to a process called nanoprecipitation, which involves rapidly mixing the lipid solution with a non-solvent, such as a nucleic acid dissolved in water, under controlled conditions of temperature, pressure, and stirring rate. During this process, the lipids self- assemble into complex nanoscale structures, which trap and protect the therapeutic nucleic acids of the invention inside. The nano particles may also be further modified with various surface coatings, such as polyethylene glycol (PEG), to improve their stability and reduce their tendency to be cleared by the immune system. The LiNPs may comprise as component (a) an mRNA, as component (b) a ionizable lipid or an ionizable lipidoid and optionally as component (c) helper lipids as defined below. Optionally, the LiNPs may comprise as component (p) a triblock copolymer which contains one poly(propylene oxide) block and two poly(ethylene oxide) blocks as described above. As component (a), the nanoparticles contained in the pharmaceutical composition of the invention, for example in the form of a formulation for intramuscular delivery or for aerosol delivery, may comprise a mRNA coding for T2_17 and/or T2_20, which provides a pharmaceutically active ingredient of the nanoparticles. In some embodiments, the pharmaceutical composition may (additionally) comprise the full-length spike protein as described herein. In some embodiments, component (a) consist of an mRNA encoding COV_S_T2_17 or consist of an mRNA encoding COV_S_T2_20. Optionally, the nanoparticles in the pharmaceutical composition comprises as component (a) an mRNA selected from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34 and/or SEQ ID NO:46 encoding COV_S_T2_17 or COV_S_T2_20 respectively. As component (b), the nanoparticles in the pharmaceutical composition may further comprise an ionizable lipid or an ionizable lipidoid. It will be understood that this encompasses the possibility that the nanoparticles comprise a combination of different ionizable lipids, a combination of different ionizable lipidoids, or a combination of one or more ionizable lipids and one or more ionizable lipidoids. The nanoparticles used in the context of the present invention typically comprise an mRNA (a) and as the ionizable lipid or as the ionizable lipidoid (b) a cationic lipid or cationic lipidoid, in the form of a mixture of these components. The pharmaceutical composition or the mRNA vaccine according to the invention optionally comprises a LiNP comprising as component (b) a ionizable lipidoid of formula (b-1):
Figure imgf000039_0001
wherein the variables a, b, p, m, n and R1A to R6A are defined as follows: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1, p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is ≥ 2; and R1A to R6A are independently of each other selected from hydrogen; -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH, -CH2-CH2-(C=O)-O-R7A, or -CH2-R7A; wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and a receptor ligand; provided that at least two residues among R1A to R6A are a group -CH2-CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2CH2(C=O)-O-R7, - CH2CH2(C=O)-NH-R7A or -CH2R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms contained in the compound of formula (b-1) are protonated to provide a compound carrying a positive charge. Optionally, the cationic lipidoid formula (b-1) comprises at least two residues among R1A to R6A, optionally at least three residues among R1A to R6A, or at least four residues among R1A to R6A are a group selected from -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH, -CH2-CH2-(C=O)-O-R7A, -CH2-CH2-(C=O)-NH-R 7A and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C- C double bond. In accordance with an optional embodiment, the compound of formula (b-1) is a compound of formula (b-1b), and component (b) comprises or consists of a lipidoid compound of the following formula (b-1b),
Figure imgf000040_0001
wherein R1A to R6A are defined as in formula (b-1), including preferred embodiments thereof; or a protonated form thereof wherein one or more of the nitrogen atoms indicated in formula (b-1b) are protonated to provide a compound carrying a positive charge. Thus, in a accordance with a particularly preferred embodiment, component (b) comprises or consists of a lipidoid of the above formula (b-1b) or a protonated form thereof, and R1A to R6A are independently selected from hydrogen and -CH2-CH(OH)-R7A, wherein R7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond, provided that at least two residues among R1A to R6A are -CH2-CH(OH)-R7A, more preferably at least three residues among R1A to R6A, and still more preferably at least four residues among R1A to R6A are -CH2-CH(OH)-R7A, wherein R7A is selected from C8-C18 alkyl and C8-C18 alkenyl having one C-C double bond. In certain embodiments, the mRNA vaccine or the pharmaceutical composition according to the invention comprises a LiNP nanoparticle comprising a cationic lipidoid of formula (b- V) and/or formula (b-VII):
Figure imgf000040_0002
As component (c), the LiNP of the pharmaceutical composition may comprise ionizable lipidoids helper lipids as described in the following. In particular, the herein described agents and reagents for delivering and/or introducing the mRNA into a target cell or a target tissue and the herein described lipids and lipidoids may be combined with one or more (e.g., two, three or four) further lipid(s) (like, for example, cholesterol, DPPC, DOPE and/or PEG-lipids (e.g. DMPE-PEG, DMG-PEG2000)). These further lipids may support the desired function of the therapeutic agents and the lipidoids (support and/or increase the delivery and/or introduction of RNA into the cell or tissue and improve transfection efficiency, respectively) and function as respective “helper lipids”. Particular examples of such “helper lipids” are cholesterol, DPPC, DOPE and/or PEG-lipids (e.g., DMPE-PEG, DMG-PEG (e.g., DMG- PEG2000). The further lipids (e.g., “helper lipids”) may also be part(s) of the herein disclosed complexes/particles. The skilled person is readily in the position to prepare complexes/particles in accordance with the invention. Examples of further lipids (e.g., “helper lipids”) are also known in the art. The skilled person is readily in the position to choose suitable further lipids (e.g., “helper lipids”) and ratios of the cationic lipidoid(s) and the further lipids (e.g. “helper lipids”). Such ratios may be molar ratios of [1-4 : 1-5], [3-4 : 4-6], [about 4 : about 5], [about 4 : about 5.3] of cationic lipidoid(s) : further lipid(s), (the more narrow ranges are preferred). For example, the cationic lipidoid may be combined with three further lipids, like DPPC, cholesterol, and DMG-PEG2000, preferably at a molar ratio of ~8.0 : ~5.3 : ~4.4 : ~0.9, respectively, or, more particularly, 8.00 : 5.29 : 4.41 : 0.88, respectively. Preferably, the lipidoids according to formula (b-1), (b-1b), (b-V), (b-VI) and (b-VII) are as described above and used with helper lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000 at the molar ratios 8.00:5.29:4.41:0.88 for formulating lipidoid nanoparticles. In some embodiments, the mRNA vaccine or the pharmaceutical composition according to the invention comprises a LiNP comprising the following components: a) a mRNA according to the invention, b) a cationic lipidoid of formula (b-1), (b-1b) (b-V), (b-VI) or (b-VIII), and c) one or more helper lipid(s), optionally selected from: c1) DPPC, and/or c2) cholesterol, and/or c3) PEG-lipid DMG-PEG2000, optionally, components b), and c1-c3), are present, optionally component b) and c1)- c3) are at the molar ratios of about 8.0: about 5.3: about 4.4: about 0.9, respectively, optionally, the NLP comprises a triblock copolymer which contains one poly(propylene oxide) block and two poly(ethylene oxide) blocks as component (p) as defined above in vehicles. A composition in which the R-isomer of formula (b-V), i.e formula (b-VI) is formulated with the lipids DPPC and cholesterol and PEG-lipid DMG-PEG2000 at the molar ratios 8.00 : 5.29 : 4.41 :0.88 is also referred herein as “Formulation I”. A composition in which the lipidoid of formula (b-VII) is formulated with the lipids DPPC and cholesterol and PEG-lipid DMG- PEG2000 at the molar ratios 8.00 : 5.29 : 4.41 : 0.88 is also referred herein as “Formulation II”. In some embodiments the LiNPs in the pharmaceutical composition of the invention comprises Formulation I and/or Formulation II. In some embodiments, the LiNP comprises Formulation I and/or Formulation II. The cationic lipidoid to mRNA ratios in the LiNP is controlled in terms of the mole ratio of nitrogen atoms of the cationic lipidoid (N) to phosphate groups in the mRNA (P) (N/P ratio). The other lipid components are calculated according the target molar lipid proportions relative to the cationic lipidoid as discussed above, and may be for example 8.00 : 5.29 : 4.41 : 0.88 for cationic lipidoid, DPPC, cholesterol and PEG-lipid DMG-PEG2000, respectively. In some embodiments, the final N/P ratio of a cationic lipidoid having formula (b-1), (b-1a), (b- V), (b-VI) and/or (b-VII) to one phosphate group of mRNA molecule, is preferably 4 to 44, preferably 4 to 16, more preferably 8 nitrogen atoms of a cationic lipidoid having formula (b- 1), (b-1a), (b-V), (b-VI) and/or (b-VII), per one phosphate group of the mRNA molecule. The lipid or lipidoid nanoparticles contained in the suspension formulation and in the aerosol in accordance with the invention preferably have a Z-average diameter in the range of 10 to 500 nm, more preferably in the range of 10 to 250 nm, still more preferably 20 to 200 nm. The indicated particle diameter is the hydrodynamic diameter of the particles, as determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C. The polydispersity index of the nanoparticles contained in the suspension formulation and in the aerosol in accordance with the invention is preferably in the range of 0.05 to 0.4, more preferably in the range of 0.05 to 0.2. The polydispersity index can be determined by dynamic light scattering (DLS). Measurements are generally carried out at 25 °C In some embodiments, the compositions comprise a pharmaceutically acceptable carrier and/or an adjuvant. For example, 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, and additional pharmaceutical agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, 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. For 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. In addition to biologically-neutral carriers, pharmaceutical 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. Optionally an mRNA vaccine of the invention is administered intramuscularly. Optionally an mRNA vaccine of the invention is administered intramuscularly, intradermally, subcutaneously by needle or by gene gun, or electroporation. Optionally, an mRNA of the invention, a vector of the invention, a pharmaceutical composition of the invention, or a vaccine of the invention is administered via the respiratory system. In some embodiments the administration is in a form which allows administration to the respiratory system via inhalation, nebulization, via a spray or droplets, e.g., a nasal spray or nasal droplets.
Figure imgf000043_0001
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. U.S.A.85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids’ Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet.6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLASTTM) (Altschul et al., J. Mol. 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. When comparing 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
Figure imgf000044_0001
, Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215: 403-410; program available from http://www.ebi.ac.uk/fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23: 2947-2948; program available from http://www.ebi.ac.uk/tools/clustalw2) and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley; programs available from http://www.ebi.ac.uk/tools/emboss/align). All programs may be run using default parameters. For example, 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. Conservative Amino Acid Substitutions A polypeptide encoded by a mRNA 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. Examples of conservative substitutions are shown below: Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The 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. Broadly Neutralising Immune Response 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. reduce), neutralise or prevent infection, and/or progress of infection, of a virus within the coronavirus family. Optionally a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of more than one type of β-coronavirus (for example, SARS- CoV, and SARS-CoV-2). Optionally a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of more than one type of β-coronavirus within the same β-coronavirus lineage (for example, more than one type of β- coronavirus within the subgenus Sarbecovirus, such as SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1). Optionally a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of coronaviruses of different β- coronavirus lineages, such as lineage B (for example, SARS-CoV, and SARS-CoV-2) and lineage C (for example, MERS-CoV). Optionally a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of most or all different β-coronaviruses. Optionally a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of most or all different viruses of the coronavirus family. Optionally a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of most or all variants of concern (VOCs) of SARS-CoV-2, including Beta, Gamma, Delta, Omicron (BA.1). Optionally a broadly neutralising immune response is sufficient to inhibit, neutralise or prevent infection, and/or progress of infection, of SARS-CoV, WIV16, RaTG13, SARS-CoV- 2, SARS-CoV-2 Beta, SARS-CoV-2 Gamma, SARS-CoV-2 Delta, SARS-CoV-2 Omicron (BA.1, BA.2, BA.2.12.1, BA.4, BA.5, XBB 1.5). The immune response may be a 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. Optionally a polypeptide encoded by an mRNA 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 are well known in the art and 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. Optionally a polypeptide encoded by an mRNA 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 mRNA has been administered (for example, expressed from an administered mRNA vaccine). Embodiments of the invention are described below, by way of example only, with reference to the accompanying drawings in which: Figure 1 illustrates in-silico design of antigen candidates: (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 (24). Human viruses are represented in green, palm civet viruses in pink and bat viruses in dark grey. The distinct two clades are coloured in red (non-ACE-2 binding) and blue (ACE-2 binding); (B) Structural models of RBD with epitope regions highlighted as spheres. The backbone of RBD is coloured according to the CONSURF (25) score calculated using the alignment used for construction of phylogenetic tree. The figure was generated and rendered using PyMol (24) using PDB (27) ids 6wps (14), 6w41 (15), and 7bz5 (11). (C) Structural representation of the different antigen designs used in the study. The epitopes that were modified to match the wild-type SARS-CoV (coloured orange) and wild-type SARS-CoV-2 (coloured grey) are represented in spheres. Further glycosylation site modification is represented in green sphere; Figure 2 shows in-vitro selection and in-vivo immunogenicity of antigens: (A) Immunisation and bleed schedule of BALB/c mice. Mice were immunised at interval of 30 days and bled every 15 days; (B) FACS binding data for the antigens. Sera from mice immunised with antigens were screened for binding to SARS-CoV, SARS-CoV-2, WIV16, and RaTG13 spike proteins. The X-axis represents the mean fluorescence intensity (MFI), and the Y-axis represents all the vaccine designs considered for screening. For each mouse sera, two replicates of MFI have been reported; (C) Elicitation of binding antibodies against SARS-CoV and SARS-CoV-2 by T2_17 was confirmed using ELISA, with SARS-CoV-2 RBD as control vaccine design. T2_17 generated cross-binding antibodies. The X-axis represents the bleeds, and the Y-axis represents the area under the curve (AUC) for ELISA binding curves. Mann-Whitney U demonstrated statistical significance (p-value: * ≤0.05, **<0.01, *** ≤ 0.001, ****≤ 0.0001); Figure 3 shows immunogenicity studies in guinea pigs and rabbits: (A) Immunisation and bleed schedule of guinea pigs. Guinea pigs were immunised with DNA delivered intradermally by the Tropis ParmaJet device at 28-day intervals and bled every 14 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) Neutralisation by guinea pig sera immunised with T2_17 and SARS2_RBD_P521N. The X-axis represents the bleed number, and the Y-axis represents the log10IC50 values for neutralisation curves; (D) Broad-neutralisation of SARS-CoV, WIV16, RaTG13, and SARS-CoV-2 by T2_17 in comparison to SARS2_RBD_P521N. Sera post 28 days after three immunisation (bleed 6) was used for comparison; (E) ACE-2 competition ELISA. Sera from Guinea pigs immunised with T2_17 and SARS2_RBD_P521N. The NIBSC standard (20/162) was used as control; (F) Immunisation and bleed schedule of rabbits. Rabbits were immunised at interval of 14 days and bled every 14 days; (G) Neutralisation by rabbit sera immunised with T2_17. The X-axis represents the bleed number, and the Y-axis represents the log10IC50 values for neutralisation curves. (H) Broad- neutralisation of SARS-CoV, WIV16, RaTG13, SARS-CoV-2, SARS-CoV-2 Beta, SARS- CoV-2 Gamma, SARS-CoV-2 Delta, and SARS-CoV-2 omicron by T2_17. Sera post 14 days after four immunisation (bleed 4) was used for comparison. NISBSC standard for SARS- CoV-2 and SARS-CoV antiserum are used as reference. Mann-Whitney U demonstrated statistical significance (p-value: * ≤0.05, **<0.01, *** ≤ 0.001, ****≤ 0.0001); Figure 4 shows immunogenicity and challenge studies in K18-hACE2 mice: (A) Immunisation, bleed, and challenge schedule of K18-hACE2 mice. K18-hACE2 mice were primed with AZD1222 vaccine and then boosted with either AZD1222, or T2_17 after four weeks. The mice were challenged after 8 weeks with either Victoria strain of SARS-CoV- 2 or the Delta variant; (B) Neutralisation of SARS-CoV, SARS-CoV-2, and delta variant of SARS-CoV-2 by K18- hACE2 mice sera. Sera of mice boosted with T2_17(DNA) and T2_17(MVA) significantly neutralised the Delta variant (B.1.617.2) in comparison to those boosted by AZD1222 at bleed 4. The X-axis represents the bleed number, and the Y-axis represents the log10IC50 values for neutralisation curves; (C) Weight loss profile of K18-hACE2 mice following challenge by the Victoria strain and the Delta variant. All the mice, except naïve were protected; (D) Immunisation, and bleed, schedule of K18-hACE2 mice for longitudinal analysis; (E) Neutralisation of SARS-CoV-2 K18-hACE2 mice sera. Neutralisation by sera of mice boosted with T2_17 (MVA) is statistically higher to those boosted by AZD1222 at bleed 2. The X-axis represents the bleed number, and the Y-axis represents the log10IC50 values for neutralisation curves; (F) Peptide micro-array analyses of the longitudinal analysis. The X-axis represents the mice sera, and the Y-axis represents the different linear peptides. The last column represents the conservation of the corresponding peptide in SARS-CoV, SARS-CoV-2, and T2_17. Mann- Whitney U test is used as statistical significance test in all the plots (p-value: * ≤0.05, **<0.01, *** ≤ 0.001); Figure 5 shows immunogenicity of mRNA in guinea pigs: (A) Immunisation, and bleed schedule of guinea pigs. The guinea pigs were immunised with mRNA at 3 weeks intervals. (B) Neutralisation of SARS-CoV, and SARS-CoV-2 by guinea pigs’ sera. The X-axis represents the bleed number, and the Y-axis represents the log10IC50 values for neutralisation curves. (C) Broad-neutralisation of SARS-CoV, WIV16, RaTG13, SARS-CoV-2, and SARS-CoV-2 omicron by T2_17. Sera post 6 weeks after boost (bleed 3) was used for comparison. Mann-Whitney U test is used as statistical significance test in all the plots (p-value: * ≤0.05, **<0.01, *** ≤ 0.001); Figure 6 shows a multiple sequence alignment of the known sarbecoviruses: Sarbecoviruses are divided into two distinct phylogenetic clades – clade 1 (boxed in blue) and clade 2. Members of clade 1 has deletions around the ACE-2 binding motif and have been reported to not bind human ACE-2 receptor. The regions corresponding to epitope region of S309, CR3022 and B38 antibodies are coloured in grey, purple, and orange respectively; Figure 7 shows neutralisation data for SARS2_RBD_P521N and SARS2_RBD in BALB/c mice: Sera from BALB/c mice immunised with SARS2_RBD_P521N and SARS-COV-2 RBD generated similar neutralising antibody response 14 days post four immunisations. The X- axis represents the antigens, and the Y-axis represents the log10IC50 values for neutralisation curves. The difference is statistically non-significant (Mann-Whitney U test, p-value = 0.4681); Figure 8 shows binding antibody data for T2_17 in guinea pigs: Elicitation of binding antibodies against SARS-CoV and SARS-CoV-2 by T2_17 and SARS2_RBD_P521N was confirmed using ELISA. 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. Mann-Whitney U test is used as statistical significance test in all the plots (p-value: * ≤0.05, **<0.01, *** ≤ 0.001); Figure 9 shows binding antibody data for T2_17 in rabbits: Elicitation of binding antibodies against SARS-CoV and SARS-CoV-2 by T2_17 was confirmed using ELISA. The X-axis represents the bleed number, and the Y-axis represents the area under the curve (AUC) for ELISA binding curve. Mann-Whitney U test is used as statistical significance test in all the plots (p-value: * ≤0.05, **<0.01, *** ≤ 0.001); Figure 10 shows 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. The X-axis represents the bleed number, and the Y-axis represents the area under the curve (AUC) for ELISA binding curve. Mann- Whitney U test is used as statistical significance test in all the plots (p-value: * ≤0.05, **<0.01, *** ≤ 0.001); Figure 11 shows immunogenicity of mRNA vaccine in BALB/c mice: (A) Immunisation, and bleed schedule of BALB/c mice. The mice were immunised with mRNA at 4 weeks intervals. (B) Elicitation of binding antibodies against SARS-CoV-2 was confirmed using ELISA for sera 2 weeks post boost (bleed 3). The X-axis represents the antigens, and the Y-axis represents the area under the curve (AUC) for ELISA binding curve; Figure 12 shows immunogenicity of mRNA vaccine in guinea pigs: Elicitation of binding antibodies against SARS-CoV and SARS-CoV-2 was confirmed using ELISA for guinea pigs. The X-axis represents the antigens, and the Y-axis represents the area under the curve (AUC) for ELISA binding curve. Mann-Whitney U test is used as statistical significance test in all the plots (p-value: * ≤0.05, **<0.01, *** ≤ 0.001); Figure 13 shows SARS S-protein architecture. The N-terminal sequence is responsible for relaying extracellular signals intracellularly. Studies show that the N-terminal region of the S protein is much more diverse than the C-terminal region, which is highly conserved (Dong et al, Genomic and protein structure modelling analysis depicts the origin and infectivity of 2019- nCoV, a new coronavirus which caused a pneumonia outbreak in Wuhan, China.2020). The figure shows the S domain, which comprises S1 and S2 domains, responsible for receptor binding and cell membrane fusion respectively; Figure 14 shows immunogenicity of mRNA vaccines in guinea pigs. The guinea pigs were immunised with 15µg T2_17_TM mRNA at 3 week intervals as per the immunisation schedule of Figure 5a. The figure shows neutralisation of SARS-CoV-2 Wuhan and SARS- CoV-2 XBB.1.5 by guinea pig sera at bleed 3 (6 weeks post boost). The boxes represent the quartiles (25th, 50th and 75th percentiles) of the distribution, and the whiskers represent the minimum and maximum of the distribution (excluding outliers) and the fliers represented as filled circle represent the outliers. Two-tailed Mann-Whitney U demonstrated statistical significance (p-value: * ≤0.05, **<0.01, *** ≤ 0.001, ****≤ 0.0001); Figure 15 shows gating strategy used to analyse flow cytometry data. (A) Preliminary FSC/SSC gates were set on the starting cell population. (B) Singlets were gated by plotting FSC-H versus FSC-A, followed by gating live cells as 7-AAD negative (C). (D) Live cells were visualized as a histogram in the RL-1 channel, with the PMT of the negative cell population set between 102 and 103. (E) The MFI values of positive control is shown. Representative flow cytometry plots are shown; Figure 16 shows immunogenicity of mRNA vaccines in guinea pigs. The guinea pigs were immunised with 15µg T2_17_TM mRNA at 3 week intervals as per the immunisation schedule of Figure 5a. The figure shows neutralisation of SARS-CoV-2 omicron VOCs by guinea pig sera at bleed 3 (6 weeks post boost). The boxes represent the quartiles (25th, 50th and 75th percentiles) of the distribution, and the whiskers represent the minimum and maximum of the distribution (excluding outliers) and the fliers represented as filled circle represent the outliers; Figure 17a shows neutralisation of sarbecovirus lentiviral pseudotypes including omicron VOCs by antisera generated in guinea pigs after immunisation with next generation optimised coronavirus T2_20 constructs, CoV_S_T3_3 (T2_20_v2)(SEQ ID NO:49), and COV_S_T3_4 (T2_17_T2_20_dimer)(SEQ ID NO:51), as well as first generation COV_S_T2_20 (SEQ ID NO:48). The data shown comprises results of study COV038 in guinea pigs. The guinea pigs were immunised twice at weeks 0 and 3, and bled four times at 3 week intervals. Data shown is for sera at SB2 (3 weeks after boost) (Figure 17b); Figure 18 shows an amino acid sequence alignment of CoV_T2_20 (SEQ ID NO:48), CoV_S_T3_3 (SEQ ID NO:49), and COV_S_T3_4 (SEQ ID NO:51) (with leader sequences). Differences between the sequences are shown as the boxed residues. In this alignment, CoV_S_T3_4 (T2_17_T2_20 dimer) is shown with the amino acid sequence of T2_20 before that of T2_17; Figure 19 shows an amino acid sequence alignment of CoV_T2_20 (SEQ ID NO:48), CoV_S_T3_3 (SEQ ID NO:49), and COV_S_T3_4 (SEQ ID NO:51) (with leader sequences). Differences between the sequences are shown as the boxed residues. In this alignment, CoV_S_T3_4 (T2_17_T2_20 dimer) is shown with the amino acid sequence of T2_17 before that of T2_20; Figure 20a shows an immunogenicity study of optimised coronavirus CoV_S_T2_20 (SEQ ID NO:48) in guinea pigs. Guinea pigs were immunised twice with mRNA at weeks 0 and 3, and bled at 3 week intervals beginning from day 0 (Figure 20b). Data shown uses sera from bleed SB2 (3 weeks after boost). The x-axis represents the pseudoviruses tested for neutralisation, and the y-axis represents the log10(IC50) values; and Figure 21 is a continuation of the study shown in Figure 20. Figure 21a shows neutralisation data for optimised coronavirus CoV_S_T2_20 (SEQ ID NO:48) antigen in guinea pigs against a more diverse panel of coronavirus pseudoviruses. Guinea pigs were immunised twice with mRNA at weeks 0 and 3, and bled at 3 week intervals beginning from day 0 (Figure 21b). Data shown uses sera from bleed SB2 (3 weeks after boost). Table of SEQ ID NOs:
Figure imgf000053_0001
1 T2_17 (also known as COV_S_T2_17) amino acid sequence 2 Transmembrane domain amino acid sequence 3 T2_20 (also known as COV_S_T2_20, or T2_17_TM) amino acid sequence (without leader sequence) 4 RNA sequence encoding T2_17 5 RNA sequence encoding transmembrane domain 6 RNA sequence encoding T2_20 7 mRNA sequence encoding T2_17 8 RNA sequence encoding T2_17 (from coding sequence of SEQ ID NO:7) 9 mRNA sequence encoding T2_20 10 RNA sequence encoding T2_20 (from coding sequence of SEQ ID NO:9) 11 5’-UTR sequence of an mRNA of the invention (Min UTR C) 12 DNA/mRNA sequence coding for 5’-GAAG-MinUTR-CT 13 DNA/mRNA sequence coding for 5’-MinUTR-CT 14 5’-UTR sequence in the vector of the invention (Min UTR CT) 15 5’-UTR sequence in the vector of the invention (5’-GAAG-Min UTR CT) 16 5’-UTR sequence in the vector of the invention (5’-GGGA-Min UTR CT) 17. DNA sequence of T7 promoter + MinUTR-C +Kozak 18. DNA sequence of T3 promoter + Min UTR-C + Kozak 19. DNA sequence of SP6 promoter + Min UTR-C + Kozak 20 DNA sequence of K11 promoter + Min UTR-C + Kozak 21 DNA sequence of T7 promoter + Min UTR-CT + Kozak 22 DNA sequence of T3 promoter + Min UTR-CT + Kozak 23 DNA sequence of SP6 promoter + Min UTR-CT + Kozak 24 DNA sequence of K11 promoter + Min UTR-CT + Kozak 25 DNA/mRNA sequence of 5´CYBA UTR 26 DNA/mRNA sequence of 3´CYBA UTR 27 RNA sequence encoding COV_S_T2_17 – ORF – 723 nt 28 RNA sequence encoding COV_S_T2_17 Min – (CAP+ 5’UTR-C+Kozak+ORF). 29 RNA sequence encoding COV_S_T2_17 Min – (CAP-5’UTR-CT+Kozak+ORF) 30 RNA sequence encoding COV_S_T2_17 Min – (CAP - 5’UTR -CT + Kozak + 3’UTR + PolyA120). 31 mRNA sequence encoding COV_S_T2_20 – (ORF) – 933 nt 32 mRNA sequence encoding COV_S_T2_20 – (5’UTR-C + Kozak+ ORF) – 946 nt 33 sequence encoding COV_S_T2_20 – (CAP-5’UTR-CT+Kozak+ORF) – 947 nt 34 sequence encoding COV_S_T2_20 – (CAP-5’UTR-CT+Kozak+ ORF + PolyA) 35 sequence of segmented polyA (1) 36 sequence of segmented polyA (2) 37 sequence of segmented polyA (3) 38 sequence of segmented polyA (4) 39 sequence of segmented polyA (5) 40 sequence of segmented polyA (6) 41 DNA sequence of ETH072T02 full-length spike (SCoV2(PP)) (T7 promoter +5’UTR+3’UTR) 42 mRNA sequence of ETH072T02 full-length spike (SCoV2(PP)) (5’ Min UTRC +3’UTR) 43 mRNA sequence of ETH072T02 full-length spike (SCoV2(PP)) (Min. 5’UTR + 3’UTR + poly(A)) 44 mRNA sequence of ETH072T02 full-length spike (SCoV2(PP)) ORF. 45 AA sequence of ETH072T02 full-length spike protein (SCoV2(PP). 46 Additional mRNA sequence encoding T2_20 47 5’-UTR sequence of an mRNA of the invention (Min UTR C) 48 T2_20 (also known as COV_S_T2_20, or T2_17_TM) amino acid sequence (with leader sequence) 49 CoV_S_T3_3 (T2_20v2) amino acid sequence (with leader sequence) 50 CoV_S_T3_3 (T2_20v2) amino acid sequence (without leader sequence) 51 CoV_S_T3_4 (T2_17_T2_20 dimer) amino acid sequence (with leader sequence) 52 CoV_S_T3_4 (T2_17_T2_20 dimer) amino acid sequence (without leader sequence) 53 CoV_S_T2_20 Scaffold Sequence 54 Leader amino acid sequence
SEQUENCES T2_17 amino acid sequence (SEQ ID NO:1): RVAPTKEVVR FPNITNLCPF GEVFNATKFP SVYAWERKKI SNCVADYSVL YNSTSFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGVI ADYNYKLPDD FTGCVIAWNT NNIDSTTGGN YNYLYRSLRK SKLKPFERDI SSDIYSPGGK PCSGVEGFNC YYPLRSYGFF PTNGTGYQPY RVVVLSFELL NAPATVCGPK LSTD Transmembrane domain amino acid sequence (SEQ ID NO:2): GGGGSGGGGS GGGGSGGGGS KSSIASFFFI IGLIIGLFLV LRVGIHLCIK LKHTKKRQIY TDIEMNRLGK T2_20 amino acid sequence (SEQ ID NO:3): RVAPTKEVVR FPNITNLCPF GEVFNATKFP SVYAWERKKI SNCVADYSVL YNSTSFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGVI ADYNYKLPDD FTGCVIAWNT NNIDSTTGGN YNYLYRSLRK SKLKPFERDI SSDIYSPGGK PCSGVEGFNC YYPLRSYGFF PTNGTGYQPY RVVVLSFELL NAPATVCGPK LSTDGGGGSG GGGSGGGGSG GGGSKSSIAS FFFIIGLIIG LFLVLRVGIH LCIKLKHTKK RQIYTDIEMN RLGK The transmembrane domain amino acid sequence is shown in bold format. RNA sequence encoding COV_S_T2_17 (SEQ ID NO:4): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUC GGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAAAAGAUC AGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGAC AGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUC GCCGAUUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACC AACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAG UCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCCCUGGCGGCAAG CCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUUCUUC CCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUG AAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACCGAC RNA sequence encoding a transmembrane domain of amino acid sequence SEQ ID NO:2 (SEQ ID NO:5): GGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUU CUAUCGCCAGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAU CCACCUGUGCAUCAAGCUGAAACACACCAAGAAGCGGCAAAUCUACACCGACAUCGAGAUGAACCGG CUGGGCAAA RNA sequence encoding T2_20 (SEQ ID NO:6): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUC GGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAAAAGAUC AGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGAC AGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUC GCCGAUUACAACUACAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACC AACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAG UCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCCCUGGCGGCAAG CCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUUCUUC CCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUG AAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAGGAUCUGGC GGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCAGC UUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCAC CUGUGCAUCAAGCUGAAACACACCAAGAAGCGGCAAAUCUACACCGACAUCGAGAUGAAC CGGCUGGGCAAA mRNA sequence encoding COV_S_T2_17 (SEQ ID NO:7): m GP G- GGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUGU UUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAA UCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAG AAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGU GCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGU GAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUAC AAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCG GCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAU CAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUAC CCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCC UGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGACUGAUGAGA AUU-Poly(~A ) RNA sequence in the above coding sequence encoding T2_17 (SEQ ID NO:8): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUCGGCGAGG UGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAGAAAAUCAGCAACUGCGUGGC CGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGUCACCCACC AAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGC GGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAAGCUGCCCGACGACUUCAC CGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUG UACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCC CUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUU CUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUGAAU GCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGAC mRNA sequence encoding COV_S_T2_20 (SEQ ID NO:9): m2 7,3´-oGP3 G- GGGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUG UUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCA AUCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAA GAAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCG UGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUA CAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACC GGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACA UCAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUA CCCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUC CUGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCG GAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGC CAGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUG UGCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCA AGUGAUGAGAAUU-Poly(~A ) ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5’UTR (minimal) =GGGAGACGCCACC 3’UTR (unspecific) = GAAUU Coding sequence = bold format text RNA sequence in the above coding sequence encoding T2_20 (SEQ ID NO:10): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUCGGCGAGG UGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAGAAAAUCAGCAACUGCGUGGC CGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGUCACCCACC AAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGC GGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAAGCUGCCCGACGACUUCAC CGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUG UACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCC CUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUU CUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUGAAU GCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAGGAUCUGGCGGAGGUGGAA GCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCAGCUUCUUCUUCAUCAUCGG CCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGUGCAUCAAGCUGAAACACACC AAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCAAG DNA/mRNA sequence coding for 5’-MinUTR-C (SEQ ID NO:11) GGAGACGCCACC DNA/mRNA sequence coding for 5’-MinUTR-CT (SEQ ID NO:12) GAAGCGCCACC DNA/mRNA sequence coding for 5’-MinUTR-CT (SEQ ID NO:13) GGGACGCCACC DNA/mRNA sequence coding for 5’-MinUTR-CT (SEQ ID NO:14) GGGAGACTGCCACC DNA/mRNA sequence coding for 5’-MinUTR-CT (SEQ ID NO:15) GAAGCTGCCACC DNA/mRNA sequence coding for 5’-MinUTR-CT (SEQ ID NO:16) GGGACTGCCACC DNA
Figure imgf000058_0002
of start codon – T7 + MinUTR-C + Kozak
Figure imgf000058_0001
ID NO:17): TAATACGACTCACTATAGGGAGA CGCCACC Cursive: T7 Promoter Underlined: Kozak DNA
Figure imgf000058_0004
of start codon – T3 + C
Figure imgf000058_0003
ID NO:18): AATTAACCCTCACTAAAGGGAGA CGCCACC Cursive: SP6 Promoter Underlined: Kozak DNA sequence upstream of start codon – SP6 + C (SEQ ID NO:19): ATTTAGGTGACACTATAGAAG CGCCACC Cursive: SP6 Promoter Underlined: Kozak DNA sequence upstream of start codon – K11 + C (SEQ ID NO:20): AATTAGGGCACACTATAGGGA CGCCACC Cursive: T3 Promoter Underlined: Kozak DNA sequence upstream of start codon – T7 + CT (SEQ ID NO:21): TAATACGACTCACTATA GGGAGACTGCCACC Cursive: T7 Promoter Underlined: Kozak DNA
Figure imgf000059_0002
of start codon – T3 + CT
Figure imgf000059_0001
AATTAACCCTCACTAAAGGGAGACTGCCACC Cursive: T3 Promoter Underlined: Kozak DNA
Figure imgf000059_0004
of start codon – SP6 + CT
Figure imgf000059_0003
: ATTTAGGTGACACTATAGAAG CTGCCACC Cursive: SP6 Promoter Underlined: Kozak DNA
Figure imgf000059_0006
of start codon – K11 + CT
Figure imgf000059_0005
: AATTAGGGCACACTATAGGGA CTGCCACC Cursive: T3 Promoter Underlined: Kozak DNA/mRNA
Figure imgf000059_0008
of 5’ UTR CYBA -37 nt-
Figure imgf000059_0007
CGCGCCUAGCAGUGUCCCAGCCGGGUUCGUGUCGCC
Figure imgf000059_0009
AUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUGUUUGUGUCUCCAU CUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUU CGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAGAAAAUCAGCAAC UGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGU CACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGA CGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAAGCUGCCCGAC GACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCGGCAACUACA ACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAU CUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGC UACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGC UGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGACUGAUGA mRNA sequence encoding COV_S_T2_17 Min – (CAP+ 5’UTR-C+Kozak+ORF) – 735 nt (SEQ ID NO:28): m GP G- GGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUGU UUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAA UCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAG AAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGU GCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGU GAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUAC AAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCG GCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAU CAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUAC CCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCC UGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGACUGAUGA mRNA sequence encoding COV_S_T2_17 Min – (CAP-5’UTR-CT+Kozak+ORF) – 737 nt (SEQ ID NO:29): m GP G- GGAGACTGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUG UUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCA AUCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAA GAAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCG UGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUA CAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACC GGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACA UCAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUA CCCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUC CUGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGACUGAUGA mRNA sequence encoding COV_S_T2_17 Min – (CAP-5’UTR- CT+Kozak+3’UTR+PolyA120) - 742 nt - (SEQ ID NO:30): m GP G- GGAGACTGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUG UUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCA AUCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAA GAAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCG UGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUA CAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACC GGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACA UCAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUA CCCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUC CUGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGACUGAUGAG AAUU-Poly(~A ) mRNA sequence encoding COV_S_T2_20 – (ORF) – 933 nt - (SEQ ID NO:31): AUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUGUUUGUGUCUCCAU CUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUU CGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAGAAAAUCAGCAAC UGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGU CACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGA CGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAAGCUGCCCGAC GACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCGGCAACUACA ACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAU CUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGC UACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGC UGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAGGAUCUGGCGG AGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCAGCUUCUUCUUC AUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGUGCAUCAAGCUGA AACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCAAGUGAUGA mRNA sequence encoding COV_S_T2_20 – (5’UTR-C + Kozak+ ORF) – 946 nt - (SEQ ID NO:32): GGGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUG UUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCA AUCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAA GAAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCG UGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUA CAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACC GGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACA UCAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUA CCCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUC CUGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCG GAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGC CAGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUG UGCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCA AGUGAUGA mRNA sequence encoding COV_S_T2_20 – (CAP-5’UTR-CT+Kozak+ORF) – 947 nt - (SEQ ID NO:33): m GP G- GGAGACTGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUG UUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCA AUCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAA GAAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCG UGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUA CAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACC GGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACA UCAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUA CCCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUC CUGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCG GAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGC CAGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUG UGCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCA AGUGAUGA mRNA sequence encoding COV_S_T2_20 – (CAP-5’UTR-CT+Kozak+ ORF + PolyA) – 952 nt -(SEQ ID NO:34): m GP G- GGAGACTGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUG UUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCA AUCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAA GAAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAG UGCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCG UGAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUA CAAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACC GGCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACA UCAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUA CCCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUC CUGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCG GAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGC CAGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUG UGCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCA AGUGAUGAGAAUU-Poly(~A ) ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5ÚTR (minimal) =GGAGACTGCCACC 3ÚTR (unspecific) = GAAUU Coding sequence = bold format text DNA/mRNA sequence of segmented polyA (1) (SEQ ID NO: 35) AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA DNA/mRNA sequence of segmented polyA (2) (SEQ ID NO: 36) AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCGATAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA DNA/mRNA sequence of segmented polyA (3) (SEQ ID NO: 37) AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGATATCAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA DNA/mRNA sequence of segmented polyA (4) (SEQ ID NO: 38) AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA DNA/mRNA sequence of segmented polyA (5) (SEQ ID NO: 39) AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA DNA/mRNA sequence of segmented polyA (6) (SEQ ID NO: 40) AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA DNA sequence of ETH072T02 full-length spike (SCoV2(PP)) T7 promoter +5’UTR+3’UTR (SEQ ID NO: 41) AAGCTTTAGCCGGCGTGGAAGGTAACAGCACCGCTGTAATACGACTCACTATAGGGAGACGCCACCATGTTCGT GTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGACCACCAGAACACAGCTGCCTCCAGCCT ACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACTCTACCCAG GACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGTCCGGCACCAATGGCACCAA GAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAACATCATCA GAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCACCAACGTG GTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTCTACTACCACAAGAACAACAAGAG CTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCAGCCTTTCC TGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCGACGGCTAC TTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCCTCAGGGCTTCTCTGCTCTGGAACC CCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAGCTACCTGA CACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGCCTAGAACC TTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTGAGCGAGAC AAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCAGCCCACCG AATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCAGATTCGCC TCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAACTCCGCCAG CTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGTGTACGCCG ACAGCTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCCGACTACAAC TACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACTCCAAAGTCGGCGG CAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGA TCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGTCCTACGGC TTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTGCATGCCCC TGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTTCAACGGCC TGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGGATATCGCC GATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTCGGCGGAGT GTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGACGTGAACTGTACCGAAG TGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATGTGTTTCAG ACCAGAGCCGGCTGTCTGATCGGAGCCGAGCACGTGAACAATAGCTACGAGTGCGACATCCCCATCGGCGCTGG CATCTGTGCCAGCTACCAGACACAGACAAACAGCCCCAGACGGGCCAGATCTGTGGCCAGCCAGAGCATCATTG CCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCACCAACTTC ACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATGTACATCTG CGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAATAGAGCCCTGA CAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACAAGACCCCT CCTATCAAGGACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAGCGGAGCTT CATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGATTGTCTGG GCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTCTGCTGACC GATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTTGGAGCTGG CGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCAGAATGTGC TGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCCTGAGCAGC ACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCAGAATGCCCAGGCACTGAACACCCTGGTCAAGCA GCTGTCCTCCAACTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCCTGAGGCCG AGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGCTGATCAGA GCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAGAGCAAGAG AGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGTGTTTCTGC ACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCAAAGCCCAC TTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTACGAGCCCCA GATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAATACCGTGT ACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACACAAGCCCC GACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGACCGGCTGAA CGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTACATCAAGT GGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGCTGTGTTGC ATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAGGACGATTC TGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGAGAATTCGAAGAGCCACCGGGCAATACGAGCT CAAGCCAGTCTCAAGCTT 5’UTR: HindIII - Random sequence of 30nts - T7 Promoter - 5´MinUTR-Kozak-Start codon 3’UTR: Stop codon-EcoRI-SapI-Random Sequence of 30nts-HindIII mRNA sequence of ETH072T02 full-length spike (SCoV2(PP)) (5’ Min UTRC +3’UTR) (SEQ ID NO: 42) m27,3´-oGP3 G- GGAGACGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGACCACCAG AACACAGCTGCCTCCAGCCTACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCA GCGTGCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTG TCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCAC CGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCG TGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTCTAC TACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGA GTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGT TTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCCTCAG GGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGC CCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGG GCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCT CTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAA CTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGT TCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCC GTGCTGTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTG CTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACAG GCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAC CTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGA GCGGGACATCTCCACCGAGATCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACT TCCCACTGCAGTCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGC TTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGT GAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGC AGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACC CCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCA GGACGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCA CCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGCACGTGAACAATAGCTACGAGTGC GACATCCCCATCGGCGCTGGCATCTGTGCCAGCTACCAGACACAGACAAACAGCCCCAGACGGGCCAGATCTGT GGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTA TCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTG GACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCAC CCAGCTGAATAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGA AGCAGATCTACAAGACCCCTCCTATCAAGGACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGC AAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAA GCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAG TGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGC GGCTGGACATTTGGAGCTGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCAT CGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGA TCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCAGAATGCCCAGGCA CTGAACACCCTGGTCAAGCAGCTGTCCTCCAACTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAG ACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACG TGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGT GTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCC TCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCT GCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAG CGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGG CATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACT TTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAG AAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAA GTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGG TCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGC AAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGA ARCA Cap = m27,3´-oGP3 G- 5’UTR (minimal) =GGAGACGCCACC ATG: Bold Stop codons: Bold mRNA sequence of ETH072T02 full-length spike (SCoV2(PP)) (Min.5’UTR + 3’UTR + poly(A)) (SEQ ID NO: 43) m27,3´-oGP3 G- GGAGACGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGACCACCAG AACACAGCTGCCTCCAGCCTACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCA GCGTGCTGCACTCTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTG TCCGGCACCAATGGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCAC CGAGAAGTCCAACATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCG TGAACAACGCCACCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTCTAC TACCACAAGAACAACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGA GTACGTGTCCCAGCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGT TTAAGAACATCGACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCCTCAG GGCTTCTCTGCTCTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGC CCTGCACAGAAGCTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGG GCTACCTGCAGCCTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCT CTGGATCCTCTGAGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAA CTTCCGGGTGCAGCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGT TCAATGCCACCAGATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCC GTGCTGTACAACTCCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTG CTTCACAAACGTGTACGCCGACAGCTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACAG GCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAAC CTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGA GCGGGACATCTCCACCGAGATCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACT TCCCACTGCAGTCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGC TTCGAACTGCTGCATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGT GAACTTCAACTTCAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGC AGTTTGGCCGGGATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACC CCTTGCAGCTTCGGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCA GGACGTGAACTGTACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCA CCGGCAGCAATGTGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGCACGTGAACAATAGCTACGAGTGC GACATCCCCATCGGCGCTGGCATCTGTGCCAGCTACCAGACACAGACAAACAGCCCCAGACGGGCCAGATCTGT GGCCAGCCAGAGCATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTA TCGCTATCCCCACCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTG GACTGCACCATGTACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCAC CCAGCTGAATAGAGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGA AGCAGATCTACAAGACCCCTCCTATCAAGGACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGC AAGCCCAGCAAGCGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAA GCAGTATGGCGATTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAG TGCTGCCTCCTCTGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGC GGCTGGACATTTGGAGCTGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCAT CGGAGTGACCCAGAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGA TCCAGGACAGCCTGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCAGAATGCCCAGGCA CTGAACACCCTGGTCAAGCAGCTGTCCTCCAACTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAG ACTGGACCCTCCTGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACG TGACCCAGCAGCTGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGT GTGCTGGGCCAGAGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCC TCACGGCGTGGTGTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCT GCCACGACGGCAAAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAG CGGAACTTCTACGAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGG CATTGTGAACAATACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACT TTAAGAACCACACAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAG AAAGAGATCGACCGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAA GTACGAGCAGTACATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGG TCACAATCATGCTGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGC AAGTTCGACGAGGACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGAGAATT- Poly(~A120) ARCA Cap = m27,3´-oGP3 G- 5ÚTR (minimal) =GGAGACGCCACC ATG: Bold 3ÚTR (unspecific) = GAATT Poly(A) tail = -poly(~A120) Stop codons: Bold mRNA sequence of ETH072T02 full-length spike (SCoV2(PP)) ORF (SEQ ID NO: 44) ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTGAACCTGACCACCAGAACACAGCTGCC TCCAGCCTACACCAACAGCTTTACCAGAGGCGTGTACTACCCCGACAAGGTGTTCAGATCCAGCGTGCTGCACT CTACCCAGGACCTGTTCCTGCCTTTCTTCAGCAACGTGACCTGGTTCCACGCCATCCACGTGTCCGGCACCAAT GGCACCAAGAGATTCGACAACCCCGTGCTGCCCTTCAACGACGGGGTGTACTTTGCCAGCACCGAGAAGTCCAA CATCATCAGAGGCTGGATCTTCGGCACCACACTGGACAGCAAGACCCAGAGCCTGCTGATCGTGAACAACGCCA CCAACGTGGTCATCAAAGTGTGCGAGTTCCAGTTCTGCAACGACCCCTTCCTGGGCGTCTACTACCACAAGAAC AACAAGAGCTGGATGGAAAGCGAGTTCCGGGTGTACAGCAGCGCCAACAACTGCACCTTCGAGTACGTGTCCCA GCCTTTCCTGATGGACCTGGAAGGCAAGCAGGGCAACTTCAAGAACCTGCGCGAGTTCGTGTTTAAGAACATCG ACGGCTACTTCAAGATCTACAGCAAGCACACCCCTATCAACCTCGTGCGGGATCTGCCTCAGGGCTTCTCTGCT CTGGAACCCCTGGTGGATCTGCCCATCGGCATCAACATCACCCGGTTTCAGACACTGCTGGCCCTGCACAGAAG CTACCTGACACCTGGCGATAGCAGCAGCGGATGGACAGCTGGTGCCGCCGCTTACTATGTGGGCTACCTGCAGC CTAGAACCTTCCTGCTGAAGTACAACGAGAACGGCACCATCACCGACGCCGTGGATTGTGCTCTGGATCCTCTG AGCGAGACAAAGTGCACCCTGAAGTCCTTCACCGTGGAAAAGGGCATCTACCAGACCAGCAACTTCCGGGTGCA GCCCACCGAATCCATCGTGCGGTTCCCCAATATCACCAATCTGTGCCCCTTCGGCGAGGTGTTCAATGCCACCA GATTCGCCTCTGTGTACGCCTGGAACCGGAAGCGGATCAGCAATTGCGTGGCCGACTACTCCGTGCTGTACAAC TCCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACAAACGT GTACGCCGACAGCTTCGTGATCCGGGGAGATGAAGTGCGGCAGATTGCCCCTGGACAGACAGGCAAGATCGCCG ACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGTGTGATTGCCTGGAACAGCAACAACCTGGACTCCAAA GTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGGAAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTC CACCGAGATCTATCAGGCCGGCAGCACCCCTTGTAACGGCGTGGAAGGCTTCAACTGCTACTTCCCACTGCAGT CCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTACAGAGTGGTGGTGCTGAGCTTCGAACTGCTG CATGCCCCTGCCACAGTGTGCGGCCCTAAGAAAAGCACCAATCTCGTGAAGAACAAATGCGTGAACTTCAACTT CAACGGCCTGACCGGCACCGGCGTGCTGACAGAGAGCAACAAGAAGTTCCTGCCATTCCAGCAGTTTGGCCGGG ATATCGCCGATACCACAGACGCCGTTAGAGATCCCCAGACACTGGAAATCCTGGACATCACCCCTTGCAGCTTC GGCGGAGTGTCTGTGATCACCCCTGGCACCAACACCAGCAATCAGGTGGCAGTGCTGTACCAGGACGTGAACTG TACCGAAGTGCCCGTGGCCATTCACGCCGATCAGCTGACACCTACATGGCGGGTGTACTCCACCGGCAGCAATG TGTTTCAGACCAGAGCCGGCTGTCTGATCGGAGCCGAGCACGTGAACAATAGCTACGAGTGCGACATCCCCATC GGCGCTGGCATCTGTGCCAGCTACCAGACACAGACAAACAGCCCCAGACGGGCCAGATCTGTGGCCAGCCAGAG CATCATTGCCTACACAATGTCTCTGGGCGCCGAGAACAGCGTGGCCTACTCCAACAACTCTATCGCTATCCCCA CCAACTTCACCATCAGCGTGACCACAGAGATCCTGCCTGTGTCCATGACCAAGACCAGCGTGGACTGCACCATG TACATCTGCGGCGATTCCACCGAGTGCTCCAACCTGCTGCTGCAGTACGGCAGCTTCTGCACCCAGCTGAATAG AGCCCTGACAGGGATCGCCGTGGAACAGGACAAGAACACCCAAGAGGTGTTCGCCCAAGTGAAGCAGATCTACA AGACCCCTCCTATCAAGGACTTCGGCGGCTTCAATTTCAGCCAGATTCTGCCCGATCCTAGCAAGCCCAGCAAG CGGAGCTTCATCGAGGACCTGCTGTTCAACAAAGTGACACTGGCCGACGCCGGCTTCATCAAGCAGTATGGCGA TTGTCTGGGCGACATTGCCGCCAGGGATCTGATTTGCGCCCAGAAGTTTAACGGACTGACAGTGCTGCCTCCTC TGCTGACCGATGAGATGATCGCCCAGTACACATCTGCCCTGCTGGCCGGCACAATCACAAGCGGCTGGACATTT GGAGCTGGCGCCGCTCTGCAGATCCCCTTTGCTATGCAGATGGCCTACCGGTTCAACGGCATCGGAGTGACCCA GAATGTGCTGTACGAGAACCAGAAGCTGATCGCCAACCAGTTCAACAGCGCCATCGGCAAGATCCAGGACAGCC TGAGCAGCACAGCAAGCGCCCTGGGAAAGCTGCAGGACGTGGTCAACCAGAATGCCCAGGCACTGAACACCCTG GTCAAGCAGCTGTCCTCCAACTTCGGCGCCATCAGCTCTGTGCTGAACGATATCCTGAGCAGACTGGACCCTCC TGAGGCCGAGGTGCAGATCGACAGACTGATCACAGGCAGACTGCAGAGCCTCCAGACATACGTGACCCAGCAGC TGATCAGAGCCGCCGAGATTAGAGCCTCTGCCAATCTGGCCGCCACCAAGATGTCTGAGTGTGTGCTGGGCCAG AGCAAGAGAGTGGACTTTTGCGGCAAGGGCTACCACCTGATGAGCTTCCCTCAGTCTGCCCCTCACGGCGTGGT GTTTCTGCACGTGACATATGTGCCCGCTCAAGAGAAGAATTTCACCACCGCTCCAGCCATCTGCCACGACGGCA AAGCCCACTTTCCTAGAGAAGGCGTGTTCGTGTCCAACGGCACCCATTGGTTCGTGACACAGCGGAACTTCTAC GAGCCCCAGATCATCACCACCGACAACACCTTCGTGTCTGGCAACTGCGACGTCGTGATCGGCATTGTGAACAA TACCGTGTACGACCCTCTGCAGCCCGAGCTGGACAGCTTCAAAGAGGAACTGGACAAGTACTTTAAGAACCACA CAAGCCCCGACGTGGACCTGGGCGATATCAGCGGAATCAATGCCAGCGTCGTGAACATCCAGAAAGAGATCGAC CGGCTGAACGAGGTGGCCAAGAATCTGAACGAGAGCCTGATCGACCTGCAAGAACTGGGGAAGTACGAGCAGTA CATCAAGTGGCCCTGGTACATCTGGCTGGGCTTTATCGCCGGACTGATTGCCATCGTGATGGTCACAATCATGC TGTGTTGCATGACCAGCTGCTGTAGCTGCCTGAAGGGCTGTTGTAGCTGTGGCAGCTGCTGCAAGTTCGACGAG GACGATTCTGAGCCCGTGCTGAAGGGCGTGAAACTGCACTACACATGATGA ATG: Bold Stop codons: Bold AA sequence of ETH072T02 Full length Spike protein (SCoV2(PP) Spike protein) (SEQ ID NO: 45) MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHA IHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQ FCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYF KIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVG YLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNL CPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVI RGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIS TEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN TSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGI CASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVD CTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQ ILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIA QYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSL SSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQ TYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQE KNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYD PLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKY EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT Additional mRNA sequence encoding COV_S_T2_20 (SEQ ID NO:46): m2 7,3´-oGP3 G- GGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUGU UUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAA UCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAG AAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGU GCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGU GAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUAC AAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCG GCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAU CAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUAC CCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCC UGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGG AGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCC AGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGU GCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCAA GUGAUGAGAAUU-Poly(~A ) ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5’UTR (minimal) =GGAGACGCCACC 3’UTR (unspecific) = GAAUU Coding sequence = bold format text DNA/mRNA sequence coding for 5’-MinUTR-C (SEQ ID NO:47) GGGAGACGCCACC Possible generic forms of poly(A) DNA/mRNA sequence of segmented polyA (8) A -S-A Wherein S is a single nucleotide selected from C, G, T or U DNA/mRNA sequence of segmented polyA (9) A -N-S N-A Wherein N a nucleotide that is not adenine. Wherein S nucleotides are any nucleotide A, C, G, T or U. >CoV_T2_20 (with leader sequence) (SEQ ID NO:48) Amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPSAARVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISN CVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPD DFTGCVIAWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRS YGFFPTNGTGYQPYRVVVLSFELLNAPATVCGPKLSTDGGGGSGGGGSGGGGSGGGGSKSSIASFFF IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK The amino acid sequence of the leader sequence (SEQ ID NO:54) is shown underlined. The amino acid sequence of the transmembrane domain (SEQ ID NO:2) is shown in bold. >CoV_S_T3_3 leader Amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPSAARVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISN CVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPD DFTGCVIAWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRS YGFFPTNGTGYQPYRVVVLSFELLHAPATVCGPKLSTDGGGGSGGGGSGGGGSGGGGSKSSIASFFF IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK The amino acid sequence of the leader sequence (SEQ ID NO:54) is shown underlined. The amino acid sequence of the transmembrane domain (SEQ ID NO:2) is shown in bold. >CoV_S_T3_3
Figure imgf000069_0001
leader
Figure imgf000069_0002
Amino acid sequence: RVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNFAPFFAFKCYGVSPT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNTNNIDSTTGGNYNYL YRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFFPTNGTGYQPYRVVVLSFELLH APATVCGPKLSTDGGGGSGGGGSGGGGSGGGGSKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHT KKRQIYTDIEMNRLGK The amino acid sequence of the transmembrane domain (SEQ ID NO:2) is shown in bold. >CoV_S_T3_4
Figure imgf000069_0003
leader
Figure imgf000069_0004
ID NO:51) Amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPSAARVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISN CVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPD DFTGCVIAWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRS YGFFPTNGTGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQRVAPTKEVVRFPNITNLCPFGEVF NATKFPSVYAWERKKISNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQ IAPGQTGVIADYNYKLPDDFTGCVIAWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPG GKPCSGVEGFNCYYPLRSYGFFPTNGTGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQGGGGSG GGGSGGGGSGGGGSKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK The amino acid sequence of the leader sequence (SEQ ID NO:54) is shown underlined. The amino acid sequence of the transmembrane domain (SEQ ID NO:2) is shown in bold. >CoV_S_T3_4
Figure imgf000069_0005
leader
Figure imgf000069_0006
Amino acid sequence: RVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTSFSTFKCYGVSPT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNTNNIDSTTGGNYNYL YRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFFPTNGTGYQPYRVVVLSFELLN APATVCGPKLSTDLIKNQRVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSV LYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVI AWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFFPTN GTGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQGGGGSGGGGSGGGGSGGGGSKSSIASFFFII GLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK The amino acid sequence of the transmembrane domain (SEQ ID NO:2) is shown in bold. >CoV_S_T2_20 Scaffold Sequence (SEQ ID NO:53) Amino acid sequence: RVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNXXXFXXFK 60 CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNT 120 NNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFF 180 PTNGTGYQPYRVVVLSFELLXAPATVCGPKLSTD 214 In this sequence X may be any amino acid residue. > Leader amino acid sequence (SEQ ID NO:54): MDAMKRGLCCVLLLCGAVFVSPSAA
Example 1 A single receptor binding domain-based antigen elicits broad humoral response against SARS-CoV-2 and related sarbecoviruses across different vaccine platforms. This example describes a novel receptor binding domain-based single antigen which elicits a pan-sarbecovirus humoral response. Abstract Of the coronaviruses that have caused zoonotic spill overs in the past two decades, the diverse group of beta-coronaviruses (β-CoVs) represent the greatest threats. Towards achieving broad vaccine protection from these viruses, vaccines capable of eliciting broad immune responses across one or multiple subgroups will be required. Utilising a novel platform (DIOSynVax©) for selecting immune optimized, and structurally engineered antigens capable of eliciting immune responses across a group of related viruses, we demonstrate proof-of-concept against the greater sarbecoviruses sub-genus with a single antigen. From an array of phylogenetically informed and epitope modified antigens, an antigen (T2_17) was selected based on broad immune responses in BALB/c mice. Immunogenicity and breadth of neutralisation of T2_17 as DNA immunogen against the SARS-CoV-2 and related viruses was confirmed in guinea pigs and rabbits using needleless intradermal immunisation. Notably, given the increasing number of mutations acquired by SARS-CoV-2 variants of concern (VOCs), the rabbit sera were tested for the capacity to neutralise VOCs - Beta, Gamma, Delta, and Omicron (BA.1). The consistent neutralising ability of the vaccine sera against the emerging VOCs validated broad specificity of the vaccine design. Further, protection against Delta in K18-hACE2 mice primed by an AZD1222 vaccine was observed on boosting with T2_17 in both DNA as well as Modified Vaccinia virus Ankara (MVA) vaccine platforms. We further validated the immunogenicity of T2_17 in mice in the mRNA vaccine platform. Here we demonstrate proof-of-concept of the DIOSynVax© antigen pipeline for the in vivo selection of a single immunogen capable of eliciting broad neutralising immune response. INTRODUCTION In the present study, to increase the coverage to all the viruses of the sarbecovirus sub- genus of β-coronaviruses, we utilised a novel platform of digitally, immune optimised synthetic vaccine antigens (DIOSynVax©). These computationally immune optimized, and structurally engineered antigens are selected in vivo to induce immune responses across a group of related viruses. First, we generated a phylogenetically informed RBD subunit-based antigen comparing all the known human and animal reservoir sarbecovirus sequences. This antigen design was further used as backbone for designing both epitope optimised and immune re-focussed designs using available structural data for spike protein in complex with RBD binding monoclonal antibodies, in this case specifically those that bound both SARS- CoV, and SARS-CoV-2, such as S309 (14) and CR3022 (15). The nucleic acid sequence of these in silico designed 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 BALB/c mice. The best-in-class immunologically optimal antigen designated T2_17, was further validated by DNA immunisation screens in mice, and subsequently in guinea pigs, and rabbits. To further validate the utility of this antigen to boost specific responses on the background of pre-existing early Wuhan isolate (used by most licensed vaccines) spike specific immune responses, the T2_17 antigen was administered as a heterologous boost using either DNA or MVA immunogens to K18-hACE2 transgenic mice previously primed with the AZD1222 vaccine. RBD specific immune responses were observed in groups immunised with the T2_17 antigen. Further immunogenicity of the T2_17 antigen was confirmed in mice and guinea pigs as an mRNA delivered immunogen based on chemically modified mRNA (16) in a lipidoid nanoparticle formulation (LNP) (17). These studies confirmed that these computational, structural antigen designs can induce broad immunogenicity using a single RBD-based antigen that generates broad neutralisation humoral response covering SARS-CoV, SARS-CoV-2 including VOCs, and related bat sarbecoviruses. RESULTS In-silico design of antigens: Sequences of spike protein of viruses belonging to the sarbecovirus lineage were compiled from NCBI virus database (18) and further pruned. The hCoV-19/Wuhan/IVDC-HB-01/2019 strain of SARS-CoV-2 was used for the analyses. The phylogenetic tree of these sequences is represented in Fig.1A. Two distinct clades are observed in the tree, separating those in clade 1, which do not interact with ACE-2 receptor (1, 19) from those in clade 2, which do. Clade 1 viruses share many of the sequence features of the members of clade 2 but possess deletions around the ACE-2 binding region (Fig.6). An optimized core sequence (T2_13) was designed, such that the novel antigen was phylogenetically closer to all the sarbecoviruses represented in the phylogenetic tree shown in Fig.1A. To further understand the importance of amino-acid composition of epitopes in generating antibody responses, we modified T2_13 to display the exact amino acid sequences of epitopes of SARS-CoV for monoclonal antibodies - S309 (14) (T2_14), and CR3022 (15) (T2_15) and of SARS-CoV-2 for monoclonal antibody - B38 (11) (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 (11) is highly divergent (Fig.1B). We further modified the epitope region for monoclonal antibody - B38 (11) by introducing a glycosylation site on the backbone of T2_14 (T2_17) and T2_15 (T2_16). This was done to mask the divergent epitope region and enhance the presentation of the conserved epitopes to the immune system. The masking of epitopes by introducing glycans has been exploited by many viruses such as Hepatitis C Virus (20), Lassa virus (21), and Influenza (22) to escape natural immunity and we use this strategy to train the immune system to the conserved epitopes. To compare the immunogenicity of soluble and membrane anchored RBD subunit-based vaccine, membrane anchored forms of T2_13 and T2_17 (T2_13_TM and T2_17_TM respectively) were generated. The structural stability of these designs was evaluated in-silico using the BUILD module of FOLDX (23) algorithm using T2_13 as the reference model. Structural models of these vaccine antigens are represented in Fig.1C. Antigen selection and immunogenicity confirmation in BALB/c mice. In vivo screening in BALB/c mice was performed by immunising with the in-silico designed antigens and SARS-CoV-2 RBD (hCoV-19/Wuhan/IVDC-HB-01/2019) as DNA immunogen (Fig.2A). The sera from immunised mice were assayed for cross-reactive antibodies against spike proteins in a flow cytometry-based cell-surface display assay. Binding against four spike proteins viz. SARS-CoV (SARS-Tor2), SARS-CoV-2 (hCoV-19/Wuhan/IVDC-HB- 01/2019), WIV16, and RaTG13 were tested. Sera taken two weeks following the second immunisation with antigen designs, demonstrated the binding profile of the vaccine candidates for different spike proteins (Fig.2B). Sera from all the antigen immunised mice showed higher binding than the PBS immunised mice across the four spike proteins, suggesting seroconversion of the mice on immunising with the antigens. Across the four spike proteins, no significant differences in binding were observed for sera from mice immunised with T2_13 and sera from mice immunised with SARS-CoV-2 RBD (all p > 0.05, MWU test), suggesting that epitopes in this design are biased towards SARS-CoV- 2 RBD. For the T2_16 design, in which the epitope region for mAb B38 was mutated to epitope region on SARS-CoV-2, binding to SARS-CoV, WIV16, and RaTG13 declined in comparison to T2_13 (p < 0.05, MWU test) without significant changes in binding to SARS- CoV-2. Matching of the epitopes of S309 and CR3022 to SARS-CoV (T2_14 and T2_15), enhanced the binding to SARS-CoV (p < 0.05, MWU test) but not to other spike proteins. Introduction of glycosylation site in design T2_17 significantly enhanced the binding of elicited antibodies to SARS-CoV and RaTG13 (p < 0.01, MWU test) in comparison to T2_14, but no difference was observed in T2_18 in comparison to T2_15. There was no statistical difference between trans-membrane anchored and soluble designs when delivered as DNA immunogen. As T2_17 has either the best (or second best) median binding to the four spike proteins, we choose T2_17 as the lead candidate for further immunological assays. Elicitation of cross-binding antibodies by T2_17 was further confirmed by ELISA with SARS- CoV RBD and SARS-CoV-2 RBD (Fig.2C), revealing robust binding antibody responses to both SARS-CoV and SARS-CoV-2 within two weeks of the second immunisation. T2_17 elicited stronger responses against SARS-CoV in comparison to SARS-CoV-2 RBD. Against SARS-CoV-2, the two antigens –SARS-CoV-2 RBD, and T2_17 generated similar binding antibody responses. Immunogenicity of T2_17 confirmed in outbred animals. To determine the breadth of antibody response and neutralisation potency of T2_17 as DNA immunogen in outbred animals, guinea pigs were immunised using the CE approved, and clinically validated Pharmajet Tropis© needleless, intradermal delivery device to ensure standardised intradermal delivery (Fig.3A). As a control we used a C-terminal glycosylation modified SARS-CoV-2 RBD (SARS2_RBD_P521N) (Fig. 3B) which we had previously evaluated in BALB/c mice (Fig.7). Generation of neutralising antibodies to both SARS-CoV and SARS-CoV-2 was confirmed using pseudoviruses expressing full-length spike proteins of SARS-CoV, and SARS-CoV-2. While both T2_17 and SARS2_RBD_P521N generated binding antibodies against both SARS-CoV and SARS-CoV-2 (Fig. 8) after one immunisation, T2_17 elicited significantly higher antibodies than SARS2_RBD_P521N to SARS-CoV and comparable antibodies against SARS-CoV-2. Higher binding antibodies were detected for T2_17 to SARS-CoV in comparison to SARS2_RBD_P521N after two immunisations while the responses were comparable for SARS-CoV-2. After three immunisations SARS2_RBD_P521N induced a higher response to SARS-CoV-2, while T2_17 had higher responses to SARS-CoV (Fig.8). Neutralising antibodies were detected for SARS-CoV-2 after first immunisation, while significant neutralising responses to SARS- CoV developed after two immunisations, though more potent for T2_17 than SARS2_RBD_P521N (Fig. 3C). Better binding and neutralising responses by SARS2_RBD_P521N to SARS-CoV-2 were expected as it differs from SARS-CoV-2 by only one amino acid. To further confirm, whether T2_17 vaccine design generates broader responses, we compared sera induced by SARS2_RBD_P521N, 28 days post 3rd immunisation for neutralisation against SARS-CoV (SARS-Tor2), SARS-CoV-2 (hCoV- 19/Wuhan/IVDC-HB-01/2019), WIV16, and RaTG13. Statistically significant higher neutralising antibody titres were generated by T2_17 against SARS-CoV, WIV16, and RaTG13 (Fig.3D). To further confirm anti-sera to T2_17 could abrogate hACE2 receptor binding, we performed an ELISA based competition assay (Fig.3E) demonstrating T2_17 and SARS2_RBD_P521N anti-sera abrogated binding to hACE-2 receptor and are comparable to the WHO standard of pooled convalescent COVID-19 patient sera (NIBSC standard - 20/162). These findings demonstrated important proof-of-concept of T2_17 as a single gene delivered, structurally engineered antigen capable of eliciting broad pan-sarbeco Coronavirus neutralising antibodies. Prior to clinical trials in humans, 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.3F). After one immunisation, binding antibodies to SARS-CoV and SARS-CoV-2 were elicited (Fig. 9), increasing on subsequent immunisations until a plateau was reached by the fourth immunisation. Robust neutralising antibodies were observed two weeks following the third immunisation (Fig. 3G) and sera post 14 days after four immunisation (bleed 4) showed broad neutralising antibody responses against the SARS-CoV, SARS-CoV-2, Beta, Gamma, Delta, and Omicron (BA.1) VOCs and bat sarbecoviruses – WIV16, and RaTG13 elicited by gene delivery of the engineered T2_17 pan-Sarbeco vaccine antigen candidate (Fig.3H). Challenge studies in mice expressing human ACE2 - K18-hACE2. As almost all the human population is seroconverted either due to natural infection, vaccination, or both, we tested the efficacy of T2_17 antigen when given as booster following AZD1222 (ChAdOx1 nCoV-19) as prime vaccine. To address this, homozygous K18-hACE2 transgenic mice were immunised with 1.4x109 vp of AZD1222 and four weeks later boosted with either T2_17, or the licensed AZD1222 vaccine (Fig. 4A), while the control group received only PBS with each immunisation. As a good neutralisation response was not observed in mice with T2_17 as DNA vaccine in prime-boost regime, we administered T2_17 either as DNA immunogen or Modified Vaccinia virus Ankara (MVA) immunogen. ChadOx- MVA prime-boost regime has been shown to be effective in Ebola (28, 29). Eight weeks post boost, all groups of mice were challenged with either a January 2020 isolate of SARS-CoV- 2 (Victoria) or the Delta variant of SARS-CoV-2 (Table 1). Table 1. Prime-boost regime of K18-hACE2 mice challenge Prime Boost Challenge PBS PBS Victoria AZD1222 AZD1222 Victoria AZD1222 T2_17(DNA) Victoria AZD1222 T2_17(MVA) Victoria PBS PBS Delta AZD1222 AZD1222 Delta AZD1222 T2_17(DNA) Delta AZD1222 T2_17(MVA) Delta Increased binding antibodies titres to both SARS-CoV and SARS-CoV-2 after boosting by either AZD1222 or T2_17 was observed (Fig. 10). Statistically significant difference in antibody titres to SARS-CoV-2 were observed four weeks after boosting with T2_17 as DNA or MVA immunogen in comparison to boosting by AZD1222 while statistically significant increase in binding antibody titres to SARS-CoV was observed on boosting with T2_17 as MVA immunogen (Fig.10). Generation of neutralising antibodies to SARS-CoV, SARS-CoV- 2 and Delta VOC was confirmed using pseudoviruses expressing full-length spike proteins of SARS-CoV, SARS-CoV-2 and Delta VOC. Neutralising antibodies for SARS-CoV-2 and the Delta VOC were detected for all the groups, except the control group prior to challenge while neutralising antibodies against SARS-CoV were detected only for T2_17 MVA boosted group (Fig. 4B). After two weeks post boost, T2_17 both as DNA and MVA immunogen neutralised the Delta variant significantly better than the sera from mice boosted with AZD1222 (Fig.4B). Mice from all the groups, except controls, survived and continued to gain weight following challenge with either the Victoria strain or Delta variant (Fig.4C). Longitudinal serology study in K18-hACE2 mice As all the group were protected in the challenge studies and similar level of neutralising antibodies were observed before and after boost for SARS-CoV and SARS-CoV-2, we explored whether this could be due to short interval between the prime and boost. For this, we primed another group of K18-hACE2 mice with AZD1222 vaccine and boosted twenty weeks afterward (Fig.4D). The groups of mice were boosted with either AZD1222, T2_17(DNA), T2_17(MVA) or PBS (Table 2). A group of mice was only primed with T2_17(MVA) as a control. The AZD1222/PBS group was included to monitor the antibodies titres over time in absence of boost. Table 2. Prime-boost regime of longitudinal serology study of K18-hACE2 mice Prime Boost PBS PBS PBS T2_17(MVA) AZD1222 PBS AZD1222 AZD1222 AZD1222 T2_17(DNA) AZD1222 T2_17(MVA) Bleeds were taken from immunised mice twelve weeks after the prime to check for the antibody titres. Only neutralising antibodies titres against SARS-CoV-2 were measured for this longitudinal analysis. In this study also, significantly higher titres were observed for T2_17(MVA) boosted group (Fig.4E). No antibody titres were observed with T2_17(MVA) primed group, suggesting MVA as weaker platform when delivered as a a prime. The antibodies levels were maintained up to 44 weeks after prime. As T2_17 is a RBD based antigen, we further explored whether higher RBD specific antibodies were generated on boosting with T2_17 in comparison to boosting with AZD1222. Terminal bleed sera from 4 mice with the highest neutralising antibody for vaccine group – PBS/PBS, PBS/T2_17(MVA), AZD1222/AZD1222, and AZD1222/T2_17(MVA) was tested against 15mer peptides with overlap of 14 from SARS-CoV RBD, SARS-CoV-2 RBD, and T2_17 using PEPperPRINT© microarray technology. The PBS/PBS mice group was used for correction of intensities for rest of the tested groups. The microarray data is shown in Fig.4F. Higher number of peptide hits were observed for T2_17(MVA) boosted group in comparison to AZD1222 boosted group, suggesting the T2_17 boosted group induced a greater number of RBD specific antibodies. Immunogenicity of the vaccine candidate in mRNA platform. To further validate the immunogenicity of the T2_17 in the mRNA platform, we immunised BALB/c mice with T2_17 as mRNA immunogen. A previous report on mRNA vaccine has shown that membrane anchored, prefusion-stabilized, full-length MERS spike antigen elicited more potent pseudovirus-neutralizing antibody responses than the soluble form (30). In the present study, in addition, T2_17 was also delivered as a trans-membrane anchored form (T2_17_TM) mRNA immunogen to mice (SEQ ID NO:46). The mRNA immunogen was delivered in a prime boost regime at four weeks interval in BALB/c at different doses viz.5µg and 10µg (Fig. 11A). Full-length spike protein with double Proline mutation in the lipid formulation like the one used for T2_17 and T2_17_TM was used as a control (SEQ ID NO:43). In addition, BNT162b2 vaccine was used as control. All the antigen immunised mice generated binding antibodies against SARS-CoV-2. The trans-membrane anchored T2_17 generated significantly higher binding antibodies at 5µg dose in comparison to soluble T2_17 (Fig. 11B). No significant difference was observed for T2_17_TM at the two test doses. T2_17 at higher dose of 10µg generated equivalent binding antibodies titres to T2_17_TM (Fig.11B). No significant difference was observed between the full length spike (SCoV2(PP) mRNA and Biontech’s BNT162b2 mRNA, i.e. no difference was observed for mRNA modified with 25% of 2-thiouridine and 25% 5-methylcytidine or 100% N1-methylpseudouridine (Fig. 11B). As higher antibody titres were observed for T2_17_TM at lower doses, we further evaluated immunogenicity of T2_17_TM in guinea pigs. Guinea pigs were immunised with mRNAs coding for T2_17_TM (SEQ ID NO:46) and full-length spike with double Proline mutations (SEQ ID NO: 43) at three-week intervals (Fig. 5A). Three weeks post prime, T2_17_TM induced binding antibodies against SARS-CoV as well as SARS-CoV-2 while the full-length spike antigen did not induce binding antibodies against SARS-CoV but induced binding antibodies against SARS-CoV-2 (Fig.12). T2_17_TM induced significantly higher binding antibody titres against SARS-CoV-2 in comparison to full-length spike after three weeks post boost (Fig. 12). Three weeks post boost higher neutralising antibody titres against SARS-CoV were observed for T2_17_TM. Few of the guinea pigs immunised with full length spike (SEQ ID NO: 43) induced neutralising titres against SARS-CoV, after three weeks post boost but the titres declined to low levels afterwards. While the neutralising titres remained high for T2_17_TM. Neutralising antibody titres were observed for both T2_17_TM and the full-length spike against SARS-CoV-2, the titres were lower for T2_17_TM in comparison to full-length spike but it must be noted that the full-length spike presents three RBD subunit as well as other epitopes in the S1 and S2 subunit. We further confirmed the breadth of the T2_17_TM by measuring the neutralising titres against RaTG13, WIV16, SARS-CoV-2 Omicron (BA.1) variant. Significantly higher neutralising titres were observed for T2_17_TM against WIV16, SARS-CoV, and SARS-CoV-2 Omicron variant (BA.1), 6 weeks post boost. The neutralising titres for the full-length spike were almost negligible for SARS-CoV-2 Omicron variant (Fig.5C). DISCUSSION Emergence of two human epidemics caused by ACE-2 using sarbecoviruses in past two decades highlights the urgent need for vaccines that can provide broad protection from SARS-CoV-2 and closely related ACE-2 receptor using sarbecoviruses that have potential to spill-over from zoonotic animal reservoirs. To achieve pan-Sarbeco/pan-Beta coronavirus protection, various vaccine strategies have been employed such as mRNA expressing chimeric version of spike proteins from different coronaviruses (31), as well as mosaic and cocktail nanoparticles expressing RBDs of different coronaviruses (32). Though these strategies have been reported to be effective in generating pan-Sarbeco/pan-Beta coronaviruses immune responses, these require synthesis, manufacturing, and formulation of multiple gene constructs which can be a huge bottleneck for large scale manufacturing. In addition to the zoonotic spill-over from the related bat or other mammal sarbecovirus, another cause of concern is the rapid accumulation of the immune escape mutation in the circulating SARS-CoV-2. Since late 2020, many mutations leading to immune escape, increased transmissibility or both have been reported with the latest circulating Omicron lineage reporting the greatest number of mutations in the SARS-CoV-2 viral genome. An effective vaccine targeting these circulating variants of concern are currently needed. An ideal candidate would be a single antigen providing protection against the diverse group of sarbecoviruses as well as VOCs. Here, we present pre-clinical data for a single antigen RBD subunit-based vaccine design that induces immune responses against SARS-CoV, SARS-CoV-2, RaTG13, WIV16, and VOCs up to BA.1 lineage. The core backbone of the antigen was designed using the novel DIOSynVax© platform. The platform integrates phylogenetic relationships between the input sequences and structural bioinformatics to generate a core antigen sequence that ideally should generate immune response against the diverse group of phylogenetically related viruses. We further modified the core antigen sequence by mutating some of the known epitopes on RBD or introducing glycosylation site or both to enhance the immunogenicity of the antigen. This resulted in a panel of antigens, referred as T2_13 to T2_17. The immunogenicity and breadth of these antigens were confirmed in BALB/c mice. From the binding profile of the sera of mice immunised with these antigens, we down-selected one of the antigens – T2_17 for further pre-clinical studies. Mice immunised with T2_17 as DNA immunogen induced significant binding titres against both SARS-CoV and SARC-CoV-2. Further neutralising antibodies were detected in out-bred guinea pigs and out-bred rabbits against both SARS-CoV and SARS-CoV-2. Notable, rabbit sera neutralised a wide panel of SARS-CoV-2 VOCs viz. Alpha, Beta, Gamma, Delta, and Omicron. These broad humoral responses validate the DIOSynVax© platform used for generation of a pan-Sarbeco RBD sub-unit-based antigen. The breadth of the antigen (T2_17) to VOCs up to BA.1 is particularly encouraging as the antigen was designed using the hCoV-19/Wuhan/IVDC-HB-01/2019 strain of SARS-CoV-2 and suggest the applicability of the platform to capture some of the future variants to an extent. Further the usability of the T2_17 as booster on the background of the non-naïve population, K18-hACE2 mice were primed with AZD1222 vaccine and boosted with AZD1222 or T2_17 as DNA immunogen or MVA immunogen at 4 weeks intervals and challenged with either Victoria or Delta strains of SARS-CoV-2. All the antigen immunised mice were protected against the challenge with increases in neutralising antibody titres against Delta in T2_17 boosted group post 4 weeks after boost. Neutralising antibodies against SARS-CoV were observed in T2_17(MVA) group. T2_17(DNA) did not induce neutralising antibodies against SARS-CoV-2. We believe this due to the difference in nature of vaccine vector between AZD1222 and DNA but has not been addressed in the present study. Further longitudinal serology study was carried out to understand the influence of boosting K18-hACE2 mice at 20 weeks interval. The antibody titres remained high for 12 weeks post prime and only T2_17(MVA) boosted group showed significant increase in the antibody titres 4 weeks post boost. Equivalent titres were observed at 44 weeks after prime across the groups for all the antigen immunised mice groups . Further, peptide microarray was performed on the terminal sera of the K18-hACE2 mice from the longitudinal study to check for differential immune response in T2_17(MVA) boosted group. Higher number of peptide hits for RBD regions were observed for T2_17(MVA) boosted group, suggesting greater elicitation of the RBD specific humoral response in T2_17 boosted group. As mRNA vaccine technology offers a competitive edge over many of the established vaccine technology with superior immunogenicity, tolerance, and faster production (33). T2_17 was tested as mRNA immunogen in mice and guinea pigs using chemically modified mRNA (16) in a lipidoid nanoparticle formulation (LNP) (17). A previous study on MERS-based vaccine has shown that membrane anchored, prefusion-stabilized, full-length MERS spike antigen elicited more potent pseudovirus-neutralizing antibody responses than the soluble form, as mRNA immunogen (30). In the present study, BALB/c mice were immunized with T2_17 and trans-membrane anchored T2_17 (T2_17_TM) at different doses viz. 5µg and 10µg. T2_17_TM showed significantly higher binding antibody titres in comparison to T2_17 at lower dose of 5µg but showed comparable binding antibodies at 10µg dose. Based on these observations, T2_17_TM was further validated as mRNA immunogen in guinea pigs. Both binding and neutralising antibodies were observed for T2_17_TM. Six weeks post boost, only guinea pigs immunised with T2_17_TM showed neutralising antibodies against SARS-CoV, and SARS-CoV-2 Omicron (BA.1) variant. The group immunised with full-length SARS-CoV- 2 spike did not show a robust neutralising immune response against SARS-CoV and SARS- CoV-2 Omicron variant. Higher antibody titres against SARS-CoV-2 and RaTG13 are observed for full-length spike, but it must be noted that full-length spike present three RBD subunits that are homologous to the SARS-CoV-2 spike tested here, and would always have higher titres in comparison to any other heterologous antigens. Moreover, the high similarity between the S2 region of SARS-CoV-2 and RaTG13 would also induces higher cross- neutralising antibody between RaTG13 and SARS-CoV-2 and would lead to higher antibody titres for full length spike in comparison to T2_17. Overall, all the studies combined supports the T2_17 as an attractive single antigen for targeting multiple sarbecoviruses and its applicability across different vaccine platforms. In conclusion, T2_17 generate a robust humoral immune response against SARS-CoV, SARS-CoV-2, RaTG13, WIV16, SARS-CoV-2 variants – Alpha, Beta, Gamma, Delta, and Omicron (BA.1). As the designs pre-dated the emergence of these VOCs and none of the sequences were included in the initial design, this demonstrates the robustness of the platform. Given the continuous emergence of new variants, it is imperative that the new vaccine antigens should be substantially different from the Wuhan strain or other variants to surpass the boosting of the immunodominant epitopes conserved in the these strains (34, 35) . As T2_17 is a novel antigen with moderate similarity with SARS-CoV-2, it may be an ideal booster vaccine candidate overcome immune imprinting by Wuhan strain-based Spike vaccines. MATERIALS AND METHODS Study design The primary aim of this study was to study the broad immune response and protective effects of the T2_17 vaccine against SARS-CoV, SARS-CoV-2, and related bat sarbecoviruses. The sample sizes were empirically estimated by considering the variations of the results and the statistical power needed while minimizing the number of animals. The animals in the study were randomly assigned for immunological readouts. Studies were not blinded. No data points were omitted from the analysis. Animal studies were approved by AWERB (Animal Welfare and Ethical Review Body), University of Cambridge and experiments carried out under an approved UK home office license. Phylogenetic analysis Protein sequences of spike proteins were downloaded from the NCBI virus database for all the known sarbecoviruses. A multiple sequence alignment (MSA) was generated using MUSCLE (36). The resulting MSA was pruned to the RBD region, filtered at 95% sequence identity, and used as input for phylogenetic tree reconstruction. The phylogenetic tree was generated using IQTREE (24) using the protein model with the best BIC score. The resultant tree was used for generation of phylogenetically optimised design using HyPhy (37). Epitope identification Available structural data (June 2020) for spike protein-antibody complexes for SARS-CoV and SARS-CoV-2 were downloaded from the Protein Databank (PDB) (27). Structural data were then pruned for antigen-antibody complexes where the epitopes were on the RBD. Amino acid residues of antigen that have at least one atom within 5Å radii of at least one atom of amino acid of antibody were defined as epitope residues, with epitope regions defined as contiguous stretches of at least 5 amino acids. Glycosylation site modification The position of the glycosylation site was determined by in-silico mutation of triplets of amino acids in the epitopes to glycosylation sequon – N-X-T (38) using the FoldX algorithm (23). Briefly, residues succeeding N-X motif, where X can be any amino acid except Pro, were mutated to either Threonine or Serine or residues preceding X-T, where X can be any amino acid except Pro, were mutated to Asn to generate novel N-X-T/S motifs. The mutations with the least energy cost, as calculated by the Build module of FoldX (23), were selected. Molecular modelling Structural models were generated for T2_13 using MODELLER (39, 40) using both SARS- CoV and SARS-CoV-2 structures as templates. The structural model with the highest DOPE score (41) was chosen as the working model for further molecular modelling. The side chains for the model were further optimised using SCWRL (42) and energy minimised using GROMACS (43). For T2_14 to T2_18, mutations were introduced using T2_13 as the reference structure using BUILD module of FOLDX algorithm (23) and checked for structural stability using FOLDX forcefield (23). Production and transformation of plasmids Sequences of antigens were gene-optimized and adapted to human codon use via the GeneOptimizer algorithm (44). These genes were cloned into pEVAC (GeneArt, Germany) via restriction digestion. Plasmids were transformed via heat-shock in chemically induced competent E. coli DH5α cells (Invitrogen 18265-017). Plasmid DNA was extracted from transformed bacterial cultures via the Plasmid Mini Kit (Qiagen 12125). All plasmids were subsequently quantified using UV spectrophotometry (NanoDrop™ -Thermo Scientific). Vaccination Experiments in Mice Eleven groups of six 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 50µl of PBS containing 50µg of plasmid DNA was administered via sub-cutaneous 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. 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 (SARS- Tor2), SARS-CoV-2 (hCoV-19/Wuhan/IVDC-HB-01/2019), WIV16 (Accession id: ALK02457), and RaTG13 (Accession id: QHR63300).48 hours after transfection, cells were transferred into V-bottom 96-well plates (50,000 cells/well). Cells were incubated with sera (diluted at 1:50 in PBS) or anti-mouse IgG Isotype negative control (Invitrogen 10400C, diluted at 20µg/mL in PBS) for 30 min, washed with FACS buffer (PBS, 1% FBS, 0.02% Tween 20) and stained with Goat anti-mouse IgG (H+L) Alexa Fluor 647 Secondary Antibody (Invitrogen A32728, diluted at 20µg/mL in FACS buffer), for 30 min in the dark. Cells were washed with FACS buffer and samples were run on a Attune NxT Flow Cytometer (Invitrogen) with a high-throughput auto sampler. Dead cells were excluded from the analysis by staining cells with 7-Aminoactinomycin D (7-AAD) and gating 7-AAD negative live cells. Enzyme-linked immunosorbent assay (ELISA) The assays were adapted from those originally described by Amanat and co-workers (45). Briefly, Nunc MaxiSorp™ flat-bottom plates were coated with 50μl per well of 1μg/ml of RBD from SARS-1 or SARS-2 DPBS (-Ca2+/-Mg2+) and incubated overnight at 4°C. The next day, the plates were blocked with 3% milk in PBST (0.1% w/v Tween20 in PBS) for 1 hour. After removing the blocking buffer, 50μl/well of serum samples diluted in PBST-NFM (1% w/w non- fat milk in PBST) were added to the plates and incubated on a plate shaker for two hours at 20°C. The plates were washed three times with 200μl of PBST, and then 50μl of HRP- conjugated goat anti Ig (H and L chains) (Jackson ImmunoResearch) was added to each well and left to incubate for one hour on a plate shaker for 1 hour. Plates were washed three times with 200μl of PBST, 50μl/well of 1-Step Ultra TMB chromogenic substrate (Sigma) was added to the plates and the chemical reaction was stopped three minutes later with 50μl 2N H2SO4. The optical density at a wavelength of 450nm (OD450) was measured using a BioRad microplate reader. Values from the dilution curve were used to determine the area under the curve. Intradermal nucleic acid immunisation with Tropis PharmaJet© delivery in Guinea pigs Two groups of eight female 7-week-old Dunkin Hartley Guinea pigs (Envigo RMS, Blackthorn, United Kingdom) were immunised a total of three times with 28 days intervals. A total volume of 200µl of PBS containing 400µg of plasmid DNA was administered by PharmaJet Tropis intradermal device, split over each hind leg. Blood was sampled from the saphenous vein at 14-day intervals. Intradermal nucleic acid immunisation with Tropis PharmaJet© delivery in in Rabbits. Ten mature (five male, five female) rabbits were immunised with a GMP lot pEVAC_T2_17 (clinical pEVAC_PS) intradermally by PharmaJet Tropis needleless delivery to the upper left and right hind limbs (300µl at 2mg/mL). For control group, ten mature (five male, five female) rabbits were injected with PBS. Arterial blood was sampled at 14 days intervals. Production of lentiviral pseudotypes Lentiviral pseudotypes were produced by transient transfection of HEK293T/17 cells with packaging plasmids p8.91 (46, 47) and pCSFLW (48) and different SARS-CoV-2 VOC spike- bearing expression plasmids using the Fugene-HD transfection reagent (49, 50). Supernatants were harvested after 48h, passed through a 0.45 µm cellulose acetate filter and titrated on HEK293T/17 cells transiently expressing human ACE-2 and TMPRSS2. Target HEK293T/17 cells were transfected 24h prior with 2 µg pCAGGS-huACE-2 and 75 ng pCAGGS-TMPRSS2 (51, 52). Pseudotype-based micro-neutralisation assay Pseudotype-based micro-neutralisation assay was performed as described previously(53). Briefly, serial dilutions of serum were incubated with SARS-CoV-2/RaTG13/SARS- CoV/WIV16/SARS-CoV-2 variant spike bearing lentiviral pseudotypes for 1 h at 37°C, 5% CO2 in 96-well white cell culture plates.1.5x104 HEK293T/17 transiently expressing human ACE-2 and TMPRSS2 were then added per well and plates incubated for 48 hrs at 37°C, 5% CO2 in a humidified incubator. Bright-Glo (Promega) was then added to each well and luminescence read after a five-minute incubation period. Experimental data points were normalised to 100% and 0% neutralisation controls and non-linear regression analysis performed in GraphPad Prism 9 to produce neutralisation curves and IC50 values. ACE-2 competition assay The SARS-CoV-2 surrogate virus neutralisation test (SVNT, Genscript, Piscataway, New Jersey, United States) was carried out as per manufacturer’s instructions. Briefly, 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. MVA production The MVA strain used in this study was MVA-CR19. Recombinant MVA that expresses SARS- CoV-2 RBD T2-17 was generated as described preciously. In brief, for in vivo recombination adherent AGE1.CR.pIX were infected with parental MVA-CR19 TK-GFP with different MOIs ranging from 0.5 to 0.006 PFU. After 2h, the cells were transfected with 0.4 µg of the shuttle vector pMVA_RBD T2_17 using Effectene (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. After 48h, the cells were harvested, lysed by three freeze/thaw- cycles and sonicated. Pure recombinant viruses were obtained by sequential plaque purification under agarose overlays and confirmed to be free of contaminating parental MVA- CR19 TK-GFP by PCR screening. This recombinant MVA encoding SARS-CoV-2 RBD T2- 17 was plaque purified for additional three rounds. The resulting recombinant MVA-CR19 RBD-T2_17 (MVA T2_17) virus stock was produced in suspension AGE1.CR.pIX cells, purified via two ultracentrifugation rounds over a 35 % sucrose cushion and titrated on DF-1 cells using crystal violet staining. The sequence of the rMVA and absence of revertant MVA was confirmed by PCR amplification and Sanger sequencing. The expression of RBD T2_17 was confirmed by Western blot analysis with monoclonal antibody CR3022 with cell lysates from HEK293 cells harvested 24 hours after infection (MOI 2) with MVA T2_17. Vaccine boost efficacy studies in K18-hACE2 mice Eight groups of six female 8–15-week-old homozygous K18-hACE2 mice (Jax) were primed with 1.4x109 viral particles of AZD1222 or PBS by intramuscular route, in a total volume of 100µl split over the two rear legs. After 28 days, two groups of six mice were boosted with PBS, AZD1222, T2_17 DNA, or T2_17 MVA . 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 40µl over both nares. Mice were weighed daily and monitored for clinical signs for a period of 14 days before being humanely culled by terminal bleed. Longitudinal serology studies in K18-hACE2 mice Six groups of six female 8–15-week-old homozygous K18-hACE2 mice (Jax) were primed with 1.4x109 viral particles of AZD1222 or PBS by intramuscular route, in a total volume of 100µl split over the two rear legs. After 20 weeks, groups of six mice were boosted with PBS, AZD1222, T2_17(DNA), or T2_17(MVA). Mice were bled at 12 weeks post prime, 24 weeks post prime and terminally bleed at week 44 post prime. Peptide microarray Four samples were selected from terminal bleed of longitudinal study of K18-hACE2 mice AZD1222 vaccine prime, T2_17 boost Vaccine efficacy study. The samples were selected based on the quality of serum and performance on pMN assay against SARS-CoV-2 Wuhan. Samples aliquots (30µL) was sent to PEPperPRINT GmbH for peptide microarray analysis. Briefly, a15-mer peptides spanning the SARS-CoV RBD (213 AA) / SARS-CoV-2 RBD (214 AA) / T2_17 RBD (214 AA) with a 14 AA overlap were printed in duplicate per array copy for a total of five array copies. HA and c-Myc control peptides were included in each array copy. The protein sequences are elongated by neutral GSGSGSG linkers to avoid truncated peptides and identical peptides are removed. In total, 1310 peptide sequences are synthesized and spotted in duplicate onto the PEPperCHIP® microarray platform. The corrected raw intensities were log transformed for all the sera samples. For each vaccine group viz. PBS/T2_17 MVA, AZD1222/AZD1222 and AZD1222/T2_17MVA, the peptides with raw intensities two-fold higher than the maximum intensity observed in the PBS/PBS group were considered as an antibody epitope hit. This allowed us to remove all the peptides with showed non-specific binding to other biomolecules in the sera. mRNA vaccine production mRNA sequences encoding the SARS-CoV-2 S protein with 2 proline mutations, T2_17, and T2_17_TM were synthesized by in-vitro transcription (IVT) from linearized plasmid DNA templates using modified nucleotides to generate partial modified mRNAs. After IVT mRNAs were dephosphorylated and enzymatically polyadenylated. Purification steps were performed by precipitation and subsequently formulated in water for injection at a concentration of 1 mg/mL. mRNAs were stored at -80°C until LNP-encapsulation. Each mRNA was LNP-encapsulated via nanoprecipitation by microfluidic mixing of mRNA in citrate buffer (pH 4.5) with ionizable-, structural-, helper-, and polyethylene glycol (PEG) lipids in ethanol, followed by buffer exchange and concentration via tangential flow filtration. mRNA/LNPs were filtered through a 0.2 μm membrane and stored at -20°C until use. The drug product was analytically characterized, and the products were evaluated as acceptable for in-vivo use. Immunisation of BALB/c mice with mRNA Seven groups of six female 8–10-week-old BALB/c mice were purchased from Charles River Laboratories (Kent, United Kingdom). Mice were immunised two times with a 21-day interval. A total volume of 50µl of vehicle containing various amount of mRNA was administered via intramuscular route in each rear hind leg. Blood was sampled from the saphenous vein at 21-day intervals, and animals were terminally bled by cardiac puncture under non-recovery anaesthesia at day 63. 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Example 2 mRNA sequence encoding COV_S_T2_17 and COV_S_T2_20 This example provides mRNA sequences encoding COV_S_T2_17 and COV_S_T2_20 polypeptide sequences. mRNA sequence encoding COV_S_T2_17 (SEQ ID NO:7): m GP G- GGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUGU UUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAA UCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAG AAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGU GCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGU GAUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUAC AAGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCG GCGGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAU CAGCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUAC CCUCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCC UGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGACUGAUGAGA AUU-Poly(~A ) Codon Optimization: GeneArt human codon optimized ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5-ÚTR (minimal) =GGAGACGCCACC 3-ÚTR (unspecific) = GAAUU Coding sequence = bold format text RNA sequence in the above coding sequence encoding T2_17 (SEQ ID NO:8): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUCGGCGAGG UGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAGAAAAUCAGCAACUGCGUGGC CGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGUCACCCACC AAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGC GGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAAGCUGCCCGACGACUUCAC CGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUG UACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCC CUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUU CUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUGAAU GCCCCUGCCACAGUGUGUGGCCCUAAGCUGAGCACCGAC mRNA sequence encoding COV_S_T2_20 (SEQ ID NO:9): m2 7,3´-oGP3 G- GGGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGU GUUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCA AUCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAA GAAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGU GCUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUG AUCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACA AGCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGC GGCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCA GCAGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCC UCUGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUG AGCUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAG GAUCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCA GCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGU GCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCAAG UGAUGAGAAUU-Poly(~A120) ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5ÚTR (minimal) =GGGAGACGCCACC 3ÚTR (unspecific) = GAAUU Coding sequence = bold format text RNA sequence in the above coding sequence encoding T2_20 (SEQ ID NO:10): AGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAAUCUGUGCCCUUUCGGCGAGGU GUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAGAAAAUCAGCAACUGCGUGGCCG ACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUGCUACGGCGUGUCACCCACCAAGC UGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGAUCAGAGGCGACGAAGUGCGGCAG AUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAAGCUGCCCGACGACUUCACCGGCUGU GUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCGGCAACUACAACUACCUGUACAGAAGCC UGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGCAGCGACAUCUAUAGCCCUGGCGGCAAG CCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUCUGCGGAGCUACGGCUUCUUCCCCACAAA UGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAGCUUCGAGCUGCUGAAUGCCCCUGCCACAG UGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGAGG AAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCAGCUUCUUCUUCAUCAUCGGCCUGAUUAUCGGCC UGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGUGCAUCAAGCUGAAACACACCAAGAAGCGGCAGAUC UACACCGACAUCGAGAUGAACCGGCUGGGCAAG Additional mRNA sequence encoding COV_S_T2_20 (SEQ ID NO:46): m27,3´-oGP3 G- GGAGACGCCACCAUGGACGCUAUGAAGAGGGGCCUGUGCUGUGUGCUGCUGCUGUGCGGAGCUGUG UUUGUGUCUCCAUCUGCCGCCAGAGUGGCCCCUACCAAAGAAGUCGUGCGGUUCCCCAACAUCACCAA UCUGUGCCCUUUCGGCGAGGUGUUCAACGCCACCAAGUUUCCCUCUGUGUACGCCUGGGAGCGCAAG AAAAUCAGCAACUGCGUGGCCGACUACAGCGUGCUGUACAACAGCACCAGCUUCAGCACCUUCAAGUG CUACGGCGUGUCACCCACCAAGCUGAACGACCUGUGCUUCACCAACGUGUACGCCGACAGCUUCGUGA UCAGAGGCGACGAAGUGCGGCAGAUUGCCCCUGGACAAACAGGCGUGAUCGCCGAUUACAACUACAA GCUGCCCGACGACUUCACCGGCUGUGUGAUCGCCUGGAACACCAACAACAUCGACAGCACCACCGGCG GCAACUACAACUACCUGUACAGAAGCCUGCGGAAGUCUAAGCUGAAGCCCUUCGAGCGGGACAUCAGC AGCGACAUCUAUAGCCCUGGCGGCAAGCCUUGUUCUGGCGUGGAAGGCUUCAACUGCUACUACCCUC UGCGGAGCUACGGCUUCUUCCCCACAAAUGGCACAGGCUACCAGCCUUACAGAGUGGUGGUCCUGAG CUUCGAGCUGCUGAAUGCCCCUGCCACAGUGUGUGGCCCUAAGCUGUCUACAGAUGGCGGCGGAGGA UCUGGCGGAGGUGGAAGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUAAAUCUUCUAUCGCCAGC UUCUUCUUCAUCAUCGGCCUGAUUAUCGGCCUGUUCCUGGUGCUGAGAGUGGGCAUCCACCUGUGC AUCAAGCUGAAACACACCAAGAAGCGGCAGAUCUACACCGACAUCGAGAUGAACCGGCUGGGCAAGU GAUGAGAAUU-Poly(~A120) ARCA Cap = m GP G- Poly(A) tail = -poly(~A120) 5ÚTR (minimal) =GGAGACGCCACC 3ÚTR (unspecific) = GAAUU Coding sequence = bold format text
Example 3 Single RBD-based antigen elicits broad humoral response against SARS-CoV-2 and related sarbecoviruses across different vaccine technologies This Example is a continuation of Example 1. In this Example, the T2_17_TM and full-length Spike (S) protein vaccines were further tested for neutralisation titre against XBB.1.5 and SARS2_Wuhan viruses in guinea pigs. T2_17_TM was also tested for neutralising titre against even further Omicron VOCs in guinea pigs. The content of the introduction, discussion, and relevant sections of the materials and methods of Example 1, applies to this Example. Immunogenicity of the vaccine candidate as mRNA Guinea pigs were immunised with either T2_17_TM at dose of 3.15μg or full-length spike with double Proline mutations at dose of 15μg at three-week intervals (Fig.5A). We further tested for neutralisation titre against one of the recent prominent variants – XBB.1.5. No neutralising titres were observed for both T2_17_TM and full-length spike vaccine. To check whether the low neutralising titre of T2_17_TM was due to low dose of 3.15µg, we tested the sera of Guinea pigs immunised with 15µg of T2_17_TM and compared it with sera of Guinea pigs immunised with 15µg of full-length spike. The sera from guinea pigs immunised with 15µg of T2_17_TM neutralised pseudoviruses expressing the XBB.1.5 variant (Fig. 14). Figure 16 shows sera from guinea pigs immunised with 15µg of T2_17_TM neutralised pseudoviruses expressing further omicron VOCs. Discussion Six weeks post boost, only guinea pigs immunised with T2_17_TM showed neutralising antibodies against SARS-CoV-2 Omicron (BA.1) variant at lower dose of 3.15µg and against XBB.1.5 at higher dose of 15µg. The group immunised with full-length SARS-CoV-2 spike did not show a robust neutralising immune response against SARS-CoV or the SARS-CoV- 2 Omicron XBB.1.5 at 15μg dose. At the time of the design of T2_17, none of the SARS- CoV-2 variants had yet been observed. Although the TM version of T2_17 still generates neutralising antibodies against VOCs including the recent XBB.1.5, the titres are lower than observed against the Wuhan strain. With the extraordinary variation due to SARS-CoV-2 global distribution in animals and humans, future updates may be needed for T2_17, such as including the sequence information of the VOCs as well as combining it with other conserved structural and non-structural antigens. Further, to understand the immunogenicity of T2_17 in the background of the current complex immunity observed in the human population, phase 1 clinical trials have now been initiated. In conclusion, all the studies combined supports T2_17 as an efficacious single antigen for targeting multiple sarbecoviruses and its applicability across different vaccine technologies. Immunisation with T2_17 generated a robust humoral immune response against SARS-CoV, SARS-CoV-2, RaTG13, WIV16, SARS-CoV-2 variants – Alpha, Beta, Gamma, Delta, and Omicron (BA.1, XBB1.5). That the design of T2_17 pre-dated the emergence of these VOCs and that none of the sequences were included in the initial design is a strong indication of the DIOSynVax technology. Furthermore, immunisation with T2_17_TM generated a robust humoral immune response against SARS-CoV, SARS-CoV-2, RaTG13, WIV16 and SARS- CoV-2 Omicron BA.1, XBB; XBB.1.5; BA.2.12.1; BA.2.75; BA.2.3.20; and BQ.1.1. Given the continuous emergence of new variants, new vaccine antigens should be substantially different from the Wuhan strain or other variants to surpass the boosting of the immunodominant epitopes conserved in these strains. All the current vaccines have full length spike as the antigen and only 16% of the antibodies generated against the spike antigen is RBD directed. As T2_17 is a novel RBD based antigen with significant difference of 14.5% with Wuhan-Hu-1 strain of SARS-CoV-2, it may be an ideal booster vaccine candidate to overcome immune imprinting by full length spike vaccines.
Figure imgf000098_0001
Next generation designed sequences of T2_20 (optimised coronavirus RBD) family of antigens We have developed next generation T2_20 (optimised coronavirus RBD) family of antigens, the amino acid sequences of which are shown below: >CoV_S_T3_3 (T2_20v2) (with leader sequence) (SEQ ID NO:49) Amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPSAARVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISN CVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPD DFTGCVIAWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRS YGFFPTNGTGYQPYRVVVLSFELLHAPATVCGPKLSTDGGGGSGGGGSGGGGSGGGGSKSSIASFFF IIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK The amino acid sequence of the leader sequence is shown underlined. The amino acid sequence of the transmembrane domain is shown in bold format. >CoV_S_T3_3 (T2_20v2) (without leader sequence) (SEQ ID NO:50) Amino acid sequence: RVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNFAPFFAFKCYGVSPT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNTNNIDSTTGGNYNYL YRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFFPTNGTGYQPYRVVVLSFELLH APATVCGPKLSTDGGGGSGGGGSGGGGSGGGGSKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHT KKRQIYTDIEMNRLGK The amino acid sequence of the transmembrane domain is shown in bold format. >CoV_S_T3_4 (T2_17_T2_20 dimer) (with leader sequence) (SEQ ID NO:51) Amino acid sequence: MDAMKRGLCCVLLLCGAVFVSPSAARVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISN CVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPD DFTGCVIAWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRS YGFFPTNGTGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQRVAPTKEVVRFPNITNLCPFGEVF NATKFPSVYAWERKKISNCVADYSVLYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQ IAPGQTGVIADYNYKLPDDFTGCVIAWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPG GKPCSGVEGFNCYYPLRSYGFFPTNGTGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQGGGGSG GGGSGGGGSGGGGSKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK The amino acid sequence of the leader sequence is shown underlined. The amino acid sequence of the transmembrane domain is shown in bold format. >CoV_S_T3_4 (T2_17_T2_20 dimer) (without leader sequence) (SEQ ID NO:52) Amino acid sequence: RVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTSFSTFKCYGVSPT KLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNTNNIDSTTGGNYNYL YRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFFPTNGTGYQPYRVVVLSFELLN APATVCGPKLSTDLIKNQRVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSV LYNSTSFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVI AWNTNNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFFPTN GTGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQGGGGSGGGGSGGGGSGGGGSKSSIASFFFII GLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK The amino acid sequence of the transmembrane domain is shown in bold format. Example 5 CoV_S_T2_20 Scaffold Sequence (SEQ ID NO:53) SEQ ID NO:53 below shows a scaffold RBD sequence for CoV_S_T2_20 (SEQ ID NO:3), CoV_S_T3_3 (SEQ ID NO:50), and CoV_S_T3_4 (SEQ ID NO:52) optimised coronavirus RBD designed structures (without leader sequence), in which the amino acid sequence of the constant regions of the scaffold is provided, with each variable amino acid residue (i.e. amino acid residues which can be varied to provide antigen which induces neutralising immune response against new and/or future SARS-CoV-2 variants) represented with an X (shown underlined in the sequence below) >CoV_S_T2_20 Scaffold Sequence (SEQ ID NO:53): RVAPTKEVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNXXXFXXFK 60 CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGVIADYNYKLPDDFTGCVIAWNT 120 NNIDSTTGGNYNYLYRSLRKSKLKPFERDISSDIYSPGGKPCSGVEGFNCYYPLRSYGFF 180 PTNGTGYQPYRVVVLSFELLXAPATVCGPKLSTD 214 Examples of sequences provided herein which are covered by this scaffold sequence are SEQ ID NOs:3 and 48 (CoV_T2_20 without and with leader sequence, respectively), SEQ ID NOs:49 and 50 (CoV_S_T3_3 (T2_20v2) with and without leader sequence, respectively), and SEQ ID NOs:51 and 52 (CoV_S_T3_4 (T2_17_T2_20 dimer) with and without leader sequence). Figures 18 and 19 show an amino acid sequence alignment of CoV_T2_20 (SEQ ID NO:48), CoV_S_T3_3 (SEQ ID NO:49), and COV_S_T3_4 (SEQ ID NO:51) (with leader sequences). Differences between the sequences are shown as the boxed residues. The amino acid residues at the variable positions in the CoV_S_T2_20, CoV_S_T3_3, and COV_S_T3_4 designed sequences are listed in the table below. The variable amino acid residue position of SEQ ID NO:53 corresponds to the amino acid residue position of SEQ ID NO:53 without a leader sequence. Variable amino acid Residue at corresponding Residue at Residue at residue position of position of CoV_S_T2_20 corresponding position corresponding position SEQ ID NO:53 (SEQ ID NO:3) of CoV_S_T3_3 of CoV_S_T3_4 (T2_20v2) (T2_17_T2_20_dimer) (SEQ ID NO:50) (SEQ ID:52) 53 S F S 54 T A T 55 S P S 57 S F S 58 T A T 201 N H N
Figure imgf000101_0001
Neutralisation of a panel of coronavirus pseudoviruses using sera from animals immunised with T2_20 family of optimised coronavirus RBD constructs Figure 17 shows neutralisation of SARS-CoV-1, SARS-CoV-2 omicron VOCs, and Wuhan spike bearing lentiviral pseudotypes (PV) by antisera generated with optimised coronavirus T2_20 family of vaccine constructs and controls in guinea pigs (study COV038), using the mRNA platform. Figure 17b shows the immunisation and bleed schedule for the guinea pigs in the study, wherein the guinea pigs were immunised twice at weeks 0 and 3, and bled four times at 3 week intervals. Data shown is for sera at SB2 (3 weeks after boost). The figure shows neutralisation of the PVs using antisera from animals immunised with next generation optimised coronavirus T2_20 antigen CoV_S_T3_3 (T2_20v2) (SEQ ID NO:49) retains neutralising activity to SARS-CoV-1 while expanding breadth to XBB.1.5, XBB.1.19.1, XBC.1, BQ.1.12, and XBB.1.9.1, at the expense of the ancestral (and extinct) Wuhan-Hu-1. Immunisation with dimeric CoV_S_T3_4 (T2_17_T2_20 dimer) (SEQ ID NO:51) antigen is comparable to immunisation with the monomeric T2_20 (CoV_S_T2_20)(SEQ ID NO:48) antigen on the Tier 1 panel of PVs. Figure 20a shows neutralisation of SARS-CoV-1, SARS-CoV-2 omicron VOCs, and Wuhan spike bearing lentiviral PVs by antisera generated with optimised coronavirus CoV_S_T2_20 (SEQ ID NO:48) using the mRNA platform (study COV038). The immunisation and bleed schedule is the same as above for Figure 17, and is illustrated in Figure 20b. Figure 21a shows further neutralisation data for the guinea pigs immunised with optimised coronavirus CoV_S_T2_20 (SEQ ID NO:48) in study COV038, wherein the guinea pigs were challenged with a more diverse panel of SARS-CoV-1 and SARS-CoV-2 S protein bearing PVs. The immunisation and bleed schedule is the same as above for Figure 17, and is also illustrated in Figure 21b. Figure 21a shows that immunisation with CoV_S_T2_20 elicits a broadly neutralising immune response against a broader panel of SARS-CoV-1 and SARS-CoV-2 S protein bearing PVs. In particular, a broadly neutralising immune response is elicited against PVs of SARS-CoV-1, related SARS1 virus WIV-16, and SARS-CoV-2 Wuhan, Alpha, Beta, Gamma, Delta, and some Omicron sub-variants, including BA.2 and BA.2.86.

Claims

Claims 1. An isolated messenger RNA (mRNA) encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:1 (T2_17), or an amino acid sequence which has at least 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:1, and an amino acid sequence of a transmembrane domain.
2. An mRNA according to claim 1, wherein the encoded transmembrane domain is linked directly to the C-terminal end of the amino acid sequence of SEQ ID NO:1, or the amino acid sequence which has at least 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:1.
3. An mRNA according to claim 1, wherein the encoded transmembrane domain comprises an amino acid sequence of SEQ ID NO:2, or an amino acid sequence which has at least 70%, 75%, 80%, 85%, 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:2.
4. An mRNA according to any preceding claim, which encodes an amino acid sequence of SEQ ID NO:1.
5. An mRNA according to claim 4, which comprises an RNA sequence of SEQ ID NO:8, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:8, SEQ ID 27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, and which encodes an amino acid sequence of SEQ ID NO:1.
6. An mRNA according to claim 4, which comprises an RNA sequence of SEQ ID NO:8, or SEQ ID 27.
7. An mRNA according to claim 4, which comprises an RNA sequence of SEQ ID NO:4, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:4 and which encodes an amino acid sequence of SEQ ID NO:1.
8. An mRNA according to claim 4, which comprises an RNA sequence of SEQ ID NO:4.
9. An mRNA according to any preceding claim, which encodes an amino acid sequence of SEQ ID NO:2.
10. An mRNA according to claim 9, which comprises an RNA sequence of SEQ ID NO:5, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:5 and which encodes an amino acid sequence of SEQ ID NO:2.
11. An mRNA according to claim 9, which comprises an RNA sequence of SEQ ID NO:5.
12. An mRNA according to any preceding claim, which encodes an amino acid sequence of SEQ ID NO:3 (T2_20), or an amino acid sequence which has at least 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:3.
13. An mRNA according to any preceding claim, which encodes an amino acid sequence of SEQ ID NO:3.
14. An mRNA according to claim 13, which comprises an RNA sequence of SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34, and which encodes an amino acid sequence of SEQ ID NO:3.
15. An mRNA according to claim 13, which comprises an RNA sequence of SEQ ID NO:10 or SEQ ID NO:31.
16. An mRNA according to claim 13, which comprises an mRNA sequence of SEQ ID NO:9, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:46.
17. An mRNA according to claim 13, which comprises an RNA sequence of SEQ ID NO:6, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:6 and which encodes an amino acid sequence of SEQ ID NO:3.
18. An mRNA according to claim 13, which comprises an RNA sequence of SEQ ID NO:6.
19. An mRNA encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:1 (T2_17), or an amino acid sequence which has at least 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:1.
20. An mRNA encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:1.
21. An mRNA according to claim 20, which comprises an RNA sequence of SEQ ID NO:8, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, or SEQ ID NO:30, or an RNA sequence which has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ribonucleic acid identity over its entire length with the RNA sequence of SEQ ID NO:8, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, or SEQ ID NO:30, and which encodes an amino acid sequence of SEQ ID NO:1.
22. An mRNA according to claim 20, which comprises an RNA sequence of SEQ ID NO:8.
23. An mRNA according to claim 20, which comprises an mRNA sequence of SEQ ID NO:7, SEQ ID 27, SEQ ID NO:28, or SEQ ID NO:29, or SEQ ID NO:30.
24. An mRNA according to any preceding claim, which comprises an Anti-Reverse Cap Analog (ARCA) at its 5’ end.
25. An mRNA according to any preceding claim, which comprises a 5’-untranslated region (5’-UTR) upstream of a coding sequence encoding the polypeptide.
26. An mRNA according to claim 25, wherein the 5’-UTR comprises, with an initiation codon sequence of the mRNA, an elongated Kozak sequence: GCCACCAUG.
27. An mRNA according to claim 25, wherein the 5’-UTR comprises any one of the following sequences: immediately upstream of an initiation codon sequence of the mRNA: a) GGAGACGCCACC (SEQ ID NO:11) b) GGGAGACGCCACC (SEQ ID NO:47), c) GAAGCGCCACC (SEQ ID NO:12), d) GGGACGCCACC (SEQ ID NO:13), e) GGGAGACTGCCACC (SEQ ID NO:14), f) GAAGCTGCCACC (SEQ ID NO:15), or g) GGGACTGCCACC (SEQ ID NO:16).
28. An mRNA according to any preceding claim, which comprises a 3’-untranslated region (3’-UTR) downstream of a coding sequence encoding the polypeptide.
29. An mRNA according to claim 28, wherein the 3’-UTR comprises a sequence selected from: a) GAAUU, or b) CCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCACCCACCTGCAATAAATGCAGCGAAGC CGGGA (SEQ ID NO:26).
30. An mRNA according to any preceding claim, which is a product of in-vitro transcription (IVT).
31. An mRNA according to any preceding claim, which comprises a polyadenylation (poly(A)) tail downstream of an open reading frame (ORF) encoding the polypeptide.
32. An mRNA according to any preceding claim, which comprises one or more modified nucleosides.
33. An mRNA according to claim 31, wherein the or each modified nucleoside is selected from any of the following: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 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-iodo- uridine, 5-methoxyuridine, 2′-O-methyl uridine, 5-iodocytidine, 5-methylcytosine, 5- methylcytidine, N1-methyladenosine, N6-methyladenosine.
34. An mRNA according to claim 32, wherein the one or more modified nucleosides comprise a 1-methylpseudouridine (m1ψ) modification.
35. An mRNA according to any of claims 32 to 34, wherein at least 50% of the uridines in the ORF have been modified.
36. An mRNA according to any of claims 32 to 34, wherein at least 50% of the uridines in the mRNA have been modified.
37. An mRNA according to claim 34, wherein at least 50 % of the uridines in the ORF have been modified to m1ψ.
38. An mRNA according to claim 34, wherein at least 50 % of the uridines in the mRNA have been modified to m1ψ.
39. An mRNA according to claim 32, wherein 5 to 50% of the uridine nucleotides are 5- iodouridine and 5 to 50% of the cytidine nucleotides are 5-iodocytidine.
40. An mRNA according to claim 32, wherein 5 to 50% of the uridine nucleotides are 2- thiouridine and 5 to 50% of the cytidine nucleotides are 5-methylcytidine.
41. An mRNA vaccine vector comprising an mRNA according to any preceding claim.
42. An mRNA vaccine, which comprises an mRNA according to any of claims 1 to 40, or an mRNA vaccine vector according to claim 41, encapsulated in a lipid nanoparticle (LNP) or a lipidoid nanoparticle (LiNP).
43. A pharmaceutical composition comprising an mRNA according to any of claims 1 to 40, an mRNA vaccine vector according to claim 41, or an mRNA vaccine according to claim 42, and a pharmaceutically acceptable carrier, excipient, or diluent.
44. A pharmaceutical composition according to claim 43, wherein the pharmaceutically acceptable vehicle solution, carrier, excipient, or diluent comprises a triblock copolymer which contains one poly(propylene oxide) block and two poly(ethylene oxide) blocks.
45. A pharmaceutical composition according to claim 43 or 44, or an mRNA vaccine according to claim 42, wherein the mRNA is complexed in the form of a LiNP nanoparticle comprising a cationic lipidoid of formula (b-1): R2A R4A R1A N {CH2 (CH2)a N [CH2 (CH2)b N]p}m [CH2 (CH2)a N]n R 6A R3A R5A (b-1), wherein the variables a, b, p, m, n and R1A to R6A are defined as follows: a is 1 and b is an integer of 2 to 4; or a is an integer of 2 to 4 and b is 1, p is 1 or 2, m is 1 or 2; n is 0 or 1 and m+n is ≥ 2; and R1A to R6A are independently of each other selected from hydrogen; -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH, -CH2-CH2-(C=O)-O-R7A, or -CH2-R7A; wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and a receptor ligand; provided that at least two residues among R1A to R6A are a group -CH2-CH(OH)-R7A, -CH(R7A)-CH2OH, -CH2CH2(C=O)-O-R7, - CH2CH2(C=O)-NH-R7A or -CH2R7 wherein R7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms contained in the compound of formula (b-1) are protonated to provide a compound carrying a positive charge.
46. An mRNA vaccine or a pharmaceutical composition according to claim 45, wherein the cationic lipidoid formula (b-1) comprises at least two residues among R1A to R6A, optionally at least three residues among R1A to R6A, or at least four residues among R1A to R6A are a group selected from -CH2-CH(OH)-R7A, -CH(R7A)-CH2-OH, -CH2-CH2-(C=O)-O-R7A, -CH2-CH2-(C=O)-NH-R 7A and -CH2-R7A, wherein R7A is selected from C3-C18 alkyl or C3-C18 alkenyl having one C- C double bond.
47. An mRNA vaccine according to claim 42, or a pharmaceutical composition according to claim 43 or 44, wherein the LiNP nanoparticle comprises a cationic lipidoid of formula (b-V) and/or formula (b-VII):
48. An mRNA vaccine according to claim 42, or a pharmaceutical composition according to claim 43 or 44, wherein the LiNP comprises: a) an mRNA according to any one of claims 1 to 40, or an mRNA vaccine vector according to claim 41, b) a cationic lipidoid of formula (b-V), and c) one or more helper lipid(s), optionally selected from: i) DPPC, and/or ii) cholesterol, and/or iii) PEG-lipid DMG-PEG2000, and optionally, components b), and c(i)-c(iii) are present, more preferably they are at the molar ratios of about 8.0: about 5.3: about 4.4: about 0.9, respectively, optionally, NLP comprises a triblock copolymer which contains one poly(propylene oxide) block and two poly(ethylene oxide) blocks as component (p) as defined above in vehicles.
49. An mRNA according to any of claims 1 to 40, an mRNA vaccine vector according to claim 41, an mRNA vaccine according to claim 42, or a pharmaceutical composition according to any one of claims 43 to 48, for use as a medicament.
50. An mRNA according to any of claims 1 to 40, an mRNA vaccine vector according to claim 41, an mRNA vaccine according to claim 42, or a pharmaceutical composition according to any one of claims 43 to 48, for use in the prevention, treatment, or amelioration of a coronavirus infection.
51. Use of an mRNA according to any of claims 1 to 40, an mRNA vaccine vector according to claim 41, an mRNA vaccine according to claim 42, or a pharmaceutical composition according to any one of claims 43 to48, in the manufacture of a medicament for the prevention, treatment, or amelioration of a coronavirus infection.
52. A method of inducing an immune response to a coronavirus in a subject, which comprises administering to the subject an effective amount of an mRNA according to any of claims 1 to 40, an mRNA vaccine vector according to claim 41, an mRNA vaccine according to claim 42, or a pharmaceutical composition according to any one of claims 43 to48.
53. A method of immunising a subject against a coronavirus, which comprises administering to the subject an effective amount of an mRNA according to any of claims 1 to 40, an mRNA vaccine vector according to claim 41, an mRNA vaccine according to claim 42, or a pharmaceutical composition according to any one of claims 43 to 48.
54. A method according to claim 52 or 53, which comprises administering an mRNA according to any of claims 1 to 40, an mRNA vaccine vector according to claim 41, an mRNA vaccine according to claim 42, or a pharmaceutical composition according to any one of claims 43 to 48, as part of a prime boost regimen.
55. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use according to claim 50, use according to claim 51, or a method according to any of claims 52 to 54, wherein the coronavirus is a beta-coronavirus.
56. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a lineage B or C beta-coronavirus.
57. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a lineage B beta-coronavirus.
58. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 56 or 57, wherein the lineage B beta-coronavirus is SARS-CoV or SARS-CoV-2.
59. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 56, wherein the lineage C beta- coronavirus is MERS-CoV.
60. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a variant of concern (VOC).
61. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 VOC.
62. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 beta, gamma, delta, or omicron VOC.
63. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 omicron XBB.1.5 virus.
64. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 omicron BA.2.12.1.
65. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 omicron BA.2.75.
66. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 omicron BA.2.3.20.
67. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 omicron BQ.1.1.
68. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 omicron XBB.
69. An mRNA, an mRNA vaccine vector, an mRNA vaccine, or a pharmaceutical composition for use, use, or a method, according to claim 55, wherein the beta-coronavirus is a SARS-CoV-2 omicron XBB.1.5.
70. An isolated polynucleotide comprising a first nucleotide sequence encoding SEQ ID NO:53 (CoV_S_T2_20 scaffold sequence), or the complement thereof, and a second nucleotide sequence encoding SEQ ID NO:53 (CoV_S_T2_20 scaffold sequence), or the complement thereof.
71. An isolated polynucleotide comprising a first nucleotide sequence encoding SEQ ID NO:1 (T2_17), or the complement thereof, and a second nucleotide sequence encoding SEQ ID NO:1 (T2_17), or the complement thereof.
72. An isolated polynucleotide according to claim 70 or 71, further comprising a nucleotide sequence encoding SEQ ID NO:2 (transmembrane domain amino acid sequence).
73. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:50 (CoV_S_T3_3), or the complement thereof.
74. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:52 (CoV_S_T3_4), or the complement thereof.
75. An isolated polynucleotide according to any of claims 70 to 74, further comprising a nucleotide sequence encoding a leader amino acid sequence, preferably SEQ ID NO:54 (leader amino acid sequence), or the complement thereof.
76. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:48 (T2_20), or the complement thereof.
77. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:49 (CoV_S_T3_3), or the complement thereof.
78. An isolated polynucleotide comprising a nucleotide sequence encoding SEQ ID NO:51 (CoV_S_T3_4), or the complement thereof.
79. An isolated polypeptide comprising first amino acid sequence of SEQ ID NO:53 (CoV_S_T2_20 scaffold sequence), and a second amino acid sequence of SEQ ID NO:53 (CoV_S_T2_20 scaffold sequence).
80. An isolated polypeptide comprising a first amino acid sequence of SEQ ID NO:1 (T2_17), and a second amino acid sequence of SEQ ID NO:1 (T2_17).
81. An isolated polypeptide according to claim 79 or 80, further comprising an amino acid sequence of SEQ ID NO:2 (transmembrane domain amino acid sequence).
82. An isolated polypeptide comprising an amino acid sequence of SEQ ID NO:50 (CoV_S_T3_3).
83. An isolated polypeptide comprising an amino acid sequence of SEQ ID NO:52 (CoV_S_T3_4).
84. An isolated polypeptide according to any of claims 79 to 83, further comprising a leader amino acid sequence, preferably SEQ ID NO:54 (leader amino acid sequence).
85. An isolated polypeptide comprising an amino acid sequence of SEQ ID NO:48 (T2_20).
86. An isolated polypeptide comprising an amino acid sequence of SEQ ID NO:49 (CoV_S_T3_3).
87. An isolated polypeptide comprising an amino acid sequence of SEQ ID NO:51 (CoV_S_T3_4).
88. A pharmaceutical composition which comprises an isolated polynucleotide according to any of claims 70 to 78, or an isolated polypeptide according to any of claims 79 to 87, and a pharmaceutically acceptable carrier, excipient, or diluent.
89. A vector which comprises an isolated polynucleotide according to any of claims 70 to 78, and a separate promoter operably linked to each different nucleotide sequence of the polynucleotide.
90. A fusion protein comprising a polypeptide according to any of claims 79 to 87.
91. A pseudotyped virus particle comprising a polypeptide according to any of claims 79 to 87.
92. An isolated polynucleotide according to any of claims 70 to 78, an isolated polypeptide according to any of claims 79 to 87, a pharmaceutical composition according to claim 88, or a vector according to claim 89, for use as a medicament.
93. An isolated polynucleotide according to any of claims 70 to 78, an isolated polypeptide according to any of claims 79 to 87, a pharmaceutical composition according to claim 88, or a vector according to claim 89, for use in the prevention, treatment, or amelioration of a coronavirus infection.
94. An isolated polynucleotide according to any of claims 70 to 78, an isolated polypeptide according to any of claims 79 to 87, a pharmaceutical composition according to claim 88, or a vector according to claim 89, for use in inducing an immune response to a coronavirus infection.
95. An isolated polynucleotide according to any of claims 70 to 78, an isolated polypeptide according to any of claims 79 to 87, a pharmaceutical composition according to claim 88, or a vector according to claim 89, for use in immunising a subject against a coronavirus infection.
96. A method of inducing an immune response to an influenza virus in a subject, which comprises administering to the subject an effective amount of: an isolated polynucleotide according to any of claims 70 to 78; an isolated polypeptide according to any of claims 79 to 87; a pharmaceutical composition according to claim 88; or a vector according to claim 89.
97. A method of immunising a subject against an influenza virus, which comprises administering to the subject an effective amount of: an isolated polynucleotide according to any of claims 70 to 78; an isolated polypeptide according to any of claims 79 to 87; a pharmaceutical composition according to claim 88; or a vector according to claim 89.
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