Modified coronavirus structure protein

Abstract

Modified coronavirus spike (S) proteins, virus-like particles (VLPs) comprising the modified S proteins, and nucleic acids encoding the modified S proteins are provided. Methods for producing the modified S proteins and VLPs in a host or host cell are also described. The modified S protein can comprise a transmembrane domain (TM) or portion of a TM and a cytosolic tail (CT) or portion of a CT, wherein the CT or portion of the CT is derived from an influenza hemagglutinin (HA) protein, and the TM or portion of the TM is heterologous to the CT or portion of the CT. Additionally, methods for inducing immunity to coronavirus infection in a subject are described, comprising administering to the subject a composition comprising a modified coronavirus (S)-protein or VLP.

Description

[Technical Field]

[0001] This disclosure relates to a modified viral structural protein. The present invention also relates to virus-like particles (VLPs) comprising a modified viral structural protein, and to a method for producing VLPs in a host or host cell. [Background technology]

[0002] Coronaviruses (CoV) are the largest group of viruses belonging to the order Nidovirales, which includes the families Coronaviridae, Arteriviridae, Mesoniviridae, and Roniviridae. The family Coronaviridae includes one of the two subfamilies of Coronaviridae, the other being Torovirinae. The family Coronaviridae is further subdivided into four genera: alpha, beta, gamma, and delta coronaviruses. Members of alpha and beta coronaviruses are found only in mammals. The genus alpha coronavirus includes two human virus species, HCoV-229E and HCoV-NL63. Important animal alpha coronaviruses are porcine gastroenteritis virus and feline peritonitis virus.

[0003] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as 2019-nCoV and HCoV-19) is a novel lineage B beta-coronavirus (beta-CoV) that causes coronavirus disease 2019 (COVID-19), a respiratory illness with high mortality and morbidity rates, causing significant public health impacts worldwide. SARS-CoV-2 outbreaks, such as the pandemic that began in 2020, present the greatest challenge to healthcare systems due to their incubation period and viral transmissibility. While treatment for COVID-19 is urgently needed, an effective vaccine is required for the long-term management of SARS-CoV-2 outbreaks.

[0004] Coronavirus virions are spherical, with a diameter of approximately 118–140 nm, as shown in recent studies using cryo-electron tomography and cryo-electron microscopy.

[0005] The most prominent structural feature of coronaviruses is the crab-shaped spike projections that arise from the surface of the virion. Coronavirus particles consist of a helical nucleocapsid structure formed by the association of a nucleocapsid (N) phosphoprotein and viral genomic RNA, surrounded by a lipid bilayer into which three or four structural proteins—spike (S), membrane (M), and envelope (E) proteins—and, in the case of some coronaviruses, a hemagglutinin-esterase (HE) protein (Masters PS. The molecular biology of coronaviruses. Adv Virus Res. 2006;66:193-292.) are inserted.

[0006] The membrane (M) protein is the most abundant structural protein in virions. It is a small (approximately 25-30 kDa) protein with three transmembrane domains and is thought to give the virion its shape. The envelope (E) protein is a short, intrinsic membrane protein of 76-109 amino acids, ranging in size from 8.4 to 12 kDa. The primary and secondary structures reveal that E has a short hydrophilic amino terminus of 7-12 amino acids, followed by a large hydrophobic transmembrane domain (TMD) of 25 amino acids, ending in a long, hydrophilic carboxyl terminus that contains the majority of the protein. The E protein is involved in several aspects of the viral life cycle, such as aggregation, budding, envelope formation, and pathogenesis.

[0007] The spike (S) protein is a glycoprotein necessary for the recognition of host receptors by many coronaviruses, as well as for the fusion of the viral membrane with the host cell membrane for viral entry into cells (Belouzard et al., Viruses 2012 Jun;4(6):1011-33). As a primary glycoprotein on the surface of the viral envelope, the S protein of Coronaviridae is a major target of neutralizing antibodies induced by natural infections, including SARS-CoV-2 infection, and is an important antigen targeted by experimental vaccine candidates.

[0008] The SARS-CoV-2 S protein, like the S proteins of other coronaviruses, is initially synthesized as a precursor protein. Individual precursor S proteins form homotrimers, undergo glycosylation within the Golgi compartment, and then undergo processing to remove the signal peptide. The S protein requires two-step protease-mediated activation to facilitate membrane fusion. The SARS-CoV-2 S protein is distinguished by the RRAR-Fulin cleavage site at the S1 / S2 junction, which is likely processed in the Golgi compartment to produce two distinct polypeptides: S1 and S2 polypeptides (or subunits), which remain non-covalently linked as S1 / S2 protomers within the homotrimer in the prefusion conformation (Walls et al. Cell 2020 181(2)p281-292; Li et al. eLife 2019;8:e51230). The furin cleavage at the S1 / S2 junction and the cleavage at the upstream S2' site of the fusion peptide occur during viral entry on the cell surface or in endosomes and can be mediated by several proteases.

[0009] This trimer is retained in a prefusion conformation before binding to the target receptor on the host cell via the receptor-binding domain (RBD) epitope. Receptor binding destabilizes the prefusion trimer, leading to the detachment of the S1 subunit and the migration of the S2 subunit to a stable post-fusion conformation via viral fusion to the cell membrane (Wrapp et al. Science, 13 Mar 2020, Vol.367, Issue 6483, pp.1260-1263). Neutralizing antibodies from individuals infected with SARS-CoV-2 have been shown to target the RBD of the S1 subunit of the S protein (Premkumar, L., 2020 Science Immunology 11 Jun 2020: Vol.5, Issue 48).

[0010] Stabilization of the S protein extradomain in prefusion conformations tends to increase recombinant expression yield, likely by preventing triggers or misfolding resulting from a tendency to adopt a more stable post-fusion structure (Hsieh et al. Science 2020, 369 p.1501-1505).

[0011] Mutations in the S protein extradomain have been shown to promote the stabilization of the prefusion conformation. International Publication No. 2018 / 081318 and its accompanying publication by Pallesen, J. et al. (PNAS August 29, 2017 114(35)) disclose double proline substitutions at or near the junction between heptad repeat 1 (HR1) and the central helix that stabilize the S extradomain trimer of the MERS-CoV spike protein in the prefusion conformation, as well as substitutions to prevent protease cleavage at the S1 / S2 cleavage site and the S2' cleavage site of the S extradomain. High-resolution structures were determined by cryo-EM using SARS-CoV-2 S proteins stabilized by double proline substitutions of homologous amino acid residues (Wrapp et al. Science 2020 367, 1260-1263; Walls et al. Cell 2020, 181, 281-292). Furthermore, disruption of the furin recognition site is thought to preserve the S protein in a prefusion conformation (Wrapp et al Science 2020 367, 1260-1263). However, even with these substitutions, the SARS-CoV-2 S protein extradomain remains unstable and difficult to reliably produce in mammalian cells, hindering the development of effective and high-yield subunit vaccines (Hsieh et al. Science 2020, 369 p. 1501-1505).

[0012] Hsieh et al. (Science 2020, 369 pp. 1501-1505) designed and expressed over 100 structural guide SARS-CoV-2 spike protein mutants in mammalian cells based on previously determined cryo-EM structures. The mutants were biochemically, biophysically, and structurally characterized to identify substitutions that resulted in increased yield and stability. Hsieh et al. report several proline, disulfide bond, salt crosslinking, and cavity-filling substitutions that increase spike expression and / or stability compared to double proline substitutions. The best-identified mutant, HexaPro, has six beneficial proline substitutions that result in 10-fold higher expression than its parent construct and can withstand heat stress, room temperature storage, and multiple freeze-thaw cycles.

[0013] The S2 subunit can be divided into three domains: a large outer domain, a transmembrane domain (TM), and a cytoplasmic end (CT). The cytoplasmic end of the S protein has been previously shown to be necessary for assembly. Two distinct retention signals can be found in the Coronaviridae CT: i) an endoplasmic reticulum retrieval signal (ERRS) and / or ii) a tyrosine-dependent localization signal (YxxI or YxxF motif). ERRS contains a dibasic KxHxx motif that binds to coatomer complex I (COPI). This motif is necessary for the localization of the SARS S protein to the ERGIC / Golgi region when co-expressed with the SARS membrane (M) protein, and localization can be disrupted by mutation of the KxHxx motif (McBride et al. J. Virol. Feb 2007, 81(5) 2418-2428). S proteins containing ERRS are recruited to COPI vesicles and then retrogradely retrieved from the Golgi apparatus to the endoplasmic reticulum (ER). Repeated cycles of S proteins between the ER and Golgi result in the intracellular retention of S proteins. Both alpha- and beta-coronavirus S proteins contain ERRS (Ujike et al. Journal of General Virology (2016), 97, 1853-1864).

[0014] The S proteins of betacoronaviruses, such as MERS-CoV, SARS-CoV, and SARS-CoV-2, possess only ERRS and cannot be retained within the cell, leading to the release of the S protein into the plasma membrane. While mutant SARS-CoV S proteins lacking ERRS are transported to the plasma membrane, the native S protein, when co-expressed with the M protein, interacts with the M protein near the budding site, resulting in intracellular retention of the S protein. This suggests that, through interaction with the M protein, SARS-CoV's ERRS contributes specifically to S protein accumulation in the posteromedial Golgi compartment, leading to S protein uptake into VLPs (Ujike et al. Journal of General Virology (2016), 97, 1853-1864). Recently, removal of ERRS has been found to promote the uptake of SARS-CoV-2 S protein into lentiviral pseudovirons (Ou et al., 2020 Nature Communications volume 11, Article number: 1620).

[0015] Yu et al. (2020 Science) constructed a set of prototype DNA vaccines expressing six variants of the SARS-CoV-2 S protein with various deletions in the cytoplasmic terminal and transmembrane domain, and evaluated their immunogenicity and protective efficacy against SARS-CoV-2 viral challenge in rhesus monkeys. Soluble fragments of the SARS-CoV-2 S protein extradomain induced reduced levels of sgmRNA (indicating viral replication), but optimal protection was achieved by the full-length S protein immunogen.

[0016] Broer et al. (2006 J.Virol. p.1302-1310) studied the roles of the transmembrane and cytoplasmic domains of the S protein in SARS-CoV infectivity and membrane fusion activity using SARS-CoV S-pseudotyped retrovirus (SARSpp). SARSpp in which the cytoplasmic domain of S was replaced with a cytoplasmic domain derived from vesicular stomatitis virus G protein (VSV-G) was up to 40% infectious compared to wild-type. In contrast, SARSpp containing both the TMD and the cytoplasmic domains of VSV-G showed significantly impaired infectivity (<5%). This suggests that the TMD of S may be involved in the SARS-CoV entry process.

[0017] Vaccination provides protection against disease by inducing subjects to initiate an immune response to a similar factor before infection. Traditionally, this has been achieved by using living, attenuated, or fully inactivated forms of infectious factors as immunogens. To avoid the risks of using whole viruses (such as dead or attenuated viruses) as vaccines, viral proteins or subunits, or recombinants thereof, have been pursued as vaccines. The main obstacle to using natural or recombinant viral proteins as vaccine agents is ensuring that the conformation of the protein mimics the antigen in its natural environment. With appropriate adjuvants, and in the case of peptides, carrier proteins can be used to enhance the immune response. Furthermore, viral proteins or subunits as vaccines may primarily induce humoral responses and therefore may not be able to induce persistent immunity. Subunit vaccines may be ineffective for diseases in which the whole inactivated virus can be demonstrated to provide superior protection.

[0018] Virus-like particles (VLPs) can be used in immunogenic compositions to express viral proteins in a preferred conformation that improves antigen presentation to the immune system. VLPs closely resemble mature virions, but they do not contain viral genomic material, are non-replicating, and are safe to administer as vaccines. Furthermore, VLPs can be manipulated to express viral glycoproteins on the surface of the VLP, which is their natural physiological configuration. Because VLPs resemble intact virions and have a multivalent particle structure, they may be more effective in inducing neutralizing antibodies against glycoproteins than soluble envelope protein antigens.

[0019] Various expression systems, including mammalian cell lines, bacteria, insect cell lines, yeast, and plant cells, are used to produce VLPs. More than 30 different viral VLPs have been generated in insect and mammalian systems for vaccine purposes (Noad, R. and Roy, P., 2003, Trends Microbiol 11:438-44). VLPs are also produced in plants (see International Publications 2009 / 076778, 2009 / 009876, 2009 / 076778, 2010 / 003225, 2010 / 003235, 2010 / 006452, 2011 / 03522, 2010 / 148511, and 2014153674, and 2012 / 083445).

[0020] VLPs are produced from native surface proteins derived from severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), including S, M, and E proteins, in insect and mammalian cells (Liu et al., 2008, J Virol., pp. 11318-11330). SARS-CoV-2 virus-like particles (VLPs) are also assembled in mammalian cells by co-expression of viral surface proteins S, M, and E (Xu et al. Front. Bioeng. Biotechnol., July 30, 2020). Further studies have shown that the M protein is essential for VLP formation (Siu et al. Journal of Virology (2008) 82:11318-11330, Huang et al. Journal of Virology (2004) 78:12557-12565). In mammalian cells, the expression of membrane protein (M) and small envelope protein (E) is essential for the efficient formation and release of SARS-CoV-2 VLPs (Xu et al. Front. Bioeng. Biotechnol., July 30, 2020). Nevertheless, the minimum requirements for SARS-CoV VLP assembly remain debatable. Y. Huang et al. (Journal of Virology (2004) 78:12557-12565) described VLP formation in transfected human cells requiring only the co-expression of M and N viral proteins, while Siu et al. (Journal of Virology (2008) 82:11318-11330) showed that both E and N proteins must be co-expressed with the M protein for efficient production and release of SARS-CoV VLPs in transfected mammalian cells.

[0021] International Publication No. 2012 / 083445 discloses the production of SARS-CoV S protein in plants in which the transmembrane domain and cytosolic terminal domain (TM / CT) of the S protein are replaced with TM / CT derived from influenza HA protein.

[0022] Some groups have proposed immunization with SARS-CoV VLPs as an effective vaccine strategy. VLPs produced in insect cells or chimeric MHV / SARS-CoV VLPs produced in mammalian cells were used in these tests (Lokugamage et al. Vaccine 2008 Feb 6;26(6):797-808, Lu et al. 2007 Immunology 122496-5024).

[0023] However, to meet the need for widespread vaccination of the world's population, efficient production of viral structural proteins and VLPs is required for the effective scale-up and manufacture of the amounts of SARS-CoV-2 VLPs necessary. SUMMARY OF THE INVENTION

[0024] The present invention relates to a modified viral structural protein. The present invention also relates to virus-like particles (VLPs) comprising the modified viral structural protein, and methods for producing VLPs in a host or host cell. More specifically, the present invention relates to a modified coronavirus S protein. The present invention also relates to virus-like particles (VLPs) comprising the modified S protein, and methods for producing VLPs in a host or host cell.

[0025] In one aspect, in order, - an external domain derived from a coronavirus S protein, - a transmembrane and cytosolic terminal domain (TMCT), wherein the TMCT is a chimeric TMCT, - a transmembrane domain (TM) or a part of the TM is a transmembrane domain (TM) derived from a coronavirus S protein, - a cytosolic terminal (CT) or a part of the CT is a cytosolic terminal (CT) derived from an influenza hemagglutinin (HA) protein, and a transmembrane and cytosolic terminal domain (TMCT) comprising, A modified coronavirus S protein is provided that comprises.

[0026] The modified S protein described herein may form trimers. Therefore, trimers containing the modified coronavirus S protein described herein are also provided.

[0027] In a further embodiment, a virus-like particle (VLP) is provided comprising the modified S protein or a trimer containing the modified S protein. Thus, the VLP comprises the modified coronavirus S protein or a trimer containing the modified S protein, and the modified S protein is -The external domain derived from the coronavirus S protein, - Transmembrane and cytosolic terminal domains (TMCTs), where the TMCT is a chimeric TMCT. - The transmembrane domain (TM) or a portion of the TM is derived from the coronavirus S protein, - The cytosolic end (CT) or a portion of the CT is derived from the influenza hemagglutinin (HA) protein, The transmembrane and cytosolic terminal domains (TMCTs) are included, Includes.

[0028] VLP may further contain plant lipids.

[0029] TM may be directly fused to CT. TM may be derived from coronavirus S protein TM, and CT may be derived from influenza HA protein CT. Furthermore, TM may be a chimeric TM containing an N-terminal sequence derived from coronavirus S protein TM and a C-terminal sequence derived from influenza HA protein TM. A chimeric TM may contain an N-terminal sequence derived from coronavirus S protein TM containing at least 20 amino acids corresponding to amino acids 1-20 of SEQ ID NO: 18 or SEQ ID NO: 169, or at least 21 amino acids corresponding to amino acids 1-21 of SEQ ID NO: 118 or 164, or at least 22 amino acids corresponding to amino acids 1-22 of SEQ ID NO: 123, and one or more amino acids from the C-terminus of influenza HA protein TM. One or more amino acids from the C-terminus of influenza HA protein TM may be selected from AGL or a conserved substitution of AGL, MAGL or a conserved substitution of MAGL. A chimeric TM may contain amino acids corresponding to amino acids 1-20 of SEQ ID NO: 18.

[0030] The CT may be a chimeric CT containing the N-terminal sequence derived from the coronavirus S protein CT and the C-terminal sequence derived from the influenza HA protein CT. The chimeric CT may contain a C-terminal sequence derived from the influenza HA protein CT containing at least 11 amino acids corresponding to amino acids 27-37 of SEQ ID NOs. 18, 126, 127, 128, 129, 130, or 131, and one or more amino acids from the N-terminus of the coronavirus S protein CT. One or more amino acids from the N-terminus of the coronavirus S protein CT may be selected from C or a conserved substitution of C, CC or a conserved substitution of CC, or CCM or a conserved substitution of CCM. The chimeric CT may contain amino acids corresponding to amino acids 27-37 of SEQ ID NOs. 18, 126, 128, 129, 130, or 131, or amino acids 27-36 of SEQ ID NOs. 127. In one embodiment, the chimeric TMCT may include a chimeric TM containing amino acids corresponding to amino acids 1-20 of SEQ ID NO: 18 or SEQ ID NO: 169, or amino acids 1-21 of SEQ ID NO: 118 or SEQ ID NO: 164, or amino acids 1-22 of SEQ ID NO: 123, a chimeric CT containing amino acids corresponding to amino acids 27-37 of SEQ ID NO: 18, 126, 127, 128, 129, 130, or 131, or a combination thereof.

[0031] CT or a portion of CT may contain 80% to 100% identity with the sequence of SEQ ID NO: 15, or amino acids 35-50 of SEQ ID NOs: 6, 8, 7, 9, 10, 12, 13, or 14, or amino acids 34-49 of SEQ ID NO: 11, or amino acids 553-568 of SEQ ID NO: 3, or amino acids 22-37 of SEQ ID NO: 18, or amino acids 21-40 of SEQ ID NO: 19, or amino acids 21-39 of SEQ ID NO: 37, or amino acids 25-36 of SEQ ID NO: 38, or amino acids 24-34 of SEQ ID NO: 39, or amino acids 22-37 of SEQ ID NOs: 126, 128, 129, 130, or 131, or amino acids 22-36 of SEQ ID NO: 127. TM or a portion of TM may contain 80% to 100% identity with the sequence of SEQ ID NO: 132 or 133.

[0032] TMCT may contain sequences that have approximately 80% to 100% identity with sequences of sequence numbers 18, 19, 37, 38, 39, 64, 126, 127, 128, 129, 130, 131, 118, 119, 120, 123, 124, 125, 134, 135, 164, 165, 166, 169, 170, 171, 172, or 173.

[0033] The modified S protein may contain an S1 subunit and an S2 subunit, and the S2 subunit may contain a chimeric TMCT.

[0034] The modified S protein can be produced as a precursor protein containing the modified S protein and the signal peptide. The precursor protein containing the modified S protein and the signal peptide may have 80% to 100% identity with amino acids 1-1234 of SEQ ID NO: 1, or amino acids 1-1234 of SEQ ID NO: 5, amino acids 1-1219 of SEQ ID NO: 21, or amino acids 1-1243 of SEQ ID NO: 30, and the amino acid sequence of CT may have 80% to 100% identity with the sequence of SEQ ID NO: 15, or amino acids 35-50 of SEQ ID NOs: 6, 8, 7, 9, 10, 12, 13, or 14, or amino acids 34-49 of SEQ ID NO: 11, or amino acids 553-568 of SEQ ID NO: 3.

[0035] The signal peptide may be native or unnatural relative to the S protein. Unnatural signal peptides may be derived from the signal peptide of protein disulfide isomerase (PDI). The modified S protein may further contain plant-specific N-glycans.

[0036] The CT or a portion of the CT in the modified S protein may be derived from influenza hemagglutinin (HA) protein from influenza B or influenza subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. The influenza hemagglutinin (HA) protein may be derived from influenza B or influenza subtypes H1, H3, H5, H6, H7, or H9.

[0037] The external domain of the modified S protein may be derived from SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43-CoV, or 229E-CoV, and the TM or part of the TM may be derived from SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43-CoV, or 229E-CoV, and both or part of the external domain and / or the TM may be derived from SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43-CoV, or 229E-CoV.

[0038] In a further embodiment, the modified S protein may contain one or more amino acid substitutions compared to the amino acid sequence of wild-type coronavirus. One or more substitutions may maintain the S protein in a prefusion state.

[0039] One or more amino acid substitutions may include i) substitutions that restrict processing at the cleavage site between the S1 subunit and the S2 subunit, ii) substitutions of one or more amino acids to one or more prolines, or iii) substitutions that restrict processing at the cleavage site between the S1 subunit and the S2 subunit and substitutions of one or more amino acids to one or more prolines.

[0040] One or more substitutions may maintain the S protein in a prefusion state, or, if expressed in a host or host cell, may result in a higher yield of the modified S protein compared to the yield of the corresponding S protein without one or more substitutions expressed in a host or host cell.

[0041] One or more amino acid substitutions may correspond to amino acids at positions 667, 668, 670, 802, 923, 927, 971, 972, or combinations thereof, when compared to the reference amino acid sequence of SEQ ID NO: 2.

[0042] In one embodiment, one or more amino acid substitutions correspond to amino acids at positions 971 and 972 when compared to the reference amino acid sequence of SEQ ID NO: 2. In another embodiment, one or more amino acid substitutions correspond to amino acids at positions 802, 927, 971, and 972 when compared to the reference amino acid sequence of SEQ ID NO: 2. Furthermore, the modified S protein may contain one or more amino acid substitutions corresponding to amino acids at positions 667, 668, and 670, or a combination thereof, when compared to the reference amino acid sequence of SEQ ID NO: 2. Therefore, the modified S protein may contain substitutions corresponding to amino acids at positions 667, 668, and 670 when compared to the reference amino acid sequence of SEQ ID NO: 2.

[0043] In one embodiment, one or more substitutions may correspond to amino acids at positions 667, 668, 670, 971, and 972 when compared to the reference amino acid sequence of SEQ ID NO: 2. The amino acid substitution corresponding to the amino acid at position 667 in SEQ ID NO: 2 may be glycine or a conserved substitution of glycine; the amino acid substitution corresponding to position 668 in SEQ ID NO: 2 may be serine or a conserved substitution of serine; the amino acid substitution corresponding to position 670 in SEQ ID NO: 2 may be serine or a conserved substitution of serine; the amino acid substitution corresponding to the amino acid at position 971 in SEQ ID NO: 2 may be proline or a conserved substitution of proline; and the amino acid substitution corresponding to the amino acid at position 972 in SEQ ID NO: 2 may be proline or a conserved substitution of proline. The modified S protein described above may further include an amino acid substitution corresponding to the amino acid at position 923 when compared to the reference amino acid sequence of SEQ ID NO: 2.

[0044] In another embodiment, one or more amino acid substitutions may correspond to amino acids at positions 667, 668, 670, 802, 927, 971, and 972 when compared to the reference amino acid sequence of SEQ ID NO: 2. The amino acid substitution corresponding to the amino acid at position 667 of SEQ ID NO: 2 may be glycine or a conserved substitution of glycine; the amino acid substitution corresponding to position 668 of SEQ ID NO: 2 may be serine or a conserved substitution of serine; the amino acid substitution corresponding to position 670 of SEQ ID NO: 2 may be serine or a conserved substitution of serine; the amino acid substitution corresponding to the amino acid at position 802 of SEQ ID NO: 2 may be proline or a conserved substitution of proline; the amino acid substitution corresponding to the amino acid at position 927 of SEQ ID NO: 2 may be proline or a conserved substitution of proline; the amino acid substitution corresponding to the amino acid at position 971 of SEQ ID NO: 2 may be proline or a conserved substitution of proline; and the amino acid substitution corresponding to the amino acid at position 972 of SEQ ID NO: 2 may be proline or a conserved substitution of proline.

[0045] In another embodiment, the modified S protein may further include an amino acid substitution corresponding to the amino acid at position 923 when compared to the reference amino acid sequence of SEQ ID NO: 2. The amino acid substitution in the modified S protein corresponding to the amino acid at position 667 of SEQ ID NO: 2 may be glycine or a conserved substitution of glycine; the amino acid substitution corresponding to position 668 of SEQ ID NO: 2 may be serine or a conserved substitution of serine; the amino acid substitution corresponding to position 670 of SEQ ID NO: 2 may be serine or a conserved substitution of serine; the amino acid substitutions corresponding to positions 802, 927, 971 and 972 of SEQ ID NO: 2 may be proline or a conserved substitution of proline; and the amino acid substitution corresponding to position 923 of SEQ ID NO: 2 may be phenylalanine or a conserved substitution of phenylalanine.

[0046] The modified S protein contains amino acids from SEQ ID NOs. 5, 21, 30, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 95, 96, 97, 108, 109, 110, 144, 145, 146, 155, 156 or 157, or amino acids 24-1259 from SEQ ID NOs. 47, amino acids 25-1259 from SEQ ID NOs. 48, and amino acids from SEQ ID NOs. 49. amino acids 25-1259, amino acids 25-1259 of SEQ ID NO 50, amino acids 25-1259 of SEQ ID NO 51, amino acids 25-1259 of SEQ ID NO 52, amino acids 25-1259 of SEQ ID NO 53, amino acids 25-1259 of SEQ ID NO 54, amino acids 25-1259 of SEQ ID NO 55, amino acids 25-1259 of SEQ ID NO 56, amino acids 25-1259 of SEQ ID NO 57, amino acids 58 This may contain 80% to 100% identity with amino acids 25-1259, amino acids 25-1262 of SEQ ID NO: 59, amino acids 25-1261 of SEQ ID NO: 60, amino acids 25-1258 of SEQ ID NO: 61 or amino acids 25-1256 of SEQ ID NO: 62, amino acids 25-1243 of SEQ ID NO: 95, amino acids 25-1240 of SEQ ID NO: 96, amino acids 25-1243 of SEQ ID NO: 97, amino acids 25-1341 of SEQ ID NO: 108, amino acids 25-1338 of SEQ ID NO: 109, amino acids 25-1341 of SEQ ID NO: 110, amino acids 25-1351 of SEQ ID NO: 144, amino acids 25-1348 of SEQ ID NO: 145, amino acids 25-1351 of SEQ ID NO: 146, amino acids 25-1159 of SEQ ID NO: 155, amino acids 25-1156 of SEQ ID NO: 156, or amino acids 25-1159 of SEQ ID NO: 157.

[0047] In another embodiment, nucleic acids comprising a nucleotide sequence encoding the modified S protein described above are provided.

[0048] In a further embodiment, a composition is provided comprising an effective amount of modified S protein, a trimer containing modified S protein, or a VLP containing modified S protein, and a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient. In yet another embodiment, a vaccine for inducing an immune response is provided. As described above, the vaccine comprises an effective amount of modified S protein, a trimer containing modified S protein, or a VLP containing modified S protein.

[0049] The composition further comprises a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient. In a further embodiment, a vaccine for inducing an immune response is provided. The vaccine comprises an effective amount of VLP containing the modified coronavirus described above. The vaccine may be a polyvalent vaccine comprising a mixture of VLPs.

[0050] In yet another embodiment, a (non-human) host or host cell containing the modified S protein, trimer, or VLP described above is provided. In yet another embodiment, a host or host cell containing the VLP described above is provided. In yet another embodiment, a composition containing an effective amount of VLP containing the modified S protein described above is provided.

[0051] In yet another embodiment, an S protein trimer is provided. The trimer comprises a modified coronavirus S protein, and the modified S protein is -The external domain derived from the coronavirus S protein, - Transmembrane and cytosolic terminal domains (TMCTs), where the TMCT is a chimeric TMCT. - The transmembrane domain (TM) or a portion of the TM is derived from the coronavirus S protein, - The cytosolic end (CT) or a portion of the CT is derived from the influenza hemagglutinin (HA) protein, The transmembrane and cytosolic terminal domains (TMCTs) are included, The modified S protein in the trimer may contain one or more amino acid substitutions when compared to the amino acid sequence of wild-type coronavirus, as described above. In a further embodiment, a composition is provided comprising the above effective amount of the trimer and a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient. In another embodiment, virus-like particles (VLPs) comprising the above trimer are also provided. The VLPs may further contain plant lipids. In another embodiment, a composition is provided comprising the above effective amount of VLPs comprising the above trimer and a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient. In a further embodiment, a vaccine for inducing an immune response is provided. The vaccine comprises the above effective amount of the trimer. In a further embodiment, a vaccine for inducing an immune response is provided. The vaccine comprises the above effective amount of VLPs comprising the above trimer. The vaccine may be a polyvalent vaccine comprising a mixture of VLPs. In yet another embodiment, a non-human host or host cell comprising the above trimer or VLPs comprising the trimer is provided.

[0052] In another embodiment, a method is provided for inducing immunity against coronavirus infection in a subject, the method comprising administering the composition or vaccine described above. The composition or vaccine may be administered to the subject once, or it may be administered to the subject multiple times. The composition or vaccine may be administered as an initial dose, and one or more subsequent doses may be administered between 1 day and 6 months after the administration of the initial dose. The subsequent dose may be administered 21 days after the administration of the initial dose.

[0053] In another embodiment, an antibody or antibody fragment prepared using the above-described composition or vaccine is provided.

[0054] In yet another embodiment, A) a method for producing virus-like particles (VLPs) in a (non-human) host or host cell, a) Introducing nucleic acids containing the nucleotide sequence encoding the modified S protein into a non-human host or host cell, or providing a non-human host or host cell containing nucleic acids containing the nucleotide sequence encoding the modified S protein. b) Incubating non-human host or host cells under conditions that enable nucleic acid expression, thereby producing VLPs, A method including this is provided.

[0055] In a further step c), non-human host cells or host cells can be collected.

[0056] In a further embodiment, B) a method for increasing the yield of coronavirus S protein production in a (non-human) host or host cell, a) Introducing nucleic acids containing the nucleotide sequence encoding the modified S protein into a non-human host or host cell, or providing a non-human host or host cell containing nucleic acids containing the nucleotide sequence encoding the modified S protein, b) Incubating non-human host or host cells under conditions that enable the expression of a modified S protein encoded by nucleic acid, thereby producing the modified S protein in a higher yield compared to a host or host cell expressing an unmodified S protein under similar or identical conditions. A method including this is provided.

[0057] In a further step c), non-human host cells or host cells can be collected.

[0058] In yet another embodiment, C) a method for increasing the yield of virus-like particles (VLPs) in a (non-human) host or host cell, a) A nucleic acid encoding a modified coronavirus S protein in a non-human host or host cell, -The external domain derived from the coronavirus S protein, - Transmembrane and cytosolic terminal domains (TMCTs), where the TMCT is a chimeric TMCT. - The transmembrane domain (TM) or a portion of the TM is derived from the coronavirus S protein, - A transmembrane and cytosolic terminal domain (TMCT) comprising a cytosolic terminal (CT) or a portion of the CT derived from the influenza hemagglutinin (HA) protein, Introducing nucleic acids, including A step of providing a non-human host or host cell containing nucleic acid encoding a modified S protein, -The external domain derived from the coronavirus S protein, Transmembrane and cytosolic terminal domains (TMCTs), wherein the TMCTs are chimeric TMCTs. - The transmembrane domain (TM) or a portion of the TM is derived from the transmembrane domain (TM) of the coronavirus S protein. - The cytosolic end (CT) or a portion of the CT is derived from the influenza hemagglutinin (HA) protein, It is a chimeric TMCT, and b) A step of incubating a non-human host or host cell under conditions that enable the expression of a modified S protein encoded by nucleic acid, thereby producing a VLP containing the modified S protein in a higher yield compared to the yield of VLPs in the host of host cells expressing an unmodified S protein under similar or identical conditions, A method is provided that includes this.

[0059] In a further embodiment, D) a method for producing virus-like particles (VLPs) in a (non-human) host or host cell, a) A nucleic acid encoding a modified coronavirus S protein in a non-human host or host cell, -The external domain derived from the coronavirus S protein, - Transmembrane and cytosolic terminal domains (TMCTs), where the TMCT is a chimeric TMCT. - The transmembrane domain (TM) or a portion of the TM is derived from the coronavirus S protein, - A transmembrane and cytosolic terminal domain (TMCT) comprising a cytosolic terminal (CT) or a portion of the CT derived from the influenza hemagglutinin (HA) protein, Introducing nucleic acids, including, or A step of providing a non-human host or host cell containing nucleic acid encoding a modified S protein, -The external domain derived from the coronavirus S protein, Transmembrane and cytosolic terminal domains (TMCTs), wherein the TMCTs are chimeric TMCTs. - The transmembrane domain (TM) or a portion of the TM is derived from the transmembrane domain (TM) of the coronavirus S protein. - The cytosolic end (CT) or a portion of the CT is derived from the influenza hemagglutinin (HA) protein, It is a chimeric TMCT, and b) Incubating non-human host or host cells under conditions that enable nucleic acid expression, thereby producing VLPs, A method is provided that includes this.

[0060] In further embodiments, the VLPs of method A), B), C), or D) may be further extracted and purified from a host or host cells. The host or host cells may include plants, plant cells, fungi, fungal cells, insects, insect cells, animals, or animal cells. The host or host cells of method A), B), C), or D) may be a plant, a part of a plant, or plant cells.

[0061] In another embodiment, VLPs manufactured by method A), B), C), or D) are provided.

[0062] Furthermore, in yet another embodiment, a composition comprising an adjuvant and virus-like particles (VLPs), wherein the VLPs comprise a modified coronavirus S protein, -The external domain derived from the coronavirus S protein, - Transmembrane and cytosolic terminal domains (TMCTs), where the TMCT is a chimeric TMCT. -The transmembrane domain (TM) or a portion of the TM is derived from the coronavirus S protein -transmembrane domain (TM), - The cytosolic end (CT) or a portion of the CT is derived from the influenza hemagglutinin (HA) protein, The transmembrane and cytosolic terminal domains (TMCTs) are included, A composition is provided comprising an adjuvant and virus-like particles (VLPs), wherein the modified S protein further comprises substitutions at positions 667, 668, 670, 971, and 972 when compared to the reference amino acid sequence of SEQ ID NO: 2.

[0063] In yet another embodiment, a composition comprising virus-like particles (VLPs), wherein the VLPs comprise a modified coronavirus S protein, -The external domain derived from the coronavirus S protein, - Transmembrane and cytosolic terminal domains (TMCTs), where the TMCT is a chimeric TMCT. - The transmembrane domain (TM) or a portion of the TM is derived from the coronavirus S protein, - A transmembrane and cytosolic terminal domain (TMCT) including a cytosolic terminal (CT) or a portion of the CT derived from influenza hemagglutinin (HA) protein, A composition is provided comprising virus-like particles (VLPs), wherein the modified S protein includes a glycine substitution at position 667, a serine substitution at position 668, a serine substitution at position 670, a proline substitution at position 971, and a proline substitution at position 972, corresponding to the reference amino acid sequence of SEQ ID NO: 2. The influenza hemagglutinin (HA) protein may be derived from influenza B or influenza subtypes H1, H3, H5, H6, H7, or H9. The composition may further comprise an adjuvant.

[0064] In a further embodiment, a composition is provided comprising virus-like particles (VLPs), wherein the VLPs comprise a modified coronavirus S protein, and the modified S protein comprises the sequence of SEQ ID NO: 21. The composition may further comprise an adjuvant.

[0065] This summary of the present invention does not necessarily describe all of its features.

[0066] These and other features of the present invention will become more apparent from the following description with reference to the accompanying drawings. [Brief explanation of the drawing]

[0067] [Figure 1] This diagram shows a schematic representation of the coronavirus S protein and the locations of the S1 / S2 (aa 685 / 686) and S2' cleavage sites. SP: Signal peptide (aa 1-15); NTD: N-terminal domain (aa 16-306); RBD: Receptor-binding domain (aa 335-527); FP: Fusion peptide (aa 816-833); HR1: Heptad repeat 1 (aa 908-991); HR2: Heptad repeat 2 (aa 1166-1207); TM: Transmembrane domain (aa 1214-1234); CT: Cytoplasmic end (aa 1235-1273). The residue numbers (aa) for each region correspond to their positions in the SARS-CoV-2 S protein (2019-nCoV).

[0068] [Figure 2]This figure shows the amino acid sequence alignments from exemplary influenza strains. The C-terminal region of the external domain, the transmembrane domain (TM), and the cytoplasmic terminal domain (CT) of hemagglutinin (HA) are shown for H1 A / California / 07 / 2009 (SEQ ID NO: 6), H2 A / Singapore / 1 / 1957 HA (SEQ ID NO: 7), H3 A / Minnesota / 41 / 2019 HA (SEQ ID NO: 8), H5 A / Indonesia / 5 / 05 HA (SEQ ID NO: 9), H6 A / Teal / Hong Kong / W312 / 97 HA (SEQ ID NO: 10), H7 A / Guangdong / 17SF003 / 2016 HA (SEQ ID NO: 11), H9 A / Hong Kong / 1073 / 99 HA (SEQ ID NO: 12), and B / Washington / 02 / 2019 HA (SEQ ID NO: 13). The consensus sequence for these sequences is also shown (sequence number 14).

[0069] [Figure 3A]SARS-CoV-2 S proteins with a native (wild-type) transmembrane domain and cytoplasmic end (wtTMCT) under the control of the following 5'UTR: nbMT78 (construct 8586), nbCSY65 (construct 8589), and nbHEL40 (construct 8591), nbMT78 (construct 8592), nbCSY65 (construct 8595), and nbHEL40 (construct 8597), where the native (wild-type) transmembrane domain and cytoplasmic end (wtTMCT) are influenza H5 This figure shows the quantified change factor difference of SARS-CoV-2 S protein accumulation in plants expressing a modified SARS-CoV-2 protein in which the TMCT of the influenza hemagglutinin (HA) of A / Indonesia / 5 / 05(H5iTMCT) is substituted; under the control of nbMT78 (construct 8610), nbCSY65 (construct 8611), and nbHEL40 (construct 8671), the natural (wild-type) cytoplasmic end (wtCT) is substituted with the CT of influenza hemagglutinin (HA) of influenza H5 A / Indonesia / 5 / 05(H5iCT). The SARS-CoV-2 S protein sequence (referred to as nCOV S (GSAS-2P)) has the following substitutions with respect to the reference sequence of Sequence ID No. 2: R667G, R668S, R670S, K971P, and V972P. The results were normalized to the SARS-CoV-2 S protein accumulation from construct 8591, and this was set to 1. [Figure 3B]This figure shows the protein separation of clarified crude extracts on a non-reducing SDS-PAGE gel. The following modified S proteins were expressed in plants: Lane 1: S protein with wild-type transmembrane domain and cytosolic terminal domain (wt TMCT) under the control of nbMT78; Lane 2: S protein with wild-type transmembrane domain and cytosolic terminal domain (wt TMCT) under the control of nbCSY65; Lane 3: S protein with wild-type transmembrane domain and cytosolic terminal domain (wt TMCT) under the control of nbHEL40; Lane 4: Modified S protein with SARS-CoV-2 external domain, transmembrane domain, and cytosolic terminal domain derived from hemagglutinin (HA) of H5 A / Indonesia / 5 / 05 (H5i TMCT) under the control of nbMT78; Lane 5: SARS-CoV-2 external domain and transmembrane domain and H5 A / Indonesia / 5 / 05 (H5i Modified S protein having a cytosolic terminal domain derived from hemagglutinin (HA) of TMCT; Lane 6: Modified S protein having the SARS-CoV-2 external domain and transmembrane domain and the H5 A / Indonesia / 5 / 05 (H5i TMCT) cytosolic terminal domain derived from hemagglutinin (HA) under the control of nbHEL40; Lane 7: Modified S protein having the SARS-CoV-2 external domain and SARS-CoV-2 transmembrane domain and the H5 A / Indonesia / 5 / 05 (H5i CT) cytosolic terminal domain derived from hemagglutinin (HA) under the control of nbMT78; Lane 8: Modified S protein having the SARS-CoV-2 external domain and SARS-CoV-2 transmembrane domain and the H5 A / Indonesia / 5 / 05 (H5i CT) cytosolic terminal domain under the control of nbCSY65 Modified S protein having a cytosolic terminal domain derived from hemagglutinin (HA) of CT); Lane 9: Modified S protein having the SARS-CoV-2 external domain and SARS-CoV-2 transmembrane domain, as well as the cytosolic terminal domain derived from hemagglutinin (HA) of H5 A / Indonesia / 5 / 05 (H5i CT), under the control of nbHEL40. The modified S protein has a molecular weight of approximately 150 kDa and is indicated by the arrow. [Figure 3C] Figure 3C shows Western blot analysis of the same series of lysates shown in Figure 3B, with the lanes corresponding to those described in Figure 3B. The upper panel shows detection with anti-SARS-CoV-2 S1 antibody (40150-R007). The lower panel shows detection with anti-SARS-CoV-2 S2 antibody (NB100-56578). The monomers of the SARS-CoV-2 protein (including the S1 and S2 subunits) have a molecular weight of approximately 150 kDa (non-reducing).

[0070] [Figure 4A]Modified S protein having a SARS-CoV-2 external domain, a SARS-CoV-2 transmembrane domain, and a cytosolic terminal domain derived from hemagglutinin (HA) of H5 A / Indonesia / 5 / 05 (H5 Indo); Modified S protein having a SARS-CoV-2 external domain, a SARS-CoV-2 transmembrane domain, and a cytosolic terminal domain derived from hemagglutinin (HA) of H1 A / California / 7 / 2009 (H1 California); Modified S protein having a SARS-CoV-2 external domain, a SARS-CoV-2 transmembrane domain, and a cytosolic terminal domain derived from hemagglutinin (HA) of H3 A / Minnesota / 41 / 2019 (H3 Minnesota); SARS-CoV-2 external domain, a SARS-CoV-2 transmembrane domain, and H6 A / Teal / Hong Kong / W312 / 97 (H6 Hong This figure shows the difference in the quantified change factor of SARS-CoV-2 S protein accumulation in plants expressing modified S proteins with a cytosolic terminal domain derived from hemagglutinin (HA) from H7 A / Guangdong / 17SF003 / 2016 (H7 Guangdong); modified S proteins with a cytosolic terminal domain derived from hemagglutinin (HA) from H9 A / Hong Kong / 1073 / 99 (H9 Hong Kong); and modified S proteins with a cytosolic terminal domain derived from hemagglutinin (HA) from B / Washington / 02 / 2019 (B Washington).The results were normalized to SARS-CoV-2 S protein accumulation from construct 8671 encoding a modified SARS-CoV-2 protein in which the native (wild-type) cytoplasmic end (wtCT) was replaced with the influenza hemagglutinin (HA) CT of influenza H5 A / Indonesia / 5 / 05 (H5iCT), under the control of nbHEL40(H5 Indo), which was set to 1. [Figure 4B]This figure shows the Western blot analysis of crude lysates from plants expressing the modified S protein derived from the following construct. Modified S protein with lane 2, SARS-CoV-2 external domain, SARS-CoV-2 transmembrane domain, and cytosolic terminal domain derived from hemagglutinin (HA) of H5 A / Indonesia / 5 / 05 (H5 Indo); Modified S protein with lane 3, SARS-CoV-2 external domain, SARS-CoV-2 transmembrane domain, and cytosolic terminal domain derived from hemagglutinin (HA) of H1 A / California / 07 / 2009 (H1 Calif); Modified S protein with lane 4, SARS-CoV-2 external domain, SARS-CoV-2 transmembrane domain, and cytosolic terminal domain derived from hemagglutinin (HA) of H3 A / Minnesota / 41 / 2019 (H3 Minn); Modified S protein with lane 5, SARS-CoV-2 external domain, SARS-CoV-2 transmembrane domain, and H6 A / Teal / Hong Kong / W312 / 97 (H6 Modified S protein having a cytosolic terminal domain derived from hemagglutinin (HA) from HK; Lane 6, Modified S protein having a SARS-CoV-2 external domain, SARS-CoV-2 transmembrane domain, and a cytosolic terminal domain derived from hemagglutinin (HA) from H7 A / Guangdong / 17SF003 / 2016 (H7 Guan); Lane 7, Modified S protein having a SARS-CoV-2 external domain, SARS-CoV-2 transmembrane domain, and a cytosolic terminal domain derived from hemagglutinin (HA) from H9 A / Hong Kong / 1073 / 99 (H9 HK); Lane 8, Modified S protein having a SARS-CoV-2 external domain, SARS-CoV-2 transmembrane domain, and a cytosolic terminal domain derived from hemagglutinin (HA) from B / Washington / 02 / 2019 (B Wash). Lane 1 is crude lysate from plants treated with simulated agroinfiltration. The Sino 400150-R007 antibody was used to detect the S1 subunit of the SARS-CoV-2 S protein. The monomers of the SARS-CoV-2 protein (including the S1 and S2 subunits) have a molecular weight of approximately 150 kDa (non-reducing). [Figure 4C] Figure 4C shows Western blot analysis of the same series of lysates shown in Figure 4B, except that the NB100-56578 antibody detects the S2 subunit of the SARS-CoV-2 S protein.

[0071] [Figure 5A] This figure shows the amino acid sequences of the C-terminal region of the natural SARS-CoV-2 S protein (wtTM / wtCT), the C-terminal region of the influenza H5 hemagglutinin (HA) (H5iTM / H5iCT), the C-terminal region of a modified SARS-CoV-2 S protein having a wild-type transmembrane domain (TM) and an influenza H5 HA cytosolic terminal (CT) domain (wtTM / H5iCT), and the C-terminal regions (wtTM / H5iCT V1-V4) of four alternative versions of the modified S protein having a variable margin between the SARS-CoV-2 transmembrane (TM) domain and the H5 A / Indonesia / 5 / 05 HA cytosolic terminal (CT) domain. The TM domain derived from the coronavirus S protein is underlined, and the CT domain derived from influenza HA is shown in bold. [Figure 5B] Figure 5A shows the quantitative difference in the rate of change of SARS-CoV-2 S protein accumulation in plants expressing four modified S proteins, each having a chimeric transmembrane domain and a cytosolic terminal domain (wtTM / H5iCT, V1-V4), as shown in Figure 5A, compared to plants expressing a modified SARS-CoV-2 S protein having a chimeric transmembrane domain and a cytosolic terminal domain (wtTM / H5iCT, V1-V4), which is set as 1.

[0072] [Figure 6A] This figure shows an electron micrograph of a virus-like particle (VLP) containing the SARS-CoV-2 S protein, which has a wild-type transmembrane domain and a cytosolic terminal domain (wtTMCT; construct 8591). [Figure 6B]This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified S protein with a SARS-CoV-2 external domain and an influenza H5 hemagglutinin transmembrane domain and cytosolic terminal domain (H5i TMCT; construct 8597). [Figure 6C] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified S protein having the SARS-CoV-2 external domain, the SARS-CoV-2 transmembrane domain, and the influenza H5 hemagglutinin cytosolic terminal domain (H5i CT; construct 8671). [Figure 6D] This figure shows an electron micrograph of a virus-like particle (VLP) containing a variant version of the modified S protein (H5i CT V1; construct 8980) that has a SARS-CoV-2 external domain and chimeric transmembrane and cytosolic terminal domains (TMCT). [Figure 6E] This figure shows an electron micrograph of a virus-like particle (VLP) containing a variant version of the SARS-CoV-2 external domain and a chimeric transmembrane and cytosolic terminal domain (TMCT) of the S protein (H5i CT V2; construct 8981). [Figure 6F] This figure shows an electron micrograph of a virus-like particle (VLP) containing an alternative version of the modified S protein (H5i CT V3; construct 8982), in which the SARS-CoV-2 external domain has both a SARS-CoV-2 external domain and chimeric transmembrane and cytosolic terminal domains (TMCT). [Figure 6G] This figure shows an electron micrograph of a virus-like particle (VLP) containing a variant version of the SARS-CoV-2 external domain and a chimeric transmembrane and cytosolic terminal domain (TMCT) of the S protein (H5i CT V4; construct 8983). [Figure 6H] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified S protein having the SARS-CoV-2 external domain, the SARS-CoV-2 transmembrane domain, and the influenza H1 hemagglutinin cytosolic terminal domain (H1 CT; construct 7390). [Figure 6I] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified S protein having the SARS-CoV-2 external domain, the SARS-CoV-2 transmembrane domain, and the influenza H3 hemagglutinin cytosolic terminal domain (H3 CT; construct 7391). [Figure 6J] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified S protein having the SARS-CoV-2 external domain, the SARS-CoV-2 transmembrane domain, and the influenza H6 hemagglutinin cytosolic terminal domain (H6 CT; construct 7392). [Figure 6K] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified S protein having the SARS-CoV-2 external domain, the SARS-CoV-2 transmembrane domain, and the influenza H7 hemagglutinin cytosolic terminal domain (H7 CT; construct 7393). [Figure 6L] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified S protein having the SARS-CoV-2 external domain, the SARS-CoV-2 transmembrane domain, and the influenza H9 hemagglutinin cytosolic terminal domain (H9 CT; construct 7394). [Figure 6M] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified S protein having the SARS-CoV-2 external domain, the SARS-CoV-2 transmembrane domain, and the influenza HA B hemagglutinin cytosolic terminal domain (HA B CT; construct 7395).

[0073] [Figure 7A] This figure shows a schematic diagram of the acceptor vector 8501. [Figure 7B] This figure shows a schematic diagram of the acceptor vector 8500. [Figure 7C] This figure shows a schematic diagram of the acceptor vector 8716.

[0074] [Figure 8A]This figure shows a schematic diagram of Vector 8586. [Figure 8B] This figure shows a schematic diagram of Vector 8589. [Figure 8C] This figure shows a schematic diagram of Vector 8591.

[0075] [Figure 9A] This figure shows a schematic diagram of Vector 8592. [Figure 9B] This figure shows a schematic diagram of Vector 8595. [Figure 9C] This figure shows a schematic diagram of Vector 8597.

[0076] [Figure 10A] This figure shows a schematic diagram of the Vector 8610. [Figure 10B] This figure shows a schematic diagram of Vector 8611. [Figure 10C] This figure shows a schematic diagram of Vector 8671.

[0077] [Figure 11A] This figure shows the quantified change multipliers of accumulation in plants expressing modified SARS-CoV-2 S protein (wtTM / H5iCT) with further substitutions. The modified SARS-CoV-2 S protein has the following substitutions: "GSAS-2P"::R667G, R668S, R670S, K971P and V972P; "GSAS-4P":R667G, R668S, R670S, K971P, V972P, F802P and A927P; and "GSAS-6P":R667G, R668S, R670S, K971P, V972P, F802P, A877P, A884P and A927P (with respect to the reference sequence of SEQ ID NO: 2). The results were normalized to the cumulative values ​​of modified SARS-CoV-2 (wtTM / H5iCT) and GSAS+2P substitution, which were set to 1. [Figure 11B]Figure 11A shows the quantified multiplicative changes in accumulation in plants expressing modified SARS-CoV-2 S protein (wtTM / H5iCT) containing the GSAS-2P, GSAS-4P, and GSAS-6P substitutions described, compared to the quantified multiplicative changes in accumulation when each modified SARS-CoV-2 S protein further incorporates the L923F substitution. The results were normalized to the accumulation of modified SARS-CoV-2 (wtTM / H5iCT) and GSAS+2P substitutions, which were set to 1.

[0078] [Figure 12A] This figure shows a schematic diagram of Vector 8980. [Figure 12B] This figure shows a schematic diagram of Vector 8981. [Figure 12C] This figure shows a schematic diagram of Vector 8982. [Figure 12D] This figure shows a schematic diagram of Vector 8983.

[0079] [Figure 13A] This is a schematic diagram of Vector 7390. [Figure 13B] This is a schematic diagram of Vector 7391. [Figure 13C] This figure shows a schematic diagram of Vector 7392. [Figure 13D] This is a schematic diagram of Vector 7393. [Figure 13E] This figure shows a schematic diagram of Vector 7394. [Figure 13F] This is a schematic diagram of Vector 7395.

[0080] [Figure 14A] This figure shows a schematic diagram of Vector 8953. [Figure 14B] This figure shows a schematic diagram of Vector 8940.

[0081] [Figure 15A] This figure shows a schematic diagram of Vector 8933. [Figure 15B]This figure shows a schematic diagram of the Vector 8960. [Figure 15C] This figure shows a schematic diagram of Vector 8947.

[0082] [Figure 16A] This figure shows the Western blot analysis of crude lysates from plants expressing the following modified S proteins: Lane 1: Modified S protein with SARS-CoV-1 external domain, transmembrane and cytosolic terminal domains ("wtTMCT", construct 9231); Lane 2: Modified S protein with SARS-CoV-1 external domain, transmembrane and cytosolic terminal domains (TMCT) derived from H5 A / Indonesia / 5 / 05 hemagglutinin (HA) ("H5iTMCT", construct 9232); Lane 3: Modified S protein with SARS-CoV-1 external domain and transmembrane domain and cytosolic terminal domain derived from H5 A / Indonesia / 5 / 05 (H5 India) hemagglutinin (HA) ("H5iCT", construct 9233); Lane 4: SARS-CoV-1 external domain and transmembrane domain, and H5 Modified S protein ("H5iCT(V4)", construct 9234) having a cytosolic terminal domain derived from hemagglutinin (HA) of A / Indonesia / 5 / 05 (H5 India) and a variable margin between the SARS-CoV-1 transmembrane (TM) domain and the H5 A / Indonesia / 5 / 05 HA cytosolic terminal (CT) domain; lane 5, modified S protein ("H1cCT", construct 9235) having an external domain and transmembrane domain derived from SARS-CoV-1 and a cytosolic terminal domain derived from hemagglutinin (HA) of H1 A / California / 7 / 2009. The primary antibody used for detection was SARS-CoV spike S1 subunit antibody (40150-MM08, 1 / 5000) from Sino Biologicals. The secondary antibody used for detection was JIR's goat anti-mouse antibody (115-035-146, 1 / 10000). The molecular weight of the modified S protein is approximately 150 kDa.

[0083] [Figure 16B] This figure shows the Western blot analysis of fractions F5 (30%), F6 (30%), F7 (25%), F8 (25%), F9 (25%), F10 (15%), and F11 (15%) from a discontinuous iodixanol density gradient. [Figure 16C] This figure shows the Western blot analysis of fractions F5 (30%), F6 (30%), F7 (25%), F8 (25%), F9 (25%), F10 (15%), and F11 (15%) from a discontinuous iodixanol density gradient. [Figure 16D] This figure shows the Western blot analysis of fractions F5 (30%), F6 (30%), F7 (25%), F8 (25%), F9 (25%), F10 (15%), and F11 (15%) from a discontinuous iodixanol density gradient. The accumulation of proteins in these fractions indicates the formation of higher molecular weight structures, i.e., VLP formation. For Western blots from the crude lysate fractions, the primary antibody used for detection was SARS-CoV Spike S1 subunit antibody from Sino Biologicals, 40150-MM08 (1 / 5000), and the secondary antibody used for detection was goat anti-mouse, JIR, 115-035-146 (1 / 10000). Figure 16B: Crude lysates were analyzed from plants expressing SARS-CoV-1 S protein (with 2P+R667A substitution) having a natural TMCT domain (wtTMCT, construct 9231). Figure 16C: Crude lysate from plants expressing a modified SARS-CoV-1 S protein (with 2P+R667A substitution) having a TMCT derived from H5 A / Indonesia / 5 / 05 HA (H5iTMCT, construct 9232). Figure 16D: Crude lysate from plants expressing a modified SARS-CoV-1 S protein (with 2P+R667A substitution) having a cytoplasmic end derived from H5 A / Indonesia / 5 / 05 HA (H5iCT, construct 9233).

[0084] [Figure 17A]This figure shows an electron micrograph of a virus-like particle (VLP) containing the SARS-CoV-1 S protein (with the 2P+R667A substitution) with a natural TMCT domain (wtTMCT, construct 9231). [Figure 17B] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified SARS-CoV-1 S protein (with a 2P+R667A substitution) with TMCT derived from H5 A / Indonesia / 5 / 05 HA (H5iTMCT, construct 9232). [Figure 17C] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified SARS-CoV-1 S protein (with a 2P+R667A substitution) with a cytoplasmic terminal derived from H5 A / Indonesia / 5 / 05 HA (H5iCT, construct 9233).

[0085] [Figure 18A] This figure shows a schematic diagram of Vector 9231. [Figure 18B] This figure shows a schematic diagram of Vector 9232. [Figure 18C] This figure shows a schematic diagram of Vector 9233. [Figure 18D] This figure shows a schematic diagram of Vector 9234. [Figure 18E] This figure shows a schematic diagram of Vector 9235.

[0086] [Figure 19A]This figure shows the Western blot analysis of crude lysates from plants expressing the modified S protein derived from the following construct. Lane 1: Modified S protein having a MERS-CoV external domain, transmembrane and cytosolic terminal domains ("wtTMCT", construct 9246); Lane 2: Modified S protein having a MERS-CoV-derived external domain, transmembrane and cytosolic terminal domains (TMCT) derived from H5 A / Indonesia / 5 / 05 hemagglutinin (HA) ("H5iTMCT", construct 9247); Lane 3: Modified S protein having a MERS-CoV-derived external domain and transmembrane domain, and a cytosolic terminal domain derived from H5 A / Indonesia / 5 / 05 (H5 India) hemagglutinin (HA) ("H5iCT", construct 9249); Lane 4: Having a MERS-CoV-derived external domain and transmembrane domain, and a cytosolic terminal domain derived from H5 A / Indonesia / 5 / 05 (H5 India) hemagglutinin (HA), with a MERS-CoV transmembrane (TM) domain and H5 Modified S protein with a variable margin between the A / Indonesia / 5 / 05 HA cytosolic terminal (CT) domain ("H5iCT(V4)", construct 9250); lane 5, modified S protein with an external domain and transmembrane domain derived from MERS-CoV and a cytosolic terminal domain derived from H1 A / California / 7 / 2009 hemagglutinin (HA) ("H1cCT", construct 9251). The primary antibody used for detection was the MERS-CoV spike protein S1 antibody (N-terminus) from Sino Biological (100208-RP02, 1 / 5000). The secondary antibody used for detection was the JIR goat anti-mouse antibody (115-035-144, 1 / 10000). The molecular weight of the modified S protein is approximately 175 kDa. [Figure 19B] This figure shows an electron micrograph of a virus-like particle (VLP) containing the MERS-COV S protein (with ASVG+2P substitution) which has a natural TMCT domain (wtTMCT, construct 9246). [Figure 19C]This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified MERS-CoV S protein (with ASVG+2P substitution) derived from H5 A / Indonesia / 5 / 05 HA (H5iTMCT, construct 9247). [Figure 19D] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified MERS-CoV S protein (with ASVG+2P substitution) with a cytoplasmic terminal derived from H5 A / Indonesia / 5 / 05 HA (H5iCT, construct 9249). [Figure 19E] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified MERS-CoV S protein (with ASVG+2P substitution) (H5iCT(V4), construct 9250) that has a cytoplasmic terminal derived from H5 A / Indonesia / 5 / 05 HA with a variable margin between the MERS-CoV transmembrane (TM) domain and the H5 A / Indonesia / 5 / 05 HA cytosolic terminal (CT) domain. [Figure 19F] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified MERS-CoV S protein (with ASVG+2P substitution) with a cytoplasmic terminal derived from H1 A / California / 7 / 2009 HA (H1cCT, construct 9251).

[0087] [Figure 20A] This figure shows a schematic diagram of Vector 9246. [Figure 20B] This figure shows a schematic diagram of Vector 9247. [Figure 20C] This figure shows a schematic diagram of Vector 9249. [Figure 20D] This figure shows a schematic diagram of the Vector 9250. [Figure 20E] This figure shows a schematic diagram of Vector 9251.

[0088] [Figure 21] This figure shows a schematic diagram of the acceptor vector 7147.

[0089] [Figure 22] This figure shows the alignment of the natural SARS-CoV-2, SARS-CoV-1, and MERS-CoV S protein sequences (SEQ ID NOs. 2, 114, and 115) from which the natural signal peptide has been removed. The RRAR fulin cleavage site (667-670) and the residues corresponding to F802P, A877P, A884P, A927P, K971P, and V972P in the natural SARS-CoV-2 S protein (SEQ ID NOs. 2), which does not contain the signal peptide, are boxed along homologous residues from the natural SARS-CoV-1 S protein (SEQ ID NOs. 114) and the natural MERS S protein (SEQ ID NOs. 115), which do not contain the signal peptide.

[0090] [Figure 23A]This figure shows the Western blot analysis of crude lysates from plants expressing the modified S protein derived from the following construct. Lane 3: Modified S protein having an OC43-CoV external domain, transmembrane and cytosolic terminal domains ("wtTMCT", construct 9269); Lane 4: Modified S protein having an OC43-CoV-derived external domain, transmembrane and cytosolic terminal domains (TMCT) derived from H5 A / Indonesia / 5 / 05 hemagglutinin (HA) ("H5iTMCT", construct 9270); Lane 5: Modified S protein having an OC43-CoV-derived external domain and transmembrane domain and a cytosolic terminal domain derived from H5 A / Indonesia / 5 / 05 (H5 India) hemagglutinin (HA) ("H5iCT", construct 9272); Lane 6: Having an OC43-CoV-derived external domain and transmembrane domain, and a cytosolic terminal domain derived from H5 A / Indonesia / 5 / 05 (H5 India) hemagglutinin (HA), with an OC43-CoV transmembrane (TM) domain and H5 Modified S protein with a variable margin between the A / Indonesia / 5 / 05 HA cytosolic terminal (CT) domain ("H5iCT(V4)", construct 9273); Lane 7, modified S protein with an external domain and transmembrane domain derived from OC43-CoV and a cytosolic terminal domain derived from H1 A / California / 7 / 2009 hemagglutinin (HA) ("H1cCT", construct 9274). The primary antibody used for detection was the anticoronavirus OC43 spike protein antibody from Antibodies-online (ABIN2754654, 1 / 1000). The secondary antibody used for detection was the goat anti-rabbit antibody from JIR (111-035-144, 1 / 10000). The molecular weight of the modified S protein is approximately 150 kDa. [Figure 23B] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified OC43-CoV S protein (with GGSGS+2P substitution) with TMCT derived from H5 A / Indonesia / 5 / 05 HA (H5iTMCT, construct 9270). [Figure 23C]This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified OC43-CoV S protein (with GGSGS+2P substitution) with a cytoplasmic terminal derived from H5 A / Indonesia / 5 / 05 HA (H5iCT, construct 9272). [Figure 23D] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified OC43-CoV S protein (with GGSGS+2P substitution) that has a cytoplasmic terminal derived from H5 A / Indonesia / 5 / 05 HA with a variable margin between the OC43-CoV transmembrane (TM) domain and the H5 A / Indonesia / 5 / 05 HA cytosolic terminal (CT) domain (H5iCT(V4), construct 9273). [Figure 23E] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified OC43-CoV S protein (with GGSGS+2P substitution) with a cytoplasmic terminal derived from H1 A / California / 7 / 2009 HA (H1cCT, construct 9274).

[0091] [Figure 24A] This figure shows a schematic diagram of Vector 9269. [Figure 24B] This figure shows a schematic diagram of Vector 9270. [Figure 24C] This figure shows a schematic diagram of Vector 9272. [Figure 24D] This is a schematic diagram of Vector 9273. [Figure 24E] This figure shows a schematic diagram of Vector 9274.

[0092] [Figure 25A] This figure shows an electron micrograph of a virus-like particle (VLP) containing the 229E-CoV S protein (with the R567A+2P substitution) which has a natural TMCT domain (wtTMCT, construct 9310). [Figure 25B]This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified 229E-CoV S protein (with R567A+2P substitution) with TMCT derived from H5 A / Indonesia / 5 / 05 HA (H5iTMCT, construct 9311). [Figure 25C] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified 229E-CoV S protein (with R567A+2P substitution) with a cytoplasmic terminal derived from H5 A / Indonesia / 5 / 05 HA (H5iCT, construct 9312). [Figure 25D] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified 229E-CoV S protein (with R567A+2P substitution) that has a cytoplasmic terminal derived from H5 A / Indonesia / 5 / 05 HA with a variable margin between the 229E-CoV transmembrane (TM) domain and the H5 A / Indonesia / 5 / 05 HA cytosolic terminal (CT) domain (H5iCT(V4), construct 9313). [Figure 25E] This figure shows an electron micrograph of a virus-like particle (VLP) containing a modified 229E-CoV S protein with a cytoplasmic terminal derived from H1 A / California / 7 / 2009 HA (with R567A+2P substitution) (H1cCT, construct 9314).

[0093] [Figure 26A] This figure shows a schematic diagram of Vector 9310. [Figure 26B] This is a schematic diagram of Vector 9311. [Figure 26C] This figure shows a schematic diagram of Vector 9312. [Figure 26D] This is a schematic diagram of Vector 9313. [Figure 26E] This figure shows a schematic diagram of Vector 9314. [Modes for carrying out the invention]

[0094] The following description is of a preferred embodiment.

[0095] Where used herein, the terms “comprising,” “having,” “including,” and “containing,” and their grammatical variations, are inclusive or open-ended and do not exclude additional elements and / or process steps not enumerated. Where used herein in relation to a use or method, the term “consisting essentially of” indicates that additional elements and / or process steps may exist, but these additions do not substantially affect the form of the enumerated method or use function. Where used herein in relation to a use or method, the term “consisting of” excludes the existence of additional elements and / or process steps. A use or method described herein as including certain elements and / or steps may also, in certain embodiments, essentially consist of these elements and / or steps, whether or not these embodiments are specifically mentioned, and in other embodiments, consist of these elements and / or steps. Furthermore, singular use includes plural use, and unless otherwise specified, “or” means “and / or.” As used herein, the term “plural” means more than one, e.g., two or more, three or more, four or more, etc. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art. As used herein, the term “about” means a variation of about + / - 10% from a given value. It should be understood that such variation is always included in any given value provided herein, whether specifically mentioned or not. The use of the words “a” or “an” as used herein in combination with the term “comprising” may mean “one,” but also coincides with the meanings of “one or more,” “at least one,” and “one or more.”

[0096] This specification relates to modified viral structural proteins in a host or host cell and their production. The modified viral structural proteins include, in order, an outer domain, a transmembrane domain (TM) or a portion of the TM, and a cytosolic terminal (CT) domain or a portion of the CT, wherein the outer domain and the TM or a portion of the TM are derived from the Coronaviridae family, and the CT or a portion of the CT is derived from the influenza hemagglutinin (HA) protein.

[0097] A modified viral structural protein could be a modified coronavirus structural protein in which the cytosolic terminal domain or a portion of the cytosolic terminal domain is replaced by the cytosolic terminal domain or a portion of the cytosolic terminal domain of the influenza hemagglutinin (HA) protein. For example, a modified viral structural protein could be a modified coronavirus spike or surface (S) protein in which the cytosolic terminal domain or a portion of the cytosolic terminal domain of the S protein is replaced by the cytosolic terminal domain or a portion of the cytosolic terminal domain of the influenza hemagglutinin (HA) protein.

[0098] This disclosure provides a modified viral structural protein in which the external domain and transmembrane domain of the modified viral structural protein may be derived from the external domain and transmembrane domain of the coronavirus S protein, and the cytosolic terminal domain may be derived from the cytosolic terminal domain of the influenza hemagglutinin (HA) protein.

[0099] A modified S protein may be a chimeric modified S protein or a chimeric S protein. “Chimera S protein” means a protein or polypeptide containing amino acid sequences and / or protein domains or portions of protein domains from two or more sources fused as a single polypeptide. For example, but not limited to, the external domain and transmembrane domain (TM) or portion of the TM of a chimeric S protein may originate from a first viral structural protein, e.g., coronavirus S protein; the cytoplasmic terminal (CT) or portion of the CT may originate from a second viral structural protein, e.g., influenza HA. ​​Furthermore, the external domain may originate from a first viral structural protein, e.g., a first coronavirus S protein; the TM or portion of the TM may originate from a second viral structural protein, e.g., a second coronavirus S protein; and the CT or portion of the CT may originate from a third viral structural protein, e.g., influenza HA. ​​Therefore, a modified S protein or a chimeric S protein may contain chimeric transmembrane and cytosolic terminal domains (TMCT).

[0100] The modified coronavirus S protein is, in order, -The external domain derived from the coronavirus S protein, - Transmembrane and cytosolic terminal domains (TMCTs), where the TMCTs may be chimeric TMCTs. - The transmembrane domain (TM) or a portion of the TM may be derived from the coronavirus S protein, - The cytosolic end (CT) or a portion of the CT is derived from the influenza hemagglutinin (HA) protein, It may include a transmembrane and cytosolic terminal domain (TMCT), Includes.

[0101] TM or a portion of TM may be directly fused to or linked to CT or a portion of CT, or TM or a portion of TM may be fused to or linked to CT or a portion of CT by an intervening peptide sequence.

[0102] Furthermore, TM may be a chimeric TM that includes an N-terminal sequence derived from coronavirus S protein TM and a C-terminal sequence derived from influenza HA protein TM. CT may be a chimeric CT that includes an N-terminal sequence derived from coronavirus S protein CT and a C-terminal sequence derived from influenza HA protein CT.

[0103] Therefore, chimeric TMCT may include natural coronavirus S protein TM, chimeric coronavirus S protein / influenza HA TM, natural influenza HA CT, chimeric influenza HA / coronavirus S protein CT, or a combination thereof. Chimeric coronavirus S protein / influenza HA TM includes a sequence derived from coronavirus S protein TM and a sequence derived from influenza HA TM. Similarly, chimeric influenza HA / coronavirus S protein CT includes a sequence derived from influenza HA CT and a sequence derived from coronavirus S protein CT.

[0104] "Chimera transmembrane and cytosolic terminal domain" or "chimeric TMCT" refers to a non-natural TMCT of the coronavirus S protein. A chimeric TMCT contains sequences that are not found together in nature. Therefore, a TMCT may contain sequences that are heterologous to the external domain of the coronavirus S protein. The term "heterologous" refers to sequences or domains that originate from different biological or synthetic sources. For example, a chimeric TMCT may contain a TM or a portion of a TM that originates from the same coronavirus S protein as the external domain, i.e., the TM may be homologous to the external domain of the S protein, or the TM or a portion of a TM may originate from a different virus TM, for example, a TM from a different coronavirus S protein as the external domain, i.e., the TM may be heterologous to the external domain of the S protein. A CT or a portion of a CT may originate from a CT that is heterologous to the external domain, TM, or both the external domain and TM of the modified S protein.

[0105] The coronavirus S protein, modified S protein, or external domain, and the transmembrane domain or part of the transmembrane domain of the modified coronavirus S protein may be derived from any member of the Coronaviridae family of viruses. For example, the coronavirus S protein, modified S protein, or the external domain and transmembrane domain of the modified coronavirus S protein may be derived from, for example, coronaviruses such as alpha-coV, beta-coV, gamma-coV, or delta-coV. For example, the coronavirus may be alpha-coV or beta-coV. The alpha-coVenus may be a duvinacovirus such as HCoV-229E (229E-CoV), or a setracovirus such as HCoV-NL63. In a preferred embodiment, the coronavirus is beta-coV. Betacoronaviruses can be A-type betacoronaviruses, such as HCoV-OC43 (OC43-CoV) or HCoV-HKU1 (HKU1-CoV); B-type betacoronaviruses, such as SARS-CoV (also called SARS-CoV-1) or SARS-CoV-2 and their variants; or C-type betacoronaviruses, such as MERS-CoV.

[0106] The coronavirus S protein, modified S protein, or external domain and the transmembrane domain or portion of the transmembrane domain of the modified coronavirus S protein may further be derived from variants of the SARS-CoV-2 lineage, including but not limited to strain B.1.1.7 ("Alpha" variant) (20I / 501Y.V1, MW531680.1), strain B.1.351 ("Beta" variant) (20H / 501Y.V2), strain P.1 ("Gamma" variant) (20J / 501Y.V3), strain B 1.617.2 ("Delta" variant), strain B.1.525, strain B.1.429 ("ETA" variant), or other variants of strains containing naturally occurring mutations in the coronavirus S protein, or naturally occurring recombinant strains thereof.

[0107] In one embodiment, the external domain and / or part of the transmembrane domain of the modified viral structural protein are derived from the spike protein (S) of a SARS-CoV-2 lineage coronavirus (also known as a SARS-CoV-2 variant). In another embodiment, the external domain and / or part of the transmembrane domain of the modified viral structural protein are derived from the spike protein (S) of SARS-CoV-1, MERS-CoV, OC43-CoV, or 229E-CoV, or a variant thereof.

[0108] With respect to modified viral structural proteins, the term “modified” as used herein may refer to replacing the cytoplasmic terminal domain (CT) or a portion of the CT in a structural protein derived from the Coronaviridae family with the CT or a portion of the CT of a different virus. For example, a modified viral structural protein may be a coronavirus S protein in which the CT or a portion of the CT of the S protein is replaced with the CT or a portion of the CT of influenza hemagglutinin (HA).

[0109] Therefore, the modified viral structural protein may be a modified coronavirus spike (S) protein containing a transmembrane domain (TM) or a portion of TM and a cytosolic end (CT) or a portion of CT, wherein the CT or a portion of CT may be derived from influenza hemagglutinin (HA) protein, and the TM or a portion of TM is heterogeneous to the CT or a portion of CT. Furthermore, the modified S protein contains a transmembrane domain (TM) or a portion of TM and a cytosolic end (CT) or a portion of CT, wherein the CT or a portion of CT may be derived from influenza hemagglutinin (HA) protein, and the CT or a portion of CT is heterogeneous to the TM or a portion of TM.

[0110] Therefore, in one embodiment, a modified coronavirus spike (S) protein is provided, comprising a transmembrane domain (TM) or a portion of the TM and a cytosolic end (CT) or a portion of the CT, where the CT or a portion of the CT is derived from the influenza hemagglutinin (HA) protein, and the TM or a portion of the TM is heterologous to the CT or a portion of the CT. The modified coronavirus spike (S) protein is also called the modified S protein.

[0111] The cytoplasmic terminal domain may be referred to as the "cytoplasmic end," "cytosolic end," "cytosolic terminal domain," "CT," "CTD," "cytoplasmic domain," "cytoplasmic domain," "CP," "CPD," or "C-terminal domain," and similar expressions. The cytoplasmic terminal domain may also encompass a portion of the cytoplasmic end.

[0112] Modified viral structural proteins, such as the modified S protein disclosed herein, have been found to possess improved properties compared to wild-type or unmodified viral structural proteins (e.g., the S protein). Examples of the improved properties of modified viral structural proteins, such as the modified S protein, include, but are not limited to, increased yield of the modified viral structural protein when expressed in a host or host cell compared to wild-type or unmodified viral structural proteins; improved integrity, stability, or both integrity and stability of the viral structural protein when expressed in a host or host cell compared to wild-type or unmodified viral structural proteins; improved integrity, stability, or both integrity and stability of virus-like particles (VLPs) containing the modified viral structural protein compared to VLPs containing the modified viral structural protein without the modifications described herein; and increased yield of VLPs containing the modified viral structural protein when expressed in host cells compared to the yield of VLPs without the modified viral structural protein expressed in the same or substantially similar host cells.

[0113] Furthermore, methods for producing virus-like particles (VLPs) containing modified viral structural proteins, such as modified S proteins, in a host or host cells are also described. When VLPs containing modified viral structural proteins, such as modified S proteins in which the natural or wild-type CT is replaced with the CT of influenza HA described herein, are produced, the yield of VLP production in the host has been observed to be increased compared to the yield of VLPs containing viral structural proteins that either i) contain the natural CT, or ii) contain a modified viral structural protein in which the transmembrane domain (TM) and CT are replaced with the TM and CT of influenza HA.

[0114] The transmembrane domain is sometimes called "TM" or "TMD". The transmembrane and cytoplasmic terminal domains may be called TMCT or TM / CT.

[0115] Figure 3A shows that when a modified S protein (e.g., modified SARS-CoV-2 S protein) was expressed in plants, the yield or protein accumulation (expressed as a multiplier change) of the modified S protein increased by approximately twofold when the natural transmembrane and cytoplasmic end (TMCT) was replaced with TMCT derived from influenza HA (constructs 8592, 8595, and 8597) compared to the yield or protein accumulation of the S protein using the natural TMCT (constructs 8586, 8589, and 8591). Furthermore, when a modified S protein (e.g., modified SARS-CoV-2 S protein) (constructs 8610, 8611, and 8671) with only the cytoplasmic end (CT) replaced with the CT of influenza HA was expressed in plants, the protein accumulation (expressed as a multiplier change) of the modified S protein using the influenza HA CT increased by approximately 1.74 to 2.14 times compared to the accumulation of the modified S protein with the TMCT replaced by the influenza HA TMCT. In response to this, the protein accumulation of the CT-modified S protein of influenza HA increased by approximately 3.57 to 4.40 times compared to the accumulation of the S protein with the native transmembrane tail and cytoplasmic end (wtTMCT).

[0116] Figure 3B shows that higher protein accumulation was observed for the modified S protein with the cytoplasmic end of influenza HA (modified SARS-CoV-2 S protein) (H5i CT) compared to the protein accumulation of the modified S protein with the cytoplasmic end of influenza HA (H5i TMCT) derived from a crude plant extract. The modified S protein with the cytoplasmic end of influenza HA (H5i CT) is visualized only by Coomassie blue staining. The band for the modified S protein with the cytoplasmic end of influenza HA (H5i CT) is more prominent and thicker compared to the bands for the S protein with the wild-type TMCT (wt TMCT) or the modified S protein with the TMCT of influenza HA (H5i TMCT). See the band of approximately 150 kDa marked as the S protein. The thickness of the band corresponds to the amount of protein present, indicating that more protein was accumulated for the H5i CT S protein. This higher protein accumulation was observed regardless of the expression enhancer used.

[0117] As will be discussed in more detail below, similar results were obtained when the modified S protein included the SARS-CoV-1 S protein with a cytoplasmic terminal derived from influenza HA (see Figure 16A) or the MERS CoV S protein with a cytoplasmic terminal derived from influenza HA (see Figure 19A).

[0118] Figure 3C shows the accumulation of S protein (SARS-CoV-2 S protein) by Western blot analysis of crude plant extracts. When a modified S protein with a cytoplasmic terminal derived from influenza HA (H5i CT) was expressed in plants, a higher accumulation of the modified S protein was observed compared to wild-type TMCT (wt TMCT) and S protein with a modified S protein in which both the transmembrane domain and the cytoplasmic terminal (TMCT) domain were replaced with influenza HA-derived TMCT (H5i TMCT). Western blot analysis in Figure 3C further shows that the SARS-CoV-2 S protein contains both the S1 domain / subunit (upper panel, detected by anti-SARS-CoV-2 S1 antibody) and the S2 domain / subunit (bottom panel, detected by anti-SARS-CoV-2 S2 antibody) and has a molecular weight of approximately 150 kDa.

[0119] This specification provides modified viral structural proteins, which may be modified coronavirus spike or surface proteins (S proteins). A modified S protein comprises, in order, an external domain, a transmembrane domain (TM) or a portion of the TM, and a cytosolic terminal (CT) domain or a portion of the CT, wherein the external domain and the transmembrane domain are derived from a coronavirus, and the CT or a portion of the CT is derived from the CT of the influenza hemagglutinin (HA) protein. The external domain and the transmembrane domain or a portion of the TM may be derived from the same coronavirus. Therefore, the external domain and the transmembrane domain or portion of the TM of the modified structural protein are homologous (i.e., not heterologous) to each other, while the CT or portion of the CT is heterologous to the external domain and the transmembrane domain.

[0120] Furthermore, the transmembrane domain (TM) or a portion of the TM of the modified S protein may originate from a different coronavirus than the external domain. Therefore, the TM or a portion of the TM in the modified S protein may be heterologous (not homologous) to both the external domain and the CT domain or a portion of the CT of the modified S protein. Similarly, the external domain may be heterologous (not homologous) to the TM or a portion of the TM and the CT domain or a portion of the CT of the modified S protein. For example, the external domain of the modified S protein may originate from a first coronavirus, the TM or a portion of the TM may originate from a second coronavirus, and the CT or a portion of the CT may originate from influenza HA. ​​The first and second coronaviruses may belong to different coronavirus families, subgroups, types, subtypes, lineages, or strains. Therefore, the first and second coronaviruses may be heterologous to each other, and also heterologous to each other with respect to the virus family from which the CT or a portion of the CT originates.

[0121] For example, the first coronavirus from which the S protein external domain originates may originate from any coronavirus, e.g., alpha-coronavirus (Alpha-CoV) or beta-coronavirus (Beta-CoV). Non-limiting examples of the first coronavirus from which the S protein external domain may originate include dubinacovirus, e.g., HCoV-229E; cetracovirus, e.g., HCoV-NL63; lineage A beta-coronavirus, e.g., HCoV-OC43 or HCoV-HKU1; lineage B beta-coronavirus, e.g., SARS-CoV or SARS-CoV-2; or lineage C beta-coronavirus, e.g., MERS-CoV. The second coronavirus from which TM originates may belong to a different coronavirus family, subgroup, type, subtype, lineage, or strain than the first coronavirus from which the external domain originates. For example, the second coronavirus from which the S protein TM originates may originate from any coronavirus, e.g., alpha-coronavirus (Alpha-CoV) or beta-coronavirus (Beta-CoV), as long as the second coronavirus is heterogeneous to the first coronavirus. Non-exclusive examples of secondary coronaviruses from which the S protein TM may originate include dubinacovirus, e.g., HCoV-229E (also known as 229E-CoV); cetracovirus, e.g., HCoV-NL63 (NL63-CoV); A-lineage betacoronavirus, e.g., HCoV-OC43 (also known as OC43-CoV) or HCoV-HKU1 (HKU1-CoV); B-lineage betacoronavirus, e.g., SARS-CoV (also known as SARS-CoV 1) or SARS-CoV 2; or C-lineage betacoronavirus, e.g., MERS-CoV (also simply known as "MERS").

[0122] Domains within coronavirus S proteins, such as those in the SARS-CoV-1 S protein, SARS-CoV-2 S protein, MERS CoV S protein, OC43-CoV S protein, or 229E-CoV S protein, can be readily identified by methods known in the art. For example, domains such as transmembrane domains can be identified by determining the degree of hydrophobicity of the protein's amino acid sequence using a transmembrane prediction program (e.g., Expert Protein Analysis System; ExPASy.org, operated by the Swiss Institute of Bioinformatics; or the Dense Alignment Surface Method, Cserzo M., et al. 1997, Prot.Eng. vol. 10, no. 6, 673-676; Lolkema JS 1998, FEMS Microbiol Rev. 22, no. 4, 305-322), by determining the hydrophobicity profile of the protein's amino acid sequence (e.g., Kyte-Doolittle Hydropathy profile), by determining the three-dimensional protein structure, and by identifying thermodynamically stable structures within the membrane (e.g., a single alpha-helix, a stable complex of several transmembrane alpha-helices, a transmembrane beta-barrel, a beta-helix, or any other thermodynamically stable structure within the membrane).

[0123] Furthermore, domains within the coronavirus S protein can be determined by comparison with known protein sequences, for example, by sequence alignment. Methods for sequence alignment for comparison are well known in the art and are described further below.

[0124] The domains and domain structure of coronavirus S proteins are well known and described. All coronavirus spike proteins (S proteins) share the same structure in two subunits or domains: an N-terminal subunit (or domain) called S1, which is involved in receptor binding, and a C-terminal S2 subunit (or domain), which is involved in viral attachment, membrane fusion, and viral entry.

[0125] Figure 1A shows a schematic diagram of the coronavirus S protein with its subunits and domains, as well as the locations of the S1 / S2 and S2' cleavage sites. The S1 subunit is distal to the viral membrane and contains a receptor-binding domain (RBD) that mediates viral attachment to the host receptor. The S2 subunit contains a fusion protein mechanism including a fusion peptide, two heptad repeat sequences (HR1 and HR2), a fusion glycoprotein, a central helix typical of a transmembrane domain, and a cytosolic terminal domain (see, for example, Kirchdoerfer et al. Nature 2016 Mar 3;531(7592):118-2, incorporated herein by reference).

[0126] The transmembrane domain (TM) and cytoplasmic terminal domain (CT) are located at the C-terminus of the S2 subunit. While these domains are conserved across all coronaviruses (see Figure 1A and Cover et al. 2009, Virol J. 2009; 6:230, incorporated herein by reference), different references, groups, and authors refer to different amino acid numbering for these domains.

[0127] For example, amino acids (aa): 1214-1234 can be assigned to TM, and aa 1235-1273 can be assigned to CT of the SARS-CoV-2 S protein (see, for example, UniProtKB-P0DTC2(SPIKE_SARS2)). When aligning the sequence of SARS-CoV-2 (SEQ ID NO: 1) with the sequence of SARS-CoV-1 from Kirchdoerfer et al. (Nature 2016 Mar 3;531(7592):118-2), SARS-CoV-2 TM corresponds to amino acids 1214-1236, and SARS-CoV-2 CT corresponds to amino acids 1237-1273.

[0128] For the purposes of this disclosure, the TM and CT of the natural (unmodified) S protein, when aligned to the coronavirus S protein reference sequence (SEQ ID NO: 1), correspond to the following amino acids: TM: amino acids 1214-1234 and CT: amino acids 1235-1273.

[0129] When 15 amino acids, including the signal peptide (SP), are removed from the S protein, TM corresponds to amino acids 1199-1219 of the reference sequence of SEQ ID NO: 2, and CT corresponds to amino acids 1220-1258 of SEQ ID NO: 2 (see also Table 1 for reference sequences and numbering).

[0130] The TM domain of the coronavirus S protein has a highly conserved N-terminal aromatic-rich extension followed by a hydrophobic sequence (see Figure 22 and Cover et al. Virology Journal volume 6, 230 (2009)). The consensus sequence of the coronavirus S protein TM domain is as follows: WYXWLGFIAGLXAXXX{X}VXXXL(sequence number 132), (where {X} is optional).

[0131] For example, the consensus sequence for the coronavirus S protein TM domain could be as follows: WY[I / V]WLGFIAGL[V / I]A[L / I][A / V][L / M]{X}V[F / T][F / I]XL(Sequence ID 133) (wherein {X} may or may not be C).

[0132] [Table 1]

[0133] Although there are differences in the numbering of residues assigned to the TM domain and the CT domain, those skilled in the art can determine the boundaries of one or more of these domains in the coronavirus S protein by using, for example, known methods described below.

[0134] In modified coronavirus S proteins, heterologous CT or a portion of CT, which may originate from influenza HA, can be directly fused to the C-terminus of the TM or a portion of the TM of the coronavirus S protein, or heterologous CT or a portion of CT can be fused to the C-terminus of the TM or a portion of the TM of the coronavirus S protein using an intervening peptide sequence (also called a linker or linker sequence). Therefore, modified S proteins may contain an intervening peptide, which fuses CT or a portion of CT to the C-terminus of TM or a portion of the TM.

[0135] Heterogeneous CT, a portion of CT, or an intervening peptide sequence containing heterogeneous CT may be fused to amino acids in the C-terminal portion of the TM domain (e.g., within the first four amino acids of the C-terminal portion of the TM domain as defined in Table 1 with reference to SEQ ID NOs: 1, 2, 21, 114, 115, 160, or 161) or the N-terminal portion of the CT domain (e.g., within the first four amino acids of the N-terminal portion of the CT domain as defined in Table 1 with reference to SEQ ID NOs: 1, 2, 21, 114, 115, 160, or 161).

[0136] For example, coronavirus™ may terminate with an amino acid residue corresponding to any one of amino acids 1230-1238 of SEQ ID NO: 1. Therefore, the C-terminus of coronavirus™ may be an amino acid corresponding to any one of amino acids 1230-1238 of SEQ ID NO: 1. In one example, coronavirus™ may terminate with an amino acid residue corresponding to amino acid 1230 of SEQ ID NO: 1. In another example, TM may terminate with an amino acid residue corresponding to amino acid 1231 of SEQ ID NO: 1. In yet another example, TM may terminate with an amino acid residue corresponding to amino acid 1232 of SEQ ID NO: 1. In yet another example, TM may terminate with an amino acid residue corresponding to amino acid 1233 of SEQ ID NO: 1. In yet another example, TM may terminate with an amino acid residue corresponding to amino acid 1234 of SEQ ID NO: 1. In yet another example, TM may terminate with an amino acid residue corresponding to amino acid 1235 of SEQ ID NO: 1. In yet another example, TM may terminate with an amino acid residue corresponding to amino acid 1236 of SEQ ID NO: 1. In yet another example, TM may terminate with an amino acid residue corresponding to amino acid 1237 of SEQ ID NO: 1. In another example, TM may be terminated with an amino acid residue corresponding to amino acid 1238 of SEQ ID NO: 1. In a preferred embodiment, TM may be terminated with an amino acid residue corresponding to amino acid 1234 of SEQ ID NO: 1.

[0137] In another example, coronavirus™ or a portion of TM may terminate with an amino acid residue corresponding to any one of amino acids 1215-1219 of SEQ ID NO: 2 or 21. Therefore, the C-terminus of coronavirus™ or a portion of TM may be an amino acid corresponding to any one of amino acids 1215-1224 of SEQ ID NO: 2 or 21. In one example, coronavirus™ or a portion of TM may terminate with an amino acid residue corresponding to amino acid 1215 of SEQ ID NO: 2 or 21. In another example, TM or a portion of TM may terminate with an amino acid residue corresponding to amino acid 1216 of SEQ ID NO: 2 or 21. In yet another example, TM or a portion of TM may terminate with an amino acid residue corresponding to amino acid 1217 of SEQ ID NO: 2 or 21. In yet another example, TM or a portion of TM may terminate with an amino acid residue corresponding to amino acid 1218 of SEQ ID NO: 2 or 21. In yet another example, TM or a portion of TM may terminate with an amino acid residue corresponding to amino acid 1219 of SEQ ID NO: 2 or 21.

[0138] Intermediate peptide sequences that can fuse a heterologous CT to the C-terminus of the TM or a portion of the TM derived from the coronavirus S protein may have a length of 0 to 10 amino acids. Therefore, the intermediate peptide sequence may have a length of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. The intermediate peptide sequence may be derived from the coronavirus protein; for example, it may be derived from the C-terminus of the TM derived from the coronavirus S protein, or the N-terminus of the CT of the coronavirus S protein, or both. The intermediate peptide sequence may further be derived from the influenza HA protein; for example, it may be derived from the C-terminus of the TM derived from the influenza HA protein, or the N-terminus of the CT of the influenza HA protein, or both. Furthermore, the intermediate peptide sequence may be heterologous to the HA portion of the coronavirus and / or modified S protein, or the intermediate peptide sequence may be an artificial sequence.

[0139] A non-restrictive example of the TM / CT domain (also called chimeric TMCT) sequence of a modified S protein is shown below. The TM domain sequence from the coronavirus S protein is underlined, and the CT domain sequence from the influenza HA is shown in bold. The italicized and bold sequences are sequences derived from the TM of influenza HA. ​​The italicized and underlined sequences are sequences derived from the CT of the coronavirus S protein. [ka] [ka]

[0140] Modified coronavirus S protein may contain a chimeric TMCT. For example, a chimeric TMCT may contain an N-terminal sequence derived from coronavirus S protein and a C-terminal sequence derived from influenza HA protein, as shown in Table 1B. TM may contain the sequence shown in the column labeled "S protein TM sequence," and CT may contain the sequence shown in the column labeled "HA protein CT sequence." CT and TM may be linked by the sequences shown in the columns labeled "S protein CT sequence" and / or "HA protein TM sequence" (also referred to as intervening sequences or linkers, as further described below).

[0141] [Table 2]

[0142] For example, the N-terminal sequence derived from coronavirus S protein TM may include at least the following: - At least 19 amino acids corresponding to amino acids 1-19 of sequence numbers 18, 19, 37, 38, 39, 118, 119, 123, 124, 164, 165, 169, or 170; At least 20 amino acids corresponding to amino acids 1-20 of sequence numbers 18, 19, 37, 38, 39, 118, 119, 123, 124, 164, 165, 169, or 170; At least 21 amino acids corresponding to amino acids 1-21 of sequence numbers 18, 19, 37, 38, 39, 118, 119, 123, 124, 164, 165, 169, or 170; At least 22 amino acids corresponding to amino acids 1-22 of sequence numbers 18, 19, 37, 38, 39, 118, 119, 123, 124, 164, 165, 169, or 170; At least 23 amino acids corresponding to amino acids 1-23 of sequence numbers 18, 19, 37, 38, 39, 118, 119, 123, 124, 164, 165, 169, or 170; At least 24 amino acids corresponding to amino acids 1-24 of sequence numbers 18, 19, 37, 38, 39, 118, 119, 123, 124, 164, 165, 169, or 170.

[0143] The N-terminal sequence derived from coronavirus S protein™ may include at least 20 amino acids corresponding to amino acids 1-20 of SEQ ID NO: 18 or 169, or at least 21 amino acids corresponding to amino acids 1-21 of SEQ ID NO: 118 or 164, or at least 22 amino acids corresponding to amino acids 1-22 of SEQ ID NO: 123, and one or more amino acids from the C-terminus of influenza HA protein™. The N-terminal sequence derived from coronavirus S protein™ may include at least 20 amino acids corresponding to amino acids 1-20 of SEQ ID NO: 18 or 169, or at least 21 amino acids corresponding to amino acids 1-21 of SEQ ID NO: 118 or 164, or at least 22 amino acids corresponding to amino acids 1-22 of SEQ ID NO: 123, and one or more amino acids from the C-terminus of influenza HA protein™. The one or more amino acids from the C-terminus of influenza HA protein™ may include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. For example, the one or more amino acids may be 2, 3, or 4 amino acids. One or more amino acids from the C-terminus of the influenza HA protein™ may be A, C, G, L, S, M, W, or conserved substitutions of A, C, G, L, S, M, W, or combinations thereof. For example, one or more amino acids from the C-terminus of the influenza HA protein™ may be selected from AG or a conserved substitution of AG, AGL or a conserved substitution of AGL, MAGL or a conserved substitution of MAGL.

[0144] The modified coronavirus S protein may also contain a chimeric CT that includes an N-terminal sequence derived from the coronavirus S protein CT and a C-terminal sequence derived from the influenza HA protein CT.

[0145] The N-terminal sequence derived from the coronavirus S protein CT may contain one or more amino acids. The one or more amino acids from the N-terminus of the coronavirus S protein CT may consist of 0, 1, 2, 3, 4, or 5 amino acids. For example, the one or more amino acids may consist of 1, 2, or 3 amino acids. The one or more amino acids from the N-terminus of the coronavirus S protein CT may be C or M or conserved substitutions of C or M. For example, the one or more amino acids from the N-terminus of the coronavirus S protein may be selected from C or a conserved substitution of C, CC or a conserved substitution of CC, CCM or a conserved substitution of CCM.

[0146] The C-terminal sequence derived from influenza HA protein CT may contain at least 11 amino acids corresponding to amino acids 27-37 of SEQ ID NO: 18. The N-terminal sequence derived from influenza HA protein CT may further contain at least 12 amino acids corresponding to amino acids 26-37 of SEQ ID NO: 18, at least 13 amino acids corresponding to amino acids 25-37 of SEQ ID NO: 18, at least 14 amino acids corresponding to amino acids 24-37 of SEQ ID NO: 18, at least 15 amino acids corresponding to amino acids 23-37 of SEQ ID NO: 18, or at least 16 amino acids corresponding to amino acids 22-37 of SEQ ID NO: 18.

[0147] In another example, the C-terminal sequence derived from the influenza HA protein CT may include at least the following: - At least 11 amino acids corresponding to amino acids 27-37 of sequence numbers 126, 127, 128, 129, 130, or 131; - At least 12 amino acids corresponding to amino acids 26-37 of sequence numbers 126, 127, 128, 129, 130, or 131; - At least 13 amino acids corresponding to amino acids 25-37 of sequence numbers 126, 127, 128, 129, 130, or 131; - At least 14 amino acids corresponding to amino acids 24-37 of sequence numbers 126, 127, 128, 129, 130, or 131; - At least 15 amino acids corresponding to amino acids 23-37 of sequence numbers 126, 127, 128, 129, 130, or 131; or - At least 16 amino acids corresponding to amino acids 22-37 of sequence numbers 126, 127, 128, 129, 130, or 131.

[0148] For example, CT may include the sequences shown in Table 1B (HA protein CT sequences). For example, CT may include amino acids 22-37 of SEQ ID NOs. 18, 126, 128, 129, 130, 131, 118, 120, 164 or 166; or amino acids 25-40 of SEQ ID NOs. 19; or amino acids 24-39 of SEQ ID NOs. 37; or amino acids 25-36 of SEQ ID NOs. 38; or amino acids 24-34 of SEQ ID NOs. 39 or 119; or amino acids 22-36 of SEQ ID NOs. 127; or amino acids 22-37 of SEQ ID NOs. 118 or 164; or amino acids 23-38 of SEQ ID NOs. 123 or 125; or amino acids 25-35 of SEQ ID NOs. 124; or amino acids 24-34 of SEQ ID NOs. 165; or amino acids 21-36 of SEQ ID NOs. 169; or amino acids 23-33 of SEQ ID NOs. 170; or amino acids 21-36 of SEQ ID NOs.

[0149] The influenza CT or a portion of the CT may be fused to or linked to the TM or a portion of the TM of an S protein having an intervening peptide sequence. For example, the intervening peptide sequence may be derived from the influenza CT, the S protein TM, or a combination thereof, or the intervening peptide sequence may be an artificial sequence. The intervening peptide sequence may be of various lengths. For example, the intervening peptide sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long, preferably 2 to 8 amino acids long. In one example, the intervening peptide sequence is 2 amino acids long and may include, for example, the sequence LC. In another example, the intervening peptide sequence is 4 amino acids long and may include, for example, the sequence LCCM. In yet another example, the intervening peptide sequence may be 5 amino acids long and may include, for example, the sequence LSLWM. In yet another example, the intervening peptide sequence may be 7 amino acids long and may include, for example, the sequence AGLSLWM. In a further example, the intervening peptide sequence may be 8 amino acids long and may include, for example, the sequence MAGLSLWM.

[0150] For example, the TMCT of a modified S protein may contain the following sequences, or sequences with 90-100% or any amount of sequence identity between them, or sequences with sequence similarity to the following: -WYIWLGFIAGLIAIVMVTIM-(X)n-CSNGSXXCXICI(Sequence ID 64), -WYVWLGFIAGLIAIVMVTIL-(X)n-CSNGSXXCXICI(Sequence ID 134), or -WYIWLGFIAGLVALALCVFFIL-(X)n-CSNGSXXCXICI(Sequence ID 135) -WYVWLLICLAGVAMLVLLFFI-(X)n-CSNGSXXCXICI(Sequence ID 172), -WWVWLCISVVLIFVVSMLLL-(X)n-CSNGSXXCXICI(Sequence ID 173), In the formula, (X) nX is an intervening peptide sequence, which may have a length of 0 to n amino acid residues, where n can be any length of 0 to 10 amino acids, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and X may contain any amino acid, for example, X may be a conserved substitution of A, C, G, F, L, S, M, W, or A, C, G, F, L, S, M, W, or a combination thereof. Non-restrictive intervening peptide sequence (X) n This may include the following: -(X)0, no intervening peptide sequence present; -(X)1, including, for example, sequences L or C, or conserved substitutions of L or C; -(X)2, including, for example, the sequence LC, or a conserved substitution of LC; -(X)3, for example, including sequence LCC, or conserved substitutions of LCC; -(X)4, for example, including the sequence LCCM, or a preserved substitution of LCCM, SLWM or a preserved substitution of SLWM, SFWM or a preserved substitution of SFWM; -(X)5, for example, including array LSLWM, or a preserved substitution of LSLWM, LSLWM or a preserved substitution of LSLWM, LSFWM or a preserved substitution of LSFWM; -(X)6, for example, including the sequence GLSLWM, or a conserved substitution of GLSLWM; -(X)7, for example, including the sequence AGLSLWM, or a conserved substitution of AGLSLWM; or -(X)8, for example, including the array MAGLSLWM, or a preserved substitution of MAGLSLWM.

[0151] Figure 5B shows the multiplicative changes in protein accumulation of modified S proteins expressed in plants, comparing the protein accumulation of a reference modified S protein in which the cytoplasmic end of the coronavirus S protein is replaced with the coronavirus S protein of H5 A / Indonesia / 5 / 05 HA wtTM / H5iCT (SEQ ID NO: 18, product of construct 8671). The modified S proteins have a C-terminal region substitution version with a variable margin (intervening peptide sequence) between the SARS-CoV-2 transmembrane (TM) domain and the H5 A / Indonesia / 5 / 05 HA cytosolic terminal (CT) domain, wtTM / H5iCT V1 (SEQ ID NO: 19, product of construct 8980), wtTM / H5iCT V2 (SEQ ID NO: 37, product of construct 8981), wtTM / H5iCT V3 (SEQ ID NO: 38, product of construct #8982), and wtTM / H5iCT V4 (SEQ ID NO: 39, product of construct 8983). All modified S proteins tested showed protein accumulation, and there were no statistically significant differences between the alternative versions and the wtTM / H5iCT reference control.

[0152] Similarly, when expressed in plants, modified S proteins, including SARS-CoV-1 S protein with the wtTM / H5iCT V4 version of TMCT (Figure 16A) or MERS S protein with the wtTM / H5iCT V4 version of TMCT (Figure 19A), showed increased protein accumulation compared to wild-type S protein (wtTMCT) or S proteins in which TMCT was replaced with H5 A / Indonesia / 5 / 05 HA TMCT (H5iTMCT). Furthermore, when expressed in plants, OC43 CoV S protein with the wtTM / H5iCT V4 version of TMCT showed increased protein accumulation compared to OC43 CoV S protein with wild-type TMCT (wtTMCT) (Figure 23A).

[0153] Therefore, the modified S protein may contain TM and CT domains (TM / CT), where CT or a portion of CT is fused to the C-terminus of TM or a portion of TM via an intervening peptide sequence, and the intervening peptide sequence is sequence X nIncludes.

[0154] Furthermore, the modified S protein may contain TM and CT domains (TM / CT) that have sequence identity or sequence similarity of approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, with sequence SEQ ID NOs. 18, 19, 37, 38, 39, 64, 126, 127, 128, 129, 130, 131, 118, 119, 120, 123, 124, 125, 134, 135, 164, 165, 166, 169, 170, 171, 172, or 173.

[0155] The modified S protein may contain a CT or a portion of a CT that has sequence identity or sequence similarity of approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount between these, including amino acids 22-37 of SEQ ID NO: 18, amino acids 21-40 of SEQ ID NO: 19, amino acids 21-39 of SEQ ID NO: 37, amino acids 25-36 of SEQ ID NO: 38 or amino acids 24-34 of SEQ ID NO: 39, amino acids 22-37 of SEQ ID NO: 126, amino acids 22-36 of SEQ ID NO: 127, amino acids 22-37 of SEQ ID NO: 128, or amino acids 22-37 of SEQ ID NO: 129 or amino acids 22-37 of SEQ ID NO: 130, or amino acids 22-37 of SEQ ID NO: 131.

[0156] The modified S protein may contain TM or a portion of TM that has sequence identity or sequence similarity of approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, between amino acids 1-20 of SEQ ID NO: 18, amino acids 1-20 of SEQ ID NO: 19, amino acids 1-20 of SEQ ID NO: 37, amino acids 1-24 of SEQ ID NO: 118, amino acids 1-23 of SEQ ID NO: 119, amino acids 1-22 of SEQ ID NO: 123, amino acids 1-24 of SEQ ID NO: 124, amino acids 1-21 of SEQ ID NO: 164, amino acids 1-23 of SEQ ID NO: 165, amino acids 1-20 of SEQ ID NO: 169, or amino acids 1-22 of SEQ ID NO: 170. Furthermore, the modified S proteins described herein may include TM or a portion of TM that have 80% to 100% identity with the sequence of SEQ ID NO: 132 or 133.

[0157] Furthermore, the modified S protein may contain 70% to 100% sequence identity or similarity with the sequences of SEQ ID NOs. 5, 59, 60, 61, 62, 95, 96, 97, 108, 109, or 110. For example, the modified S protein may contain sequences having 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, sequence identity or similarity with the sequences of SEQ ID NOs. 5, 59, 60, 61, 62, 95, 96, 97, 108, 109, or 110.

[0158] For example, the cytoplasmic terminal domain (CT) or a portion of the CT of a viral structural protein such as the coronavirus S protein may be replaced with a CT or a portion of the CT derived from influenza hemagglutinin (HA), as described later, and the resulting protein is called a modified viral structural protein. Therefore, a coronavirus S protein in which the natural CT or a portion of the natural CT is replaced with a CT or a portion of the CT derived from HA may be called a modified coronavirus S protein or a modified S protein. Furthermore, as described above, the HA CT or a portion of the HA CT may be directly fused to the N-terminus of the coronavirus TM domain, or fused to the N-terminus of coronavirus TM or a portion of TM via an intervening peptide sequence. Therefore, the HA CT or a portion of the HA CT may be fused to the C-terminus of the S protein TM or a portion of the S protein TM via an intervening peptide sequence.

[0159] Influenza hemagglutinin, or HA, is a homotrimeric type I membrane glycoprotein that generally contains a signal peptide, an HA1 domain, and an HA2 domain with a transmembrane anchor site at the C-terminus and a small cytoplasmic end (see, e.g., Figures 1C and 2). The amino acid sequences of HA from various influenza strains are well known in the art. Furthermore, the amino acid and nucleotide sequences encoding HA are well known and available. See, for example, the BioDefence Public Health base (Influenza Virus; see URL:biohealthbase.org) or the National Center for Biotechnology Information (see URL:ncbi.nlm.nih.gov), both of which are incorporated herein by reference. Exemplary amino acid sequences of the cytoplasmic domains of HA from different influenza strains are shown in Figure 2.

[0160] Although different criteria and groups assign different lengths to the CT of HA, the N-terminal sequence of the CT is conserved among HAs from different influenza subtypes and strains, and it has been shown that at least five residues have sequence identity with at least 10 of the 13 HA subtypes (Simpson and Lamb 1992, Journal of Virology, 790-803). Figure 2 shows the alignment of the amino acid sequences from exemplary influenza strains with the conserved sequences of the N-terminal portion of the HA protein. The consensus sequence of the influenza cytoplasmic terminal (CT) domain is as follows: XXWMCSNGSXXCXICI (Sequence ID 15) (See also Figure 2, C-terminus of Sequence ID 14)

[0161] The CT sequence corresponding to the HA cytoplasmic terminal domain consensus sequence can be fused to the C-terminus of the TM of the coronavirus S protein, either directly or via an intervening peptide sequence (linker sequence), as described above.

[0162] Furthermore, amino acid residues located at the N-terminus or C-terminus of the natural influenza HA TM / CT boundary may also be included in the CT sequence that is fused directly or via an intervening peptide sequence to the TM or part of the TM of the modified coronavirus S protein.

[0163] Therefore, the CT sequence or a portion of the CT sequence may begin with an amino acid residue corresponding to any one of amino acids 30-40 of, for example, SEQ ID NO: 14. Thus, the N-terminus of a CT sequence may be an amino acid corresponding to any one of amino acids 30-40 of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 14. In one example, the CT sequence may begin with an amino acid residue corresponding to amino acid 30 of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 14. In another example, the CT sequence may begin with an amino acid residue corresponding to amino acid 31 of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 14. In yet another example, the CT sequence may begin with an amino acid residue corresponding to amino acid 32 of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 14. In yet another example, the CT sequence may begin with an amino acid residue corresponding to amino acid 33 of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 14. In further examples, the CT sequence may begin with an amino acid residue corresponding to amino acid 34 of SEQ ID NOs. 6, 7, 8, 9, 10, 11, 12, 13, or 14. In another example, the CT sequence may begin with an amino acid residue corresponding to amino acid 35 of SEQ ID NOs. 6, 7, 8, 9, 10, 11, 12, 13, or 14. In further examples, the CT sequence may begin with an amino acid residue corresponding to amino acid 36 of SEQ ID NOs. 6-13 or 14. In another example, the CT sequence may begin with an amino acid residue corresponding to amino acid 37 of SEQ ID NOs. 6, 7, 8, 9, 10, 11, 12, 13, or 14. In further examples, the CT sequence may begin with an amino acid residue corresponding to amino acid 38 of SEQ ID NOs. 6-13 or 14. In another example, the CT sequence may begin with an amino acid residue corresponding to amino acid 39 of SEQ ID NOs. 6, 7, 8, 9, 10, 11, 12, 13, or 14. In further examples, the CT sequence may begin with an amino acid residue corresponding to amino acid 40 of sequence numbers 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0164] The cytoplasmic end (CT) or portion of the CT of the modified S protein may be derived from the CT or portion of the CT of hemagglutinin (HA) of any one influenza type, subtype, or strain. For example, the CT may be derived from HA derived from influenza A or influenza B. For example, the CT may be derived from HA of influenza subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. For example, the CT may be derived from HA of subtypes H1, H2, H3, H5, H6, H7, or H9. Furthermore, the CT or portion of the CT may be derived from HA of influenza B. Influenza B may be derived from the B / Yamagata or B / Victoria lineage.

[0165] For example, the CT or part of the CT of a modified S protein may be derived from the CT of hemagglutinin (HA) influenza H1, H3, H5, H6, H7, H9, or B strains. Non-limiting examples of influenza strains from which HA CT may be derived include influenza H1 California / 7 / 2009, H2 A / Singapore / 1 / 1957, H3 A / Minnesota / 41 / 2019, H5 A / Indonesia / 5 / 05, H6 A / Teal / Hong Kong / W312 / 97, H7 A / Guangdong / 17SF003 / 2016, H9 A / Hong Kong / 1073 / 99, or B / Washington / 02 / 2019. Non-limiting examples of HA CT amino acid sequences are shown in Figure 2.

[0166] As shown in Figure 4A, when the native cytoplasmic end (CT) of the SARS-CoV-2 S protein was replaced with CTs derived from influenza HA H1 California / 7 / 2009 (H1 Calif), H3 A / Minnesota / 41 / 2019 (H3 Minn), H6 A / Teal / Hong Kong / W312 / 97 (H6 HK), H7 A / Guangdong / 17SF003 / 2016 (H7 Guan), H9 A / Hong Kong / 1073 / 99 (H9 HK), or B / Washington / 02 / 2019 (B Wash), similar multiplicative changes in protein accumulation were observed in these modified SARS-CoV-2 S proteins compared to SARS-CoV-2 S with CTs derived from H5 A / Indonesia / 5 / 05 (H5 Indo). These observations were confirmed by Western blot analysis (see Figures 4B and 4C).

[0167] Similar results were obtained when the native cytoplasmic end (CT) of the SARS-CoV-1 S protein, the native CT of the MERS S protein, or the native CT of the OC43 CoV S protein was replaced with CTs derived from influenza HA H1 California / 7 / 2009 (H1cCT) (see Figures 16A, 19A, and 23A).

[0168] Therefore, the cytoplasmic terminal domain (CT) or a portion of the CT consists of the sequence of SEQ ID NO: 15, or amino acids 30-50 of SEQ ID NOs: 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 31-50 of SEQ ID NOs: 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 32-50 of SEQ ID NOs: 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 33-50 of SEQ ID NOs: 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 34-50 of SEQ ID NOs: 6, 7, 8, Amino acids 35-50 of 9, 10, 12, 13, 14, or amino acids 36-50 of SEQ ID NOs. 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 37-50 of SEQ ID NOs. 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 38-50 of SEQ ID NOs. 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 39-50 of SEQ ID NOs. 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 40-50 of SEQ ID NOs. 6, 7, 8, 9, 10, 12, 13, 14, or amino acids 31-49 of SEQ ID NO. 1 The amino acids 32-49 of 1, or the amino acids 33-49 of SEQ ID NO: 11, or the amino acids 34-49 of SEQ ID NO: 11, or the amino acids 35-49 of SEQ ID NO: 11, or the amino acids 36-49 of SEQ ID NO: 11, or the amino acids 37-49 of SEQ ID NO: 11, or the amino acids 38-49 of SEQ ID NO: 11, or the amino acids 39-49 of SEQ ID NO: 11, or the amino acids 548-568 of SEQ ID NO: 3, or the amino acids 549-568 of SEQ ID NO: 3, or the amino acids 550-568 of SEQ ID NO: 3, or the amino acids 551-568 of SEQ ID NO: 3, Alternatively, amino acids 552-568 of SEQ ID NO: 3, or amino acids 553-568 of SEQ ID NO: 3, or amino acids 554-568 of SEQ ID NO: 3, or amino acids 555-568 of SEQ ID NO: 3, or amino acids 556-568 of SEQ ID NO: 3, or amino acids 557-568 of SEQ ID NO: 3, or amino acids 558-568 of SEQ ID NO: 3, may have sequence identity or similarity of approximately 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between.

[0169] Furthermore, the modified S protein may contain 70% to 100% sequence identity or similarity with the sequences of SEQ ID NOs. 5, 53, 54, 55, 56, 57, or 58. For example, the modified S protein may contain sequences having 70%, 75%, 80%, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, sequence identity or similarity with the sequences of SEQ ID NOs. 5, 53, 54, 55, 56, 57, or 58, or amino acids 25 to 1259 of SEQ ID NOs. 53, 54, 55, 55, 56, 57, or 58.

[0170] In further embodiments, the modified S protein external domain and / or transmembrane domain can be obtained from coronavirus S proteins other than the SARS-CoV-2 S protein, such as the SARS-CoV-1 S protein, MERS-CoV S protein, OC43-CoV S protein, 229E-CoV S protein, etc.

[0171] As shown in Figure 16A, when comparing the protein accumulation of S protein using wild-type TMCT (wt TMCT) or modified S protein using TMCT of influenza H5 HA derived from crude plant extracts (H5i TMCT), higher protein accumulation was observed for modified SARS-CoV-1 S protein using influenza H5 HA-derived CT (H5iCT), influenza H1 HA-derived CT (H1cCT), or influenza H5 HA-derived CT ("H5iCT(V4)") which has a variable margin between SARS-CoV-1 TM and H5 HA CT. The modified S protein assembled into a high molecular weight structure which was confirmed to be a VLP (see Figures 17A-17C) (see Figures 16B-16D). The amount of protein accumulation of SARS-CoV-1 protein with natural TMCT was below the detection limit of Western blot analysis when presented on the same gel as SARS-CoV-1 S protein with modified TMCT and / or CT (see Figure 16A). However, when only SARS-CoV-1 protein with natural TMCT was present on the gel, a signal could be detected by Western blot analysis (see Figure 16B), and VLPs were observed by electron microscopy (see Figure 17B).

[0172] Similar results were obtained for the modified MERS-CoV S protein (see Figure 19A). Compared to the protein accumulation of S proteins with wild-type TMCT (wt TMCT) or modified S proteins with influenza H5 HA TMCT (H5i TMCT), higher protein accumulation was observed for modified MERS-CoV S proteins with influenza H5 HA-derived CT (H5iCT), influenza H1 HA-derived CT (H1cCT), or influenza H5 HA-derived CT ("H5iCT(V4)") with a variable margin between MERS-CoV TM ​​and H5 HA CT (see the protein band at approximately 175 kDa). The highest accumulation was observed for modified MERS-CoV with influenza H1 HA CT (H1cCT). Smaller bands observed at approximately 100 kDa are most likely proteolytic cleavage fragments of the S protein. While we do not wish to be bound by theory, it is thought that substitution of the natural CT by influenza HA CT stabilizes the MERS S protein and reduces S protein cleavage.

[0173] As further shown in Figure 23A, low protein yields were observed in plants expressing OC43 CoV S protein with the natural OC43 CoV S protein TMCT. However, when the natural OC43 CoV S protein TMCT was replaced with influenza H5 HA-derived TMCT (H5iTMCT), influenza H5 HA-derived CT (H5iCT), influenza H1 HA-derived CT (H1cCT), or influenza H5 HA-derived CT ("H5iCT(V4)") which has a variable margin between OC43-CoV TM ​​and H5 HA CT, higher protein accumulation was observed compared to OC43 CoV S protein with the natural TMCT (see the band around 150 kDa; larger bands shown on the gel are thought to be protein trimers). Similar results were observed with the modified 229E-CoV S protein (data not shown).

[0174] Furthermore, MERS-CoV S proteins, OC43-CoV S proteins, and 229E-CoV S proteins possessing influenza H5 HA-derived TMCT (H5iTMCT), influenza H5 HA-derived CT (H5iCT), or influenza H1 HA-derived CT were observed to form VLPs, as shown in Figures 19B-19F, 23B-23E, and 25A-25E.

[0175] Accordingly, this disclosure provides a “modified viral structural protein,” a “viral structural fusion protein,” or a “chimeric viral structural protein,” wherein the external domain and transmembrane domain (TM) or part of the TM of the viral structural protein are derived from a coronavirus, and the cytosolic end (CT) or part of the CT is derived from an influenza protein. For example, the external domain and transmembrane domain may be derived from a coronavirus spike (S) protein, and the cytosolic end (CT) or part of the CT may be derived from an influenza HA protein. The modified S protein may, in order, include: i) an external domain derived from a coronavirus S protein (including the FP, HR1, and HR2 domains of the S1 subunit and the S2 subunit); ii) a coronavirus transmembrane domain (TM) or part of the coronavirus TM; and iii) an influenza HA cytoplasmic end domain (CT) or part of the HA CT. Accordingly, in the modified S protein, the CT or part of the CT is heterogeneous with respect to the TM and the external domain. Similarly, the TM (and external domain) of the modified S protein is heterogeneous with respect to the CT. The external domain and the transmembrane domain (TM) may originate from the same coronavirus (i.e., the external domain and TM may be homologous to each other), or the external domain may originate from a first coronavirus and the TM from a second coronavirus (i.e., the external domain and TM may be heterogeneous to each other).

[0176] A "chimeric protein" or "chimeric polypeptide," also called a "fusion protein," means a protein or polypeptide that is fused as a single polypeptide from two or more sources, for example, an external domain and transmembrane domain derived from a first viral structural protein derived from, for example, the coronavirus S protein, and an amino acid sequence derived from the cytoplasmic terminal (CT) of a second viral structural protein, for example, the CT derived from influenza HA.

[0177] Modified coronavirus S protein may include a transmembrane and cytosolic terminal domain (TMCT), where the TMCT is a chimeric TMCT. A chimeric TMCT may include a TMCT in which the transmembrane domain (TM) or part of the TM is derived from coronavirus S protein, and the cytosolic terminal (CT) or part of the CT is derived from influenza hemagglutinin (HA) protein. Furthermore, a chimeric TMCT may include natural coronavirus S protein TM, chimeric coronavirus S protein / influenza HA TM, natural influenza HA CT, chimeric influenza HA / coronavirus S protein CT, or a combination thereof. For example, a modified coronavirus S protein may include a chimeric TMCT having a natural influenza HA CT and a chimeric TM, where the chimeric TM includes an N-terminal sequence derived from the coronavirus S protein TM and a C-terminal sequence derived from the influenza HA protein TM. In another example, a modified coronavirus S protein may include a chimeric TMCT with natural coronavirus S protein TM and a chimeric CT, where the chimeric CT includes an N-terminal sequence derived from coronavirus S protein and a C-terminal sequence derived from influenza HA protein. In a further example, the modified coronavirus S protein may include a chimeric TMCT with a chimeric TM, where the chimeric TM comprises an N-terminal sequence derived from the TM of the coronavirus S protein and a C-terminal sequence derived from the TM of the influenza HA protein, and the chimeric CT comprises an N-terminal sequence derived from the coronavirus S protein and a C-terminal sequence derived from the influenza HA protein.

[0178] In this disclosure, when a modified S protein or modified coronavirus spike (S) protein is referred to, it means a modified coronavirus spike (S) protein comprising the transmembrane domain (TM) or a portion thereof of the S protein TM and the cytosolic end (CT) or a portion thereof, wherein the CT is derived from the influenza hemagglutinin (HA) protein and the TM is heterogeneous to the CT.

[0179] The modified S protein contains 70% to 100% sequence identity or similarity to the sequences of SEQ ID NOs. 5, 21, 30, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 95, 96, 97, 108, 109, 110, 144, 145, 146, 155, 156, or 157. For example, the modified S protein contains 70% to 100% sequence identity or similarity to the sequences of SEQ ID NOs. 5, 21, 30, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 95, 96, The sequences 97, 108, 109, 110, 144, 145, 146, 155, 156 or 157, or amino acids 25-1259 of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 Amino acids 25-1259, amino acids 25-1259 of SEQ ID NO: 57, amino acids 25-1259 of SEQ ID NO: 58, amino acids 25-1262 of SEQ ID NO: 59, amino acids 25-1261 of SEQ ID NO: 60, amino acids 25-1258 of SEQ ID NO: 61, amino acids 25-1256 of SEQ ID NO: 62, amino acids 25-1243 of SEQ ID NO: 95, amino acids 25-1240 of SEQ ID NO: 96, amino acids 25-1243 of SEQ ID NO: 97, amino acids 25-1341 of SEQ ID NO: 108, amino acids 25-1338 of SEQ ID NO: 109, SEQ ID NO: The sequence may contain amino acids 25-1341 of 110, amino acids 25-1351 of SEQ ID NO: 144, amino acids 25-1348 of SEQ ID NO: 145, amino acids 25-1351 of SEQ ID NO: 146, amino acids 25-1159 of SEQ ID NO: 155, amino acids 25-1156 of SEQ ID NO: 156, or amino acids 25-1159 of SEQ ID NO: 157, and sequences having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount of sequence identity or similarity between these.

[0180] The modified S protein may be further produced or synthesized as a modified S protein precursor (also called a precursor S protein), which comprises the modified S protein and a signal peptide, the signal peptide being either natural to coronavirus (i.e., a homolog to the external domain) or unnatural or heterologous to the external domain. In non-limiting examples, the natural signal peptide may be replaced with a signal peptide derived from protein disulfide isomerase (PDI).

[0181] The modified S protein precursor may contain a signal peptide that is unnatural or heterologous to the external domain. The unnatural signal peptide may replace the entire natural signal peptide or a portion of the natural signal peptide of the coronavirus S protein. Furthermore, the unnatural or heterologous signal peptide may be directly fused to the N-terminus of the modified S protein, or it may be fused to the N-terminus of the modified S protein using an intervening peptide sequence.

[0182] Signal peptides (also called signal sequences, targeting signals, localization signals, localization sequences, transport peptides, leader sequences, or leader peptides) are short peptides located at the N-terminus of most newly synthesized proteins destined for the secretory pathway. Signal peptides are involved in the targeting of proteins to the inner membrane system, including the endoplasmic reticulum and Golgi apparatus, and are removed by co-translation by signal peptidases located in the ER lumen, generating mature proteins. Because experimental methods for identifying targeting sequences are time-consuming and cumbersome, different computational approaches to predict targeting signals have been developed and are well known in the art. Signal peptides generally have low sequence similarity but share several characteristic properties. To predict signal sequences and their cleavage sites, many prediction methods have been developed that take these features into account, such as SignalP (Bendtsen et al., J Mol Biol. 2004 Jul 16;340(4):783-95.; Petersen et al., Nature Methods volume 8, pages 785-786 (2011)), Signal-CF (Chou and Shen, Biochem Biophys Res Commun. 2007 Jun 8;357(3):633-40), and Signal-BLAST (Frank and Sippl, Bioinformatics, 2008 Oct 1;24(19):2172-6), which are incorporated herein by reference.

[0183] The SignalP prediction program is used to predict the signal peptide cleavage site of the SARS-CoV-2 S protein between positions 15 and 16 of the sequence corresponding to sequence SEQ ID NO: 1. However, the signal peptide cleavage site of the SARS-CoV-2 S protein may be predicted or occur between other consecutive positions of the sequence corresponding to sequence SEQ ID NO: 1. For example, the signal peptide cleavage site of the SARS-CoV-2 S protein may also be predicted or occur between positions 13 and 14 of the sequence corresponding to sequence SEQ ID NO: 1.

[0184] The N-terminal region of the native SARS-CoV-2 S protein (including the native signal peptide sequence) is shown below. MFVFLVLLPLVSSQC VNLTTRTQLPPAYTNS (SEQ ID NO: 63)

[0185] Underline the predicted signal peptide sequence (SP). The sequences shaded in gray correspond to the sequences shown in Table 2. The first amino acid residue of the mature SARS-CoV-2 S protein can be valine (V) having that position named 1 (+1), which corresponds to V16 of the precursor S protein (native SARS-CoV-2 S protein with native signal peptide). The first amino acid residue of the mature SARS-CoV-2 S protein can be other residues of SEQ ID NO: 1 or SEQ ID NO: 63 shown in Table 2. For example, the first amino acid residue of the mature SARS-CoV-2 S protein can be glutamine (Q) having that position named 14 (-2).

Table 3

[0186] Signal peptides or peptide sequences for directing the localization of an expressed protein or polypeptide to the apoplast include native (protein-related) signals or leader sequences, or heterologous signal sequences, such as, but not limited to, the rice amylase signal peptide (McCormick 1999, Proc Natl Acad Sci USA 96:703-708) or the protein disulfide isomerase signal peptide (PDI). Thus, as described herein, the modified S protein can be produced as a precursor protein comprising the modified S protein and a heterologous amino acid signal peptide sequence. For example, the modified S protein precursor can include a signal peptide derived from protein disulfide isomerase (PDI SP; nucleotides 32-103 of accession number Z11499).

[0187] Thus, the present disclosure also provides a modified S protein precursor comprising a modified S protein and a native or non-native signal peptide, and a nucleic acid encoding such a protein.

[0188] The modified viral structural protein can be a modified S protein, and the modified S protein can be a monomeric or single-stranded modified S protein. The monomeric or single-stranded modified S protein can comprise an S1 domain (subunit) and an S2 domain (subunit), and the S2 domain (subunit) is modified to replace the native CT of the S protein with the CT of the influenza HA protein, and the modified S protein is a single continuous polypeptide chain. The monomeric or single-stranded modified S protein can trimerize to form a trimer called a trimeric modified S protein. A trimer is a macromolecular complex formed by three, usually non-covalently bound, proteins.

[0189] The S protein is cleaved at a conserved activation cleavage site into two polypeptide chains, an S1 subunit and an S2 subunit, which remain associated as an S1 / S2 protomer within the homotrimer. Without wishing to be bound by theory, cleavage of the S protein into subunits may be important for viral infectivity, but may not be essential for protein trimerization.

[0190] The modified S protein can further comprise one or more substitutions, replacements or mutations. For example, the modified S protein can comprise one or more substitutions, replacements or mutations in an external domain to increase expression, yield and stability, or to increase the expression, yield, stability of the modified S protein in a suitable expression system.

[0191] For example, the modified S protein can comprise substitutions or mutations at the S1 / S2 and / or S2' protease cleavage sites to prevent protease cleavage at these sites. Thus, when produced in a host or host cell, the modified S protein is not cleaved into separate S1 and S2 subunits or polypeptide chains.

[0192] Modified viral structural proteins, such as modified S proteins, can be further assembled into trimers of modified viral structural proteins. Therefore, coronavirus protein trimers containing modified S proteins, as described herein, are further provided. The trimer may contain a single-stranded modified S protein, which comprises an S1 subunit and an S2 subunit, wherein the CT of the S2 subunit is replaced with the CT of influenza hemagglutinin (HA).

[0193] The trimer can be further stabilized in the prefusion conformation. Therefore, modified viral structural proteins, such as the modified S protein, may further include one or more substitutions, substitutions, or mutations to inhibit the conformational change of the S protein from the prefusion conformation to the post-fusion conformation, thereby stabilizing the S protein or the S protein trimer in the prefusion conformation.

[0194] "Amino acid substitution" or "substitution" means replacing an amino acid in the amino acid sequence of a protein with a different amino acid. The terms amino acid, amino acid residue, or residue are used interchangeably in this disclosure. One or more amino acids may be replaced or substituted with one or more amino acids different from the original amino acid or wild-type amino acid at that position without changing the entire length of the amino acid sequence of the protein.

[0195] For example, modified viral structural proteins such as modified S proteins can be stabilized by proline substitutions, substitutions that enable the formation of disulfide bonds and salt crosslinks, and / or cavity-filling substitutions.

[0196] Hsieh et al. (incorporated herein by reference, Science 2020, 369 pp. 1501-1505) designed and expressed various SARS-CoV-2 spike protein mutants in mammalian cells. A S protein mutant with six proline substitutions, called HexaPro, was expressed 9.8 times more highly than the S protein mutant with only double proline substitutions, exhibited an approximately 5°C increase in Tm, and retained the trimer prefusion conformation in mammalian cell lines. The HexaPro mutant is considered by Hsieh et al. to be the best mutant.

[0197] In this disclosure, the highest yield was observed with a combination of four proline substitutions ("4P") corresponding to positions 802, 927, 971, and 972 of SEQ ID NO: 2, and an additional single amino acid substitution at position 923. Furthermore, a higher yield was also observed with a combination of six proline substitutions ("6P") corresponding to positions 802, 877, 884, 927, 971, and 972, and a further single amino acid substitution at position 923.

[0198] As provided herein, the modified S protein may further comprise one or more substitutions, substitutions, or mutations for increasing the stability, yield, or stability and yield of the modified protein in a host or cost cell, such as a plant or plant cell.

[0199] The modified S proteins described herein may include one or more mutations, modifications, or substitutions in the amino acid sequence of any one or more amino acids corresponding to the amino acids in the reference sequences described below.

[0200] The terms "corresponds to an amino acid," "corresponds to an amino acid," or "corresponds to a sequence" mean that an amino acid (or nucleotide) corresponds to an amino acid (or nucleotide) in a sequence alignment with the reference coronavirus sequence described below. The corresponding amino acid position in the coronavirus sequence can be determined by alignment of the coronavirus S protein to a known sequence. Sequence alignment methods for comparison are well known in the art and are described further below. Examples of corresponding amino acids are shown in Table 3.

[0201] [Table 4]

[0202] For example, a modified S protein may have one or more (e.g., two consecutive) proline substitutions at or near the boundary between the HR1 domain and the central helix domain that stabilize the S external domain trimer in a prefusion conformation, as described in International Publication No. 2018 / 081318, incorporated herein by reference. Furthermore, one or more substitutions may restrict and / or prevent processing or cleavage at the cleavage site between the S1 and S2 subunits.

[0203] The modified S protein may contain one or more substitutions at the positions shown in Table 3. For example, the modified S protein may contain one or more substitutions at positions 667, 668, 670, 802, 877, 884, 923, 927, 971, 972, or combinations thereof, of the reference sequence of SEQ ID NO: 2 (SARS-CoV-2). The corresponding positions in the S proteins of SARS-CoV-1, MERS-CoV, OC43-CoV, and 229E-CoV are shown in Table 3. The corresponding amino acid positions in the S proteins of other coronaviruses can be determined by methods known in the art.

[0204] GSAS-2P (971 and 972) For example, the modified S protein may have one or more substitutions in one or more amino acids corresponding to the amino acids at positions 667, 668, 670, 971, or 972 of the amino acid sequence of SEQ ID NO: 2.

[0205] In one embodiment, the modified S protein may include substitutions, modifications, or mutations corresponding to positions 667, 668, 670, or combinations thereof (numbered according to Sequence ID No. 2). For example, the amino acid corresponding to position 667 may be a substitution of glycine (G) or conserved glycine (G), the amino acid corresponding to position 668 may be a substitution of serine (S) or conserved serine (S), and the amino acid corresponding to position 670 may be a substitution of serine (S) or conserved serine (S).

[0206] The modified S protein may further include substitutions, modifications, or mutations corresponding to positions 971, 972, or positions 971 and 972 (numbered according to Sequence ID No. 2). For example, the amino acids corresponding to positions 971 and / or 972 may be proline (P) substitutions or conserved proline (P) substitutions.

[0207] The modified S protein may contain one or more substitutions, where one or more substitutions include, or consist of, one or more amino acid substitutions corresponding to the amino acids at positions 667, 668, 670, 971, and 972 of SEQ ID NO: 2. The modified S protein having one or more substitutions may be stabilized by prefusion confirmation. Furthermore, the modified S protein may form a trimer stabilized by prefusion confirmation.

[0208] For example, the modified S protein may include the following substitutions (numbering according to SEQ ID NO: 2): R667G, R668S, R670S (referred to herein as "GSAS"). The modified S protein may also have the following substitutions (numbering according to SEQ ID NO: 2): K971P and V972P (referred to herein as "2P"). Further, the modified S protein may have the following substitutions (numbering according to SEQ ID NO: 2): R667G, R668S, R670S, K971P and V972P (referred to herein as "GSAS-2P").

[0209] For example, the modified S protein may have an amino acid sequence of the sequence of SEQ ID NO: 47 or amino acids 25 to 1259 of SEQ ID NO: 47 and an amino acid sequence having about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount between them of sequence identity or sequence similarity, where the amino acid sequence has a glycine (G) or a conserved substitution of glycine (G) at position 667, a serine (S) or a conserved substitution of serine (S) at position 668, a serine (S) or a conserved substitution of serine (S) at position 670, and a proline (P) or a conserved substitution of proline (P) at positions 971 and 972, and where the modified S protein forms VLPs when expressed.

[0210] In another example, the modified S protein may have one or more substitutions in one or more amino acids corresponding to the amino acids at positions 654, 955 or 956 of the amino acid sequence of SEQ ID NO: 114 or at positions 730, 733, 1043 or 1044 of the amino acid sequence of SEQ ID NO: 115.

[0211] For example, the modified S protein may contain the following substitutions: R654A (numbered according to SEQ ID NO: 114) or R730A and / or R733G (numbered according to SEQ ID NO: 115). The modified S protein may also have the following substitutions: K955P and / or V956P (numbered according to SEQ ID NO: 114) or V1043P and / or L1044P (numbered according to SEQ ID NO: 115). Furthermore, the modified S protein may have the following substitutions: R654A, K955P and V956P (numbered according to SEQ ID NO: 114) or R730A, R733G, V1043P, L1044P (numbered according to SEQ ID NO: 115).

[0212] GSAS-4P (802, 927, 971, and 972) The modified S protein may further have amino acid substitutions corresponding to the amino acids at positions 667, 668, and 670, and one or more further substitutions (numbered by SEQ ID NO: 2) at one or more residues corresponding to positions 802, 927, 971, and 972. For example, the amino acids corresponding to positions 802, 927, 971, and 972 may be proline (P) substitutions or conserved proline (P) substitutions.

[0213] As shown in Figure 11A, the modified S protein with the "GSAS" modification and the following modifications: F802P, A927P, K971P, V972P (referred to as "GSAS-4P" and expressed from construct 8953) showed a 2.47-fold increase in yield of the modified S protein compared to the yield of the "GSAS-2P" S protein (expressed from construct 8671).

[0214] Therefore, the modified S protein may contain one or more substitutions, where one or more substitutions include or consist of one or more amino acid substitutions corresponding to the amino acids at positions 667, 668, 670, 802, 927, 971 and 972 in SEQ ID NO: 2.

[0215] For example, a modified S protein may have an amino acid sequence having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount of sequence identity or similarity between the amino acid sequence of SEQ ID NO: 48 or amino acids 25-1259 of SEQ ID NO: 48, where the amino acid sequence has glycine (G) or a conserved substitution of glycine (G) at position 667, serine (S) or a conserved substitution of serine (S) at position 668, serine (S) or a conserved substitution of serine (S) at position 670, and proline (P) or a conserved substitution of proline (P) at positions 802, 927, 971, and 972, where the modified S protein forms a VLP when expressed.

[0216] In another example, the modified S protein may have one or more substitutions in one or more amino acids corresponding to amino acids at positions 654, 786, 911, 955, or 956 of the amino acid sequence of SEQ ID NO: 114, or at positions 730, 733, 872, 999, 1043, or 1044 of the amino acid sequence of SEQ ID NO: 115.

[0217] For example, the modified S protein may contain the following substitutions: R654A (numbered according to SEQ ID NO: 114) or R730A and / or R733G (numbered according to SEQ ID NO: 115). The modified S protein may also have the following substitutions: F786P, S911P, K955P and / or V956P (numbered according to SEQ ID NO: 114) or A872P, N999P, V1043P and / or L1044P (numbered according to SEQ ID NO: 115). Furthermore, the modified S protein may have the following substitutions: R654A, F786P, S911P, K955P and V956P (numbered according to SEQ ID NO: 114) or R730A, R733G, A872P, N999P, V1043P, L1044P (numbered according to SEQ ID NO: 115).

[0218] GSAS-6P (802, 877, 884, 927, 971 and 972) The modified S protein may further have amino acid substitutions corresponding to the amino acids at positions 667, 668, and 670, and one or more further substitutions (numbered by SEQ ID NO: 2) at one or more residues corresponding to positions 802, 877, 884, 927, 971, and 972. For example, the amino acids corresponding to positions 802, 877, 884, 927, 971, and 972 may be proline (P) substitutions or conserved proline (P) substitutions (numbered by SEQ ID NO: 2).

[0219] As shown in Figure 11A, the modified S proteins with the "GSAS" modification and the following modifications: F802P, A877P, A884P, A927P, K971P, V972P (referred to as "GSAS-6P" and expressed from construct 8940) showed a 2.11-fold increase in S protein yield compared to the "GSAS-2P" S protein (expressed from construct 8671).

[0220] Therefore, the modified S protein may contain one or more substitutions, where one or more substitutions include or consist of one or more amino acid substitutions corresponding to the amino acids at positions 667, 668, 670, 802, 877, 884, 927, 971, and 972 in SEQ ID NO: 2.

[0221] For example, a modified S protein may have an amino acid sequence having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount of sequence identity or similarity between these, where the amino acid sequence has a conserved substitution of glycine (G) or glycine (G) at position 667, a conserved substitution of serine (S) or serine (S) at position 668, a conserved substitution of serine (S) or serine (S) at position 670, and a conserved substitution of proline (P) or proline (P) at positions 802, 802, 877, 884, 927, 971, and 972, where the modified S protein forms a VLP when expressed.

[0222] In another example, the modified S protein may have one or more substitutions in one or more amino acids corresponding to amino acids at positions 654, 786, 861, 868, 911, 955, or 956 in the amino acid sequence of SEQ ID NO: 114, or at positions 730, 733, 872, 949, 956, 999, 1043, or 1044 in the amino acid sequence of SEQ ID NO: 115.

[0223] For example, the modified S protein may contain the following substitutions: R654A (numbered by SEQ ID NO: 114) or R730A and / or R733G (numbered by SEQ ID NO: 115). The modified S protein may also have the following substitutions: F786P, A861P, A868P, S911P, K955P and / or V956P (numbered by SEQ ID NO: 114) or A872P, S949P, A956P, N999P, V1043P and / or L1044P (numbered by SEQ ID NO: 115). Furthermore, the modified S protein may have the following substitutions: R654A, F786P, A861P, A868P, S911P, K955P and V956P (numbering according to sequence number 114) or R730A, R733G, A872P, S949P, A956P, N999P, V1043P and L1044P (numbering according to sequence number 115).

[0224] Replacement at position 923 The modified S proteins described herein may further include substitutions, modifications, or mutations corresponding to position 923 (numbered by SEQ ID NO: 2). For example, the amino acid corresponding to position 923 may be a substitution of phenylalanine (F) or a conserved substitution of phenylalanine (F).

[0225] As shown in Figure 11B, the modified S protein with the "GSAS-2P" modification and L923F substitution (expressed from construct 8933) showed a 1.36-fold increase in yield compared to the "GSAS-2P" S protein without the L923F substitution (expressed from construct 8671). The modified S protein with the "GSAS-4P" modification and L923F substitution (expressed from construct 8960) showed a 2.88-fold increase in yield compared to the "GSAS-2P" S protein without the L923F substitution (expressed from construct 8671). The modified S protein with the "GSAS-6P" modification and L923F substitution (expressed from construct 8947) showed a 2.47-fold increase in yield compared to the "GSAS-2P" S protein without the L923F substitution (expressed from construct 8671).

[0226] Therefore, the modified S protein may contain one or more substitutions, one or more of which are amino acid substitutions corresponding to amino acids at positions 667, 668, 670, 927, 971, 972, 802, 877, 884, 923, or combinations thereof, in SEQ ID NO: 2. For example, a modified S protein may contain one or more substitutions, one or more of which are amino acid substitutions corresponding to the amino acids at positions 667, 668, 670, 971, 972, 923 or combinations thereof in SEQ ID NO: 2 (GSAS-2P-923), or 667, 668, 670, 927, 971, 972, 802, 923 or combinations thereof in SEQ ID NO: 2 (GSAS-4P-923), or 667, 668, 670, 927, 971, 972, 802, 877, 884, 923 or combinations thereof in SEQ ID NO: 2 (GSAS-6P-923).

[0227] For example, a modified S protein may contain one or more substitutions, each of which may consist of one or more substitutions of amino acids corresponding to the amino acids at the position. - Sequence ID 2, 667, 668, 670, 971, 972 and 923 (GSAS-2P-923), - Sequence numbers 667, 668, 670, 927, 971, 972, 802 and 923 of Sequence ID No. 2 (GSAS-4P-923), or - Sequence numbers 667, 668, 670, 927, 971, 972, 802, 877, 884 and 923 of sequence number 2 (GSAS-6P-923).

[0228] For example, the modified S protein has the amino acid sequence of SEQ ID NO: 50 or amino acids 25-1259 of SEQ ID NO: 50 (the amino acid sequence has a conserved substitution of glycine (G) or glycine (G) at position 667, a conserved substitution of serine (S) or serine (S) at positions 668 and 670, a conserved substitution of proline (P) or proline (P) at positions 971 and 972, and a conserved substitution of phenylalanine (F) or phenylalanine (F) at position 923) and SEQ ID NO: 51 or amino acids 25-1259 of SEQ ID NO: 51 (the amino acid sequence has a conserved substitution of glycine (G) or glycine (G) at position 667, a conserved substitution of serine (S) or serine (S) at positions 668 and 670, a conserved substitution of proline (P) or proline (P) at positions 927, 971, 972 and 802, and at position 923 The modified S protein may have an amino acid sequence having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount of sequence identity or similarity between these, and when expressed, the modified S protein forms a VLP.

[0229] Therefore, modified coronavirus S proteins that may include the following are provided: 1. Chimeric transmembrane domain and cytoplasmic terminal domain (TMCT); 2. One or more substitutions corresponding to amino acids at positions 667, 668 and / or 670 (numbered according to SEQ ID NO: 2) when compared to the corresponding wild-type coronavirus S protein; 3. One or more substitutions corresponding to amino acids at positions 802, 877, 884, 927, 971 and / or 972 (numbered according to SEQ ID NO: 2) when compared to the corresponding wild-type coronavirus S protein; 4. Substitution at position 923 (numbered according to SEQ ID NO: 2) when compared to the corresponding wild-type coronavirus S protein; 5. Or any combination of the modifications and / or substitutions described in 1-4.

[0230] As used herein, the terms “conserved substitution” or “conservative substitution” and its grammatical variations refer to amino acids of the same class as the described substitution or residue, but distinct from the reference amino acid (substitution) (i.e., nonpolar residues substituting nonpolar residues, aromatic residues substituting aromatic residues, polar uncharged residues substituting polar uncharged residues, and charged residues substituting charged residues). Further information on conserved substitutions can be found, for example, in Sahin-Toth et al. (Protein ScL, 3:240-247, 1994), Hochuli et al. (Bio / Technology, 6:1321-1325, 1988), Henikoff S, and Henikoff JG (Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992), and in widely used genetics and molecular biology textbooks.

[0231] Modified viral structural proteins can be further glycosylated. Coronavirus S protein, coronavirus M protein, and coronavirus E protein are glycosylated, with both N-linked and O-linked glycosylation occurring.

[0232] Modified viral structural proteins may contain glycosylation patterns specific to the host or host cell in which the modified viral structural protein is expressed. For example, when expressed in a plant or plant cell, the modified viral structural protein may contain plant-specific N-glycans. Therefore, modified viral structural proteins having plant-specific N-glycans are also provided.

[0233] As described herein, the cytosolic terminal domain (CT) of the modified viral structural protein may be replaced with a CT derived from influenza hemagglutinin (HA). The external domain and transmembrane domain (TM) of the above viral structural protein are fused to the influenza HA cytosolic terminal domain (CT) such that the CT is heterogeneous with respect to the external domain and transmembrane domain of the viral structural protein, such as the S protein. The modified S protein may self-assemble to form a virus-like particle (VLP).

[0234] Therefore, this specification further relates to virus-like particles (VLPs). More specifically, this specification relates to VLPs containing modified viral structural proteins such as modified S protein, and to methods for generating VLPs in a host or host cell using modified viral structural proteins such as modified S protein. The VLPs include modified viral structural proteins such as modified S protein as described herein.

[0235] As shown in Figures 6C, 17A, 17B, and 17C, the modified viral structural proteins are exemplified by modified S proteins (modified SARS-CoV-2 or modified SARS-CoV-1 S proteins), and the natural or wild-type CT is replaced by a CT derived from the influenza HA protein that self-assembles into VLPs when expressed in plants. The VLPs are similar to those produced in the same plant expression system using S proteins with natural TM / CT sequences (see Figures 6A and 17A) or modified S proteins with H5 influenza TM / CT sequences (see Figures 6B and 17B).

[0236] Furthermore, as shown in Figures 6D, 6E, 6F, and 6G, modified S proteins having a variable margin or boundary (intervening peptide sequence) between the TM domain and the influenza CT domain also self-assemble into VLPs when expressed in plants.

[0237] Furthermore, as shown in Figures 6H, 6I, 6J, 6K, 6L, and 6M, modified S proteins in which the natural or wild-type CT is replaced by CT derived from influenza HA proteins from H1, H3, H6, H7, H9, and B influenza, respectively, also self-assemble into VLPs when expressed in plants.

[0238] Furthermore, as shown in Figures 19B-19F, 23B-23E, and 25A-25E, the modified S proteins derived from MERS-CoV, OC43-CoV, and 229E-CoV possessed influenza H5 HA-derived TMCT (H5iTMCT), influenza H5 HA-derived CT (H5iCT), or influenza H1 HA-derived CT, and also formed VLPs.

[0239] The term "virus-like particle (VLP)" or "VLPs" refers to a virus-like structure that is generally morphologically and antigenically similar to a virion produced in infection but lacks sufficient genetic information to replicate and is therefore non-infectious. A VLP is a self-assembling structure containing one or more structural proteins, such as a modified viral structural protein, such as a modified S protein, but not limited to modified S proteins. Thus, a VLP may contain a modified S protein. A VLP may further contain viral structural proteins, which consist of modified S proteins. Thus, in some embodiments, a VLP may lack or not contain coronavirus M protein and / or coronavirus E protein. Thus, in some embodiments, a VLP produced from a modified viral structural protein described herein does not contain coronavirus M protein, coronavirus E protein, or coronavirus M protein and coronavirus E protein. Furthermore, in some embodiments, the VLP does not contain structural or non-structural proteins derived from viruses that are heterologous to the Coronaviridae family or influenza viruses. For example, the VLP does not contain structural or non-structural proteins derived from viruses that do not belong to the Coronaviridae family.

[0240] In another embodiment, the VLP may comprise coronavirus E protein, coronavirus M protein, and modified coronavirus S protein. In another embodiment, the VLP may comprise coronavirus E protein and modified coronavirus S protein. In another embodiment, the VLP may comprise coronavirus M protein and modified coronavirus S protein. Furthermore, the VLP may comprise coronavirus E protein, modified coronavirus M protein, and modified coronavirus S protein. The VLP may further comprise modified coronavirus E protein, modified coronavirus M protein, and modified coronavirus S protein. In another embodiment, the VLP may comprise modified coronavirus E protein and modified coronavirus S protein. In another embodiment, the VLP may comprise modified coronavirus M protein and modified coronavirus S protein.

[0241] VLPs can be produced in suitable hosts or host cells, including plants and plant cells. After extraction from the host or host cells, VLPs can be recovered as intact structures during isolation and further purification under appropriate conditions.

[0242] VLPs can be purified or extracted using any suitable method, such as chemical or biochemical extraction. VLPs are relatively sensitive to drying, heat, pH, surfactants, and detergents. Therefore, it may be useful to use a method that maximizes yield, minimizes contamination of the VLP fraction with cellular proteins, maintains the integrity of the protein or VLP, and, if necessary, relaxes the relevant lipid envelope or membrane, cell wall, and releases the protein or VLP. Minimizing or eliminating the use of detergents or surfactants such as SDS or Triton® X-100 may be beneficial to improve the yield of VLP extraction. The VLPs can then be evaluated for structure and size, for example, by electron microscopy (see Figure 4B) or by size exclusion chromatography.

[0243] In the case of enveloped viruses such as coronaviruses, it may be advantageous for the lipid layer or membrane to be retained by the virus. The composition of the lipids may vary depending on the system (for example, plant-produced enveloped viruses may contain plant lipids or plant sterols in their envelope), and may contribute to an improved immune response.

[0244] Therefore, VLPs produced in a host or host cell may contain lipids from the host or host cell's plasma membrane. For example, VLPs produced in plants may contain plant-derived lipids ("plant lipids"), VLPs produced in insect cells may contain lipids derived from the insect cell's plasma membrane (generally called "insect lipids"), and VLPs produced in mammalian cells may contain lipids derived from the mammalian cell's plasma membrane (generally called "mammalian lipids").

[0245] Plant lipids or plant-derived lipids may be in the form of a lipid bilayer and may further include an envelope surrounding the VLP. Plant-derived lipids may include lipid components of the plasma membrane of the plant from which the VLP is produced, including phospholipids, tri-, di-, and monoglycerides, and lipid-soluble sterols or sterol-containing metabolites. Examples include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol, phosphatidylserine, sphingoglycolipids, plant sterols, or combinations thereof. Examples of plant sterols include campesterol, stigmasterol, ergosterol, brassicasterol, δ-7-stigmasterol, δ-7-avenasterol, daunosterol, sitosterol, 24-methylcholesterol, cholesterol, or beta-sitosterol. As those skilled in the art will understand, the lipid composition of the plasma membrane of a cell may vary depending on the culture or growth conditions of the cell or organism or species from which the cell is obtained. Generally, beta-sitosterol is the most abundant plant sterol.

[0246] While we do not wish to be bound by theory, plant-derived VLPs containing plant-based lipids may induce a stronger immune response than VLPs produced using other manufacturing systems, and the immune response induced by these plant-derived VLPs may be stronger than the immune response induced by live or attenuated whole-virus vaccines.

[0247] Furthermore, in addition to the potential adjuvant effect of plant lipids, the ability of plant N-glycans to promote the capture of glycoprotein antigens by antigen-presenting cells may be advantageous for VLP production in plants.

[0248] VLPs produced within plants may contain modified viral structural proteins containing plant-specific N-glycans. Therefore, this disclosure also provides VLPs containing modified viral structural proteins having plant-specific N-glycans. Furthermore, VLPs comprising plant lipids and modified viral structural proteins having plant-specific N-glycans are provided.

[0249] A method for producing virus-like particles (VLPs) containing a modified structural protein in a host or host cell is also provided. Furthermore, a method for increasing the yield of VLP production containing a modified structural protein in a host or host cell is also provided. The method comprises introducing a nucleic acid containing a sequence encoding the modified structural protein into a host or host cell, and incubating the host or host cell under conditions that enable nucleic acid expression, thereby producing VLPs. The modified viral structural protein can be produced in a higher yield compared to a host or host cell expressing an unmodified viral structural protein.

[0250] For example, as shown in Figure 3A, the yield of VLPs expressed in plants may increase if the cytoplasmic end (CT) of the viral structural protein is replaced with the CT of influenza HA to produce a modified viral structural protein, such as a modified S protein. As further shown in Figures 11A and 11B, if the modified S protein further includes one or more substitutions, one or more of which include or consist of amino acid substitutions corresponding to the amino acids at positions 667, 668, 670, 802, 923, 927, 971 and / or 972 of SEQ ID NO: 2, the yield of VLPs containing the modified S protein when expressed in plants may increase further.

[0251] The yield of a modified viral structural protein (e.g., modified S protein) or the yield of a VLP containing a modified viral structural protein produced in a host or host cells, such as a plant or plant cells, may increase by 1.1 to 10 times, or any amount in between, compared to the yield of the corresponding unmodified viral structural protein or the yield of a VLP containing the corresponding unmodified viral structural protein. Compared to the yield of the corresponding unmodified viral structural protein or VLP containing the corresponding unmodified viral structural protein when produced in the host or host cells under the same conditions, for example, the yield of the modified viral structural protein (e.g., modified S protein) or the yield of the VLP (containing the modified viral structural protein) in the host or host cells is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3. 7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 times or any amount in between.

[0252] The modified viral structural proteins described herein are the amino acid sequences of SEQ ID NOs: 1, 2, 5, 21, 30, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 95, 96, 97, 108, 109, 110, 112, 113, 114, 115, 144, 145, 146, 155, 156, 157, 158, 159, 160, or 161, or amino acids 25-1259 of SEQ ID NO: 47, amino acids 25-1259 of SEQ ID NO: 48 , amino acids 25-1259 of SEQ ID NO: 49, amino acids 25-1259 of SEQ ID NO: 50, amino acids 25-1259 of SEQ ID NO: 51, amino acids 25-1259 of SEQ ID NO: 52, amino acids 25-1259 of SEQ ID NO: 53, amino acids 25-1259 of SEQ ID NO: 54, amino acids 25-1259 of SEQ ID NO: 55, amino acids 25-1259 of SEQ ID NO: 56, amino acids 25-1259 of SEQ ID NO: 57, amino acids 25-1259 of SEQ ID NO: 58, amino acids 25-1262 of SEQ ID NO: 59, SEQ ID NO: 6 Amino acids 25-1261 for SEQ ID NO: 0, 25-1258 for SEQ ID NO: 61, 25-1256 for SEQ ID NO: 62, 25-1243 for SEQ ID NO: 95, 25-1240 for SEQ ID NO: 96, 25-1243 for SEQ ID NO: 97, 25-1341 for SEQ ID NO: 108, 25-1338 for SEQ ID NO: 109, 25-1341 for SEQ ID NO: 110, 25-1351 for SEQ ID NO: 144, 25-1348 for SEQ ID NO: 145, SEQ ID NO: 14 The modified S protein comprises amino acid sequences having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between sequence identity or similarity between amino acids 25-1351 of SEQ ID NO: 6, amino acids 25-1159 of SEQ ID NO: 155, amino acids 25-1156 of SEQ ID NO: 156, or amino acids 25-1159 of SEQ ID NO: 157, and an amino acid sequence having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, sequence identity or similarity, wherein the modified S protein forms a VLP when expressed in a host or host cell.The amino acid sequences of the external domain and transmembrane domain of the modified S protein are as follows: amino acids 1-1234 of SEQ ID NO: 1, amino acids 1-1219 of SEQ ID NO: 2, amino acids 1-1234 of SEQ ID NO: 5, amino acids 1-1219 of SEQ ID NO: 21, amino acids 1-1243 of SEQ ID NO: 30, amino acids 25-1243 of SEQ ID NO: 47, amino acids 25-1243 of SEQ ID NO: 48, amino acids 25-1243 of SEQ ID NO: 49, amino acids 25-1243 of SEQ ID NO: 50, amino acids 25-1243 of SEQ ID NO: 51, amino acids 25-1243 of SEQ ID NO: 52, and amino acids 1-1243 of SEQ ID NO: 53. Acids 25-1243, amino acids 25-1243 of SEQ ID NO: 54, amino acids 25-1243 of SEQ ID NO: 55, amino acids 25-1243 of SEQ ID NO: 56, amino acids 25-1243 of SEQ ID NO: 57, amino acids 25-1243 of SEQ ID NO: 58, amino acids 25-1242 of SEQ ID NO: 59, amino acids 25-1242 of SEQ ID NO: 60, amino acids 25-1246 of SEQ ID NO: 61, or amino acids 25-1245 of SEQ ID NO: 62, amino acids 25-1227 of SEQ ID NO: 95, amino acids 25-1227 of SEQ ID NO: 96, amino acids 25-1227 of SEQ ID NO: 97, SEQ ID NO: 1 Amino acids 25-1325 of 08, amino acids 25-1325 of SEQ ID NO: 109, amino acids 25-1325 of SEQ ID NO: 110, amino acids 25-1335 of SEQ ID NO: 144, amino acids 25-1335 of SEQ ID NO: 145, amino acids 25-1335 of SEQ ID NO: 146, amino acids 25-1143 of SEQ ID NO: 155, amino acids 25-1143 of SEQ ID NO: 156, or amino acids 25-1143 of SEQ ID NO: 157, and approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between. The modified S protein has either identity or sequence similarity, and the amino acid sequence of the cytoplasmic terminal domain (CT) of the modified S protein has approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, sequence identity or sequence similarity with the sequence of SEQ ID NO: 15, amino acids 35-50 of SEQ ID NOs: 6, 8, 7, 9, 10, 12, 13, 14, amino acids 34-49 of SEQ ID NO: 11, or amino acids 553-568 of SEQ ID NO: 3, and the modified S protein forms VLPs when expressed in a host or host cells.

[0253] Furthermore, the modified viral structural protein may be encoded by a nucleotide sequence of SEQ ID NOs: 22, 26, 29, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 90, 91, 92, 95, 96, 97, 103, 104, 105, 139, 140, 141, 150, 151, or 152, and a nucleotide sequence having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, where the nucleotide sequence encodes a modified S protein that forms a VLP when expressed in a host or host cell.

[0254] Further provided are nucleotide sequences encoding a modified S protein having an amino acid sequence of SEQ ID NOs: 1, 2, 5, 21, 30, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 95, 96, 97, 108, 109, 110, 144, 145, 146, 155, 156 or 157 and an amino acid sequence having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, sequence identity or sequence similarity, wherein the modified S protein forms a VLP when expressed in a host or host cell.The nucleotide sequences are as follows: amino acids 1-1234 for SEQ ID NO: 1, amino acids 1-1219 for SEQ ID NO: 2, amino acids 1-1234 for SEQ ID NO: 5, amino acids 1-1219 for SEQ ID NO: 21, amino acids 1-1243 for SEQ ID NO: 30, amino acids 25-1243 for SEQ ID NO: 47, amino acids 25-1243 for SEQ ID NO: 48, amino acids 25-1243 for SEQ ID NO: 49, amino acids 25-1243 for SEQ ID NO: 50, amino acids 25-1243 for SEQ ID NO: 51, amino acids 25-1243 for SEQ ID NO: 52, amino acids 25-1243 for SEQ ID NO: 53, and amino acids 25-12 for SEQ ID NO: 54. 43, amino acids 25-1243 of SEQ ID NO: 55, amino acids 25-1243 of SEQ ID NO: 56, amino acids 25-1243 of SEQ ID NO: 57, amino acids 25-1243 of SEQ ID NO: 58, amino acids 25-1242 of SEQ ID NO: 59, amino acids 25-1242 of SEQ ID NO: 60, amino acids 25-1246 of SEQ ID NO: 61, amino acids 25-1245 of SEQ ID NO: 62, amino acids 25-1227 of SEQ ID NO: 95, amino acids 25-1227 of SEQ ID NO: 96, amino acids 25-1227 of SEQ ID NO: 97, amino acids 25-1325 of SEQ ID NO: 108, amino acids 25-109 The external domain and membrane of a modified S protein having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount between these, sequence identity or similarity between amino acids 1325, 25-1325 of SEQ ID NO: 110, 25-1335 of SEQ ID NO: 144, 25-1335 of SEQ ID NO: 145, 25-1335 of SEQ ID NO: 146, 25-1143 of SEQ ID NO: 155, 25-1143 of SEQ ID NO: 156, or 25-1143 of SEQ ID NO: 157, and the external domain and membrane of the modified S protein having sequence identity or similarity of approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount between these. The amino acid sequence of the transtransforward domain may be encoded, and the amino acid sequence of the cytoplasmic terminal domain of the modified S protein has approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount between them of sequence identity or similarity with the sequence of SEQ ID NO: 15, amino acids 35-50 of SEQ ID NOs: 6, 8, 7, 9, 10, 12, 13, 14, amino acids 34-49 of SEQ ID NO: 11, or amino acids 553-568 of SEQ ID NO: 3, and the modified S protein forms VLPs when expressed in a host or host cells.

[0255] The amino acid sequences of SEQ ID NOs. 5, 21, 30, or 47-62, or amino acids 24-1259 of SEQ ID NOs. 47, amino acids 25-1259 of SEQ ID NOs. 48, amino acids 25-1259 of SEQ ID NOs. 49, amino acids 25-1259 of SEQ ID NOs. 50, amino acids 25-1259 of SEQ ID NOs. 51, amino acids 25-1259 of SEQ ID NOs. 52, amino acids 25-1259 of SEQ ID NOs. 53, amino acids 25-1259 of SEQ ID NOs. 54, Amino acids 25-1259 of sequence number 55, amino acids 25-1259 of sequence number 56, amino acids 25-1259 of sequence number 57, amino acids 25-1259 of sequence number 58, amino acids 25-1262 of sequence number 59, amino acids 25-1261 of sequence number 60, amino acids 25-1258 of sequence number 61, amino acids 25-1256 of sequence number 62, amino acids 25-1243 of sequence number 95, amino acids 2 of sequence number 96 Further provided are nucleotide sequences encoding a modified S protein having an amino acid sequence with approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, sequence identity or sequence similarity between amino acids 5-1240, 25-1243 of SEQ ID NO: 97, 25-1341 of SEQ ID NO: 108, 25-1338 of SEQ ID NO: 109, 25-1341 of SEQ ID NO: 110, 25-1351 of SEQ ID NO: 144, 25-1348 of SEQ ID NO: 145, 25-1351 of SEQ ID NO: 146, 25-1159 of SEQ ID NO: 155, 25-1156 of SEQ ID NO: 156, or 25-1159 of SEQ ID NO: 157, and an amino acid sequence having approximately 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount in between, sequence identity or sequence similarity between amino acids 5-1240, 25-1243 of SEQ ID NO: 97, 25-1341 of SEQ ID NO: 108, 25-1338 of SEQ ID NO: 109, 25-1341 of SEQ ID NO: 110, 25-1351 of SEQ ID NO: 144, 25-1348 of SEQ ID NO: 145, 25-135

[0256] The terms “similarity percentage,” “sequence similarity,” “identity percentage,” or “sequence identity” are used when referring to specific sequences, for example, as described in the University of Wisconsin GCG software program, or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 supplement). Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved, for example, using the Smith & Waterman algorithm (1981, Adv. Appl. Math. 2:482), the Needleman & Wunsch alignment algorithm (1970, J. Mol. Biol. 48:443), the Pearson & Lipman similarity search method (1988, Proc. Natl. Acad. Sci. USA 85:2444), or by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA, the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.).

[0257] Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST2.0 algorithms described in Altschul et al., (1977, Nuc. Acids Res. 25:3389-3402) and Altschul et al., (1990, J. Mol. Biol. 215:403-410), respectively. BLAST and BLAST2.0, along with the parameters described herein, are used to determine the sequence identity percentage of nucleic acids and proteins in this disclosure. For example, the BLASTN program (for nucleotide sequences) may use, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program can use, by default, a word length of 3, an expected value (E) of 10, a BLOSUM62 score matrix (Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915), alignment (B) of 50, an expected value (E) of 10, M=5, N=-4, and a comparison of both strands. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (see URL: ncbi.nlm.nih.gov / ).

[0258] Nucleic acid sequences or nucleotide sequences referred to herein may be “substantially homologous,” “substantially similar,” or “substantially identical” to a sequence or a complement of a sequence if the nucleic acid sequence or nucleotide sequence hybridizes with one or more nucleotide sequences or complements of a nucleic acid sequence or nucleotide sequence as defined herein under strict hybridization conditions. A sequence is “substantially homologous,” “substantially similar,” or “substantially identical” if at least about 70%, or 70–100%, or any amount in between, of the nucleotides, e.g., 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100%, or any amount in between, matches over a defined length of the nucleotide sequence, provided that such homologous sequence exhibits one or more of the characteristics of the sequence or encoded product described herein.

[0259] Many organisms exhibit a bias in the use of specific codons to encode particular amino acid insertions in growing peptide chains. Codon priority, or codon bias, which is the difference in codon usage frequency between organisms, is caused by the degeneracy of the genetic code and is well documented across many organisms. Codon bias often correlates with the translation efficiency of messenger RNA (mRNA), which is thought to depend, among other things, on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Therefore, based on codon optimization, genes can be tuned for optimal gene expression in a given organism. The process of optimizing the nucleotide sequence encoding a heterologously expressed protein can be a crucial step in improving expression yield. Optimization requirements may include steps to improve the host's ability to produce the foreign protein.

[0260] There are various codon optimization techniques known in the art to improve the translation kinetics of translationally inefficient protein-coding regions. These techniques primarily rely on identifying the codon usage frequency of a particular host organism. If a particular gene or sequence is to be expressed in this organism, the coding sequence of such gene and sequence is modified to replace the codons of the target sequence with codons that are more frequently used in the host organism.

[0261] "Codon optimization" is defined as modifying a nucleic acid sequence by replacing at least one, two or more, or a significant number of, codons in the native sequence with codons that are more or most frequently used in the genes of another organism or species, in order to enhance expression in a target host or host cell. Different species exhibit specific biases for specific codons of specific amino acids.

[0262] This disclosure includes codon-optimized synthetic polynucleotide sequences, for example, synthetic polynucleotide sequences optimized for human codon use frequency or plant codon use frequency. The codon-optimized polynucleotide sequences can then be expressed in a host, such as a plant. More specifically, sequences optimized for human codon use frequency or plant codon use frequency can be expressed in plants. While we do not wish to be constrained by theory, it is thought that sequences optimized for human codons, when plants are used as hosts, will increase the guanine-cytosine (GC) content of the sequence and improve expression yield.

[0263] As used herein, the terms “construct,” “vector,” or “expression vector” refer to recombinant nucleic acids used to transfer an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding a modified viral structural protein described herein) into a host cell (e.g., a plant cell) and to direct the expression of the exogenous nucleic acid sequence in the host cell. “Expression cassette” refers to a nucleic acid containing the nucleotide sequence of interest, operably (or actionably) ligated thereto, under the control of an appropriate promoter or other regulatory element for the transcription of the nucleic acid of interest in the host cell. As those skilled in the art will understand, an expression cassette may include a terminator sequence that is any sequence active in the host cell (e.g., a plant host). For example, in plants, the terminator sequence may be derived from a bipartite RNA virus, e.g., a comovirus RNA-2 genomic segment, the terminator sequence may be an NOS terminator, or the terminator sequence may be obtained from the 3'UTR of an alfalfa plastocyanin gene.

[0264] The nucleic acid comprising the nucleotide sequence encoding the modified viral structural protein described herein may further comprise sequences that enhance the expression of the viral structural protein in a host, a part of a host, or a host cell. The expression-enhancing sequences may comprise a 5'UTR enhancer element or a plant-derived expression enhancer operably associated with the nucleic acid encoding the modified viral structural protein. The sequence encoding the modified viral structural protein may also be optimized to increase expression, for example, by optimizing human codon usage frequency, increasing GC content, or a combination thereof.

[0265] The terms “regulatory region,” “regulatory element,” or “promoter” typically refer to a portion of nucleic acid upstream of the protein-coding region of a gene, which may, but not always, consist of either DNA or RNA, or both DNA and RNA. If a regulatory region is active and operationally related to or operationally linked to a nucleotide sequence of interest, it can result in the expression of that nucleotide sequence. Regulatory elements may mediate organ specificity or control developmental or temporal gene activation. “Regulatory regions” include promoter elements, core promoter elements exhibiting basal promoter activity, elements that can be induced in response to external stimuli, elements that mediate promoter activity, such as negative regulatory elements or transcriptional enhancers. As used herein, “regulatory regions” also include post-transcriptionally active elements, such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability determinants, which regulate gene expression. Some of these latter elements may be located proximal to the coding region.

[0266] In the context of this disclosure, the terms “regulatory element” or “regulatory region” typically refer to a sequence of DNA upstream (5') of the coding sequence of a structural gene, which regulates the expression of the coding region by providing recognition for RNA polymerase and / or other factors necessary for transcription to begin at a particular site. However, it should be understood that other nucleotide sequences located within introns or within the 3' of the sequence may also contribute to the regulation of the expression of the coding region of interest. An example of a regulatory element that provides recognition for RNA polymerase or other transcription factors to ensure initiation at a particular site is a promoter element. Most, though not all, eukaryotic promoter elements contain a TATA box, which is a conserved nucleic acid sequence consisting of adenosine and thymidine nucleotide base pairs, typically located approximately 25 base pairs upstream of the transcription start site. Promoter elements may include basic promoter elements responsible for transcription initiation, as well as other regulatory elements that modify gene expression.

[0267] Several types of regulatory regions exist, including developmentally regulated, inducible, and constitutive. Regulatory regions that are developmentally regulated or that control differential gene expression under their control are activated within an organ or tissue at a specific point in time during its development. However, some developmentally regulated regulatory regions may be preferentially active within a specific organ or tissue at a particular developmental stage, and may be active in a developmentally regulated manner, or similarly at a basal level, in other organs or tissues within the plant. Examples of tissue-specific regulatory regions include the napine promoter and the curciferin promoter (Rask et al., 1998, J. Plant Physiol. 152:595-599; Bilodeau et al., 1994, Plant Cell 14:125-130). An example of a leaf-specific promoter is the plastocyanin promoter (see U.S. Patent No. 7,125,978, incorporated herein by reference).

[0268] An inducible regulatory region is capable of directly or indirectly activating the transcription of one or more DNA sequences or genes in response to an inducer. In the absence of the inducer, the DNA sequences or genes are not transcribed. Typically, a protein factor that specifically binds to the inducible regulatory region and activates transcription may exist in an inactive form and then be directly or indirectly converted to an active form by the inducer. However, the protein factor may not be present. The inducer may be a chemical substance such as a protein, metabolite, growth regulator, herbicide, or phenolic compound, or a physiological stress imposed directly by heat, cold, salt, or toxic elements, or indirectly through the action of a pathogen such as a virus or disease agent. Plant cells containing an inducible regulatory region may be exposed to the inducer by external application of the inducer to the cell or plant, such as by spraying, watering, heating, or similar methods. Inducible regulatory elements may originate from either plant or non-plant genes (e.g., Gatz, C. and Lenk, IRP, 1998, Trends Plant Sci. 3, 352-358). Examples of potential inducible promoters include, but are not limited to, tetracycline-inducible promoters (Gatz, C., 1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 89-108), steroid-inducible promoters (Aoyama, T. and Chua, NH, 1997, Plant J. 2, 397-404), and ethanol-inducible promoters (Salter, MG, et al, 1998, Plant Journal 16, 127-132; Caddick, MX, et al, 1998, Nature Biotech. 16, 177-180), as well as cytochanin-inducible IB6 and CKI1 genes (Brandstatter, I. and Kieber, JJ, 1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science). Examples include 274, 982-985) and the auxin-inducible element DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971).

[0269] Constitutive regulatory regions sequentially direct gene expression across various parts of the plant and throughout plant development. Examples of known constitutive regulatory elements include the CaMV 35S transcript (p35S; Odell et al., 1985, Nature, 313:810-812; incorporated herein by reference), rice actin 1 (Zhang et al., 1991, Plant Cell, 3:1155-1165), actin 2 (An et al., 1996, Plant J., 10:107-121) or tms2 (U.S. Patent No. 5,428,147), and the triose phosphate isomerase 1 gene (Xu et al., 1994, Plant Physiol. 106:459-467), and the maize ubiquitin 1 gene (Cornejo et al., 1993, Plant Examples include the ubiquitin 1 and 6 genes in Arabidopsis (Moltorf et al, 1995, Plant Mol. Biol. 29:637-646), the tobacco translation initiation factor 4A gene (Mandel et al, 1995, Plant Mol. Biol. 29:995-1004), the cassava vein mosaic virus promoter, pCAS (Verdaguer et al., 1996); the promoter of the small subunit of ribulose diphosphate carboxylase, pRbcS (Outchkourov et al., 2003); and promoters associated with pUbi (in monocots and dicots).

[0270] As used herein, the term “constituent” does not necessarily mean that nucleotide sequences under the control of a constitutive regulatory region are expressed at the same level in all cell types, but rather that the sequences are expressed in a wide range of cell types, despite frequent fluctuations in their abundance.

[0271] One or more gene constructs of this disclosure may also include further enhancers of either translational or transcriptional enhancers, as may be specified. The enhancers may be located at 5' or 3' relative to the sequence being transcribed. Enhancer regions are well known to those skilled in the art and may include an ATG start codon, an adjacent sequence, etc. The start codon may be homeophase with the reading frame ("in-frame") of the coding sequence, if present, to provide correct translation of the transcribed sequence.

[0272] The terms "5'UTR" or "5' untranslated region," "5' leader sequence," or "5'UTR enhancer element" refer to a region of mRNA that is not translated. The 5'UTR typically begins at the transcription start site and ends immediately before the start codon of the translation start site or coding region. The 5'UTR can regulate the stability and / or translation of mRNA transcripts.

[0273] As used herein, the term “plant-derived expression enhancer” refers to a nucleotide sequence derived from a plant that encodes the 5'UTR. Examples of plant-derived expression enhancers are incorporated herein by reference in U.S. Provisional Patent Application No. 62 / 643,053 (filed March 14, 2018) and International Patent Application No. PCT / CA2019 / 050319 (filed March 14, 2019), or incorporated herein by reference in Diamos AGet al. (2016, Front Plt This is described in Sci.7:1-15. Plant-derived expression enhancers may be selected from nbEPI42, nbSNS46, nbCSY65, nbHEL40, nbSEP44, nbMT78, nbATL75, nbDJ46, nbCHP79, nbEN42, atHSP69, atGRP62, atPK65, atRP46, nb30S72, nbGT61, nbPV55, nbPPI43, nbPM64, and nbH2A86, as described in U.S. Patent Application Publication No. 62 / 643,053 and PCT / CA2019 / 050319. Plant-derived expression enhancers may be used in a plant expression system that includes a plant-derived expression enhancer sequence and a regulatory region operably linked to the nucleotide sequence of interest, such as the nucleotide sequence encoding the modified S protein.

[0274] RNA stability and / or translation efficiency can be further improved by including a 3' untranslated region (3'UTR). Therefore, one or more gene constructs described herein may further include a 3'UTR.

[0275] The 3' untranslated region may contain polyadenylation signals and any other regulatory signals that can perform mRNA processing or gene expression. Polyadenylation signals are typically characterized by the addition of a polyadenylate track to the 3' end of the mRNA precursor. Polyadenylation signals are generally recognized by the presence of homology to the canonical form 5'AATAAA-3', although variability is not uncommon. Appropriate non-limiting examples of the 3' region include plant genes such as Agrobacterium tumorigenic (Ti) plasmid genes and soybean storage protein genes, such as nopalin synthase (Nos gene), the small subunit of the ribulose-1,5-bisphosphate carboxylase gene (ssRUBISCO; U.S. Patent No. 4,962,028 incorporated herein by reference), promoters used to regulate plastocyanin expression as described in U.S. Patent No. 7,125,978 (incorporated herein by reference), 3'UTR derived from Arracacha virus B isolation gene (AvB) (SEQ ID NO: 40), 3'UTR derived from Beet necrotic yellow vein virus (trBNYVV) (SEQ ID NO: 41), 3'UTR derived from Southern bean mosaic virus (SBMV) (SEQ ID NO: 42), and Turnip ringspot virus. The 3'UTR contains a polyadenylation signal from the following 3'UTRs: TuRSV (SEQ ID NO: 43), Cowpea Mosaic Virus (CPMV) (SEQ ID NO: 44), Broad bean true mosaic virus (BBTMV) (SEQ ID NO: 45), or Ourmia melon virus (trOUMV) (SEQ ID NO: 46). The 3'UTR may be used in conjunction with a heterologous 5'UTR to regulate expression levels.

[0276] Therefore, a “construct,” “vector,” “expression vector,” or “expression cassette” is provided, which comprises a nucleic acid containing a target nucleotide sequence (such as a modified viral structural protein) that is under the control of the 3'UTR and operably (or capable of acting) ligated to the 3'UTR. Furthermore, the nucleic acid may contain a 3'UTR operably (or capable of acting) ligated to the target nucleotide sequence (such as a modified viral structural protein).

[0277] Modified viral structural proteins may, as desired, target any intracellular or extracellular space, organelle, or tissue of a host cell, such as a plant or plant cell. To localize the expressed protein to a specific location, the nucleic acid encoding the protein can be ligated to a nucleic acid sequence encoding a signal peptide or a leader sequence. The signal peptide may also be called a transport peptide, signal sequence, leader sequence, targeting signal, localization signal, localization sequence, transport peptide, or leader peptide.

[0278] One or more of the modified gene constructs of this specification may be expressed in any suitable host or host cell transformed by the nucleic acids, nucleotide sequences, constructs, or vectors of this disclosure. The host or host cell may originate from any source, including plants, fungi, bacteria, insects, and animals, such as mammals. Thus, the host or host cell may be selected from plants or plant cells, fungi or fungal cells, bacteria or bacterial cells, insects or insect cells, and animals or animal cells. The mammal or animal may not be human. In a preferred embodiment, the host or host cell is a plant, a plant, or a part of a plant cell.

[0279] As used herein, the terms “plant,” “plant part,” “plant component,” “plant substance,” “plant biomass,” “plant material,” “plant extract,” or “plant leaf” may include whole plants, tissues, cells, or any fraction thereof, intracellular plant components, extracellular plant components, plant liquid or solid extracts, or combinations thereof, which can provide transcription, translation, and post-translational mechanisms for the expression of one or more nucleic acids described herein, and / or which expressed proteins or VLPs can be extracted and purified. Plants may include, but are not limited to, herbaceous plants. Herbaceous plants may be annual, biennial, or perennial plants. Plants include, for example, canola, Brassica genus, maize, Nicotiana genus (nicotiana spp.), (tobacco), for example Nicotiana benthamiana, Nicotiana rustica, Nicotiana, tabacum, Nicotiana alata, Arabidopsis thaliana, alfalfa, for example potato, sweet potato (Ipomoea batatus), ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton, maize, rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum This may also include, but is not limited to, crops such as vulgare and safflower (Carthamus tinctorius).

[0280] As used herein, the term “plant part” refers to any part of a plant, including but not limited to leaves, stems, roots, flowers, fruits, plant cells obtained from leaves, stems, roots, flowers, fruits, plant extracts obtained from leaves, stems, roots, flowers, fruits, or combinations thereof. In one embodiment, a plant part refers to, for example, an area portion of a plant such as leaves, stems, flowers, and fruits. As used herein, the term “plant extract” refers to a plant-derived product obtained after treating a plant, a part of a plant, plant cells, or a combination thereof physically (e.g., by freezing and then extraction in a suitable buffer), mechanically (e.g., by grinding or homogenizing the plant or part of a plant and then extraction in a suitable buffer), enzymatically (e.g., using cell wall-degrading enzymes), chemically (e.g., using one or more chelating agents or buffers), or a combination thereof. Plant extracts can be further processed to remove undesirable plant components, such as cell wall residues. Plant extracts may be obtained to aid in the recovery of one or more components from plants, parts of plants or plant cells, such as proteins (including protein complexes, protein surface structures and / or VLPs), nucleic acids, lipids, carbohydrates, or combinations thereof from plants, parts of plants or plant cells. If a plant extract contains proteins, it may be called a protein extract. A protein extract may be a crude plant extract, a partially purified plant or protein extract, or a purified product, containing one or more proteins, protein complexes, such as protein trimers, protein superstructures, and / or VLPs from plant tissue. If desired, protein extracts or plant extracts may be partially purified using techniques known to those skilled in the art, for example, the extract may be subjected to salt or pH precipitation, centrifugation, gradient density centrifugation, filtration, chromatography, such as size exclusion chromatography, ion exchange chromatography, affinity chromatography, or a combination thereof. Protein extracts may also be purified using techniques known to those skilled in the art.

[0281] The constructs disclosed herein can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For a review of such techniques, see, for example, Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. DT. Dennis, DH. Turpin, DD. Lefebvre, DB. Layzell (eds), Addison Wesly, Langmans Ltd., London, pp. 561-579 (1997). Other methods include direct DNA incorporation, the use of liposomes, such as electroporation using protoplasts, microinjection, microprojectiles or whiskers, and vacuum filtration.For example, Bilang, et al. (Gene 100:247-250(1991), Scheid et al. (Mol. Gen. Genet. 228:104-112, 1991), Guerche et al. (Plant Science 52:111-116, 1987), Neuhause et al. Genet.75:30-36,1987), Klein et al.,Nature 327:70-73(1987);Howell et al.(Science 208:1265,1980),Horsch et al.(Science 227:1229-1231,1985),DeBlock et al.,Plant Physiology 91:694-701,1989), Methods for Plant Molecular Biology(Weissbach and See Weissbach, eds., Academic Press Inc., 1988), Methods in Plant Molecular Biology (Schuler and Zielinski, eds., Academic Press Inc., 1989), Liu and Lomonossoff (J. Virol Meth, 105:343-348, 2002), U.S. Patent Nos. 4,945,050; 5,036,006; and 5,100,792, U.S. Patent Application No. 08 / 438,666 (filed May 10, 1995), and U.S. Patent Application No. 07 / 951,715 (filed September 25, 1992) (all of which are incorporated herein by reference).

[0282] The constructs of this disclosure can be expressed using transient expression methods as described below (see Liu and Lomonossoff, 2002, Journal of Virological Methods, 105:343-348, incorporated herein by reference). Alternatively, a vacuum-based transient expression method, such as that described by Kapila et al. 1997 (incorporated herein by reference), may be used. These methods may include, for example, agroinoculation or agroinfiltration, syringe infiltration, but other transient methods as described above may also be used. By agroinoculation, agroinfiltration, or syringe infiltration, a mixture of agrobacteria containing the desired nucleic acid enters the intercellular space of tissue, e.g., leaves, aerial parts of a plant (including stems, leaves, and flowers), other parts of a plant (stem, roots, flowers), or the entire plant. After crossing the epidermis, the agrobacteria infect and transfer t-DNA copies into the cells. t-DNA is transcribed into episomes, and mRNA is translated, leading to the production of the target protein in infected cells, but the passage of t-DNA through the nucleus is transient.

[0283] To aid in the identification of transformed plant cells, constructs of the present disclosure may be further modified to include plant-selectable markers. Useful selection markers include enzymes that provide resistance to antibiotics, such as gentamicin, hygromycin, kanamycin, or chemicals such as herbicides like phosphinothricin, glyphosate, and chlorosulfuron. Similarly, color-identifiable compounds such as GUS (β-glucuronidase) or luminescent enzymes such as luciferase or GFP can be used.

[0284] Transgenic plants, plant cells, or seeds containing the gene constructs of this disclosure, which can be used as platform plants suitable for transient protein expression as described herein, are also considered part of this disclosure. Methods for regenerating whole plants from plant cells are also known in the art (see, for example, Guerineau and Mullineaux (1993, Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148)). Generally, transformed plant cells are cultured in a suitable medium that may contain selective agents such as antibiotics, and selective markers are used to facilitate the identification of transformed plant cells. Once callus is formed, shoot formation can be promoted by using appropriate plant hormones according to known methods, and the shoots are transferred to rooting medium for plant regeneration. The plants can then be used to establish repeat generations from seeds or using vegetative propagation techniques. Transgenic plants can also be produced without using tissue culture. Methods for the stable transformation and regeneration of these organisms are established in the art and are known to those skilled in the art. Available techniques include Vasil et al. (Cell Culture and Somatic Cell Genetics of Plants, Vol. I, Il and III, Laboratory Procedures and Their This is outlined in Applications, Academic Press, 1984, and Weissbach and Weissbach (Methods for Plant Molecular Biology, Academic Press, 1989). The methods for obtaining transgenic and regenerated plants are not important to this disclosure.

[0285] When a plant, plant part, or plant cell is transformed or co-transformed by two or more nucleic acid constructs, the nucleic acid constructs may be introduced into Agrobacterium in a single transfection event so that the nucleic acids are pooled and bacterial cells are transfected. Alternatively, the constructs may be introduced sequentially. In this case, the first construct is introduced into Agrobacterium as described, and the cells are grown under selective conditions (e.g., in the presence of an antibiotic) in which only the individually transformed bacteria can grow. Following this first selection step, the second nucleic acid construct is introduced into Agrobacterium as described, and the cells are grown under double-selective conditions so that only double-transformed bacteria can grow. The double-transformed bacteria can then be used to transform a plant, a plant or part of a plant cell as described herein, or to be subjected to further transformation steps to accommodate a third nucleic acid construct.

[0286] Alternatively, if a plant, plant part, or plant cell is transformed or co-transformed by two or more nucleic acid constructs, the nucleic acid constructs may be introduced into the plant by co-infiltrating the plant, plant part, or plant cell into a mixture of Agrobacterium cells, each Agrobacterium cell may contain one or more constructs introduced into the plant. During the infiltration process, the concentrations of various Agrobacterium populations containing the desired constructs can be varied to alter the relative expression levels of the target nucleotide sequences within the constructs, plant, plant part, or plant cells.

[0287] Modified viral surface proteins or VLPs containing modified viral surface proteins described herein may be used to induce an immune response in a subject.

[0288] "Immune response" generally refers to the response of the adaptive immune system of a target. The adaptive immune system generally includes humoral responses and cell-mediated responses. Humoral responses are aspects of immunity mediated by secreted antibodies produced by cells of the B lymphocyte lineage (B cells). Secreted antibodies bind to antigens on the surface of invading microorganisms (such as viruses or bacteria) and risk destruction. Humoral immunity is generally used to refer to antibody production and the associated processes, as well as the effector functions of antibodies, including Th2 cell activation and cytokine production, memory cell generation, phagocytic opsonization, and pathogen elimination. Terms such as "regulate" or "modulate" refer to an increase or decrease in a particular response or parameter determined by one of several assays that are commonly known or used, some of which are exemplified herein.

[0289] Cell-mediated responses are immune responses that do not involve antibodies, but rather involve the activation of macrophages, natural killer cells (NKs), antigen-specific cytotoxic T lymphocytes, and the release of various cytokines in response to antigens. The term "cell-mediated immunity" is generally used to refer to some Th cell activation, Tc cell activation, and T cell-mediated responses. Cell-mediated immunity can be particularly important in responses to viral infections.

[0290] For example, the induction of antigen-specific CD8-positive T lymphocytes can be measured using an ELISA assay, and the stimulation of CD4-positive T lymphocytes can be measured using a proliferation assay. Anticoronavirus antibody titers can be quantified using an ELISA assay, and the isotype of antigen-specific or cross-reactive antibodies can also be measured using anti-isotype antibodies (e.g., anti-IgG, IgA, IgE, or IgM). Methods and techniques for performing such assays are well known in the art.

[0291] The presence or level of cytokines can also be quantified. For example, the T helper cell response (Th1 / Th2) is characterized by the measurement of IFN-γ and IL-4 secreting cells by ELISA (e.g., BD Biosciences OptEIA kit). Peripheral blood mononuclear cells (PBMCs) or splenocytes obtained from the subject may be cultured and the supernatant analyzed. T lymphocytes can also be quantified by fluorescence-activated cell sorting (FACS) using marker-specific fluorescent labeling and methods known in the art.

[0292] Microneutralization assays can also be performed to characterize the immune response in a subject (see, for example, the method of Rowe et al., 1973). Virus neutralizing titers can be quantified by a number of methods, including: 1) counting of lysed plaques after vigorous cell crystal fixation / staining (plaque assay); 2) microscopic observation of cell lysis in in vitro culture; and 3) ELISA and spectrophotometric detection of coronaviruses.

[0293] As used herein, the term “epitope” or “epitopes” refers to a structural portion of an antigen to which an antibody specifically binds.

[0294] A method for producing antibodies or antibody fragments is provided, comprising administering a VLP containing a modified viral structural protein, a modified viral structural protein as described herein, a trimer or a trimer-modified viral structural protein, or a modified viral structural protein to a subject or host animal to produce antibodies or antibody fragments. Antibodies or antibody fragments produced by this method are also provided.

[0295] Accordingly, this disclosure also provides the use of viral structural proteins or VLPs, including the modified viral structural proteins described herein, for inducing immunity against coronavirus infection in subjects. Antibodies or antibody fragments prepared by administering the modified viral structural proteins or VLPs containing the modified viral structural proteins to subjects or host animals are also disclosed herein.

[0296] Further provided are compositions comprising an effective amount of a modified viral structural protein or VLP containing the modified viral structural protein described herein, and a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient, for inducing an immune response in a subject. Also provided are vaccines for inducing a second immune response to coronavirus in a subject, comprising an effective amount of a modified viral structural protein or a VLP containing the modified viral structural protein.

[0297] Further compositions are provided that may contain a mixture of VLPs, insofar as at least one of the VLPs in the composition contains the modified coronavirus S protein described herein. For example, each coronavirus S protein containing one or more modified S proteins can be expressed from one or more coronavirus families, subgroups, types, subtypes, lineages, or strains, and the corresponding VLPs can be purified. Virus-like particles obtained from two or more coronavirus families, subgroups, types, subtypes, lineages, or strains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more coronavirus families, subgroups, types, subtypes, lineages, or strains) can be combined as desired to produce a mixture of VLPs, provided that one or more VLPs in the mixture of VLPs contain the modified S protein described herein. The VLPs can be combined or produced in a desired ratio, for example, approximately equal ratios, or they can be combined such that one coronavirus family, subgroup, type, subtype, lineage, or strain constitutes the majority of the VLPs in the composition. Further provided are compositions of VLPs comprising one or more modified S proteins having an external domain and / or TM or a portion of TM derived from one or more Coronaviridae, subgroups, types, subtypes, lineages, or strains, respectively, such that a mixture of different modified S proteins provided in this disclosure may be present in any individual VLP of the composition.

[0298] A composition or vaccine may include a VLP containing a modified viral structural protein, for example, a modified S protein derived from one type of coronavirus family, subgroup, type, subtype, lineage, or strain; or a composition or vaccine may include multiple VLP types, each VLP type containing a modified S protein, and the modified S protein in the same VLP may be derived from one type of coronavirus family, subgroup, type, subtype, lineage, or strain; that is, a composition or vaccine may include a mixture of different coronavirus VLPs, each VLP may contain a modified S protein derived from the same coronavirus family, subgroup, type, subtype, lineage, or strain. For example, a composition or vaccine may include a first VLP containing a first modified S protein derived from a first coronavirus family, subgroup, type, subtype, lineage, or strain, and a second VLP containing a second modified S protein derived from a second coronavirus family, subgroup, type, subtype, lineage, or strain. Furthermore, the composition may also include a third VLP comprising a third modified S protein derived from a third coronavirus family, subgroup, type, subtype, lineage, or strain, and / or the composition or vaccine may include a fourth VLP comprising a fourth modified S protein derived from a fourth coronavirus family, subgroup, type, subtype, lineage, or strain. Thus, this description also provides compositions or vaccines that are monovalent or univalent or multivalent. A monovalent composition or vaccine may immunize a subject against a single type of coronavirus strain, while a multivalent composition or vaccine may immunize a subject against two or more coronavirus strains. For example, the composition or vaccine may be a bivalent composition or vaccine that, upon administration, immunizes a subject against two different types of coronavirus family, subgroup, type, subtype, lineage, or strain. Furthermore, the composition or vaccine may be a trivalent composition, or the vaccine or composition may be a tetravalent or quadrivalent composition or vaccine.

[0299] Furthermore, the polyvalent composition may include a VLP containing one or more modified S proteins having different HA cytoplasmic terminals. For example, the polyvalent composition may include a VLP or a group of VLPs, each containing two or more modified S proteins, each containing an S protein external domain, an S protein transmembrane domain, and a cytoplasmic terminal derived from HA from influenza H1, H3, H5, H6, H7, H9, or B strains. Non-limiting examples of influenza strains include, for example, H1 California / 7 / 2009, H3 A / Minnesota / 41 / 2019, H5 A / Indonesia / 5 / 05, H6 A / Teal / Hong Kong / W312 / 97, H7 A / Guangdong / 17SF003 / 2016, H9 A / Hong Kong / 1073 / 99, or B / Washington / 02 / 2019.

[0300] A polyvalent composition or vaccine having multiple types of VLPs may further comprise a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient for inducing an immune response in a subject.

[0301] Adjuvants for enhancing the target immune response to vaccine antigens are well known and can be used in conjunction with the vaccines or pharmaceutical compositions described herein. Many types of adjuvants are available for use. Common adjuvants for human use are aluminum hydroxide, aluminum phosphate, and calcium phosphate. Many adjuvants also exist based on oil emulsions (oil-in-water or water-in-oil emulsions such as Freund's incomplete adjuvant (FIA), Montanide®, Adjuvant65, and Lipovant®), bacterial products (or their synthetic derivatives), endotoxins, fatty acids, paraffin or vegetable oils, cholesterol, and aliphatic amines or natural organic compounds, such as squalene. Examples of non-limiting adjuvants that may be used include oil-in-water emulsions of squalene oil (e.g., MF-59 or AS03), adjuvants consisting of synthetic TLR4 agonist glucopyranosyllipid A (GLA) incorporated into a stable emulsion (SE) (GLA-SE), or the Toll-like receptor (TLR9) agonist adjuvant CpG1018.

[0302] Therefore, a vaccine or pharmaceutical composition may contain one or more adjuvants. For example, a vaccine or pharmaceutical composition may contain aluminum hydroxide, aluminum phosphate, calcium phosphate, an oil-in-water or water-in-oil emulsion, an emulsion containing squalene (e.g., MF-59 or AS03), an emulsion containing GLA-SE, or the CpG 1018 adjuvant.

[0303] The pharmaceutical compositions, vaccines, or formulations described herein can be manufactured by methods known in themselves, for example, by conventional mixing, dissolution, granulation, sugar-coated tablet production, polishing, emulsification, encapsulation, encapsulation, or tableting processes.

[0304] Pharmaceutical compositions, vaccines, or formulations may be manufactured by mixing or pre-mixing any components before administration, for example, by manually or mechanically assisting the mixing of two or more vaccine suspensions, pharmaceutically acceptable carriers, adjuvants, vehicles, or excipients as a step performed before the final formulation, vaccine, or pharmaceutical composition is administered.

[0305] Pharmaceutical compositions, vaccines, or formulations may be administered orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously to the target.

[0306] Injectable preparations can be prepared in conventional forms, as liquid solutions or suspensions, as solid forms suitable for liquid solutions or suspensions before injection, or as emulsions. Suitable excipients include, for example, water, physiological saline, dextrose, mannitol, lactose, lecithin, albumin, monosodium glutamate, and cysteine ​​hydrochloride. Furthermore, if desired, the injectable pharmaceutical composition may contain small amounts of non-toxic auxiliary substances such as wetting agents and pH buffers. Physiologically compatible buffers include, but are not limited to, Hanks' solution, Ringer's solution, or physiological saline buffer. If desired, absorption-enhancing preparations (e.g., liposomes) may be used.

[0307] A composition or vaccine may be administered to a subject in a single dose. Furthermore, a vaccine or composition may be administered to a subject in multiple doses. Therefore, a composition, formulation, or vaccine may be administered to a subject in a single dose to induce an adverse immune response, or it may be administered in multiple doses. For example, a composition or vaccine may be administered in two, three, four, or five doses. Thus, a composition or vaccine may be administered to a subject in an initial dose, and one or more doses may be administered thereafter. The doses may be temporally separated from each other. For example, after the initial dose, one or more subsequent doses may be administered 1, 2, 3, 4, 5, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after the initial dose, or at any point in between. Furthermore, a composition or vaccine may be administered annually. For example, a composition or vaccine may be administered as a seasonal vaccine.

[0308] This disclosure further provides the following sequences. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0309] The present invention will be further explained in the following examples.

[0310] example Example 1: Preparation of modified structural protein constructs SARS-CoV-2 S protein constructs were prepared using techniques well known in the art. For example, SARS-CoV-2 spike protein constructs with wtTMCT (construct numbers 8586, 8589, 8591, see Figures 8A-8C) were cloned as described below. SARS-CoV-2 spike protein constructs with H5iTMCT (construct numbers 8592, 8595, 8597, see Figures 9A-9C) and SARS-CoV-2 spike protein constructs with H5iCT (construct numbers 8610, 8611, 8671, see Figures 10A-10C) were obtained using similar techniques and sequence primers, templates, and products, and are listed in Table 5.

[0311] SARS-CoV-2 spike protein with wtTMCT (construction numbers 8586, 8589, 8591) The sequence encoding mature SARS-CoV-2 spike (S) protein 2 (SEQ ID NO: 23), which has the GSAS+K971P+V972P external domain mutation and is fused to alfalfa PDI secretory signaling peptide (PDISP), and possesses a native transmembrane domain and a native cytoplasmic end (wtTMCT) derived from SARS-CoV-2, was cloned into three different expression systems using the following PCR-based method. Using primers IF(PDI)-CoV(opt2).c (SEQ ID NO: 24) and IF(AVB)-CoV(opt2).r (SEQ ID NO: 25), the fragment containing the SARS-CoV-2 spike protein (wtTMCT) encoding sequence was amplified using PDISP-SARS-CoV-2 spike protein with the wtTMCT gene sequence (SEQ ID NO: 22) as a template. The PCR products were cloned into three different expression systems using an in-fusion cloning system (Clontech, Mountain View, CA).

[0312] For the initial expression system, construct number 8501 (Figure 7A) was digested with AatII and StuI restriction enzymes, and the linearized plasmid was used for the initial in-fusion assembly reaction. Construct number 8501 is an acceptor plasmid intended for "in-fusion" cloning of the target gene in a 2X35S(+C) / nbMT78 / PDI / AvB / NOS-based expression cassette. This acceptor plasmid also incorporates gene constructs for the co-expression of the alfalfa plastocyanin gene promoter and the TBSV P19 suppressor for silencing under the terminator. The backbone is a pCAMBIA binary plasmid, and the sequence of the t-DNA boundary from left T-DNA to right is shown in SEQ ID NO: 31. The resulting construct was assigned the number 8586 (SEQ ID NO: 32). The amino acid sequence of the SARS-CoV-2-derived mature spike protein fused to alfalfa PDI secretion signal peptide (PDISP) is shown in SEQ ID NO: 23. The representation of plasmid 8586 is shown in Figure 8A.

[0313] For the second expression system, construct number 8500 (Figure 7B) was also digested with AatII and StuI restriction enzymes, and the linearized plasmid was used in the second in-fusion assembly reaction. Construct number 8500 is an acceptor plasmid intended for "in-fusion" cloning of the target gene in a 2X35S(+C) / nbCSY65 / PDI / AvB / NOS-based expression cassette. This acceptor plasmid also incorporates gene constructs for the co-expression of the alfalfa plastocyanin gene promoter and the TBSV P19 suppressor for silencing under the terminator. The backbone is a pCAMBIA binary plasmid, and the sequence of the t-DNA boundary from left T-DNA to right is shown in SEQ ID NO: 33. The resulting construct was assigned the number 8589 (SEQ ID NO: 34). The amino acid sequence of the SARS-CoV-2-derived mature spike protein fused to alfalfa PDI secretion signal peptide (PDISP) is shown in SEQ ID NO: 23. The representation of plasmid 8589 is shown in Figure 8B.

[0314] For the third expression system, construct number 8716 (Figure 7C) was also digested with AatII and StuI restriction enzymes, and the linearized plasmid was used in the third in-fusion assembly reaction. Construct number 8716 is an acceptor plasmid intended for "in-fusion" cloning of the target gene in a 2X35S(+C) / nbHEL40 / PDI / AvB / NOS-based expression cassette. This acceptor plasmid also incorporates gene constructs for the co-expression of the alfalfa plastocyanin gene promoter and the TBSV P19 suppressor for silencing under the terminator. The backbone is a pCAMBIA binary plasmid, and the sequence of the t-DNA boundary from left T-DNA to right is shown in SEQ ID NO: 35. The resulting construct was assigned the number 8591 (SEQ ID NO: 36). The amino acid sequence of the SARS-CoV-2-derived mature spike protein fused to alfalfa PDI secretion signal peptide (PDISP) is shown in SEQ ID NO: 23. The representation of plasmid 8591 is shown in Figure 8C.

[0315] SARS-CoV-2 spike protein containing H5iTMCT (construction numbers 8592, 8595, 8597) A sequence encoding the mature spike (S) protein of SARS-CoV-2 (SEQ ID NO: 27), possessing the GSAS+K971P+V972P external domain mutation and having the H5 A / Indonesia / 5 / 05 HA (H5iTMCT) transmembrane domain and cytoplasmic terminal, was fused to alfalfa PDI secretion signal peptide (PDISP) and cloned into the same three expression systems as above using a similar PCR-based method (see Table 5 for primers and Example 3 for the sequences used). Architect number 8592 (Figure 9A) is derived from acceptor construct 8501, construct number 8595 (Figure 9B) is derived from acceptor construct 8500, and construct number 8597 (Figure 9C) is derived from acceptor construct 8716 using the same technique as above. Primers, templates, and products are provided in Table 5 below.

[0316] SARS-CoV-2 spike protein containing H5iCT (construction counts 8610, 8611, 8671) Sequences encoding the GSAS+K971P+V972P external domain mutation and the mature spike (S) protein of SARS-CoV-2 (SEQ ID NO: 30) with the cytoplasmic terminal of H5 A / Indonesia / 5 / 05 HA (H5iCT) were fused to alfalfa PDI secretion signal peptide (PDISP) and cloned into the same three expression systems as above using a similar PCR-based method (see Table 5 for primers and Example 3 for sequences used). Architect number 8610 (Figure 10A) is derived from acceptor construct 8501, construct number 8611 (Figure 10B) is derived from acceptor construct 8500, and construct number 8671 (Figure 10C) is derived from acceptor construct 8716 using the same technique as above. Primers, templates, and products are provided in Table 5 below.

[0317] SARS-CoV-2 spike proteins with alternative TM-CT fusion sequences (construction numbers 8980, 8981, 8982, 8983) The sequence encoding the mature spike (S) protein from SARS-CoV-2 with the GSAS+K971P+V972P external domain mutation, as shown in Sequence ID No. 19, and the cytoplasmic terminal (H5iCT) sequence from H5 A / Indonesia / 5 / 05 HA, was fused to alfalfa PDI secretion signaling peptide (PDISP). This was cloned into the same expression system described for construct 8671 to obtain construct 8980 (Figure 12A). Similar constructs were created for the mature spike (S) protein from SARS-CoV-2 with the GSAS+K971P+V972P external domain mutation and the cytoplasmic terminal (H5iCT) sequence from H5 A / Indonesia / 5 / 05 HA, as shown in Sequence ID No. 37 (construct 8981, Figure 12B), Sequence ID No. 38 (construct 8982, Figure 12C), and Sequence ID No. 39 (construct 8983, Figure 12D).

[0318] SARS-CoV-2 spike proteins with CT derived from other HA strains (construction numbers 7390, 7391, 7392, 7393, 7394, and 7395) The mature spike (S) protein derived from SARS-CoV-2 with the GSAS+K971P+V972P external domain mutation and the sequence encoding the cytoplasmic end (H1CT) from H1 A / California / 7 / 2009 HA were fused to alfalfa PDI secretion signal peptide (PDISP), and cloned into the same expression system as described for construct 8671 above by a similar PCR-based method (see Table 5 for primers and Example 3 for sequences used). Thus, the resulting construct 7390 encodes a modified S protein containing the H1 A / California / 7 / 2009 HA cytoplasmic end (H1CT) (Figure 13A). Similar structures were fabricated for H3 A / Minnesota / 41 / 2019 (structure 7391, H3 CT) (Figure 13B), H6 A / Teal / Hong Kong / W312 / 97 (structure 7392, H6 CT) (Figure 13C), H7 A / Guangdong / 17SF003 / 2016 (structure 7393, H7 CT) (Figure 13D), H9 A / Hong Kong / 1073 / 99 (structure 7394, H9h CT) (Figure 13E), or B / Washington / 02 / 2019 (structure 7395, HA B CT) (Figure 13F).

[0319] SARS-CoV-2 spike protein with substitutions (construction numbers 8933, 8960, 8947) Modified SARS-CoV-2 S protein constructs, including combinations of mutations in the S protein such as R667G, R668S, R670S, F802P, A877P, A884P, A927P, K971P, V972P, and L923F, were prepared using techniques well known in the art, essentially as described above. The constructs have the following substitutions: construct 8933: R667G, R668S, R670S, K971P, V972P and L923F ("GSAS-2P-923"); construct 8960: R667G, R668S, R670S, F802P, A927P, K971P, V972P and L923F ("GSAS-4P-923") and construct 8947: R667G, R668S, R670S, F802P, A877P, A884P, A927P, K971P, V972P and L923F ("GSAS-6P-923").

[0320] SARS-CoV-1 spike protein with wtTMCT and modified TMCT (construction numbers 9231, 9232, 9233, 9234, 9235) A sequence (SEQ ID NO: 88) encoding a mature SARS-CoV-1 spike (S) protein with a native transmembrane domain and native cytoplasmic end (wtTMCT) derived from SARS-CoV-1, fused to alfalfa PDI secretory signal peptide (PDISP) and possessing the R654A+K955P+V956P external domain mutation, was cloned into the following expression systems by PCR. Fragments containing the PDISP-SARS-COV-1 spike protein (wtTMCT) encoding sequence were amplified using PDISP-SARS-COV-1 spike protein with the wtTMCT gene sequence (SEQ ID NO: 88) as a template, using primers IF(nbHEL40)-PDI.c (SEQ ID NO: 86) and IF(AvB+wtCT).r (SEQ ID NO: 87). The PCR products were cloned into the following expression systems using an In-Fusion Cloning System (Clontech, Mountain View, CA).

[0321] Architect #7147 (Figure 21) was digested with AatII and StuI restriction enzymes, and the linearized plasmid was used in the first in-fusion assembly reaction. Architect #7147 is an acceptor plasmid intended for "in-fusion" cloning of the target gene in a 2X35S(+C) / nbHEL40 / AvB / NOS-based expression cassette. This acceptor plasmid also incorporates gene constructs for the co-expression of the alfalfa plastocyanin gene promoter and the TBSV P19 suppressor for silencing under the terminator. The backbone is a pCAMBIA binary plasmid, and the sequence of the t-DNA boundary from left T-DNA to right is shown in SEQ ID NO: 111. The resulting construct was assigned the number 9231. The amino acid sequence of the SARS-CoV-1-derived mature spike protein fused to alfalfa PDI secretion signal peptide (PDISP) is shown in SEQ ID NO: 93. Plasmid 9231 is shown in Figure 18A.

[0322] The sequence encoding the mature spike (S) protein from SARS-CoV-1 with the R654A+K955P+V956P external domain mutation, and one of the following—i) the transmembrane domain and the cytoplasmic end from H5 A / Indonesia / 5 / 05 HA (H5iTMCT), ii) the cytoplasmic end from H5 A / Indonesia / 5 / 05 HA (H5iCT and mutant H5iCT(V4)), or iii) the cytoplasmic end from H1 A / California / 7 / 2009 HA (H1cCT)—were fused to alfalfa PDI secretion signal peptide (PDISP), and cloned into the same expression system as described for construct 9231 above by a similar PCR-based method (see Table 5 for primers and Example 3 for the sequences used). Therefore, the resulting constructs 9232, 9233, 9234, and 9235 encode a modified S protein containing H5 A / Indonesia / 5 / 05 TMCT (H5iTMCT) (Figure 18B, SEQ ID NO: 94), a modified S-COV-1 S protein containing H5 A / Indonesia / 5 / 05 CT (H5iCT) (Figure 18C, SEQ ID NO: 95), a modified S protein containing the H5 A / Indonesia / 5 / 05 CT variant (H5iCT(V4)) (Figure 18D, SEQ ID NO: 96), or a modified S protein containing H1 A / California / 7 / 2009 CT (H1cCT) (Figure 18E, SEQ ID NO: 97).

[0323] MERS-CoV spike proteins containing wtTMCT and modified TMCT (construction numbers 9246, 9247, 9249, 9250, 9251) A sequence (SEQ ID NO: 101) encoding a mature MERS-CoV spike (S) protein with the R730A+R733G+V1043P+L1044P external domain mutation and a native transmembrane domain and native cytoplasmic end (wtTMCT) derived from MERS-CoV fused to alfalfa PDI secretory signal peptide (PDISP) was cloned into the following expression systems by PCR. The fragment containing the PDISP-MERS-COV spike protein (wtTMCT) encoding sequence was amplified using the PDISP-MERS-COV spike protein template (wtTMCT gene sequence, SEQ ID NO: 101) with primers IF(nbHEL40)-PDI.c (SEQ ID NO: 86) and IF(AvB+wtCT-MERS).r (SEQ ID NO: 98). The PCR products were cloned into the following expression systems using an In-Fusion Cloning System (Clontech, Mountain View, CA).

[0324] Architect #7147 (Figure 21) was digested with AatII and StuI restriction enzymes, and the linearized plasmid was used in the first in-fusion assembly reaction. Architect #7147 is an acceptor plasmid intended for "in-fusion" cloning of the target gene in a 2X35S(+C) / nbHEL40 / AvB / NOS-based expression cassette. This acceptor plasmid also incorporates gene constructs for the co-expression of the alfalfa plastocyanin gene promoter and the TBSV P19 suppressor for silencing under the terminator. The backbone is a pCAMBIA binary plasmid, and the sequence of the t-DNA boundary from left T-DNA to right is shown in SEQ ID NO: 111. The resulting construct was assigned the number 9246. The amino acid sequence of the MERS-COV-derived mature spike protein fused to alfalfa PDI secretion signal peptide (PDISP) is shown in SEQ ID NO: 106. Plasmid 9246 is shown in Figure 20A.

[0325] A sequence encoding a mature spike (S) protein derived from MERS-CoV with the R730A+R733G+V1043P+L1044P external domain mutation, and one of the following—i) the transmembrane domain and the cytoplasmic end derived from H5 A / Indonesia / 5 / 05 HA (H5iTMCT), ii) the cytoplasmic end derived from H5 A / Indonesia / 5 / 05 HA (H5iCT and mutant H5iCT(V4)), or iii) the cytoplasmic end derived from H1 A / California / 7 / 2009 HA (H1cCT)—was fused to alfalfa PDI secretion signal peptide (PDISP), and cloned into the same expression system described for construct 9246 above by a similar PCR-based method (see Table 5 for primers and Example 3 for sequences used). Therefore, the resulting constructs 9247, 9249, 9250, and 9251 encode a modified MERS-COVS protein containing H5 A / Indonesia / 5 / 05 TMCT (H5iTMCT) (Figure 20B, SEQ ID NO: 107), a modified S protein containing H5 A / Indonesia / 5 / 05 CT (H5iCT) (Figure 20C, SEQ ID NO: 108), a modified S protein containing the H5 A / Indonesia / 5 / 05 CT variant (H5iCT(V4)) (Figure 20D, SEQ ID NO: 109), or a modified S protein containing H1 A / California / 7 / 2009 CT (H1cCT) (Figure 20E, SEQ ID NO: 110).

[0326] OC43-CoV spike protein with wtTMCT and modified TMCT (construction numbers 9269, 9270, 9272, 9273, and 9274) A sequence (SEQ ID NO: 137) encoding a mature OC43-CoV spike (S) protein with the R761G+R762G+R764G+R765S+A1077P+L1078P external domain mutation and a native transmembrane domain and native cytoplasmic end (wtTMCT) derived from OC43-CoV fused to alfalfa PDI secretion signal peptide (PDISP) was cloned into the following expression system by PCR. The fragment containing the PDISP-OC43-COV spike protein (wtTMCT) encoding sequence was amplified using the wtTMCT gene sequence (SEQ ID NO: 137) as a template for the PDISP-OC43-COV spike protein, using primers IF(nbHEL40)-PDI.c (SEQ ID NO: 86) and IF(AvB+wtCT-OC43).r (SEQ ID NO: 136). PCR products were cloned into the following expression systems using an in-fusion cloning system (Clontech, Mountain View, CA).

[0327] Architect #7147 (Figure 21) was digested with AatII and StuI restriction enzymes, and the linearized plasmid was used in the first in-fusion assembly reaction. Architect #7147 is an acceptor plasmid intended for "in-fusion" cloning of the target gene in a 2X35S(+C) / nbHEL40 / AvB / NOS-based expression cassette. This acceptor plasmid also incorporates gene constructs for the co-expression of the alfalfa plastocyanin gene promoter and the TBSV P19 suppressor for silencing under the terminator. The backbone is a pCAMBIA binary plasmid, and the sequence of the t-DNA boundary from left T-DNA to right is shown in SEQ ID NO: 111. The resulting construct was assigned the number 9269. The amino acid sequence of the mature spike protein derived from OC43-COV fused to alfalfa PDI secretion signal peptide (PDISP) is shown in SEQ ID NO: 142. Plasmid 9269 is shown in Figure 24A.

[0328] The sequence encoding the mature spike (S) protein from OC43-CoV with the R761G+R762G+R764G+R765S+A1077P+L1078P external domain mutation, and one of the following—i) the transmembrane domain and the cytoplasmic end from H5 A / Indonesia / 5 / 05 HA (H5iTMCT), ii) the cytoplasmic end from H5 A / Indonesia / 5 / 05 HA (H5iCT and mutant H5iCT(V4)), or iii) the cytoplasmic end from H1 A / California / 7 / 2009 HA (H1cCT)—was fused to alfalfa PDI secretion signal peptide (PDISP), and cloned into the same expression system as described for construct 9269 above by a similar PCR-based method (see Table 5 for primers and Example 3 for the sequences used). Therefore, the resulting constructs 9270, 9272, 9273, and 9274 encode a modified OC43-COVS protein containing H5 A / Indonesia / 5 / 05 TMCT (H5iTMCT) (Figure 24B, SEQ ID NO: 143), a modified S protein containing H5 A / Indonesia / 5 / 05 CT (H5iCT) (Figure 24C, SEQ ID NO: 144), a modified S protein containing the H5 A / Indonesia / 5 / 05 CT variant (H5iCT(V4)) (Figure 24D, SEQ ID NO: 145), or a modified S protein containing H1 A / California / 7 / 2009 CT (H1cCT) (Figure 24E, SEQ ID NO: 146).

[0329] 229E-CoV spike protein with wtTMCT and modified TMCT (construction numbers 9310, 9311, 9312, 9313, and 9314) A sequence (SEQ ID NO: 148) encoding a mature 229E-CoV spike (S) protein with the R567A+T871P+I872P external domain mutation and a native transmembrane domain and native cytoplasmic end (wtTMCT) derived from 229E-CoV fused to alfalfa PDI secretion signal peptide (PDISP) was cloned into the following expression systems by PCR. The fragment containing the PDISP-229E-COV spike protein (wtTMCT) encoding sequence was amplified using the PDISP-OC43-COV spike protein, which uses the wtTMCT gene sequence (SEQ ID NO: 148) as a template, with primers IF(nbHEL40)-PDI.c (SEQ ID NO: 86) and IF(CoV229EwtCT).r (SEQ ID NO: 147). The PCR products were cloned into the following expression systems using an In-Fusion Cloning System (Clontech, Mountain View, CA).

[0330] Architect #7147 (Figure 21) was digested with AatII and StuI restriction enzymes, and the linearized plasmid was used in the first in-fusion assembly reaction. Architect #7147 is an acceptor plasmid intended for "in-fusion" cloning of the target gene in a 2X35S(+C) / nbHEL40 / AvB / NOS-based expression cassette. This acceptor plasmid also incorporates gene constructs for the co-expression of the alfalfa plastocyanin gene promoter and the TBSV P19 suppressor for silencing under the terminator. The backbone is a pCAMBIA binary plasmid, and the sequence of the t-DNA boundary from left T-DNA to right is shown in SEQ ID NO: 111. The resulting construct was assigned the number 9310. The amino acid sequence of the mature spike protein derived from 229E-COV fused to alfalfa PDI secretion signal peptide (PDISP) is shown in SEQ ID NO: 153. Plasmid 9310 is shown in Figure 26A.

[0331] The sequence encoding the mature spike (S) protein from 229E-CoV with the R567A+T871P+I872P external domain mutation, and one of the following—i) the transmembrane domain and the cytoplasmic end from H5 A / Indonesia / 5 / 05 HA (H5iTMCT), ii) the cytoplasmic end from H5 A / Indonesia / 5 / 05 HA (H5iCT and mutant H5iCT(V4)), or iii) the cytoplasmic end from H1 A / California / 7 / 2009 HA (H1cCT)—were fused to alfalfa PDI secretion signal peptide (PDISP), and cloned into the same expression system as described for construct 9310 above by a similar PCR-based method (see Table 5 for primers and Example 3 for the sequences used). Therefore, the resulting constructs 9311, 9312, 9313, and 9314 encode a modified 229E-COVS protein containing H5 A / Indonesia / 5 / 05 TMCT (H5iTMCT) (Figure 26B, SEQ ID NO: 154), a modified S protein containing H5 A / Indonesia / 5 / 05 CT (H5iCT) (Figure 26C, SEQ ID NO: 155), a modified S protein containing the H5 A / Indonesia / 5 / 05 CT variant (H5iCT(V4)) (Figure 26D, SEQ ID NO: 156), or a modified S protein containing H1 A / California / 7 / 2009 CT (H1cCT) (Figure 26E, SEQ ID NO: 157). [Table 6-1] [Table 6-2]

[0332] Example 2: Method Agrobacterium tumefaciens transfection The Agrobacterium tumefaciens strain AGL1 was transfected by electroporation with a SARS-CoV-2 modified S protein expression vector using the method described by D'Aoust et al., 2008 (Plant Biotech. J. 6: 930-40). The transfected Agrobacterium were then cultured in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 μM acetosyringone, 50 μg / ml kanamycin, and 25 μg / ml carbenicillin pH 5.6 to an OD of 0.6–1.6. 600 The Agrobacterium was allowed to grow until it reached the specified stage. The Agrobacterium suspension was centrifuged before use and resuspended in immersion medium (10 mM MgCl2 and 10 mM MES pH 5.6).

[0333] Preparation of plant biomass, inoculants, and agroinfiltration N. benthamiana plants were grown from seeds in a flat bed filled with commercially available peat moss substrate. The plants were grown in a greenhouse under a 16 / 8 photoperiod and a temperature system of 25°C day / 20°C night. Three weeks after sowing, individual plantlets were removed, transplanted into pots, and grown in the greenhouse for another three weeks under the same environmental conditions.

[0334] Agrobacteria transfected with each expression vector were placed in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 μM acetosyringone, 50 μg / ml kanamycin, and 25 μg / ml carbenicillin pH 5.6, and OD levels of 0.6–1.6 were measured. 600The bacteria were allowed to grow until they reached a certain size. The Agrobacterium suspension was centrifuged before use, resuspended in a solubilicate medium (10 mM MgCl2 and 10 mM MES pH 5.6), and stored overnight at 4°C. On the day of solubilization, the culture batch was diluted to 2.5 culture volumes and warmed before use. Whole N. benthamiana plants were placed upside down in a bacterial suspension in an airtight stainless steel tank under a vacuum of 20–40 Tor for 2 minutes. The plants were returned to the greenhouse for an incubation period of 6 or 9 days until harvest.

[0335] Leaf harvesting and extraction of total protein and VLPs After incubation, the above-ground parts of the plants were harvested, frozen at -80°C, and crushed into fragments. Each sample of the freeze-pulverized plant material was mechanically homogenized (Polytron) in two volumes of cold 50 mM Tris buffer containing pH 8.0 + 500 mM NaCl, 0.4 μg / ml metanisulfite, and 1 mM phenylmethanesulfonyl fluoride to extract all soluble proteins. After homogenization, the slurry was centrifuged at 10,000 g for 10 minutes at 4°C, and these clarified crude extracts (supernatant) were retained for analysis.

[0336] The total protein content of the clarified crude extract was determined by the Bradford assay (Bio-Rad, Hercules, California) using bovine serum albumin as a reference standard. Proteins were separated by SDS-PAGE under reducing conditions using Criterion® TGX Stain-Free® precast gels (Bio-Rad Laboratories, Hercules, CA). Proteins were visualized by staining the gel with Coomassie Brilliant Blue. Alternatively, proteins were visualized using the Gel Doc® EZ imaging system (Bio-Rad Laboratories, Hercules, CA) and electrotransferred onto polyvinyl difluoride (PVDF) membranes (Roche Diagnostics Corporation, Indianapolis, Indiana) for immunodetection. Prior to immunoblotting, the membranes were blocked at 4°C for 16–18 hours in 5% skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS-T).

[0337] For VLP purification, proteins were extracted from frozen biomass by mechanical extraction using a blender containing two volumes of extraction buffer (50 mM Tris buffer at pH 7.0 + 500 mM NaCl), and the pH was reduced to 6.1 using 0.5 M citrate. The slurry was filtered through a large-pore nylon filter to remove large fragments and centrifuged at 5000 g at 4°C for 5 minutes. The supernatant was collected and centrifuged again at 5000 g for 30 minutes (4°C) to remove further fragments and passed through a clarification filter. The supernatant was then loaded with a discontinuous iodixanol density gradient. Analytical density gradient centrifugation was performed as follows: 38 mL tubes were prepared containing discontinuous iodixanol density gradients in Tris buffer (3 ml at 35%, 3 ml at 30%, 3 ml at 25%, 3 ml at 15%, and 5 ml at 10% iodixanol), and 22 ml of extract containing virus-like particles was overlaid. The gradient was centrifuged at 120,000 g for 2 hours (4°C). After centrifugation, 1 mL fractions were collected from the bottom to the top, and the fractions were analyzed by protein staining or SDS-PAGE combined with Western blotting. Fractions 6-9 were pooled and buffer was exchanged using an Amicon centrifuge. Protein content was determined by the Bradford assay.

[0338] Protein analysis and immunoblotting Immunoblotting was performed in the first incubation with primary mAbs (anti-S1, Sino Biological, catalog no. 40150-R007 or anti-S2, Novus biological, catalog no. NB100-56578) diluted in 2% skim milk in 0.1% TBS-Tween20. A peroxidase-conjugated goat anti-rabbit antibody (Jackson Immunoresearch, catalog no. 115-035-144) was used as a secondary antibody for chemiluminescent detection in 2% skim milk in 0.1% TBS-Tween20. The immunoreactive conjugate was detected by chemiluminescence using luminol as a substrate (Roche Diagnostics Corporation). Human IgG antibody was conjugated with horseradish peroxidase using the EZ-Link Plus® Activated Peroxidase conjugation kit (Pierce, Rockford, Ill.).

[0339] In plants, yields were evaluated for clarified crude extracts and analyzed using capillary-based electrophoresis (Protein Simple, BioTechne) and a WES analysis system. Briefly, soluble proteins from crude extracts were separated by molecular weight in capillaries and immobilized on a matrix. S protein amounts were determined using a standard curve with purified VLPs, and an anti-S2 antibody (Novus Biological, catalog no. NB100-56578) was used for detection according to the manufacturer's instructions. The yields were then normalized using a comparator structure set to 1.

[0340] The primary antibody used for detecting the SARS-CoV S protein was the SARS-CoV Spike S1 subunit antibody from Sino Biologicals, 40150-MM08 (1 / 5000), and the secondary antibody used for detection was the goat anti-mouse antibody, JIR, 115-035-146 (1 / 10000). The primary antibody used for detecting the MERS-CoV S protein was the MERS-CoV spike protein S1 antibody (N-terminus) from Sino Biological (100208-RP02, 1 / 5000). The secondary antibody used for detection was the goat anti-mouse antibody from JIR (115-035-144, 1 / 10000). The primary antibody used for detection was the anti-coronavirus OC43 spike protein antibody from Antibodies-online (ABIN2754654, 1 / 1000). The secondary antibody used for detection was JIR(111-035-144,1 / 10000) goat anti-rabbit antibody.

[0341] Electron microscopy To determine whether the expressed S protein assembled on the VLP, transmission electron microscopy (TEM) of immunocaptured particles was performed on purified VLP. A glow discharge carbon / copper grid (10 seconds, 0.3 mbar) was placed on 20 μL of purified VLP (100 μg / mL) for 5 minutes, and then washed four times with sterile distilled water. The grid was floated in 20 μL of 2% uranyl acetate for 1 minute, then excess solution was removed by touching it with moistened filter paper, and after drying on the filter paper for 24 hours, it was observed with a TEM (Tecnai Microscope).

[0342] Example 3: Array The following array was used in the example above. Natural SARS-CoV-2 S protein wtTM / CT AA(P0DTC2) (SEQ ID NO: 1) Natural SARS-CoV-2 S protein wt™ / CT AA(P0DTC2) (SEQ ID NO: 2) without signal peptide (SP) H5 A / Indonesia / 5 / 05 Hemagglutinin (HA)AA (A5A5L7) (Sequence ID 3) MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTNDLCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHE ASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSE LEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGRNSPQRESRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENL NKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESIRNGTYNYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI H5 A / Indonesia / 5 / 05 hemagglutinin (HA) virus cDNA (EF541394.1) (SEQ ID NO: 4) Modified SARS-CoV-2 (SEQ ID NO: 5) with H5 A / Indonesia / 5 / 05 hemagglutinin CT AA H1 A / California / 7 / 2009 Hemagglutinin™ / CT AA (SEQ ID NO: 6) IDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI H2 A / Singapore / 1 / 1957 Hemagglutinin™ / CT AA (SEQ ID NO: 7) IKGVKLSSMGVYQILAIYATVAGSLSLAIMMAGISFWMCSNGSLQCRICI H3 A / Minnesota / 41 / 2019 Hemagglutinin™ / CT AA (SEQ ID NO: 8) IKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI H5 A / Indonesia / 5 / 05 Hemagglutinin™ / CT AA (SEQ ID NO: 9) ISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI H6 A / Teal / Hong Kong / W312 / 97 Hemagglutinin™ / CT AA (SEQ ID NO: 10) IESVKLENLGVYQILAIYSTVSSSLVLVGLIMAMGLWMCSNGSMQCRICI H7 A / Guangdong / 17SF003 / 2016 Hemagglutinin™ / CT AA (SEQ ID NO: 11) IDPVKLSSGYKDVILWFSFGASCFILLAIVMGLVFICVKNGNMRCTICI H9 A / Hong Kong / 1073 / 99 Hemagglutinin™ / CT AA (SEQ ID NO: 12) IEGVKLESEGTYKILTIYSTVASSLVLAMGFAAFLFWAMSNGSCRCNICI B / Washington / 02 / 2019 Hemagglutinin™ / CT AA (SEQ ID NO: 13) AASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL Consensus sequence of the C-terminal region of influenza hemagglutinin (SEQ ID NO: 14) IXGVKLXSXGXYXILXIYSTVASSLXLXXXXXXXXXXWMCSNGSXXCXICI Consensus sequence of the CT domain of influenza hemagglutinin (SEQ ID NO: 15) XXWMCSNGSXXCXICI C-terminal region of natural SARS-CoV-2 S protein wtTM / CT (SEQ ID NO: 16) WYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT C-terminal region of H5 A / Indonesia / 5 / 05 hemagglutinin (SEQ ID NO: 17) ISGVKLESIGTYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI C-terminal region of modified SARS-CoV-2 S protein containing H5i hemagglutinin CT (SEQ ID NO: 18) WYIWLGFIAGLIAIVMVTIMLSLWMCSNGSLQCRICI Modified SARS-CoV-2 S protein with H5i hemagglutinin CT in its C-terminal region, mutation 1 (SEQ ID NO: 19) WYIWLGFIAGLIAIVMVTIMMAGLSLWMCSNGSLQCRICI (SP-free) SARS-CoV-2 S protein GSAS+PP wtTM / CT AA (SEQ ID NO: 20) (SP-free) Modified SARS-CoV-2 S protein GSAS+PP with H5i hemagglutinin CT AA (SEQ ID NO: 21) PDI-SARS-CoV-2 S protein GSAS+PP wtTM / CT-DNA (SEQ ID NO: 22) PDI-SARS-CoV-2 S protein GSAS+PP wtTM / CT-AA (SEQ ID NO: 23) IF(PDI)-CoV(opt2).c (SEQ ID NO: 24) TCTCAGATCTTCGCGGTGAATCTTACGACGCGAACACAGTTACCACCCGCAT IF(AVB)-CoV(opt2).r(SEQ ID NO: 25) ACGACACGACTAAGGCCTCTAAGTATAGTGCAGCTTCACGCCCTTCAGGAC PDI-modified SARS-CoV-2 S protein GSAS+PP H5i™ / CT-DNA (SEQ ID NO: 26) PDI-modified SARS-CoV-2 S protein GSAS+PP H5iTM / CT-AA (SEQ ID NO: 27) IF(Avb)-H5I.r(Sequence ID 28) ACGACACGACTAAGGCCTTTAAATGCAAATTCTGCATTGTAACGATCC PDI-modified SARS-CoV-2 S protein GSAS+PP wtTM / H5iCT-DNA (SEQ ID NO: 29) PDI-modified SARS-CoV-2 S protein GSAS+PP wtTM / H5iCT-AA (SEQ ID NO: 30) Cloning vector 8501 (SEQ ID NO: 31) from left T-DNA to right T-DNA Construction 8586 (Sequence ID 32) from 2X35S Promoter to NOS Terminator Cloning vector 8500 (SEQ ID NO: 33) from left T-DNA to right T-DNA Construction 8589 (Sequence ID 34) from 2X35S Promoter to NOS Terminator Cloning vector 8716 (SEQ ID NO: 35) from left T-DNA to right T-DNA Construction 8591 (Sequence ID 36) from 2X35S Promoter to NOS Terminator Modified SARS-CoV-2 S protein with H5i hemagglutinin CT in its C-terminal region, mutation 2 (SEQ ID NO: 37) WYIWLGFIAGLIAIVMVTIMAGLSLWMCSNGSLQCRICI Modified SARS-CoV-2 S protein with H5i hemagglutinin CT in its C-terminal region, mutation 3 (SEQ ID NO: 38) WYIWLGFIAGLIAIVMVTIMLCCMCSNGSLQCRICI Modified SARS-CoV-2 S protein with H5i hemagglutinin CT in its C-terminal region, mutation 4 (SEQ ID NO: 39) WYIWLGFIAGLIAIVMVTIMLCCSNGSLQCRICI 3'UTR AvB (Arakacha virus B) (SEQ ID NO: 40) TAGTCGTGTCGTTTTTCAAATAATAATCCTTTTAGGGTTTTAGTTAGTTTAAATTTTCTGTTGCTCCTGTTTAGCAGGTCGTGCCTTCAGCAAGCACACAAAAACAGAGTGTTTATTTTAAGTTGTTTGTTTAGTGATTCAAAAAAAAA 3'UTR trBNYVV (root disease) (Sequence ID 41) CCTATCTTGATGAAGGTTGTTGTGGTTTTCTCATTACTGTTTTATTATTGTTTGAGTTGCTTATGTCGGTTCTTGATTATGTGGTGCATAATTATTGAACTAATTGTTTGTTGGGTTGTAATGTACTGACTGGGTGTGAATTGTACCAGTCGTTAAAGGGTTTACTATCAGTATATTGATAT 3'UTR SBMV (Southern Bean Mosaic Virus) (SEQ ID NO: 42) TGAGGAGTTGTATAATAATACCTGCACCCTTCTCTTTGGCAGGGAGGGTGTTTCGTTTTCACAATGCCACGCGCTTGAGGGAGAATGCACGTTAATCATCCCTCCGCTAGTGATGGAGCGTAATCCAAAAGT 3'UTR TuRSV (Cubling Spot Virus) (SEQ ID NO: 43) TGATTTATAATAGCCATAGATTAAGTTTAAATGTATTACGTTTGTATTTTATTCTCTTTTTTAAGTTTCCTATGTTGTTTTAAATTAAATATCTGTATAATTAGTAGATGTAAATCTGCTTTGTGCGTTTGACAGTCTTGG GAAACGCACTGGTTCATGAGATAGGACCACCTAGGAGGTAGGACTCTGGGTTTTAATTATCTCATTTCTTAGTTATACCGTATTATATATATGATTTAGTAGTAATTGTTTTCTCTTGATATGTATTATTACTTTTTTATT 3'UTR CPMV (Cowpi Mosaic Virus) (SEQ ID NO: 44) ATTTTCTTTAGTTTGAATTTACTGTTATTCGGTGTGCATTTCTATGTTTGGTGAGCGGTTTTCTGTGCTCAGAGTGTGTTTATTTTATGTAATTTAATTTCTTTGTGAGCTCCTGTTTAGCAGGTCGTCCCTTCAGCAAGGACACAAAAAGATTTTAATTTTATT 3'UTR BBTMV (Broad Bean True Mosaic Virus) (SEQ ID NO: 45) TAGTTTTCTTCCGCTTTTCTTTTGTAGTGTGTGGTTTTCTTTGTTTCTTCTTTTCTTTTCTCTTTCCTTTTCTCTTACTCCTGCCTGGCAGGTCGTGCCTTCAGTAAGCACAACAAAAATATGCATTTATTAGAGTA TTTCTTTCTTCTTTAGCATAAAGGTATTGAAGACCTATAAACTTCGTCCGGGTTGGGGAAAGTACCAGCTTAGCATATCTTTAGAAAACTATATAGAGCTCTTTACCTTGAGTTGTTTCCTAAAGTTTATGCAAAAAA 3'UTR trOUMV (Omiya Melon Virus) (SEQ ID NO: 46) CTCACGTCTGGGGTGAGCCCTAGCCAAATAGGAAAACGATAAGCGCTTTGCATGCAAAATGAGTTGGGCCACAAGTGCCACTCGCAGCGAAGGCGGTCTGAGGTTTCCCCCTGGCGGTTACTTCCATATCTTTGGGAGATAACTGGG Modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 47) containing (PDI)H5i hemagglutinin CT AA Modified SARS-CoV-2 S protein GSAS+4P (SEQ ID NO: 48) containing (PDI)H5i hemagglutinin CT AA Modified SARS-CoV-2 S protein GSAS+6P (SEQ ID NO: 49) containing (PDI)H5i hemagglutinin CT AA Modified SARS-CoV-2 S protein GSAS+PP+923 (SEQ ID NO: 50) containing (PDI)H5i hemagglutinin CT AA Modified SARS-CoV-2 S protein GSAS+4P+923 (SEQ ID NO: 51) containing (PDI)H5i hemagglutinin CT AA Modified SARS-CoV-2 S protein GSAS+6P+923 (SEQ ID NO: 52) containing (PDI)H5i hemagglutinin CT AA (PDI)H1 Cal-hemagglutinin CT AA-containing modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 53) (PDI)H3 Minn hemagglutinin CT AA-containing modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 54) Modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 55) containing (PDI)H6 HK hemagglutinin CT AA (PDI)H7 Guangdong hemagglutinin CT AA-containing modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 56) Modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 57) containing (PDI)H9 HK hemagglutinin CT AA Modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 58) containing (PDI)B / Wash hemagglutinin CT AA Modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 59) containing (PDI)H5i hemagglutinin CT (alternative 1)AA Modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 60) containing (PDI)H5i hemagglutinin CT (alternative 2)AA Modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 61) containing (PDI)H5i hemagglutinin CT (alternative 3)AA Modified SARS-CoV-2 S protein GSAS+PP (SEQ ID NO: 62) containing (PDI)H5i hemagglutinin CT (alternative 4)AA N-terminal region of the natural SARS-CoV-2 S protein (natural signal peptide sequence is underlined) (SEQ ID NO: 63) MFVFLVLLLPLVSSQCVNLTTRTQLPPAYTNS Intermediate peptide sequence (X) n Modified TMCT (SEQ ID NO: 64) WYIWLGFIAGLIAIVMVTIM(X) n CSNGSXXCXICI PDI-S protein GSAS+4P-DNA (SEQ ID NO: 65) PDI-S protein GSAS+6P-DNA (SEQ ID NO: 66) PDI-S protein GSAS+2P+L923F-DNA (SEQ ID NO: 67) PDI-S protein GSAS+4P+L923F-DNA (SEQ ID NO: 68) PDI-S protein GSAS+6P+L923F-DNA (SEQ ID NO: 69) PDI-modified S protein (V1) DNA (SEQ ID NO: 70) containing H5i hemagglutinin CT PDI-modified S protein (V2) DNA (SEQ ID NO: 71) containing H5i hemagglutinin CT PDI-modified S protein (V3) DNA (SEQ ID NO: 72) containing H5i hemagglutinin CT PDI-modified S protein (V4) DNA (SEQ ID NO: 73) containing H5i hemagglutinin CT PDI-S protein + H1 Cal DNA (SEQ ID NO: 74) PDI-S protein + H3 Minn DNA (SEQ ID NO: 75) PDI-S protein + H6 HK DNA (SEQ ID NO: 76) PDI-S protein + H7 Guangdong DNA (SEQ ID NO: 77) PDI-S protein + H9 HK DNA (SEQ ID NO: 78) PDI-S protein + B / Wash DNA (SEQ ID NO: 79) IF-H1HawaiiCT.r (Sequence ID 80) acgacacgactaaggcctttaaatacatattctacactgtagagaccc IF-H3MinnesotaCT.r (Sequence ID 81) acgacacgactaaggcctttagatgcagatgttgcatctgatgttgcccttctg IF-HongKongCT.r (Sequence ID 82) acgacacgactaaggcctttatatacatatcctgcactgcattgaaccattt IF-GuangdongCT.r (SEQ ID NO: 83) acgacacgactaaggcctttatatacaaatagtgcaccgcatgtttcca IF-H9HKCT.r (Sequence ID 84) acgacacgactaaggcctttatatacaaatgttgcatctgcaagatccat IF-BWashCT.r(SEQ ID NO: 85) acgacacgactaaggcctttatagacaaatggagcaagaaacattgtctc IF(nbHEL40)-PDI.c(Sequence ID 86) ccaaaacacattgagcaaaatggcgaaaaacgttgcgattttcggcttat IF(AvB+wtCT).r(Sequence ID 87) ACGACACGACTAAGGCCTTTAGGTATAATGGAGTTTCACCCCCTTCAGAA PDI-SARS-COV-1 wtTMCT-DNA (SEQ ID NO: 88) PDI-SARS-COV-1 H5iTMCT-DNA (SEQ ID NO: 89) PDI-SARS-COV-1 H5iCT-DNA (SEQ ID NO: 90) PDI-SARS-CoV-1 H5iCT(V4)-DNA (matching number 91) PDI-SARS-COV-1 H1cCT-DNA (SEQ ID NO: 92) PDI-SARS-COV-1 wtTMCT-AA (SEQ ID NO: 93) PDI-SARS-COV-1 H5iTMCT-AA (SEQ ID NO: 94) PDI-SARS-COV-1 H5iCT-AA (SEQ ID NO: 95) PDI-SARS-COV-1 H5iCT(V4)-AA (Pairing number 96) PDI-SARS-COV-1 H1cCT-AA (Sequence ID 97) IF(AvB+wtCT-MERS).r(Sequence ID 98) ACGACACGACTAAGGCCTTCAGTGAACGTGGACCTTGTGAGGCTCAAGGTCATACTCCTC IF(H1cCT-wtTM).r(Sequence ID 99) ACGACACGACTAAGGCCTTCAAATACATATTCTACACTGTAGAGACCCA IF(H5ITMCT).r(Sequence ID 100) ACGACACGACTAAGGCCTTCAAATGCAAATTCTGCATTGTAACGATCC PDI-MERS-wtTMCT-DNA (SEQ ID NO: 101) PDI-MERS-H5iTMCT-DNA (SEQ ID NO: 102) PDI-MERS-H5iCT-DNA (SEQ ID NO: 103) PDI-MERS-H5iCT(V4)-DNA(SEQ ID NO: 104) PDI-MERS-H1cCT-DNA (SEQ ID NO: 105) PDI-MERS-wtTMCT-AA (SEQ ID NO: 106) PDI-MERS-H5iTMCT-AA (Sequence ID 107) PDI-MERS-H5iCT-AA (Sequence ID 108) PDI-MERS-H5iCT(V4)-AA(Sequence ID 109) PDI-MERS-H1cCT-AA (Sequence ID 110) Cloning vector 7147 (sequence number 111) from left T-DNA to right T-DNA Natural SARS-CoV-1 S protein wt™ / CT AA (P59594) (SEQ ID NO: 112) Natural MERS S protein wt™ / CT AA AFY13307 (SEQ ID NO: 113) Natural SARS-CoV-1 S protein wt™ / CT AA P59594 (SEQ ID NO: 114) without signal peptides Natural MERS S protein wt™ / CT AA AFY13307 (SEQ ID NO: 115) without signal peptides TMCT region (SEQ ID NO: 116) of modified PDI-SARS-COV-1 wtTMCT-AA WYVWLGFIAGLIAIVMVTILLCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT TMCT region (sequence number 117) of modified PDI-SARS-COV-1 H5iTMCT-AA WYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI TMCT region (sequence number 118) of modified PDI-SARS-COV-1 H5iCT-AA WYVWLGFIAGLIAIVMVTILLSLWMCSNGSLQCRICI Modified PDI-SARS-COV-1 H5iCT(V4)-AA TMCT region (SEQ ID NO: 119) WYVWLGFIAGLIAIVMVTILLCCSNGSLQCRICI Modified PDI-SARS-COV-1 H1cCT-AA TMCT region (SEQ ID NO: 120) WYVWLGFIAGLIAIVMVTILLSFWMCSNGSLQCRICI TMCT region of modified PDI-MERS-wtTMCT-AA (SEQ ID NO: 121) WYIWLGFIAGLVALALCVFFILCCTGCGTNCMGKLKCNRCCDRYEEYDLEPHKVHVH TMCT region (sequence number 122) of modified PDI-MERS-H5iTMCT-AA WYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI TMCT region (sequence number 123) of modified PDI-MERS-H5iCT-AA WYIWLGFIAGLVALALCVFFILSLWMCSNGSLQCRICI Modified PDI-MERS-H5iCT(V4)-AA TMCT region (sequence number 124) WYIWLGFIAGLVALALCVFFILCCSNGSLQCRICI TMCT region of modified PDI-MERS-H1cCT-AA (SEQ ID NO: 125) WYIWLGFIAGLVALALCVFFILSFWMCSNGSLQCRICI Modified PDI-S protein + H1 Cal TMCT region (SEQ ID NO: 126) WYIWLGFIAGLIAIVMVTIMLSFWMCSNGSLQCRICI Modified PDI-S protein + TMCT region of H3 Minn (SEQ ID NO: 127) WYIWLGFIAGLIAIVMVTIMLMWACQKGNIRCNICI Modified PDI-S protein + H6 HK TMCT region (SEQ ID NO: 128) WYIWLGFIAGLIAIVMVTIMLGLWMCSNGSMQCRICI Modified PDI-S protein + TMCT region of H7 Guangdong (SEQ ID NO: 129) WYIWLGFIAGLIAIVMVTIMLVFICVKNGNMRCTICI Modified PDI-S protein + H9 HK TMCT region (SEQ ID NO: 130) WYIWLGFIAGLIAIVMVTIMLLFWAMSNGSCRCNICI Modified PDI-S protein + B / Wash TMCT region (SEQ ID NO: 131) WYIWLGFIAGLIAIVMVTIMLVVYMVSRDNVSCSICL Consensus sequence of the TM domain of the coronavirus S protein (SEQ ID NO: 132) WYXWLGFIAGLXAXXX{X}VXXXL({X} may not exist) Consensus sequence of the TM domain of the coronavirus S protein (SEQ ID NO: 133) WY[I / V]WLGFIAGL[V / I]A[L / I][A / V][L / M]{X}V[F / T][F / I]XL (wherein {X} may or may not be C). Intermediate peptide sequence X n Modified SARS-CoV-1 S protein TM / CT region containing (SEQ ID NO: 134) WYVWLGFIAGLIAIVMVTIL-(X)n-CSNGSXXCXICI Intervening peptide sequence X n TM / CT region (SEQ ID NO: 135) of the modified MERS S protein having WYIWLGFIAGLVALALCVFFIL-(X)n-CSNGSXXCXICI IF(AvB+wtCT-OC43).r(SEQ ID NO: 136) ACGACACGACTAAGGCCTTCAGTCGTCATGCGAGGTCTTAATGACAAGC PDI-OC43-wtTMCT-DNA(SEQ ID NO: 137) PDI-OC43-H5iTMCT-DNA (SEQ ID NO: 138) PDI-OC43-H5iCT-DNA (SEQ ID NO: 139) PDI-OC43-H5iCT(V4)-DNA(SEQ ID NO: 140) PDI-OC43-H1cCT-DNA (SEQ ID NO: 141) PDI-OC43-wtTMCT-AA (Sequence ID 142) PDI-OC43-H5iTMCT-AA (Sequence ID 143) PDI-OC43-H5iCT-AA (Sequence ID 144) PDI-OC43-H5iCT(V4)-AA(Sequence ID 145) PDI-OC43-H1cCT-AA (Sequence ID 146) IF(CoV229EwtCT).r(Sequence ID 147) ACGACACGACTAAGGCCTTCACTGTATGTGGATCTTTTCGACATCGTA PDI-229E-wtTMCT-DNA (SEQ ID NO: 148) PDI-229E-H5iTMCT-DNA (SEQ ID NO: 149) PDI-229E-H5iCT-DNA (SEQ ID NO: 150) PDI-229E-H5iCT(V4)-DNA (SEQ ID NO: 151) PDI-229E-H1cCT-DNA (SEQ ID NO: 152) PDI-229E-wtTMCT-AA (Sequence ID 153) PDI-229E-H5iTMCT-AA (Sequence ID 154) PDI-229E-H5iCT-AA (Sequence ID 155) PDI-229E-H5iCT(V4)-AA(Sequence ID 156) PDI-229E-H1cCT-AA (Sequence ID 157) Natural OC43-CoV S protein wt™ / CT AA (AVR40344) (SEQ ID NO: 158) Natural 229E S protein wt™ / CT AA (P15423) (SEQ ID NO: 159) Natural OC43-CoV S protein wt™ / CT AA (AVR40344) (SEQ ID NO: 160) without signal peptides Natural 229E S protein wt™ / CT AA (P15423) (SEQ ID NO: 161) without signal peptides TMCT region (SEQ ID NO: 162) of modified PDI-OC43-COV wtTMCT-AA WYVWLLICLAGVAMLVLLFFICCCTGCGTSCFKKCGGCCDDYTGYQELVIKTSHDD TMCT region (sequence number 163) of modified PDI-OC43-COV H5iTMCT-AA WYQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI TMCT region (sequence number 164) of modified PDI-OC43-COV H5iCT-AA WYVWLLICLAGVAMLVLLFFISLWMCSNGSLQCRICI Modified PDI-OC43-COV H5iCT(V4)-AA TMCT region (SEQ ID NO: 165) WYVWLLICLAGVAMLVLLFFICCSNGSLQCRICI TMCT region of modified PDI-OC43-COV H1cCT-AA (SEQ ID NO: 166) WYVWLLICLAGVAMLVLLFFISFWMCSNGSLQCRICI TMCT region of modified PDI-229E-wtTMCT-AA (SEQ ID NO: 167) WWVWLCISVVLIFVVSMLLLCCCSTGCCGFFSCFASSIRGCCESTKLPYYDVEKIHIQ TMCT region (sequence number 168) of modified PDI-229E-H5iTMCT-AA WWQILSIYSTVASSLALAIMMAGLSLWMCSNGSLQCRICI TMCT region (sequence number 169) of modified PDI-229E-H5iCT-AA WWVWLCISVVLIFVVSMLLLSLWMCSNGSLQCRICI TMCT region (sequence number 170) of modified PDI-229E-H5iCT(V4)-AA WWVWLCISVVLIFVVSMLLLCCSNGSLQCRICI TMCT region of modified PDI-229E-H1cCT-AA (SEQ ID NO: 171) WWVWLCISVVLIFVVSMLLLSFWMCSNGSLQCRICI Intermediate peptide sequence X n The TM / CT region of the modified OC43-CoV S protein (SEQ ID NO: 172) WYVWLLICLAGVAMLVLLFFI-(X)n-CSNGSXXCXICI Intermediate peptide sequence X n The TM / CT region of the modified OC43-CoV S protein (SEQ ID NO: 173) WWVWLCISVVLIFVVSMLLL-(X)n-CSNGSXXCXICI

[0343] All quotations are incorporated herein by reference.

[0344] The present invention has been described with respect to one or more embodiments. However, it will be apparent to those skilled in the art that many modifications and alterations can be made without departing from the scope of the invention as defined in the claims. [Sequence Listing Free Text]

[0345] Sequence Listing 5 <223> Modified SARS-CoV-2 with H5 A / Indonesia / 5 / 05 hemagglutinin CT AA Sequence Listing 14 <223> Consensus sequence of the C-terminal region of influenza hemagglutinin Sequence Listing 14 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 15 <223> Consensus sequence of the CT domain of influenza hemagglutinin Sequence Listing 15 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 18 <223> C-terminal region of modified SARS-CoV-2 S protein containing H5i hemagglutinin CT Sequence Listing 19 <223> C-terminal region of modified SARS-CoV-2 S protein with H5i hemagglutinin CT, mutation 1 Sequence Listing 20 <223> (No SP) SARS-CoV-2 S protein GSAS+PP wtTM / CT AA Sequence Listing 21 <223> (No SP) Modified SARS-CoV-2 S protein with H5i hemagglutinin CT AA GSAS+PP Sequence Listing 22 <223> PDI-SARS-CoV-2 S protein GSAS+PP wtTM / CT-DNA Sequence Listing 23 <223> PDI-SARS-CoV-2 S protein GSAS+PP wtTM / CT-AA Sequence Listing 26 <223> PDI-modified SARS-CoV-2 S protein GSAS+PP H5i™ / CT-DNA Sequence Listing 27 <223> PDI-modified SARS-CoV-2 S protein GSAS+PP H5iTM / CT-AA Sequence Listing 29 <223> PDI-modified SARS-CoV-2 S protein GSAS+PP wtTM / H5iCT-DNA Sequence Listing 30 <223> PDI-modified SARS-CoV-2 S protein GSAS+PP wtTM / H5iCT-AA Sequence Listing 31 <223> Cloning vector 8501 from left T-DNA to right T-DNA Sequence Listing 32 <223> 2X35S Promoter to NOS Terminator Construction 8586 Sequence Listing 33 <223> Cloning vector 8500 from left T-DNA to right T-DNA Sequence Listing 34 <223> 2X35S Promoter to NOS Terminator Construction 8589 Sequence Listing 35 <223> Cloning vector 8716 from left T-DNA to right T-DNA Sequence Listing 36 <223> 2X35S Promoter to NOS Terminator Construction 8591 Sequence Listing 37 <223> C-terminal region of modified SARS-CoV-2 S protein with H5i hemagglutinin CT, mutation 2 Sequence Listing 38 <223> C-terminal region of modified SARS-CoV-2 S protein with H5i hemagglutinin CT, mutation 3 Sequence Listing 39 <223> C-terminal region of modified SARS-CoV-2 S protein with H5i hemagglutinin CT, mutation 4 Sequence Listing 47 <223> Modified SARS-CoV-2 S protein with (PDI)H5i hemagglutinin CT AA GSAS+PP Sequence Listing 48 <223> Modified SARS-CoV-2 S protein GSAS+4P containing (PDI)H5i hemagglutinin CT AA Sequence Listing 49 <223> Modified SARS-CoV-2 S protein GSAS+6P containing (PDI)H5i hemagglutinin CT AA Sequence Listing 50 <223> Modified SARS-CoV-2 S protein GSAS+PP+923 containing (PDI)H5i hemagglutinin CT AA Sequence Listing 51 <223> Modified SARS-CoV-2 S protein GSAS+4P+923 containing (PDI)H5i hemagglutinin CT AA Sequence Listing 52 <223> Modified SARS-CoV-2 S protein GSAS+6P+923 containing (PDI)H5i hemagglutinin CT AA Sequence Listing 53 <223> (PDI)H1 Modified SARS-CoV-2 S protein with Cal-hemagglutinin CT AA GSAS+PP Sequence Listing 54 <223> (PDI)H3 Minn hemagglutinin CT AA modified SARS-CoV-2 S protein GSAS+PP Sequence Listing 55 <223> Modified SARS-CoV-2 S protein with (PDI)H6 HK hemagglutinin CT AA GSAS+PP Sequence Listing 56 <223> (PDI)H7 Guangdong hemagglutinin CT AA modified SARS-CoV-2 S protein GSAS+PP Sequence Listing 57 <223> (PDI)H9 HK hemagglutinin CT AA-containing modified SARS-CoV-2 S protein GSAS+PP Sequence Listing 58 <223> Modified SARS-CoV-2 S protein GSAS+PP with (PDI)B / Wash hemagglutinin CT AA Sequence Listing 59 <223> Modified SARS-CoV-2 S protein GSAS+PP with (PDI)H5i hemagglutinin CT (alternative 1)AA Sequence Listing 60 <223> Modified SARS-CoV-2 S protein GSAS+PP with (PDI)H5i hemagglutinin CT (alternative 2)AA Sequence Listing 61 <223> Modified SARS-CoV-2 S protein GSAS+PP with (PDI)H5i hemagglutinin CT (alternative 3)AA Sequence Listing 62 <223> Modified SARS-CoV-2 S protein GSAS+PP with (PDI)H5i hemagglutinin CT (alternative 4)AA Sequence Listing 64 <223> Modified TMCT having intervening peptide sequence (X)n Sequence Listing 64 <223> Xaa can be any combination of 0 to 10 amino acids. Sequence Listing 64 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 64 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 65 <223> PDI-S protein GSAS+4P-DNA Sequence Listing 66 <223> PDI-S protein GSAS+6P-DNA Sequence Listing 67 <223> PDI-S protein GSAS+2P+L923F-DNA Sequence Listing 68 <223> PDI-S protein GSAS+4P+L923F-DNA Sequence Listing 69 <223> PDI-S protein GSAS+6P+L923F-DNA Sequence Listing 70 <223> PDI-modified S protein containing H5i hemagglutinin CT(V1)DNA Sequence Listing 71 <223> PDI-modified S protein containing H5i hemagglutinin CT(V2)DNA Sequence Listing 72 <223> PDI-modified S protein containing H5i hemagglutinin CT(V3)DNA Sequence Listing 73 <223> PDI-modified S protein containing H5i hemagglutinin CT(V4)DNA Sequence Listing 74 <223> PDI-S-protein + H1 Cal DNA Sequence Listing 75 <223> PDI-S protein + H3 Minn DNA Sequence Listing 76 <223> PDI-S-protein + H6 HK DNA Sequence Listing 77 <223> PDI-S-protein + H7 Guangdong DNA Sequence Listing 78 <223> PDI-S protein + H9 HK DNA Sequence Listing 79 <223> PDI-S protein + B / Wash DNA Sequence Listing 111 <223> Cloning vector 7147 from left T-DNA to right T-DNA Sequence Listing 116 <223> TMCT region of modified PDI-SARS-COV-1 wtTMCT-AA Sequence Listing 118 <223> TMCT region of modified PDI-SARS-COV-1 H5iCT-AA Sequence Listing 119 <223> TMCT region of modified PDI-SARS-COV-1 H5iCT(V4)-AA Sequence Listing 120 <223> TMCT region of modified PDI-SARS-COV-1 H1cCT-AA Sequence Listing 121 <223> TMCT region of modified PDI-MERS-wtTMCT-AA Sequence Listing 122 <223> TMCT region of modified PDI-MERS-H5iTMCT-AA Sequence Listing 123 <223> TMCT region of modified PDI-MERS-H5iCT-AA Sequence Listing 124 <223> TMCT region of modified PDI-MERS-H5iCT(V4)-AA Sequence Listing 125 <223> TMCT region of modified PDI-MERS-H1cCT-AA Sequence Listing 126 <223> TMCT region of modified PDI-S-protein + H1 Cal Sequence Listing 127 <223> TMCT region of modified PDI-S protein + H3 Minn Sequence Listing 128 <223> TMCT region of modified PDI-S protein + H6 HK Sequence Listing 129 <223> Modified PDI-S-protein + H7 Guangdong TMCT region Sequence Listing 130 <223> TMCT region of modified PDI-S-protein + H9 HK Sequence Listing 131 <223> TMCT region of modified PDI-S-protein + B / Wash Sequence Listing 132 <223> Consensus sequence of the TM domain of coronavirus S protein Sequence Listing 132 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 132 <223> Xaa may or may not exist. Sequence Listing 133 <223> Consensus sequence of the TM domain of coronavirus S protein Sequence Listing 133 <223> Xaa can be Ile or Val. Sequence Listing 133 <223> Xaa can be Val or Ile. Sequence Listing 133 <223> Xaa can be Leu or Ile. Sequence Listing 133 <223> Xaa can be Ala or Val. Sequence Listing 133 <223> Xaa can be Leu or Met. Sequence Listing 133 <223> Xaa may or may not be Cys. Sequence Listing 133 <223> Xaa can be Phe or Thr. Sequence Listing 133 <223> Xaa can be Phe or Ile. Sequence Listing 133 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 134 <223> TM / CT region of modified SARS-CoV-1 S protein containing intervening peptide sequence Sequence Listing 134 <223> X can be any combination of 0 to 10 amino acids. Sequence Listing 134 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 134 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 135 <223> TM / CT region of modified MERS S protein containing intervening peptide sequence Sequence Listing 135 <223> X can be any combination of 0 to 10 amino acids. Sequence Listing 135 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 135 <223> Xaa could be any naturally occurring amino acid. Sequence Listing 162 <223> TMCT region of modified PDI-OC43-COV wtTMCT-AA Sequence Listing 163 <223> TMCT region of modified PDI-OC43-COV H5iTMCT-AA Sequence Listing 164 <223> TMCT region of modified PDI-OC43-COV H5iCT-AA Sequence Listing 165 <223> TMCT region of modified PDI-OC43-COV H5iCT(V4)-AA Sequence Listing 166 <223> TMCT region of modified PDI-OC43-COV H1cCT-AA Sequence Listing 167 <223> TMCT region of modified PDI-229E-wtTMCT-AA Sequence Listing 168 <223> TMCT region of modified PDI-229E-H5iTMCT-AA Sequence Listing 169 <223> TMCT region of modified PDI-229E-H5iCT-AA Sequence Listing 170 <223> TMCT region of modified PDI-229E-H5iCT(V4)-AA Sequence Listing 171 <223> TMCT region of modified PDI-229E-H1cCT-AA Sequence Listings 172-173 <223> TM / CT region of modified OC43-CoV S protein containing intervening peptide sequence Xn Sequence Listings 172-173 <223> Xaa can be any combination of 0 to 10 amino acids. Sequence Listings 172-173 <223> Xaa could be any naturally occurring amino acid.

Claims

1. These are modified coronavirus S proteins, in order: - The external domain derived from the coronavirus S protein, - Transmembrane and cytosolic terminal domains (TMCTs), wherein the TMCTs are chimeric TMCTs. - A transmembrane domain (TM) derived from a chimeric TM comprising an N-terminal sequence derived from coronavirus S protein or coronavirus S protein TM and a C-terminal sequence derived from influenza HA protein TM, - A cytosolic end (CT) derived from a chimeric CT that includes an N-terminal sequence derived from influenza hemagglutinin (HA) protein or coronavirus S protein CT, and a C-terminal sequence derived from influenza HA protein CT, The transmembrane and cytosolic terminal domains (TMCTs) are included, Includes, The chimera™ comprises an N-terminal sequence derived from the coronavirus S protein™ containing at least 20 amino acids corresponding to amino acids 1 to 20 of SEQ ID NO: 18 or SEQ ID NO: 169, or at least 21 amino acids corresponding to amino acids 1 to 21 of SEQ ID NO: 118 or 164, or at least 22 amino acids corresponding to amino acids 1 to 22 of SEQ ID NO: 123, and one or more amino acids from the C-terminus of the influenza HA protein™, wherein the one or more amino acids from the C-terminus of the influenza HA protein™ are selected from AG or a conserved substitution of AG, AGL or a conserved substitution of AGL, MAGL or a conserved substitution of MAGL, The chimeric CT comprises a C-terminal sequence derived from influenza HA protein CT containing amino acids corresponding to amino acids 27-37 of SEQ ID NOs. 18, 126, 128, 129, 130, or 131, or amino acids corresponding to amino acids 27-36 of SEQ ID NO. 127, and one or more amino acids from the N-terminus of coronavirus S protein CT, wherein the one or more amino acids from the N-terminus of coronavirus S protein CT are selected from conserved substitutions of C or C, conserved substitutions of CC or CC, or conserved substitutions of CCM or CCM. Modified coronavirus S protein.

2. The modified coronavirus S protein according to claim 1, wherein the TM is directly fused to the CT, or the TM is fused to the CT by an intervening peptide sequence, wherein the intervening peptide sequence is preferably L, LCCM, LSLWM, AGLSLWM, or MAGLSLWM.

3. The modified coronavirus S protein according to claim 1, wherein the chimera™ contains amino acids corresponding to amino acids 1 to 20 of SEQ ID NO: 18 or SEQ ID NO: 169, or amino acids 1 to 21 of SEQ ID NO: 118 or SEQ ID NO: 164, or amino acids 1 to 22 of SEQ ID NO:

123.

4. The modified coronavirus S protein according to claim 1, wherein the chimeric CT contains amino acids corresponding to amino acids 27 to 37 of SEQ ID NO: 18, 126, 128, 129, 130, or 131, or amino acids 27 to 36 of SEQ ID NO:

127.

5. The modified coronavirus S protein according to claim 1, wherein the chimeric TMCT comprises a chimeric TM containing amino acids corresponding to amino acids 1 to 20 of SEQ ID NO: 18 or SEQ ID NO: 169, or amino acids 1 to 21 of SEQ ID NO: 118 or SEQ ID NO: 164, or amino acids 1 to 22 of SEQ ID NO: 123, and a chimeric CT containing amino acids corresponding to amino acids 27 to 37 of SEQ ID NO: 18, 126, 128, 129, 130 or 131, amino acids 27 to 36 of SEQ ID NO: 127, or a combination thereof.

6. The modified S protein according to any one of claims 1 to 5, wherein the S protein includes one or more amino acid substitutions when compared to the amino acid sequence of the wild-type coronavirus S protein.

7. The modified S protein according to claim 6, wherein the one or more amino acid substitutions correspond to amino acids at positions 667, 668, 670, 802, 923, 927, 971, 972, or combinations thereof, when compared with the reference amino acid sequence of SEQ ID NO:

2.

8. The modified S protein according to claim 7, wherein the amino acid substitution corresponding to the amino acid at position 667 of SEQ ID NO: 2 is glycine or a conserved substitution of glycine, the amino acid substitution corresponding to position 668 of SEQ ID NO: 2 is serine or a conserved substitution of serine, the amino acid substitution corresponding to position 670 of SEQ ID NO: 2 is serine or a conserved substitution of serine, the amino acid substitutions corresponding to the amino acids at positions 802, 927, 971 and 972 of SEQ ID NO: 2 are proline or a conserved substitution of proline, and the amino acid substitution corresponding to position 923 of SEQ ID NO: 2 is phenylalanine or a conserved substitution of phenylalanine.

9. The modified S protein according to any one of claims 6 to 8, wherein, when the one or more substitutions maintain the S protein in a prefusion state or are expressed in a host or host cell, the modified S protein yields a higher yield of the modified S protein compared to the yield of the corresponding S protein without the one or more substitutions expressed in the host or host cell.

10. The modified S protein according to any one of claims 1 to 9, wherein the influenza HA protein is derived from influenza type B or influenza subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16.

11. The modified S protein according to any one of claims 1 to 10, wherein the external domain is derived from SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43-CoV, or 229E-CoV.

12. The modified S protein according to any one of claims 1 to 11, wherein the TMCT includes a sequence having 80% to 100% identity with the sequence of SEQ ID NOs: 18, 19, 37, 38, 39, 64, 126, 127, 128, 129, 130, 131, 118, 119, 120, 123, 124, 125, 134, 135, 164, 165, 166, 169, 170, 171, 172, or 173.

13. A modified S protein according to any one of claims 1 to 12, comprising a plant-specific N-glycan.

14. The amino acid sequence of the aforementioned S protein is the amino acids of SEQ ID NOs. 5, 21, 30, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 95, 96, 97, 108, 109, 110, 144, 145, 146, 155, 156 or 157, or amino acids 24-1259 of SEQ ID NO. 47, amino acids 25-1259 of SEQ ID NO. 48, and amino acids of SEQ ID NO.

49. amino acids 25-1259, amino acids 25-1259 of SEQ ID NO: 50, amino acids 25-1259 of SEQ ID NO: 51, amino acids 25-1259 of SEQ ID NO: 52, amino acids 25-1259 of SEQ ID NO: 53, amino acids 25-1259 of SEQ ID NO: 54, amino acids 25-1259 of SEQ ID NO: 55, amino acids 25-1259 of SEQ ID NO: 56, amino acids 25-1259 of SEQ ID NO: 57, amino acids 25-1259 of SEQ ID NO: 58 59. The modified S protein according to claim 1, comprising 80% to 100% identity with amino acids 25 to 1262 of SEQ ID NO: 59, amino acids 25 to 1261 of SEQ ID NO: 60, amino acids 25 to 1258 of SEQ ID NO: 61 or amino acids 25 to 1256 of SEQ ID NO: 62, amino acids 25 to 1243 of SEQ ID NO: 95, amino acids 25 to 1240 of SEQ ID NO: 96, amino acids 25 to 1243 of SEQ ID NO: 97, amino acids 25 to 1341 of SEQ ID NO: 108, amino acids 25 to 1338 of SEQ ID NO: 109, amino acids 25 to 1341 of SEQ ID NO: 110 or amino acids 25 to 1351 of SEQ ID NO: 144, amino acids 25 to 1348 of SEQ ID NO: 145, amino acids 25 to 1351 of SEQ ID NO: 146, amino acids 25 to 1159 of SEQ ID NO: 155, amino acids 25 to 1156 of SEQ ID NO: 156, or amino acids 25 to 1159 of SEQ ID NO:

157.

15. The modified S protein according to any one of claims 1 to 13, wherein the CT has 80% to 100% identity with the sequence of SEQ ID NO: 15, or amino acids 35 to 50 of SEQ ID NO: 6, 8, 7, 9, 10, 12, 13 or 14, or amino acids 34 to 49 of SEQ ID NO: 11, or amino acids 553 to 568 of SEQ ID NO: 3, or amino acids 22 to 37 of SEQ ID NO: 18, or amino acids 21 to 40 of SEQ ID NO: 19, or amino acids 21 to 39 of SEQ ID NO: 37, or amino acids 25 to 36 of SEQ ID NO: 38, or amino acids 24 to 34 of SEQ ID NO: 39, or amino acids 22 to 37 of SEQ ID NO: 126, 128, 129, 130 or 131, or amino acids 22 to 36 of SEQ ID NO:

127.

16. The aforementioned S protein is produced as a precursor, and the precursor protein contains amino acids 1-1234 of SEQ ID NO: 1, or amino acids 1-1234 of SEQ ID NO: 5, amino acids 1-1243 of SEQ ID NO: 30, amino acids 1-1227 of SEQ ID NO: 95, amino acids 1-1325 of SEQ ID NO: 108, amino acids 1-1216 of SEQ ID NO: 112, amino acids 1-1318 of SEQ ID NO: 113, amino acids 1-1335 of SEQ ID NO: 144, amino acids 1-155 The modified S protein according to claim 1, wherein the amino acid sequence of CT contains 80% to 100% identity with amino acids 1 to 1325 of SEQ ID NO: 158 and amino acids 1 to 1135 of SEQ ID NO: 159, and the amino acid sequence of CT contains 80% to 100% identity with the sequence of SEQ ID NO: 15, or amino acids 35 to 50 of SEQ ID NO: 6, 8, 7, 9, 10, 12, 13 or 14, or amino acids 34 to 49 of SEQ ID NO: 11, or amino acids 553 to 568 of SEQ ID NO:

3.

17. A nucleic acid comprising a nucleotide sequence encoding the modified S protein according to any one of claims 1 to 16.

18. A virus-like particle (VLP) comprising the modified S protein according to any one of claims 1 to 16.

19. A vaccine for inducing an immune response, comprising an effective amount of the modified S protein described in any one of claims 1 to 16 or the VLP described in claim 18.

20. Use of a modified S protein according to any one of claims 1 to 16 or a VLP according to claim 18 for the manufacture of a pharmaceutical or vaccine for inducing an immune response to coronavirus infection in a subject.

21. A non-human host or host cell comprising a modified S protein according to any one of claims 1 to 16, a nucleic acid according to claim 17, or a VLP according to claim 18.

22. A method for producing virus-like particles (VLPs) in a non-human host or host cell, a) Introducing the nucleic acid described in claim 17 into the non-human host or host cell, or providing the non-human host or host cell containing the nucleic acid described in claim 17, b) Incubating the non-human host or host cell under conditions that enable the expression of the nucleic acid, thereby producing the VLP, Methods that include...

23. The method according to claim 22, wherein the non-human host or host cell includes a plant, a part of a plant, a plant cell, a fungus, a fungal cell, an insect, an insect cell, an animal, or an animal cell.

24. The non-human host or host cell according to claim 21, wherein the non-human host or host cell includes a plant, a part of a plant, a plant cell, a fungus, a fungal cell, an insect, an insect cell, an animal, or an animal cell.

25. A composition comprising virus-like particles (VLPs), wherein the VLPs comprise the modified coronavirus S protein described in claim 1. (i) The modified S protein includes substitutions at positions 667, 668, 670, 971 and 972 when compared to the reference amino acid sequence of SEQ ID NO: 2; (ii) The S protein includes a glycine substitution at position 667, a serine substitution at position 668, a serine substitution at position 670, a proline substitution at position 971, and a proline substitution at position 972, the positions of which correspond to the reference amino acid sequence of SEQ ID NO: 2; (iii) The modified S protein contains the sequence of SEQ ID NO: 21 or amino acids 25-1259 of SEQ ID NO: 51; or (iv) A composition wherein the modified S protein includes substitutions at positions 667, 668, 670, 802, 923, 927, 971 and 972 when compared to the reference amino acid sequence of SEQ ID NO: 2.

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