Virus vector and vaccine composition comprising same

Abstract

The present invention provides a viral vector comprising, at an insertion site in a viral genome, a nucleic acid sequence encoding a modified extracellular domain of an SARS-CoV-2 spike protein wherein the modified extracellular domain comprises an N-terminal signal peptide that allows the spike protein to enter the secretion system of a host cell wherein the N-terminal signal peptide is a non-linear signal peptide that allows the spike protein to enter the secretion system of the host cell, and wherein the non-linear signal peptide is a non-linear signal peptide that allows the spike protein to enter the secretion system of the host cell. The modified extracellular domain comprises a C-terminal deletion of at least the heptapeptide repeat region 2 (HR2), transmembrane fragment (TM) and cytoplasmic tail region (CT) of the spike protein. The invention also provides a vaccine composition comprising the virus vector.

Description

Technical Field

[0001] This invention relates to coronavirus vaccines comprising viral vectors. In particular, this invention provides a vaccine against SARS-CoV-2. Background Technology

[0002] SARS-CoV-2, which emerged in December 2019, is the cause of the COVID-19 pandemic, having a devastating impact on global health and the economy. As of February 28, 2023, a total of 758.3 million people have been confirmed infected, and at least 6.8 million have reportedly died from the disease. There is an urgent need for effective vaccines against new variants to mitigate the global burden of mortality and morbidity associated with SARS-CoV-2 infection and to enable societies to gradually recover from lockdowns.

[0003] Currently, over 200 candidate vaccines are under development, and promising and compelling data have been obtained from inactivated virus vaccines, protein subunit vaccines, and RNA-based and adenovirus vector-based vaccines expressing the SARS-CoV-2 spike protein. The European Union (EU) has approved 12 COVID-19 vaccines for marketing: 6 mRNA-based vaccines; 2 adenovirus vector-based vaccines; 1 inactivated, adjuvanted vaccine; and 3 subunit vaccines. All of these vaccines are administered intramuscularly, primarily inducing a systemic antibody response, with limited induction of airway mucosal immunity, a prerequisite for inactivated immunity. A booster vaccine designed to address this limitation is urgently needed. Summary of the Invention

[0004] This invention is defined by the features of the independent claims. Specific embodiments are defined in the dependent claims.

[0005] According to a first aspect of the present invention, a viral vector is provided, the viral vector comprising, at an insertion site in the viral genome, a nucleic acid sequence encoding a modified extracellular domain of the SARS-CoV-2 spike protein. The modified extracellular domain includes an N-terminal signal peptide that allows the spike protein to enter the host cell's secretory system. The modified extracellular domains include the deletion of at least the C-terminus of the heptapeptide repeat region 2 (HR2), the transmembrane fragment (TM), and the cytoplasmic tail region (CT) of the spike protein.

[0006] According to a second aspect of the present invention, a vaccine composition is provided, the vaccine composition comprising a carrier as defined in this disclosure, preferably in the form of a mucosal administration formulation.

[0007] According to a third aspect of the invention, a method for preventing COVID-19 is provided, the method comprising administering to a mammal, preferably a human, a preventive or therapeutically effective amount of a vaccine composition defined in this disclosure.

[0008] According to a fourth aspect of the invention, the use of the viral vector defined in this disclosure in the preparation of a vaccine composition for inducing an immune response in mammals, preferably humans, is provided. Attached Figure Description

[0009] Figure 1 A schematic diagram of an adenovirus vector vaccine construct, which includes a modified extracellular domain encoding the SARS-CoV-2 spike protein at the E1 insertion site, as well as an IL-3 signaling sequence and an SV40 polyadenylate (polyA) signaling sequence regulated by the human cytomegalovirus (CMV) promoter.

[0010] Figure 2. Schematic diagram of the SARS-CoV-2 spike protein and its modified extracellular domain. (A) Full-length SARS-CoV-2 spike protein, corresponding to SEQ ID NO:1. Amino acids 1-13 correspond to the signal sequence (SS), amino acids 14-1205 correspond to the extracellular domain of the spike protein, amino acids 1209-1229 correspond to the transmembrane domain (TM) of the spike protein, and amino acids 1230-1268 correspond to the cytoplasmic tail region (CT) of the spike protein. NTD, N-terminal domain; RBD, receptor-binding domain; SD1, subdomain 1; SD2, subdomain 2; S1 / S2, S1 / S2 cleavage site; S2', S2' cleavage site; FP, fusion peptide; HR1, heptapeptide repeat region 1; CH, central helical region; CD, linker domain; HR2, heptapeptide repeat region 2. (B) The extracellular domain of the spike protein, excluding the TM and CT regions (amino acids 1-1203). (C) The extracellular domain of the spike protein, excluding the HR2, TM, and CT regions.

[0011] Figure 3 Western blot analysis of the supernatant collected from HEK293 cells transfected with pFCV2.1 is shown. pGFP was also transfected and used to control antibody specificity. 25 nm of purified spike protein was used as a positive control.

[0012] Figure 4 shows the vaccination and blood collection procedures for Syrian golden hamsters (A), and the immune response at 14 days (B) and 28 days (C) post-infection. The intranasal vaccination dose for hamsters was 2.5 × 10⁻⁶. 5 Up to 2.5×10 8Four vaccine doses of each virus particle / agent were administered, and immune IgG responses against the original strain receptor-binding domain (RBD) and omicron BA.4 / 5 RBD were measured by ELISA.

[0013] Figure 5 Schematic diagram of the modified extracellular domains of the SARS-CoV-2 spike protein, with different fusion domains attached to the C-terminus. (A) Modified extracellular domain of spike protein with a trimerizing domain fused to the C-terminus. (B) Modified extracellular domain of spike protein with a transmembrane domain fused to the C-terminus. (C) Modified extracellular domain of spike protein with both a trimerizing domain and a transmembrane domain fused to the C-terminus. (D) Modified extracellular domain of spike protein with one or more receptor-binding domains (RBDs) from other immunologically different SARS-CoV-2 spike proteins fused to the C-terminus. (E) Modified extracellular domain of spike protein with one or more receptor-binding domains (RBDs) and a trimerizing domain fused to the C-terminus from other immunologically different SARS-CoV-2 spike proteins. (F) Modified extracellular domain of spike protein with one or more receptor-binding domains (RBDs) and a trimerizing domain fused to the C-terminus from other immunologically different SARS-CoV-2 spike proteins. (G) A modified extracellular domain of a spike protein with one or more receptor-binding domains (RBDs) and transmembrane domains fused to the C-terminus of an immunologically distinct SARS-CoV-2 spike protein. (H) A modified extracellular domain of a spike protein with one or more receptor-binding domains (RBDs), trimerizing domains, and transmembrane domains fused to the C-terminus of an immunologically distinct SARS-CoV-2 spike protein. TRD, trimerizing domain; TMD, transmembrane domain; RBD, receptor-binding domain.

[0014] Figure 6. Intranasal vaccination with FCV2.1 protects hamsters from SARS-CoV-2 infection. A.) Immunization and challenge protocol. The results were obtained from vaccination with FCV2.1 (2 doses, 1x10). 10 The viral load (B.), infectivity titer (C.), and immunohistochemical analysis of the viral nucleocapsid in the nasal turbinate tissues of hamsters vaccinated with VP / dose and unvaccinated hamsters (D.) were measured. In hamsters vaccinated with FCV2.1, SARS-CoV-2 virus levels decreased very rapidly, approaching the detection limit. This suggests that, in addition to protecting vaccinated individuals, FCV2.1 may significantly reduce transmission and viral spread.

[0015] Figure 7. Schematic diagram of the neutralizing effect of FCV2.3 and its application as a booster after initial administration of Comirnaty (original). A.) FCV2.3 consists of a modified extracellular domain of the spike protein fused to its C-terminus with a receptor-binding domain (RBD1) derived from other immunologically distinct SARS-CoV-2 spike proteins. The receptor-binding domain in FCV2.3 is derived from the XBB variant. B.) Neutralizing antibody (NAb) responses in mice receiving two intramuscular doses of Comirnaty (Com(w)) and mice receiving one intramuscular dose of Comirnaty followed by an intranasal dose of FCV2.3 (FCV2.3). Compared to two doses of Comirnaty, a booster dose of FCV2.3 induced a strong NAb response against all tested variants (original bead, BA.4 / 5, and XBB). Administering FCV2.3 as a booster after initial vaccination with Comirnay resulted in strong neutralizing antibody responses against multiple SARS-CoV-2 variants, including the XBB variant. This suggests that the addition of RBD from immunologically different variants can amplify the NAb response, thereby providing protection against a wider range of SARS-CoV-2 variants. Detailed Implementation

[0016] In this document, the term "vector" is used to refer to a nucleic acid molecule capable of mediating the entry (e.g., transport, delivery, etc.) of another nucleic acid molecule into a cell. The transferred nucleic acid is typically linked to, for example, inserted into, the vector nucleic acid molecule. The vector may include a sequence that directs autonomous replication, or may include a sequence sufficient to enable integration into the host cell's DNA. Commonly used vectors include, for example, plasmids, granules, and viral vectors. Commonly used viral vectors include, for example, replication-defective retroviruses, adenoviruses, adeno-associated viruses, and lentiviruses. It will be apparent to those skilled in the art that viral vectors may include various viral components in addition to one or more nucleic acids that mediate the entry of the transferred nucleic acid. Therefore, the term "viral vector" may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself.

[0017] In this paper, the term "signal sequence" refers to a signal peptide, which is part of the N-terminus of a secretory protein that is secreted extracellularly and thus crosses the cell membrane. Signal peptides typically consist of approximately 10 to 30 amino acids and are subsequently cleaved and removed by cell membrane-specific proteases, leaving only the secretory protein transported extracellularly. Signal peptides act as targeting signals, enabling cellular transport mechanisms to direct proteins to specific intracellular or extracellular locations. To date, over 4000 signal peptides are known to exist in eukaryotic cells. DNA libraries encoding signal peptides are disclosed, for example, in WO 2021045541A1 and KR20210028116A.

[0018] In this paper, the term "transmembrane domain" refers to a hydrophobic α-helical structure that crosses the host cell membrane. Transmembrane domains can be directly fused to the C-terminal portion of a fusion protein encoded by a vector. For example, transmembrane domains can originate from membrane-embedded proteins (e.g., receptors, differentiation molecule clusters, enzymes, transport proteins, cell adhesion molecules, etc.). A specific example is the transmembrane domain derived from a type I transmembrane protein such as human VCAM-1 (vascular cell adhesion molecule 1). Type I transmembrane proteins are anchored to the lipid membrane via a stop-transfer anchor sequence, while their N-terminal domain targets the extracellular space, where the mature form of the protein resides on the cell membrane.

[0019] This invention is based on a viral vector that produces a modified form of the SARS-CoV-2 spike protein, which exhibits increased yield when expressed by a viral vector, preferably an adenovirus vector. This increased spike protein expression provides a stronger immunogenicity for the vaccine. Furthermore, the C-terminal deletion of the modified spike protein removes a dominant immunotope that potentially competes with the neutralizing epitope in the RBD region for an immune response. Therefore, the modified spike protein of this invention directs the anti-spike protein immune response more towards the remaining spike protein extracellular domain containing the neutralizing epitope in the RBD region.

[0020] Viral vectors are widely used platforms in vaccine design and have shown great potential in combating a variety of infectious diseases. Currently, many viral vector-based vaccines against SARS-CoV-2 are in preclinical and clinical development, including two vaccines that have already gained market access in the EU.

[0021] The vaccine of the present invention has been developed as a booster vaccine for individuals who have completed a full course of vaccination, such as Comirnay, Spikevax, or ADZ1222 (one or more doses), and as a primary vaccine for individuals who have not yet received any vaccine against SARS-CoV-2 infection. The viral vector in the vaccine produces a secreted form of a C-terminated truncated SARS-CoV-2 spike protein. In a preferred embodiment, the C-terminated spike protein includes a mutated furin cleavage site with single amino acid modifications R677G, R678S, and R680S (corresponding to positions 685, 686, and 688 in SEQ ID NO:2, respectively). In an embodiment, the C-terminated spike protein is modified by proline substitution at residues 981 and 982 (corresponding to positions 989 and 990 in SEQ ID NO:2, respectively).

[0022] The spike protein mediates SARS-CoV-2 entry into host cells and is the primary target of neutralizing antibodies produced by infected individuals, as well as a major target of antibody-mediated immunity (Chaudhary et al., 2021). Therefore, aside from inactivated and attenuated whole-virus methods, almost all ongoing COVID-19 vaccine programs utilize the spike protein as an immunogen. All COVID-19 vaccines approved in the EU and the US are based on the SARS-CoV-2 spike protein.

[0023] The spike protein of Omicron BA.5 strain has the following amino acid sequence (SEQ ID NO:1): MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVT 60 WFHAISGTNGIKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATN 120 VVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN 180 FKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALH 240 RSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS 300 FTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADY 360 SVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKL 420 PDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAG 480 VNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG 540 LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS 600 NQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIP 660 IGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTE 720 ILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQV 780 KQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIA 840 ARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYR 900 FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQ 960 LSSKFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLA 1020 ATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG 1080 KAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPEL 1140 DSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK 1200 YEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL 1260 KGVKLHYT 1268 In the above sequence (SEQ ID NO:1), amino acids at positions 1-13 correspond to the signal sequence, amino acids at positions 14-1203 correspond to the extracellular domain of the spike protein, amino acids at positions 1209-1229 correspond to the transmembrane domain of the spike protein, and amino acids at positions 1230-1268 correspond to the cytoplasmic tail region (CT) of the spike protein.

[0024] In a preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein has the following amino acid sequence (SEQ ID NO:2): MSRLPVLLLLQLLVRPGLQAPQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLF 60 LPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSL 120 LIVNNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLM 180 DLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITR 240 FQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLS 300 ETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKR 360 ISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGN 420 IADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGN 480 KPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK 540 CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSV 600 ITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTTPWRVYSTGSNVFQTRAGCLIGAEYVN 660 NSYECDIPIGAGICASYQTQTKSHGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTN 720 FTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCCTQLKRALTGIAVEQDKN 780 TQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY 840 GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP 900 FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQ 960 ALNTLVKQLSSKFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAE 1020 IRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTT 1080 APAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTV 1140 YD 1142 In the above sequence (SEQ ID NO:2), amino acids 1-19 correspond to the signal sequence, and amino acids 22-1142 correspond to the extracellular domain of the spike protein, which is deleted at the C-terminus. In another embodiment, the C-terminal deletion site is between the linker domain CD (amino acids 1030-1062 of SEQ ID NO:1) and the heptapeptide repeat region 2 (HR2) (amino acids 1158-1197 of SEQ ID NO:1). In another embodiment, the C-terminal deletion site is in the linker domain CD (amino acids 1030-1062 of SEQ ID NO:1). In another preferred embodiment, the C-terminal deletion site is located in the region corresponding to positions 1030-1157 of the spike protein of Omeprone BA.5 strain in SEQ ID NO:1.

[0025] Therefore, this invention relates to a viral vector comprising a nucleic acid sequence encoding a modified extracellular domain of the SARS-CoV-2 spike protein at an insertion site in the viral genome. The modified extracellular domain includes an N-terminal signal peptide that allows the spike protein to enter the host cell's secretory system. The modified extracellular domains include the deletion of at least the C-terminus of the heptapeptide repeat region 2 (HR2), the transmembrane fragment (TM), and the cytoplasmic tail region (CT) of the spike protein.

[0026] In a preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein (corresponding to the C-terminal deleted spike extracellular domain) comprises the amino acid sequence of SEQ ID NO:2, amino acids 22-1142 of SEQ ID NO:2, or a sequence having at least 80% sequence identity with the sequence of SEQ ID NO:2 or amino acids 22-1142 of SEQ ID NO:2. In a more preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein (corresponding to the C-terminal deleted spike extracellular domain) comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the sequence of SEQ ID NO:2 or amino acids 22-1142 of SEQ ID NO:2.

[0027] In the case of two or more amino acid sequences, the term "sequence identity" means that two or more sequences or subsequences are identical. Two sequences are said to be "substantially identical" if, when compared and aligned to the maximum extent within a comparison window or specified region, they show a certain percentage of identical amino acid residues as determined by one of the following sequence comparison algorithms or by manual alignment and visual inspection (i.e., 29% identity in a specified region, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity, or the entire sequence if not specified).

[0028] Two examples of algorithms suitable for determining sequence identity and sequence similarity percentages are the BLAST and BLAST2.0 algorithms, described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. For amino acid sequences, the BLASTP program uses the following default values: word length of 3, expected value (E) of 10, and BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915-10919], alignment (B) of 50, expected value (E) of 10, M=5, N=-4, and compares the two strands. For short amino acid sequences, the PAM30 scoring matrix can be applied.

[0029] In another preferred embodiment, the N-terminal signal peptide is the human interleukin-3 secretory signal peptide or the natural signal peptide of the SARS-CoV-2 spike protein (i.e., amino acids 1-13 of SEQ ID NO:1).

[0030] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein includes a mutated furin protease cleavage site, a single amino acid modification at positions R677G, R678S, and R680S (corresponding to positions 685, 686, and 688 in SEQ ID NO:2, respectively), and / or a proline substitution at residues at positions 981 and 982 (corresponding to positions 989 and 990 in SEQ ID NO:2, respectively).

[0031] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein includes the amino acid sequence of SEQ ID NO:2 or amino acids at positions 22-1142 of SEQ ID NO:2.

[0032] In another preferred embodiment, the vector is selected from the group consisting of: adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, herpes simplex virus vector, poxvirus vector, preferably vaccinia virus vector and orthomyxovirus vector, and preferably influenza virus vector.

[0033] In a more preferred embodiment, the vector is an adenovirus vector, most preferably a non-replicating adenovirus serotype 5 vector. In some embodiments, the adenovirus vector as described herein is adenovirus type 5 (Ad5), which may include, for example, Ad5 with deletions in the E1 / E3 region and Ad5 with deletions in the E4 region. Other suitable adenovirus vectors include type 2 strains, orally tested types 4 and 7 strains, enterovirus types 40 and 41, and other human adenovirus strains (e.g., Ad34), as well as chimpanzee adenovirus type 36 and serotype Y25, and vectors derived from group C chimpanzee adenoviruses, which are sufficient to deliver antigens and elicit an adaptive immune response to the transgenic antigen. In embodiments, the insertion site in the adenovirus serotype 5 vector is selected from the group consisting of E1 insertion sites, E3 insertion sites, and E4 insertion sites.

[0034] Various promoters can be used in the adenoviral vectors described herein. For example, commonly used promoters and enhancers are derived from, for example, β-actin, adenovirus, simian virus (SV40), and human cytomegalovirus (CMV). For example, the vector enables protein expression under the regulation of CMV promoters, β-actin promoters, early SV40 promoters, late SV40 promoters, metallothionein promoters, and murine mammary tumor virus promoters.

[0035] In another preferred embodiment, the nucleic acid sequence encoding the modified extracellular domain is as shown in SEQ ID NO:3, or is a nucleotide sequence having at least 70%, 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:3.

[0036] In a preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein is encoded by the following nucleotide sequence (SEQ ID NO:3): ATGAGCCGCCTGCCCGTCCTGCTCCTGCTCCAACTCCTGGTCCGCCCCGGACTCCAAGCT 60 CCCCAGTGCGTCAACCTGATCACCAGAACCCAAAGCTACACCAATAGCTTCACACGGGGG 120 GTGTACTACCCTGATAAGGTGTTCCGGAGTTCTGTGCTGCACTCCACACAGGACCTGTTT 180 CTGCCTTTTTTTTCCAATGTGACATGGTTCCACGCCATCAGCGGCACCAACGGCACAAAA 240 AGGTTTGACAACCCCGTGCTGCCATTTAATGATGGGGTGTACTTCGCTTCCACCGAGAAA 300 TCCAACATCATTAGGGGCTGGATCTTCGGCACTACCCTGGATTCTAAGACTCAGTCCCTG 360 CTGATCGTGAATAACGCCACCAACGTGGTGATCAAGGTGTGCGAATTTCAGTTCTGCAAC 420 GATCCATTCCTGGACGTGTACTACCACAAGAATAACAAGAGCTGGATGGAGAGCGAGTTC 480 CGCGTGTACTCTTCCGCAAACAACTGCACCTTTGAATATGTGAGCCAGCCCTTCCTGATG 540 GACCTGGAGGGAAAGCAGGGCAATTTCAAAAACCTCAGGGAATTTGTCTTCAAGAATATC 600 GATGGCTATTTCAAGATCTACAGCAAGCATACTCCCATCAACCTGGGCAGAGACCTGCCC 660 CAGGGCTTCAGCGCTCTGGAGCCTCTGGTGGACCTGCCCATCGGCATCAACATTACCAGA 720 TTCCAGACCCTGCTGGCTCTGCATCGGTCTTACCTGACTCCCGGAGATAGCAGCAGCGGC 780 TGGACCGCCGGCGCCGCTGCCTATTATGTGGGGTACCTGCAGCCTAGAACCTTCCTGCTG 840 AAGTACAACGAGAACGGCACCATCACAGATGCCGTGGACTGCGCTCTGGACCCACTCTCC 900 GAGACCAAATGCACCCTGAAGTCCTTTACCGTGGAGAAAGGCATCTACCAGACCTCCAAT 960 TTCAGAGTGCAGCCTACAGAGAGCATCGTGCGCTTCCCCAACATCACCAACCTGTGCCCT 1020 TTCGACGAGGTGTTCAATGCAACACGGTTTGCCAGCGTGTACGCTTGGAATCGGAAGCGG 1080 ATCAGCAACTGCGTGGCCGACTACTCTGTGCTGTATAACTTCGCCCCCTTTTTCGCCTTT 1140 AAATGTTACGGAGTGTCCCCAACTAAGCTGAACGACCTGTGCTTCACAAACGTGTATGCT 1200 GATAGCTTCGTGATTCGGGGCAACGAAGTCAGCCAGATCGCTCCCGGACAGACTGGAAAC 1260 ATCGCCGACTACAACTACAAGCTGCCTGACGACTTCACCGGCTGCGTGATCGCCTGGAAC 1320 TCCAACAAGCTGGATAGCAAGGTGGGCGGCAATTACAACTACCGGTATCGCCTGTTCAGA 1380 AAGTCTAATCTGAAGCCTTTTGAGCGCGACATCAGCACCGAGATCTATCAGGCCGGAAAC 1440 AAGCCCTGCAACGGAGTGGCCGGGGTGAACTGCTACTTTCCTCTGCAGAGCTATGGCTTT 1500 CGGCCCACCTACGGGGTGGGGCACCAGCCCTACCGGGTGGTGGTCCTGTCCTTCGAGCTG 1560 CTGCACGCCCCCGCCACCGTGTGCGGCCCCAAGAAATCCACCAACCTGGTGAAGAATAAG 1620 TGTGTGAACTTCAACTTCAACGGCCTCACCGGCACCGGCGTGCTGACCGAGTCTAATAAG 1680 AAATTCCTGCCCTTCCAGCAGTTCGGGAGGGACATCGCCGATACCACAGATGCCGTCAGA 1740 GACCCACAGACCCTGGAGATCCTGGACATCACCCCCTGCTCATTCGGCGGCGTGAGCGTC 1800 ATCACACCTGGCACCAACACCAGCAATCAGGTTGCCGTGCTGTACCAGGGGGTGAACTGC 1860 ACCGAGGTGCCTGTGGCCATTCACGCCGACCAGCTCACCCCTACTTGGCGGGTGTACAGC 1920 ACAGGCTCCAACGTGTTCCAGACCAGGGCCGGATGCCTGATCGGCGCCGAGTACGTGAAT 1980 AATTCCTATGAGTGCGACATCCCCATCGGAGCCGGGATTTGCGCCAGCTACCAGACCCAG 2040 ACAAAGAGCCACGGCAGCGCCAGCTCCGTCGCCAGCCAGAGCATCATCGCCTACACCATG 2100 TCCCTGGGGGCCGAGAATAGCGTCGCATACAGTAATAATTCCATTGCCATCCCAACCAAC 2160 TTCACCATCAGCGTGACCACCGAAATTCTGCCCGTGTCAATGACCAAAACCTCCGTGGAC 2220 TGTACCATGTACATCTGTGGCGACAGCACCGAGTGCAGCAACCTGCTGCTGCAGTACGGA 2280 TCCTTTTGCACCCAGCTGAAGCGGGCCCTGACAGGAATCGCCGTGGAGCAGGACAAGAAC 2340 ACCCAGGAGGTGTTCGCCCAGGTGAAACAGATCTACAAGACCCCCCCCATCAAGTACTTC 2400 GGTGGGTTCAACTTCAGCCAGATCCTGCCAGACCCCTCTAAGCCCTCCAAGAGGAGCTTT 2460 ATCGAAGACCTGCTGTTCAACAAGGTCACCCTGGCCGATGCAGGCTTCATCAAGCAGTAC 2520 GGGGATTGTCTGGGCGACATCGCCGCCAGAGACCTGATCTGTGCACAGAAGTTTAACGGC 2580 CTGACAGTCCTGCCTCCACTGCTGACTGACGAGATGATCGCCCAGTATACCTCCGCCCTG 2640 CTGGCCGGAACTATTACTAGCGGCTGGACCTTCGGCGCCGGTGCCGCTCTGCAGATCCCC 2700 TTTGCCATGCAGATGGCCTATCGCTTCAACGGCATTGGAGTGACCCAGAACGTGCTGTAT 2760 GAGAACCAGAAGCTGATCGCCAACCAGTTCAACTCCGCCATCGGCAAAATTCAGGACTCT 2820 CTGAGCTCCACTGCCAGTGCCCTGGGCAAGCTGCAGGATGTGGTGAATCACAACGCCCAG 2880 GCCCTGAACACACTCGTGAAACAGCTGTCCTCCAAGTTCGGCGCTATTAGCAGCGTGCTG 2940 AATGATATCCTGAGCCGGCTGGATCCCCCCGAGGCCGAAGTGCAGATTGATAGACTGATT 3000 ACAGGCAGACTGCAGAGTCTGCAGACCTACGTGACTCAGCAGCTGATCCGCGCTGCCGAG 3060 ATCAGAGCCTCCGCCAACCTGGCCGCCACCAAGATGTCCGAGTGCGTGCTGGGCCAGTCC 3120 AAAAGAGTGGATTTTTGCGGCAAGGGCTACCACCTGATTGTCATTTCCTCAGTCTGCTCCT 3180 CACGGCGTGGTGTTCCTGCACGTGACCTATGTGCCCGCCCAGGAGAAGAACTTTACCACT 3240 GCCCCTGCCATCTGCCACGACGGCAAGGCCCACTTTCCCCGCGAGGGCGTGTTCGTGTCT 3300 AACGGAACCCACTGGTTCGTCACCCAGAGGAATTTTTACGAGCCACAGATCATTACCACA 3360 GATAACACCTTTGTCAGTGGCAATTGCGATGTGGTGATCGGCATCGTCAACAATACCGTG 3420 TACGAC 3426 In another preferred embodiment, the SARS-CoV-2 spike protein expressed by the vector is secreted from the host cells infected by the viral vector in the form of soluble monomers.

[0037] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein includes at its C-terminus one or more additional receptor-binding domains (RBDs) from the SARS-CoV-2 spike protein that are immunologically different from the modified extracellular domain of the SARS-CoV-2 spike protein. For example, in an embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein is immunologically corresponding to the spike protein of Omeprón BA.5 strain, wherein the one or more additional receptor-binding domains (RBDs) from the SARS-CoV-2 spike protein are immunologically corresponding to one or more spike proteins derived from the SARS-CoV-2 variant group consisting of: XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.1.9.2, XBB.1.5.1, FD.2, XBB, BQ.1.1, CH.1.1, BQ.1, BN.1, and BJ.1.

[0038] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein includes a trimerizing domain at its C-terminus, preferably a T4 fibritin trimerizing domain, a human collagen trimerizing domain, a leucine zipper domain, or any other additional C-terminal amino acid sequence that promotes the trimerization of the spike protein.

[0039] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein includes a transmembrane domain at its C-terminus, or any other additional C-terminal amino acid sequence that facilitates transmembrane anchoring of the spike protein.

[0040] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein includes, in any order, the following in the C-terminal region: i) a trimerizing domain, preferably a T4 fibroin trimerizing domain, a human collagen trimerizing domain, a leucine zipper domain, or any other additional C-terminal amino acid sequence that promotes the trimerization of the spike protein; and ii) a transmembrane domain, or any other additional C-terminal amino acid sequence that promotes the transmembrane anchoring of the spike protein.

[0041] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein includes, in any order at its C-terminus: i) one or more receptor-binding domains (RBDs) of the SARS-CoV-2 spike protein that are immunologically different from the modified extracellular domain of the SARS-CoV-2 spike protein, and ii) a trimerization domain, preferably a T4 fibroin trimerization domain, a human collagen trimerization domain, a leucine zipper domain, or any other additional C-terminal amino acid sequence that promotes the trimerization of the spike protein.

[0042] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein includes, in any order at its C-terminus: i) one or more receptor-binding domains (RBDs) of the SARS-CoV-2 spike protein that are immunologically different from the modified extracellular domain of the SARS-CoV-2 spike protein, and ii) a transmembrane domain, or any other additional C-terminal amino acid sequence that facilitates transmembrane anchoring of the spike protein.

[0043] In another preferred embodiment, the modified extracellular domain of the SARS-CoV-2 spike protein comprises, in any order at its C-terminus: i) one or more receptor-binding domains (RBDs) of the SARS-CoV-2 spike protein that are immunologically different from the modified extracellular domain of the SARS-CoV-2 spike protein; ii) a trimerizing domain, preferably a T4 fibroin trimerizing domain, a human collagen trimerizing domain, a leucine zipper domain, or any other additional C-terminal amino acid sequence that promotes the trimerization of the spike protein; and iii) a transmembrane domain, or any other additional C-terminal amino acid sequence that promotes the transmembrane anchoring of the spike protein.

[0044] In an embodiment, the present invention also relates to a vaccine composition comprising a viral vector as defined in this disclosure, preferably in the form of a mucosal administration formulation.

[0045] In a preferred embodiment, the mucosal application formulation is selected from the group consisting of nasal drops, aerosols, sprays, powder sprays, gels, microspheres, liposomes, membranes, and suspensions. More preferably, the vaccine composition is a liquid spray or a sprayable composition. In one embodiment, the vaccine composition is administered intranasally.

[0046] In a preferred embodiment, the vaccine further comprises a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient. In a more preferred embodiment, the vaccine composition comprises A195 buffer, Tris, sodium chloride, magnesium chloride, histidine, sucrose, polysorbate-80, EDTA, and ethanol. In the most preferred embodiment, the vaccine composition comprises A195 buffer; 10 mM Tris, 75 mM NaCl, 1 mM MgCl2, 10 mM histidine, 5% (wt / vol) sucrose, 0.02% (wt / vol) polysorbate-80, 0.1 mM EDTA, and 0.5% (vol / vol) ethanol at pH 7.4.

[0047] In another embodiment, the present invention relates to a method for preventing COVID-19, the method comprising administering to a mammal, preferably a human, a preventive or therapeutically effective amount of a vaccine composition defined in this disclosure.

[0048] In yet another embodiment, the present invention relates to the use of the viral vector defined in this disclosure in the preparation of pharmaceutical compositions for inducing an immune response in mammals, wherein the vector encodes a modified extracellular domain of the SARS-CoV-2 spike protein. The modified extracellular domain includes an N-terminal signal peptide that allows the spike protein to enter the host cell's secretory system. The modified extracellular domains include the deletion of at least the C-terminus of the heptapeptide repeat region 2 (HR2), the transmembrane fragment (TM), and the cytoplasmic tail region (CT) of the spike protein.

[0049] It should be understood that the embodiments of the present invention disclosed herein are not limited to the specific structures, processes, or materials disclosed herein, but extend to equivalents recognized by those skilled in the art. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be limiting.

[0050] Throughout this specification, references to "one embodiment" or "implementation" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment.

[0051] As used herein, for convenience, multiple items, structural elements, constituent elements, and / or materials may be presented in a public list. However, these lists should be interpreted as if each member in the list were individually identified as a separate and unique member. Therefore, no single member in such a list should be construed as being factually equivalent to any other member in the same list, based solely on their performance within the common group, without any indication of the contrary. Furthermore, various embodiments and examples of the invention, along with alternatives to its various components, may be mentioned herein. It should be understood that such embodiments, examples, and alternatives should not be construed as factual equivalents of each other, but should be regarded as separate and autonomous representations of the invention.

[0052] Furthermore, in one or more embodiments, the described features, structures, or characteristics can be combined in any suitable manner. Numerous specific details, such as examples of length, width, shape, etc., are provided in the following description to provide a thorough understanding of embodiments of the invention. However, those skilled in the art will recognize that the invention can be practiced without one or more specific details, or using other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring various aspects of the invention.

[0053] While the foregoing embodiments illustrate the principles of the invention in one or more specific applications, it will be apparent to those skilled in the art that various modifications, uses, and implementation details can be made without inventiveness and without departing from the principles and concepts of the invention. Therefore, it is not intended to be limiting except as set forth in the following claims.

[0054] The verbs “comprise” and “include” are used in this document as disclosure restrictions, neither excluding nor requiring the presence of any unlisted features. Unless otherwise expressly stated, the features listed in the dependent claims may be freely combined with each other. Furthermore, it should be understood that the use of “a” or “an” throughout this document, i.e., the singular form, does not exclude the plural form.

[0055] Experimental Section This experimental section presents results obtained using the investigational drug product (IMP) FINCoVac 2.1 (FCV2.1), which is based on a non-replicating Ad5 vector and expresses a modified extracellular domain (extracellular domain) of the SARS-CoV-2 spike protein from the Omeprone BA.5 strain. Figure 1 FINCoVac 2.1 is designed for intranasal administration, and when administered intranasally, the modified spike extracellular domain encoded is expected to generate a host immune response, thereby inducing immunity against SARS-CoV-2. Furthermore, this section also provides results obtained using a derivative of the FCV2.1 vaccine (referred to as FCV2.3).

[0056] A truncated extracellular domain of the SARS-CoV-2 spike protein was cloned into pRLF1 to obtain the non-replicating adenovirus vaccine vector plasmid pFCV2.1. The truncated spike protein extracellular domain gene was synthesized into eBlocks™ gene fragments (Integrated DNA Technologies), and then assembled using Gibson assembly master mix (NEB, E2611) to produce a longer fragment containing the CMV promoter and enhancer regions, the truncated spike protein extracellular domain, WPRE elements, and a polyadenylation signal sequence. The assembled fragment was then amplified by PCR. To assemble the newly prepared truncated spike protein extracellular domain fragment into an adenovirus vector, the adenovirus vector genome backbone plasmid pRLF1 was first linearized using the BstZ17I restriction endonuclease, followed by ethanol precipitation. pRLF1 is an adenovirus vector genome backbone plasmid developed by the Rokote laboratory in Finland, used for Gibson assembly of recombinant transgenes with adapted E1 regions missing.

[0057] To obtain pFCV2.1, the truncated spike protein extracellular domain fragment was assembled into the digested viral backbone pRLF1 using Gibson assembly master mix (NEB, E2611) according to the manufacturer's instructions. The Gibson assembly reactant was then transformed into NEB® 5-α high-efficiency E. coli (ESC) according to the manufacturer's instructions. E. coli(NEB, C2987H). Positive clones were screened by PCR, and the correct recombination events were further confirmed by sequencing the constructs.

[0058] The final viral vaccine product will be stored at below -60°C. Long-term stability studies will be conducted to provide data for determining batch shelf life. Based on preliminary data from adenovirus stored in the intended final formulation buffer, stability is expected for at least 3 years.

[0059] In vitro characterization of the truncated spike protein extracellular domain HEK293 cell line Human embryonic kidney HEK293 cell lines were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin. Cells were cultured at 37°C in a humidified environment of 5% CO2.

[0060] HEK293 transfection According to the manufacturer's instructions, 2 µg of the full-length adenovirus vaccine vector plasmid pFCV2.1 was transfected into HEK293 cells using the Trans-IT (MirusBio) transfection reagent.

[0061] Western blot analysis The supernatant of HEK293 cells transfected with pFCV2.1 was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane (Bio-Rad). After blocking with a solution of phosphate-buffered saline containing 5% skim milk powder and 0.05% Tween 20 for 30 min at room temperature (RT), the membrane was treated with serum from recovered COVID-19 patients as the primary antibody, followed by treatment with an IRdye-labeled secondary antibody against human immunoglobulins (LI-COR Biosciences). The blots were visualized using an Odyssey infrared imaging system and Image Studio v3.1 software (LI-COR Biosciences).

[0062] Virus production The pFCV2.1 adenovirus vaccine vector plasmid was digested with PacI (New England Biolabs) to release the adenovirus genome. The digested viral genome was purified by ethanol precipitation and dissolved in TE buffer. Then, 2 µg of the linearized viral genome was transfected into HEK293 cells using Trans-IT (MirusBio) transfection reagent according to the manufacturer's instructions.

[0063] The virus recovered from transfection was then further amplified in HEK293 cells and purified using the Adeno-X Maxi Purification Kit (Takara Bio) according to the manufacturer's instructions.

[0064] Animal research Feeding conditions and animal care The temperature in the laboratory animal housing was maintained between +19°C and +25°C. Relative humidity was at least 30% and did not exceed 70%. Lighting was artificial, with a 12-hour light-12-hour dark cycle, consistent with the natural diurnal cycle. Animals were housed individually (1 animal / cage). Animal care followed the Standard Operating Procedure (SOP) of the University of Turku Central Animal Laboratory. All animals were observed daily for general health, signs of morbidity, and mortality. Particular attention was paid to any clinical signs that might indicate adverse cardiovascular, respiratory, or central nervous system reactions. Detailed clinical observation was conducted outside the living cages on the day of administration, as follows: once before administration, immediately after administration upon the animal's awakening, and 3–4 hours and approximately 24 hours after administration. Detailed clinical observation was conducted weekly after administration and once on the day of dissection (day 28 of the study).

[0065] Study design and dosage set Use Syrian golden hamsters (golden hamsters) Mesocricetus auratus The HsdHan® strain:AURA evaluated the immunogenicity of FCV2.1 in two female and two male animals. FCV2.1 was administered intranasally at four different concentrations to two female and two male animals. Animals received their initial vaccination on day 0 of the study, followed by a booster dose on day 14. Animals were weighed and randomly assigned to groups, with males and females in separate groups. At the start of the study, animals were 34–36 weeks old, and the weight change of the animals used in the study was less than ±25% of the mean weight for each sex. Blood samples for IgG analysis were collected on day -7 (baseline), day 14 (two weeks after initial administration), and day 28 (two weeks after booster administration). Results are shown in... Figure 3 And in Figure 4.

[0066] Efficacy study of FCV2.1 in Syrian golden hamsters challenged with the Omeprone BA.5 variant of SARS-CoV-2 A total of 27 Syrian golden hamsters (approximately 50% male and 50% female) were divided into two groups as follows: Group 1 (G1), positive control group (n=12): unvaccinated and treated with 104 The SARS-CoV-2 omega BA.5 variant was attacked with a TCID50 dose.

[0067] Group 2 (G2), the group vaccinated with FCV2.1 (two intranasal administrations, each dose being 1x10). 10 10 viral particles, spaced 3 weeks apart (n=15), were administered two weeks after the second immunization. 4 The SARS-CoV-2 omega BA.5 variant was attacked with a TCID50 dose.

[0068] Clinical signs (including weight) of all animals were recorded daily from the day of SARS-CoV-2 infection. Four or five animals from each group were sacrificed at each time point on days 2, 4, and 7 post-infection (dpi). Autopsies were performed, and oropharyngeal (OP) swabs, blood, nasal turbinates, and lung tissue were collected for viral RNA and infectious viral particles analysis. Immunohistochemical analysis of SARS-CoV-2 nucleocapsid proteins was performed on the nasal turbinates.

[0069] Vaccination studies using FCV2.3 in Balb / c mice Eight Balb / c mice were first intramuscularly vaccinated with Comirnaty (original, 2 μg / dose) and then divided into two groups of four animals each. Group 1 received a second intramuscular dose of Comirnaty (original) 20 days after the first dose, while Group 2 received an intranasal booster dose of FCV2.3 20 days after the first dose of Comirnaty (original). Blood samples were collected three weeks later, and neutralizing antibodies against the original strain, BA.1, BA.4 / 5, and XBB were assessed.

[0070] Citation List Patent documents WO 2021045541A1 KR20210028116A Non-patent literature Chaudhary, J. K., Yadav, R., Chaudhary, P. K., Maurya, A., Kant, N.,Rugaie, O. A., Haokip, H. R., Yadav, D., Roshan, R., Prasad, R., Chatrath,A., Singh, D., Jain, N.,&Dhamija, P. (2021).Insights into COVID-19 VaccineDevelopment Based on Immunogenic Structural Proteins of SARS-CoV-2, HostImmune Responses, and Herd Immunity. Cells , 10 (11)。

Claims

1. A viral vector comprising, at an insertion site in the viral genome, a nucleic acid sequence encoding a modified extracellular domain of the SARS-CoV-2 spike protein. in, The modified extracellular domain includes an N-terminal signal peptide that allows the spike protein to enter the host cell's secretory system. The modified extracellular domain includes the deletion of at least the C-terminus of the heptapeptide repeat region 2 (HR2), the transmembrane fragment (TM), and the cytoplasmic tail region (CT) of the spike protein.

2. The viral vector according to claim 1, wherein, The C-terminal deletion site is located in the region corresponding to positions 1030-1157 of the spike protein of Omecron BA.5 strain SEQ ID NO:

1.

3. The viral vector according to claim 1 or 2, wherein, The C-terminal deletion includes a portion, but not all, of the linker domain (CD) of the spike protein.

4. The viral vector according to any one of claims 1-3, wherein, The N-terminal signal peptide is either the human interleukin-3 secreted signal peptide or the natural signal peptide of the SARS-CoV-2 spike protein.

5. The viral vector according to any one of claims 1-4, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes a mutated furin cleavage site with single amino acid modifications at positions R677G, R678S, and R680S, wherein the positions correspond to the corresponding numbered amino acid positions of the spike protein of strain Omecron BA.5 of SEQ ID NO:

1.

6. The viral vector according to claim 5, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes the amino acid sequence of SEQ ID NO:2, amino acids 22-1142 of SEQ ID NO:2, or a sequence having at least 80% sequence identity with the sequence of SEQ ID NO:2 or amino acids 22-1142 of SEQ ID NO:

2.

7. The viral vector according to any one of claims 1-6, wherein, The vector is selected from the group consisting of: adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, herpes simplex virus vector, poxvirus vector, preferably vaccinia virus vector and orthomyxovirus vector, and preferably influenza virus vector.

8. The viral vector according to claim 7, wherein, The vector is an adenovirus vector, preferably a non-replicating adenovirus serotype 5 vector.

9. The viral vector according to any one of claims 1-8, wherein, The modified extracellular domains of the SARS-CoV-2 spike protein include at least the N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptapeptide repeat 1 (HR1), and central helix (CH).

10. The viral vector according to any one of claims 1-9, wherein, The nucleic acid sequence encoding the modified extracellular domain is as shown in SEQ ID NO:3, or is a nucleotide sequence having at least 70%, 80%, 85%, 90% or 95% sequence identity with the nucleotide sequence shown in SEQ ID NO:

3.

11. The viral vector according to claim 9, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes at its C-terminus one or more additional receptor-binding domains (RBDs) from the SARS-CoV-2 spike protein that are immunologically different from the modified extracellular domain of the SARS-CoV-2 spike protein.

12. The viral vector according to any one of claims 1-11, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes a trimerization domain at its C-terminus, preferably a T4 fibroin trimerization domain, a human collagen trimerization domain, a leucine zipper domain, or any other additional C-terminal amino acid sequence that promotes the trimerization of the spike protein.

13. The viral vector according to any one of claims 1-12, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes a transmembrane domain at its C-terminus, or any other additional C-terminal amino acid sequence that facilitates transmembrane anchoring of the spike protein.

14. The viral vector according to claim 12 or 13, wherein, The modified extracellular domains of the SARS-CoV-2 spike protein include, in any order, the following in the C-terminal region: i) a trimerizing domain, preferably a T4 fibroin trimerizing domain, a human collagen trimerizing domain, a leucine zipper domain, or any other additional C-terminal amino acid sequence that promotes the trimerization of the spike protein; and ii) a transmembrane domain, or any other additional C-terminal amino acid sequence that promotes the transmembrane anchoring of the spike protein.

15. The viral vector according to claim 11 or 12, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes, in any order at its C-terminus: i) one or more receptor-binding domains (RBDs) of the SARS-CoV-2 spike protein that are immunologically different from the modified extracellular domain of the SARS-CoV-2 spike protein, and ii) a trimerization domain, preferably a T4 fibroin trimerization domain, a human collagen trimerization domain, a leucine zipper domain, or any other additional C-terminal amino acid sequence that promotes the trimerization of the spike protein.

16. The viral vector according to claim 11 or 13, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes, in any order at its C-terminus: i) one or more receptor-binding domains (RBDs) of the SARS-CoV-2 spike protein that are immunologically different from the modified extracellular domain of the SARS-CoV-2 spike protein, and ii) a transmembrane domain, or any other additional C-terminal amino acid sequence that facilitates transmembrane anchoring of the spike protein.

17. The viral vector according to any one of claims 11-13, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes, in any order at its C-terminus: i) one or more receptor-binding domains (RBDs) of the SARS-CoV-2 spike protein that are immunologically different from the modified extracellular domain of the SARS-CoV-2 spike protein; ii) a trimerizing domain, preferably a T4 fibroin trimerizing domain, a human collagen trimerizing domain, a leucine zipper domain, or any other additional C-terminal amino acid sequence that promotes the trimerization of the spike protein; and iii) a transmembrane domain, or any other additional C-terminal amino acid sequence that promotes the transmembrane anchoring of the spike protein.

18. A vaccine composition comprising a carrier according to any one of claims 1-17, preferably in the form of a mucosal administration formulation.

19. The vaccine composition according to claim 18, wherein, The mucosal application formulation is selected from the group consisting of: nasal drops, aerosols, sprays, powder sprays, gels, microspheres, liposomes, membranes, and suspensions.

20. The vaccine composition according to claim 19, wherein, The vaccine composition is a liquid spray or a sprayable composition.

21. The vaccine composition according to any one of claims 18-20, wherein the vaccine composition further comprises a pharmaceutically acceptable adjuvant, a loading agent, a diluent, or an excipient.

22. The vaccine composition of claim 21, wherein the vaccine composition comprises A195 buffer, Tris, sodium chloride, magnesium chloride, histidine, sucrose, polysorbate-80, EDTA and ethanol.

23. The vaccine composition according to claim 22, wherein the vaccine composition comprises A195 buffer; 10 mM Tris, 75 mM NaCl, 1 mM MgCl2, 10 mM histidine, 5% (wt / vol) sucrose, 0.02% polysorbate-80 (wt / vol), 0.1 mM EDTA, and 0.5% (vol / vol) ethanol at pH 7.

4.

24. The vaccine composition according to any one of claims 18-23, wherein, The composition is administered intranasally.

25. A method for preventing COVID-19, the method comprising administering to a mammal, preferably a human, a preventive or therapeutically effective amount of a vaccine composition according to any one of claims 18-23, preferably intranasally.

26. Use of the viral vector according to any one of claims 1-18 in the preparation of a vaccine composition for inducing an immune response in mammals, preferably humans.

27. The use according to claim 26, wherein, The N-terminal signal peptide is either the human interleukin-3 secretory signal peptide or the natural signal peptide of the SARS-CoV-2 spike protein.

28. The use according to claim 26 or 27, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein includes a mutated furin cleavage site with single amino acid modifications at positions R677G, R678S, and R680S, wherein the positions correspond to the corresponding numbered amino acid positions of the spike protein of strain Omecron BA.5 of SEQ ID NO:

1.

29. The use according to claim 28, wherein, The modified extracellular domain of the SARS-CoV-2 spike protein has the amino acid sequence of SEQ ID NO:

2.

30. The use according to any one of claims 26-29, wherein, The vector is selected from the group consisting of: adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, herpes simplex virus vector, poxvirus vector, preferably vaccinia virus vector and orthomyxovirus vector, and preferably influenza virus vector.

31. The use according to claim 30, wherein, The vector is an adenovirus vector, preferably a non-replicating adenovirus serotype 5 vector.

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