Coronavirus vaccine

Abstract

Designed coronavirus polypeptide sequences and their use as vaccines against viruses of the coronavirus family are described. The designed sequences include designed coronavirus spike (S) proteins and fragments thereof, including designed full-length S protein sequences, SEQ ID NOs: 88, 87, and 53. Designed coronavirus envelope (E), membrane (M), and nucleocapsid (N) protein sequences are also described, as are their use as vaccines. Nucleic acid molecules encoding the polypeptides, vectors, fusion proteins, pharmaceutical compositions, cells, and their use as vaccines against viruses of the coronavirus family are also described.

Description

[Technical Field]

[0001] The present invention relates to nucleic acid molecules, polypeptides, vectors, cells, fusion proteins, pharmaceutical compositions, combined preparations and their use as vaccines against viruses of the coronavirus family. [Background technology]

[0002] Coronaviruses (CoVs) cause a wide variety of animal and human diseases. Notable human diseases caused by CoVs are zoonotic diseases such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Viruses within this family generally cause mild, self-limiting respiratory infections in immunocompetent humans, but can also cause severe, fatal illness characterized by the onset of fever, extreme fatigue, dyspnea, anoxia, and pneumonia. CoVs are transmitted through close contact via respiratory droplets from infected subjects, with varying degrees of infectivity within each strain.

[0003] CoVs belong to the Coronaviridae family of viruses, all of which are enveloped. CoVs contain a single-stranded, forward-oriented RNA genome, 25 to 31 kilobases in length (Siddell S.G. 1995, The Coronaviridae), the largest genome yet found in an RNA virus. Based on phylogenetic clustering, the Coronaviridae family is subtyped into four genera: α, β, γ, and δ coronaviruses, and each genus is further subdivided into clusters depending on the viral strain. For example, the genus β-CoV (group 2 CoV), four lineages (a, b, c, and d) are commonly recognized. Type A (subgenus Envecovirus) includes HCoV-OC43 and HCoV-HKU1 (various species). Type B (subgenus Sarbecovirus) includes SARSr-CoV (including all strains of SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1). Type C (subgenus Merbecovirus) includes Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4), Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), and MERS-CoV (various species). Type D (subgenus Novecovirus) includes the Rousettus bat coronavirus HKU9 (BtCoV-HKU9).

[0004] CoV virions are spherical with characteristic club-shaped spikes emanating from their surface. Virions contain four major structural proteins: spike (S); membrane (M); envelope (E); and nucleocapsid (N) proteins, all of which are encoded by the viral genome. Some subsets of β-CoVs contain a fifth structural protein, hemagglutinin esterase (HE), which enhances S protein-mediated cell entry and viral spread through mucosal membranes through its acetylesterase activity. Homotrimers of S glycoproteins constitute the unique spike structure on the viral surface. These trimers are class I fusion proteins that mediate viral binding to host receptors through the interaction of the S protein and its receptor. In most CoVs, S is cleaved by host cell proteases into two separate polypeptides—S1 and S2. S1 contains the receptor-binding domain (RBD) of the S protein (the exact positioning of the RBD varies depending on the virus strain), and S2 forms the stem of the spike molecule.

[0005] Figure 1 shows the SARS S-protein architecture. The N-terminal sequence is involved in relaying extracellular signals into the cell. Studies have shown that the N-terminal region of the S protein is much more diverse than the highly conserved C-terminal region (Dong et al., Genomic and protein structure modeling analysis depicts the origin and infectivity of 2019-nCoV, a new coronavirus which caused a pneumonia outbreak in Wuhan, China. 2020). The diagram shows the S domain, including the S1 and S2 domains involved in receptor binding and cell membrane fusion, respectively.

[0006] RNA viruses generally have a much higher mutation rate than DNA viruses because viral RNA polymerase lacks the editing capabilities of DNA polymerase. This explains why viruses can spread from their natural host reservoir to other species and from person to person, and why it is difficult to create effective vaccines to prevent diseases caused by RNA viruses. Current vaccine candidates for RNA viruses are often limited by the viral strain used as the vaccine insert, which is often selected based on wild-type strain availability rather than informed design. Technical challenges for developing vaccines for enveloped RNA viruses include: i) viral variability in wild-type field isolate glycoproteins (GPs) limits the breadth of protection offered by vaccine antigens; ii) selection of vaccine antigens expressed by vaccine inserts is highly experimental, and immunogen selection is a slow, trial-and-error process; and iii) developing new vaccine candidates in the face of evolving or unexpected viral epidemics can be time-consuming, potentially delaying vaccine development.

[0007] Before 2002, CoVs were thought to cause only mild respiratory problems and became endemic in the human population, causing 15–30% of respiratory tract infections annually. Since their initial discovery in the 1960s, the CoV family has expanded extensively, causing numerous outbreaks in both humans and animals. The SARS epidemic that occurred in Guangdong Province, China, in 2002–2003 was the most severe disease caused by any coronavirus known at the time. During that period, approximately 8,098 cases occurred, with 774 deaths (an overall mortality rate of approximately 9.6%). The mortality rate was approximately 50% in individuals over the age of 90. The virus identified as SARS-CoV is a β-CoV of family 2b and originated in bats. Two novel virus isolates from bats are more similar to human SARS-CoV than any other virus identified at the time, and bind to the same cellular receptor, angiotensin-converting enzyme 2 (ACE2), as human-derived SARS-CoV.

[0008] The SARS-CoV epidemic was brought under control in 2003, and a novel human CoV, the 2c β-CoV family, emerged in the Middle East in 2012. MERS is the causative agent of a series of highly pathogenic respiratory tract infections in the Middle East, with an initial mortality rate of 50%. An estimated 2,494 cases and 858 deaths caused by MERS have been reported since its emergence, with an overall estimated case fatality rate of 34.4% according to the World Health Organization (WHO). Along with SARS-CoV, this novel CoV likely originated in bats, with intermediate hosts such as dromedaries likely contributing to the spread of the pandemic. This virus utilizes another peptidase receptor, dipeptidyl peptidase 4 (DPP4), as its receptor. The reason why CoVs utilize host peptidases as their binding receptors is currently unknown, as entry occurs even in the absence of enzymatic activity.

[0009] Shortly before the end of 2019, another novel CoV, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged. A major outbreak began in Wuhan, China, in late 2019. As the virus spread to more than 25 countries within one month of its emergence, the WHO declared a global public health emergency on January 30, 2020. The number of SARS-CoV-2 (SARS2) infections increased exponentially across many countries around the world. Efforts to halt the spread of the virus reduced the number of infected cases and deaths caused by the virus. However, second and third waves of the virus occurred in many countries, resulting in a global figure of over 142 million confirmed cases and over 3 million confirmed deaths (as of April 22, 2021, according to the WHO).

[0010] Since the first described human infection with SARS-CoV-2 in December 2019, nine vaccines have been approved for use in humans (Craven, 2021, Regulatory Focus, News Articles, 2020, 3, COVID-19 Vaccine Tracker: https: / / www.raps.org / news-and-articles / news-articles / 2020 / 3 / covid-19-vaccine-tracker). As of October 2022, more than 37 vaccines have been approved for use in humans, with many more in development (Craven, 2022, Regulatory Focus, News Articles, 2020, 3, COVID-19 Vaccine Tracker: https: / / www.raps.org / news-and-articles / news-articles / 2020 / 3 / covid-19-vaccine-tracker). The AstraZeneca / Oxford COVID-19 vaccine (AZD1222) uses an adenoviral vector. Two of the vaccines currently in use worldwide, BNT162b2 (Pfizer) and mRNA-1273 (Moderna), are based on lipid nanoparticle delivery of mRNA encoding a stabilized pre-fusion form of the spike protein derived from SARS-CoV-2, isolated early in the epidemic from Wuhan, China.Both of these vaccines demonstrated >94% efficacy in preventing coronavirus disease 2019 (COVID-19) in Phase III clinical trials conducted in late 2020 in multiple countries (Polack et al., C4591001 Clinical Trial Group (2020). Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 383, 2603-2615; Baden et al., COVE Study Group (2021). Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N. Engl. J. Med. 384, 403-416). However, the recent emergence of novel widespread variants has significantly raised concerns about the effectiveness of current vaccines, especially in countries such as South Africa and Brazil where the epidemic is dominated by variant strains (Garcia-Beltran et al., 2021, Cell 184, 2372-2383: Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity).

[0011] One of the earliest variants to emerge and rapidly become dominant worldwide was D614G. In the United Kingdom, a novel lineage designated B.1.1.7 (also known as VOC-202012 / 01 or 501Y.V1) rapidly emerged. B.1.1.7 contains not only D614G but also N501Y in the ACE2 receptor-binding domain (RBD), three amino acid deletions in the spike, and seven missense mutations, and has been reported to be more infectious than D614G. In Denmark, there have also been reports of SARS-CoV-2 transmission between humans and mink due to a variant designated mink cluster 5 or B.1.1.298, which contains Y453F in the RBD, two amino acid deletions, and four missense mutations. Another variant recently emerged in California, designated B.1.429, contains four missense mutations in the spike, one of which is a single L452R RBD mutation. The ability of the B.1.1.298 and B.1.429 variants to evade neutralizing humoral immunity from previous infection or vaccination remains to be determined. Novel variants arising from the B.1.1.28 lineage first described in Brazil and Japan, designated P.2 (with three spike missense mutations) and P.1 (also called gamma variants, with 12 spike missense mutations), contain the E484K mutation, while P.1 also contains K417T and N501Y mutations in the RBD. These strains are spreading rapidly, and both P.2 and P.1 have recently been found in documented cases of SARS-CoV-2 reinfection. Of greatest concern is the emergence of multiple strains of the B.1.351 lineage (also known as 501Y.V2), which was first reported in South Africa and has since spread globally. This lineage contains three RBD mutations, K417N, E484K, and N501Y, in addition to several mutations outside the RBD. The B.1.617.2 (delta variant), which contains increased transmissibility, subsequently emerged. The variant first detected in India in December 2020 contains four mutations in the RBD: L452R, T478K, K417N, and E484K.More recently, the B.1.1.529 (BA.1 / Omicron) variant emerged, containing 30 mutations in the S protein, 15 of which are in the RBD, and has been shown to cause significant humoral immune evasion and increased transmissibility. Since then, several Omicron subvariants have emerged, including BA.2, BA.3, BA.4, and BA.5. Some of these subvariants also contain the BA.2.12.1 subvariant. The emergence of novel variants that appear to escape immune responses has prompted vaccine manufacturers to develop boosters for these spike variants.

[0012] Human cases or outbreaks of hemorrhagic fever caused by coronaviruses occur sporadically and irregularly. Outbreaks cannot be easily predicted. With few exceptions, there is no cure or established drug treatment for CoV infection. Vaccines have been approved for only some CoVs, but these vaccines are not always used because they are not highly effective or, in some cases, have been reported to promote the selection of novel pathogenic CoVs through recombination of circulating strains. As of April 2020, several potential vaccines for SARS-CoV had been developed but were not approved for use. A year later, several novel vaccines had received regulatory approval, and mass vaccination programs were underway. Even one year later, more vaccines are still receiving regulatory approval. The first mass vaccination program began in early December 2020, and as of February 15, 2021, the WHO estimates that 175.3 million vaccine doses had been administered. At least seven different vaccines are in use worldwide. The WHO issued an Emergency Use Listing (EUL) for the Pfizer-BioNTech COVID-19 vaccine (BNT162b2) on December 31, 2020. The WHO issued an EUL for two versions of the AstraZeneca / Oxford COVID-19 vaccine (AZD1222) on February 15, 2021. As of February 18, 2021, the UK had administered the first dose of either the Pfizer-BioNTech or AstraZeneca / Oxford vaccine to 12 million people. Both the Pfizer and Moderna vaccines use mRNA platforms that encode the S protein. Pfizer uses a nanoparticle vector for nucleic acid delivery, while AstraZeneca uses an adenovirus vector.

[0013] Many hurdles remain to overcome in the development of effective CoV vaccines. First, immunity, whether natural or artificial, does not necessarily protect against subsequent infection (Fehr et al. Methods Mol Biol. 2015, 1282:1-23). ​​Second, the tendency of viruses to recombine can pose a problem by increasing viral genetic diversity and thereby rendering vaccines ineffective. Furthermore, vaccination with viral S-protein has been shown to result in enhanced disease in the case of FIPV (feline infectious peritonitis virus), a highly pathogenic strain of feline CoV. This enhanced disease virulence is caused by non-neutralizing antibodies that facilitate viral entry into host cells in a process called antibody-dependent enhancement (ADE). Following primary infection with one virus strain, neutralizing antibodies are produced against that same strain of virus. However, if a different strain infects a host in a second infection, the secondary non-neutralizing antibodies produced during the first infection do not neutralize the virus but instead bind to the virus, which then binds to IgG Fc receptors on immune cells and mediates viral entry into these cells (Wan et al. Journal of Virology. 2020, 94(5):1-13). When developing vaccines against viruses capable of ADE (or that induce ADE-like proinflammatory responses), it is important that epitopes involved in eliciting non-neutralizing antibodies are identified and that these epitopes are either masked by modification or removed from the vaccine. These non-neutralizing epitopes on the S-protein can also lead to immune conversion, where non-neutralizing epitopes exclude neutralizing epitopes for binding to antibodies. Neutralizing epitopes are ignored by the immune system, which is unable to neutralize the antigen. In the case of recombinant RBD vaccines, previously buried surfaces containing non-neutralizing immunodominant epitopes can be newly exposed, eliminating epitopes involved in neutralization by the immune system. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Dong et al,Genomic and protein structure modeling analysis depicts the origin and infectivity of 2019-nCoV, a new coronavirus which caused a pneumonia outbreak in Wuhan,China.2020 [Non-licensed document 2] Craven, 2021, Regulatory Focus, News Articles, 2020, 3, COVID-19 Vaccine Tracker: https: / / www.raps.org / news-and-articles / news-articles / 2020 / 3 / covid-19-vaccine-tracker [Non-licensed document 3] Polack et al., C4591001 Clinical Trial Group (2020). Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N. Engl. J. Med. 383, 2603-2615

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[0015] Therefore, there is a need to provide effective vaccines that induce broadly neutralizing immune responses to protect against emerging and re-emerging diseases caused by CoVs, particularly β-CoVs such as SARS-CoV and recently SARS-CoV-2, and in particular vaccines that lack non-neutralizing epitopes that can lead to viral immune evasion and disease progression through ADE (or ADE-like pro-inflammatory responses).

[0016] There is also a need to provide improved coronavirus vaccines that elicit broadly neutralizing antibodies against SARS-CoV-2 variants, particularly those of current and recent concern, and in particular to provide effective vaccines that induce broadly neutralizing immune responses that protect against Delta and some Omicron strains.

[0017] Furthermore, there is a need to provide vaccines that successfully combat vaccine escape of new SARS-CoV-2 variants.

[0018] Designed coronavirus spike (S) protein sequences (full-length, truncated, and receptor-binding domain, RBD) Figure 2 shows a multiple sequence alignment of the S-protein (region surrounding cleavage site 1) comparing a SARS-CoV isolate (SARS-CoV-1) with a closely related bat betacoronavirus (RaTG13) isolate with four SARS-CoV-2 isolates. The SARS-CoV S-protein (1269 amino acid residues) shares high sequence identity (approximately 73%) with the SARS-CoV-2 S-protein (1273 amino acid residues). Expansion of cleavage site 1 (shown as the boxed region in the figure) has been observed in all SARS-CoV-2 strains to date. The majority of insertions / substitutions are observed in subunit 1, with minimal substitutions in subunit S2 compared to SARS-CoV-1. The C-terminus contains epitopes that elicit non-neutralizing antibodies and are involved in antibody-dependent enhancement.

[0019] The applicant has generated a novel amino acid sequence of the S protein, designated CoV_T2_1 (hereinafter also referred to as Wuhan-Node-1), which has improved immunogenicity (enabling the protein and its derivatives to induce a broadly neutralizing immune response).

[0020] The amino acid sequences of the full-length S protein (SEQ ID NO: 13) (CoV_T2_1, Wuhan-Node-1), the truncated S protein (tr, lacking the C-terminal portion of the S2 sequence) (SEQ ID NO: 15) (CoV_T2_4; Wuhan_Node1_tr), and the receptor binding domain (RBD) (SEQ ID NO: 17) (CoV_T2_7; Wuhan_Node1_RBD) (and their respective encoding nucleic acid sequences, SEQ ID NOs: 14, 16, 18) are provided in the Examples below.

[0021] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:17.

[0022] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:17.

[0023] SEQ ID NO: 17 is the amino acid sequence of a novel S-protein RBD designed by the present applicant.

[0024] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:15.

[0025] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0026] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:13 or an amino acid sequence having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:13.

[0027] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:13.

[0028] Examples 6 and 7 below provide amino acid sequence alignments of the novel S protein RBD amino acid sequence Wuhan_Node1_RBD(CoV_T2_7) (SEQ ID NO: 17) with the RBD amino acid sequences of SARS-TOR2 isolate AY274119 (AY274119_RBD(CoV_T2_5) (SEQ ID NO: 5)) and SARS_CoV_2 isolate hCov-19 / Wuhan / LVDC-HB-01 / 2019 (EPI_ISL_402119) (EPI_ISL_402119_RBD(CoV_T2_6) (SEQ ID NO: 11)), respectively.

[0029] As described in Example 9 below, Figure 4 shows the Wuhan_Node1_RBD (CoV_T2_7) amino acid sequence (SEQ ID NO: 17) with amino acid residue differences highlighted in bold and underlined from respective alignments with the AY 274119_RBD (CoV_T2_5) (SEQ ID NO: 5) and EPI_ISL_402119_RBD (CoV_T2_6) (SEQ ID NO: 11) amino acid sequences (Examples 6 and 7, respectively). The amino acid residue differences between the two alignments are listed in the table below (the numbering of the residue positions corresponds to the position in the Wuhan_Node1_RBD(CoV_T2_7) (SEQ ID NO: 17) amino acid sequence). The common differences between the two alignments are at amino acid residues: 3, 6, 7, 21, 22, 38, 42, 48, 67, 70, 76, 81, 83, 86, 87, 92, 121, 122, 123, 125, 126, 128, 134, 137, 138, 141, 150, 152, 153, 154, 155, 167, 171, 178, 180, 181, 183, 185, 187, 188, 189, 191, 194, 195, 219 (shown in grey highlighting in Figure 4 and the table below). [Table 1-1] [Table 1-2]

[0030] The amino acid insertions are at positions 167-172 (compared to AY274119_RBD) and 163-167 (compared to EPI_ISL_402119_RBD) (boxed in Figure 4).

[0031] Optionally, an isolated polypeptide of the invention comprises at least one of the amino acid residues at a position corresponding to the amino acid residue position of SEQ ID NO: 17, as shown in Table 2 below. [Table 2]

[0032] Optionally, an isolated polypeptide of the invention comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0033] Optionally, an isolated polypeptide of the invention comprises at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0034] Optionally, an isolated polypeptide of the invention comprises at least 15 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0035] Optionally, an isolated polypeptide of the invention comprises at least 20 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0036] Optionally, an isolated polypeptide of the invention comprises at least 25 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0037] Optionally, an isolated polypeptide of the invention comprises at least 30 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0038] Optionally, an isolated polypeptide of the invention comprises at least 35 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0039] Optionally, an isolated polypeptide of the invention comprises at least 40 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0040] Optionally, an isolated polypeptide of the invention comprises all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 2.

[0041] Optionally, an isolated polypeptide of the invention comprises at least one of the amino acid residues at a position corresponding to the amino acid residue position of SEQ ID NO: 17, as shown in Table 3 below. [Table 3-1] [Table 3-2]

[0042] Optionally, an isolated polypeptide of the invention comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0043] Optionally, an isolated polypeptide of the invention comprises at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0044] Optionally, an isolated polypeptide of the invention comprises at least 15 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0045] Optionally, an isolated polypeptide of the invention comprises at least 20 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0046] Optionally, an isolated polypeptide of the invention comprises at least 25 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0047] Optionally, an isolated polypeptide of the invention comprises at least 30 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0048] Optionally, an isolated polypeptide of the invention comprises at least 35 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0049] Optionally, an isolated polypeptide of the invention comprises at least 40 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0050] Optionally, an isolated polypeptide of the invention comprises at least 45 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0051] Optionally, an isolated polypeptide of the invention comprises at least 50 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0052] Optionally, an isolated polypeptide of the invention comprises at least 55 amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0053] Optionally, an isolated polypeptide of the invention comprises at least 60 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0054] Optionally, an isolated polypeptide of the invention comprises all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 3.

[0055] Optionally, an isolated polypeptide of the invention comprises at least one of the amino acid residues at a position corresponding to the amino acid residue position of SEQ ID NO: 17, as shown in Table 4 below. [Table 4-1] [Table 4-2]

[0056] Optionally, an isolated polypeptide of the invention comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0057] Optionally, an isolated polypeptide of the invention comprises at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0058] Optionally, an isolated polypeptide of the invention comprises at least 15 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0059] Optionally, an isolated polypeptide of the invention comprises at least 20 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0060] Optionally, an isolated polypeptide of the invention comprises at least 25 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0061] Optionally, an isolated polypeptide of the invention comprises at least 30 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0062] Optionally, an isolated polypeptide of the invention comprises at least 35 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0063] Optionally, an isolated polypeptide of the invention comprises at least 40 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0064] Optionally, an isolated polypeptide of the invention comprises at least 45 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0065] Optionally, an isolated polypeptide of the invention comprises at least 50 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0066] Optionally, an isolated polypeptide of the invention comprises at least 55 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0067] Optionally, an isolated polypeptide of the invention comprises at least 60 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0068] Optionally, an isolated polypeptide of the invention comprises all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:17, as shown in Table 4.

[0069] The present invention also provides an isolated polypeptide comprising a coronavirus S protein RBD domain having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in Table 5 below. [Table 5-1] [Table 5-2]

[0070] The present invention also provides an isolated polypeptide comprising a coronavirus S protein RBD domain having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in Table 6 below. [Table 6-1] [Table 6-2]

[0071] The present invention also provides an isolated polypeptide comprising a coronavirus S protein RBD domain having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in Table 7 below. [Table 7-1] [Table 7-2]

[0072] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:5.

[0073] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:11.

[0074] The novel S protein RBD sequences are further referred to herein as CoV_S_T2_13-CoV_S_T2_18 (SEQ ID NOS: 27-32, respectively). CoV_S_T2_13 is the direct output of our design algorithm, and CoV_S_T2_14-CoV_S_T2_18 are epitope-enriched versions of CoV_S_T2_13. The amino acid sequences of these designed sequences are provided below and in Example 12. [ka] [ka]

[0075] An alignment of these sequences with the SARS2 reference sequence (EPI_ISL_402119_RBD(CoV_T2_6) (SEQ ID NO: 11)) is shown in Example 12 below.

[0076] The amino acid differences between the SARS2 reference sequence and the designed sequences are shown below in Table 8.1 (differences from the reference sequence are highlighted in bold, differences common to all designed sequences are underlined). [Table 8-1-1] [Table 8-1-2]

[0077] The amino acid changes common to all of the designed sequences are summarized below in Table 8.2. [Table 8-2]

[0078] Optional additional changes are summarized in Table 8.3 below. [Table 8-3]

[0079] Additional changes listed in Table 8.3 are found in SEQ ID NOs: 27-29, 31, and 32. Tables 8.4 to 8.6 below summarize further optional additional changes. [Table 8-4] [Table 8-5] [Table 8-6]

[0080] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 27 (COV_S_T2_13), or an amino acid sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 27.

[0081] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 28 (COV_S_T2_14), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 28 over its entire length.

[0082] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 29 (COV_S_T2_15), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 29.

[0083] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 30 (COV_S_T2_16), or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 30.

[0084] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 31 over its entire length.

[0085] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 32 over its entire length.

[0086] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 27 (COV_S_T2_13) or an amino acid sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 27 comprises at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in Table 8.2 above.

[0087] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 28 (COV_S_T2_14) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 28 comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.2 above.

[0088] Optionally, polypeptides of the invention, including isolated polypeptides comprising the amino acid sequence of SEQ ID NO: 29 (COV_S_T2_15) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 29, comprise at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.2 above.

[0089] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 30 (COV_S_T2_16) or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 30 comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.2 above.

[0090] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31 comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.2 above.

[0091] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32 comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.2 above.

[0092] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 27 (COV_S_T2_13) or an amino acid sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 27 further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.3 above.

[0093] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 28 (COV_S_T2_14) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 28 further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.3 above.

[0094] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 29 (COV_S_T2_15) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 29 further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.3 above.

[0095] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31 further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.3 above.

[0096] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32, further comprises at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in Table 8.3 above. Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 28 (COV_S_T2_14), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 28, further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.4 above.

[0097] Optionally, polypeptides of the invention, including isolated polypeptides comprising the amino acid sequence of SEQ ID NO: 29 (COV_S_T2_15) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 29, further comprise at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.5 above.

[0098] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31 further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.4 above.

[0099] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31 further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.6 above.

[0100] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32 further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.5 above.

[0101] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18) or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32 further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 8.6 above.

[0102] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 27 (COV_S_T2_13).

[0103] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 28 (COV_S_T2_14).

[0104] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 29 (COV_S_T2_15).

[0105] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 30 (COV_S_T2_16).

[0106] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17).

[0107] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18).

[0108] The present invention also provides an isolated polypeptide comprising a coronavirus S protein RBD domain having at least one of the amino acid residues at a position corresponding to the amino acid residue position of SEQ ID NO:11, as shown in Table 8.2 above.

[0109] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain having at least one amino acid residue at a position corresponding to an amino acid residue position of SEQ ID NO:11, as shown in Table 8.2 above, comprises at least five amino acid residues at positions corresponding to an amino acid residue position of SEQ ID NO:11, as shown in Table 8.2 above.

[0110] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain having at least one amino acid residue at a position corresponding to an amino acid residue position of SEQ ID NO:11, as shown in Table 8.2 above, comprises at least 10 amino acid residues at positions corresponding to an amino acid residue position of SEQ ID NO:11, as shown in Table 8.2 above.

[0111] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain having at least one amino acid residue at a position corresponding to an amino acid residue position of SEQ ID NO:11, as shown in Table 8.2 above, comprises at least 15 amino acid residues at positions corresponding to an amino acid residue position of SEQ ID NO:11, as shown in Table 8.2 above.

[0112] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain having at least one amino acid residue at a position corresponding to an amino acid residue position of SEQ ID NO:11, as shown in Table 8.2 above, comprises all of the amino acid residues at the positions corresponding to the amino acid residue positions of SEQ ID NO:11, as shown in Table 8.2 above.

[0113] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain having at least 1, 5, 10, 15, or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11, as shown in Table 8.2 above, further comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11, as shown in Table 8.3 above.

[0114] Optionally, the coronavirus S protein RBD domain having at least 1, 5, 10, 15, or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11, as set forth in Table 8.2 above, and the isolated polypeptides of the invention comprising at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11, as set forth in Table 8.3 above, further comprise at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11, as set forth in any of Tables 8.4-8.6 above.

[0115] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:5.

[0116] Optionally, an isolated polypeptide of the invention comprising a coronavirus S protein RBD domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:11.

[0117] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 92 (CoV_S_T2_17+tPA signal sequence).

[0118] The discontinuous epitope sequence of the designed S protein RBD sequence COV_S_T2_14-18 (SEQ ID NOs: 28-32) The sequence alignment below shows the alignment of the designed S protein RBD sequences COV_S_T2_13-18. The colored boxes indicate residues of discontinuous epitopes present in sequences COV_S_T2_14-18, which are shown in different colors. Changes made to the COV_S_T2_13 sequence to provide discontinuous epitopes that elicit a broader or stronger immune response are indicated by the boxed regions. [ka]

[0119] Residues of the discontinuous epitope present in COV_S_T2_14 and COV_S_T2_17 (marked in black) are as follows: i) NITNLCPFGEVFNATK (SEQ ID NO: 57) - residues 13-28; ii) KKISN (SEQ ID NO: 58) - residues 38-42; iii) NI (SEQ ID NO: 59) - residues 122-123

[0120] The residues of the discontinuous epitope present in COV_S_T2_15 and COV_S_T2_18 (marked in purple) are as follows: i) YNSTFFSTFKCYGVSPTKLNDLCFS (SEQ ID NO: 60) - residues 51-75; ii) DDFM (SEQ ID NO: 61) - residues 109-112 iii) FELLN (SEQ ID NO: 62) - residues 197-201

[0121] The residues of the discontinuous epitope present in COV_S_T2_16 (marked in orange) are as follows: i) RGDEVRQ (SEQ ID NO: 63) - residues 85-91, ii) TGKIADY (SEQ ID NO: 64) - residues 97-103; iii) YRLFRKSN (SEQ ID NO: 65) - residues 135-142; iv) YQAGST (SEQ ID NO: 66) - residues 155-160 v) FNCYFPLQSYGFQPTNGVGY (SEQ ID NO: 67) - residues 168-187

[0122] The residues of the discontinuous epitopes present in COV_S_T2_13, COV_S_T2_15, COV_S_T2_16, and COV_S_T2_18 (vertically adjacent to the epitopes marked in black) are as follows: (i) NITNLCPFGEVFNATR (SEQ ID NO: 68) - residues 13-28; (ii) KRISN (SEQ ID NO: 69) - residues 38-42; (iii) NL (SEQ ID NO: 70)—residues 122 to 123

[0123] The residues of the discontinuous epitopes present in COV_S_T2_13, COV_S_T2_14, COV_S_T2_16, and COV_S_T2_17 (vertically adjacent to the epitopes marked in purple) are as follows: (i) YNSTSFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 71) - residues 51 to 75; (ii) DDFT (SEQ ID NO: 72)—residues 109-112 (iii) FELLN (SEQ ID NO: 62) - residues 197-201

[0124] The residues of the discontinuous epitopes present in COV_S_T2_13, COV_S_T2_14, and COV_S_T2_15 (vertically adjacent to the epitope marked in orange) are as follows: i) RGDEVRQ (SEQ ID NO: 63) - residues 85-91, (ii) TGVIADY (SEQ ID NO: 73)—residues 97-103; (iii) YRSLRKSK (SEQ ID NO: 74)—residues 135-142; (iv) YSPGGK (SEQ ID NO: 75) - residues 155 to 160 (V) FNCYYPLRSYGFFPTNGVGY (SEQ ID NO: 76) - residues 168 to 187

[0125] The residues of the discontinuous epitopes present in COV_S_T2_17 and COV_S_T2_18 (vertically adjacent to the epitope marked in orange) are as follows: i) RGDEVRQ (SEQ ID NO: 63) - residues 85-91, (ii) TGVIADY (SEQ ID NO: 73)—residues 97-103; (iii) YRSLRKSK (SEQ ID NO: 74)—residues 135-142; (iv) YSPGGK (SEQ ID NO: 75) - residues 155 to 160 (V) FNCYYPLRSYGFFPTNGTGY (SEQ ID NO: 77) - residues 168 to 187

[0126] In accordance with the present invention, there is provided an isolated polypeptide comprising an amino acid sequence having the following non-contiguous amino acid sequence: i) NITNLCPFGEVFNATK (SEQ ID NO: 57); ii) KKISN (SEQ ID NO: 58); iii) NI (SEQ ID NO: 59).

[0127] In accordance with the present invention, there is provided an isolated polypeptide comprising an amino acid sequence having the following non-contiguous amino acid sequence: i) YNSTFFSTFKCYGVSPTKLN DLCFS (SEQ ID NO: 60); ii) DDFM (SEQ ID NO: 61); iii) FELLN (SEQ ID NO: 62).

[0128] In accordance with the present invention, there is provided an isolated polypeptide comprising an amino acid sequence having the following non-contiguous amino acid sequence: i) RGDEVRQ (SEQ ID NO: 63), ii) TGKIADY (SEQ ID NO: 64); iii) YRLFRKSN (SEQ ID NO: 65); iv) YQAGST (SEQ ID NO: 66); v) FNCYFPLQSYGFQPTNGVGY (SEQ ID NO: 67).

[0129] If desired, one or more of the amino acid residues of SEQ ID NOs: 63-67 in a polypeptide of the invention comprising a discontinuous amino acid sequence of SEQ ID NOs: 63-67 may be altered (e.g., by substitution or deletion) to provide a glycosylation site.

[0130] The present invention also provides an isolated polypeptide comprising an amino acid sequence having the following non-contiguous amino acid sequence: (i) NITNLCPFGEVFNATR (SEQ ID NO: 68), (ii) KRISN (SEQ ID NO: 69), (iii) NL (SEQ ID NO: 70)

[0131] In accordance with the present invention, there is provided an isolated polypeptide comprising an amino acid sequence having the following non-contiguous amino acid sequence: (i) YNSTSFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 71), (ii) DDFT (SEQ ID NO: 72) (iii) FELLN (SEQ ID NO: 62)

[0132] In accordance with the present invention, there is provided an isolated polypeptide comprising an amino acid sequence having the following non-contiguous amino acid sequence: (i) RGDEVRQ (SEQ ID NO: 63), (ii) TGVIADY (SEQ ID NO: 73), (iii) YRSLRKSK (SEQ ID NO: 74), (iv) YSPGGK (SEQ ID NO: 75) (v) FNCYYPLRSYGFFPTNGVGY (SEQ ID NO: 76)

[0133] In accordance with the present invention, there is provided an isolated polypeptide comprising an amino acid sequence having the following non-contiguous amino acid sequence: (i) RGDEVRQ (SEQ ID NO: 63), (ii) TGVIADY (SEQ ID NO: 73), (iii) YRSLRKSK (SEQ ID NO: 74), (iv) YSPGGK (SEQ ID NO: 75) (v) FNCYYPLRSYGFFPTNGTGY (SEQ ID NO: 77)

[0134] Optionally, the discontinuous amino acid sequences of each polypeptide of the invention are present in the order listed.

[0135] Optionally, each non-contiguous amino acid sequence is separated from an adjacent non-contiguous amino acid sequence by at least three amino acid residues.

[0136] Optionally, each non-contiguous amino acid sequence is separated from an adjacent non-contiguous amino acid sequence by a maximum of 100 amino acid residues.

[0137] Optionally, polypeptides of the invention comprising the recited non-contiguous amino acid sequences are up to 250, 500, 750, 1,000, 1,250, or 1,500 amino acid residues in length.

[0138] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:28 comprises the following non-contiguous amino acid sequence: i) NITNLCPFGEVFNATK (SEQ ID NO: 57); ii) KKISN (SEQ ID NO: 58); iii) NI (SEQ ID NO: 59).

[0139] Optionally, the discontinuous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 13-28, (ii) residues 38-42, and (iii) residues 122-123 of SEQ ID NO:28, respectively.

[0140] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:29 comprises the following non-contiguous amino acid sequence: i) YNSTFFSTFKCYGVSPTKLNDLCFS (SEQ ID NO: 60); ii) DDFM (SEQ ID NO: 61); iii) FELLN (SEQ ID NO: 62).

[0141] Optionally, the discontinuous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO:29, respectively.

[0142] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 30 comprises the following non-contiguous amino acid sequence: i) RGDEVRQ (SEQ ID NO: 63), ii) TGKIADY (SEQ ID NO: 64); iii) YRLFRKSN (SEQ ID NO: 65); iv) YQAGST (SEQ ID NO: 66); v) FNCYFPLQSYGFQPTNGVGY (SEQ ID NO: 67).

[0143] Optionally, the discontinuous amino acid sequences (i), (ii), (iii), (iv), and (v) are at amino acid residue positions corresponding to (i) residues 85-91, (ii) residues 97-103, (iii) residues 135-142, (iv) residues 155-160, and (v) residues 168-187 of SEQ ID NO: 30, respectively.

[0144] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31 comprises the following non-contiguous amino acid sequence: i) NITNLCPFGEVFNATK (SEQ ID NO: 57); ii) KKISN (SEQ ID NO: 58); iii) NI (SEQ ID NO: 59).

[0145] Optionally, the non-contiguous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 13-28, (ii) residues 38-42, and (iii) residues 122-123 of SEQ ID NO: 31, respectively.

[0146] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32 comprises the following non-contiguous amino acid sequence: i) YNSTFFSTFKCYGVSPTKLNDLCFS (SEQ ID NO: 60); ii) DDFM (SEQ ID NO: 61); iii) FELLN (SEQ ID NO: 62).

[0147] Optionally, the non-contiguous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO: 32, respectively.

[0148] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:29 comprises the following non-contiguous amino acid sequence: (i) NITNLCPFGEVFNATR (SEQ ID NO: 68), (ii) KRISN (SEQ ID NO: 69), (iii) NL (SEQ ID NO: 70)

[0149] Optionally, the discontinuous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 13-28, (ii) residues 38-42, and (iii) residues 122-123 of SEQ ID NO:29, respectively.

[0150] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 30 comprises the following non-contiguous amino acid sequence: (i) NITNLCPFGEVFNATR (SEQ ID NO: 68), (ii) KRISN (SEQ ID NO: 69), (iii) NL (SEQ ID NO: 70)

[0151] Optionally, the discontinuous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 13-28, (ii) residues 38-42, and (iii) residues 122-123 of SEQ ID NO: 30, respectively.

[0152] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32 comprises the following non-contiguous amino acid sequence: (i) NITNLCPFGEVFNATR (SEQ ID NO: 68), (ii) KRISN (SEQ ID NO: 69), (iii) NL (SEQ ID NO: 70)

[0153] Optionally, the discontinuous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 13-28, (ii) residues 38-42, and (iii) residues 122-123 of SEQ ID NO: 32, respectively.

[0154] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:28 comprises the following non-contiguous amino acid sequence: (i) YNSTSFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 71), (ii) DDFT (SEQ ID NO: 72) (iii) FELLN (SEQ ID NO: 62)

[0155] Optionally, the discontinuous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO:28, respectively.

[0156] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 30 comprises the following non-contiguous amino acid sequence: (i) YNSTSFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 71), (ii) DDFT (SEQ ID NO: 72) (iii) FELLN (SEQ ID NO: 62)

[0157] Optionally, the non-contiguous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO: 30, respectively.

[0158] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31 comprises the following non-contiguous amino acid sequence: (i) YNSTSFSTFKCYGVSPTKLNDLCFT (SEQ ID NO: 71), (ii) DDFT (SEQ ID NO: 72) (iii) FELLN (SEQ ID NO: 62)

[0159] Optionally, the discontinuous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO: 31, respectively.

[0160] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:28 comprises the following non-contiguous amino acid sequence: (i) RGDEVRQ (SEQ ID NO: 63), (ii) TGVIADY (SEQ ID NO: 73), (iii) YRSLRKSK (SEQ ID NO: 74), (iv) YSPGGK (SEQ ID NO: 75) (v) FNCYYPLRSYGFFPTNGVGY (SEQ ID NO: 76)

[0161] Optionally, the non-contiguous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO:28, respectively.

[0162] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:29 comprises the following non-contiguous amino acid sequence: (i) RGDEVRQ (SEQ ID NO: 63), (ii) TGVIADY (SEQ ID NO: 73), (iii) YRSLRKSK (SEQ ID NO: 74), (iv) YSPGGK (SEQ ID NO: 75) (v) FNCYYPLRSYGFFPTNGVGY (SEQ ID NO: 76)

[0163] Optionally, the discontinuous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO:29, respectively.

[0164] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31 comprises the following non-contiguous amino acid sequence: (i) RGDEVRQ (SEQ ID NO: 63), (ii) TGVIADY (SEQ ID NO: 73), (iii) YRSLRKSK (SEQ ID NO: 74), (iv) YSPGGK (SEQ ID NO: 75) (v) FNCYYPLRSYGFFPTNGTGY (SEQ ID NO: 77)

[0165] Optionally, the non-contiguous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO: 31, respectively.

[0166] Optionally, an isolated polypeptide of the invention comprising an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32 comprises the following non-contiguous amino acid sequence: (i) RGDEVRQ (SEQ ID NO: 63), (ii) TGVIADY (SEQ ID NO: 73), (iii) YRSLRKSK (SEQ ID NO: 74), (iv) YSPGGK (SEQ ID NO: 75) (v) FNCYYPLRSYGFFPTNGTGY (SEQ ID NO: 77)

[0167] Optionally, the non-contiguous amino acid sequences (i), (ii), and (iii) are at amino acid residue positions corresponding to (i) residues 51-75, (ii) residues 109-112, and (iii) residues 197-201 of SEQ ID NO: 32, respectively.

[0168] Designed coronavirus S protein RBD sequence with altered glycosylation sites Epitope masking / unmasking has been shown to alter immune responses by masking non-neutralizing epitopes or unmasking important epitopes in MERS (Du L et al., Nat. Comm., volume 7, Article number: 13473 (2016)). We prepared additional designed S protein RBD sequences (SARS2 RBD designs M7, M8, M9, and M10) in which we either deleted or introduced glycosylation sites into the SARS2 RBD sequence. The changes made are shown in FIG. 13 and discussed in Example 14 below. Designs M7 and M9 contain an introduced glycosylation site at the position indicated by the circled number 4 (residue position 203) in FIG. 13. Designs M8 and M10 contain deleted glycosylation sites at the positions indicated by circled numbers 1 and 2 (residue positions 13 and 25, respectively) in Figure 13. The M8 design also contains an introduced glycosylation site at the position indicated by circled number 3 (residue position 54).

[0169] The amino acid sequences of SARS2 RBD designs M7, M8, M9, and M10 are shown below and in Example 14. [ka]

[0170] Such polypeptides are particularly advantageous because they induce broadly neutralizing antibody responses against a diverse panel of coronavirus VOCs, as demonstrated by the results in Figures 55-59 and described in Example 38. Notably, heterologous immunization using an M7 DNA prime followed by an M7 MVA boost results in significantly higher titers of neutralizing antibodies against a panel of VOCs (Wuhan-1 B, alphaB.1.1.7, betaB.1.351, gammaP.1, deltaB.1.617.2, and omicronB.A.1) compared to homologous immunization with an M7 DNA prime followed by an M7 DNA boost (Figure 57C). The strongest nAb responses could be observed in MVA RBD M7-boosted mice against the Wuhan-1 B, alphaB.1.1.7, gammaP.1, and deltaB.1.617.2 variants. Furthermore, M7 DNA prime followed by M7 MVA boost induced significantly higher titers of neutralizing antibodies against Wuhan-1 B, alpha B.1.1.7, gamma P.1, delta B.1.617.2, and comparable neutralization against beta B.1.351 and omicron BA.1 and BA.2 compared to heterologous DNA prime / MVA boost with WT RBD.

[0171] An alignment of these sequences with the SARS2 reference sequence (EPI_ISL_402119_RBD(CoV_T2_6) (SEQ ID NO: 11)) is shown in Example 14 below.

[0172] The amino acid differences between the SARS2 reference sequence and the designed sequence are shown in Table 9 below (with differences from the reference sequence highlighted in bold). [Table 9-1] [Table 9-2] [Table 9-3]

[0173] According to the present invention there is provided an isolated polypeptide comprising an amino acid sequence according to SEQ ID NO: 33 (designed S protein RBD sequence M7).

[0174] According to the present invention there is provided an isolated polypeptide comprising an amino acid sequence according to SEQ ID NO: 34 (designed S protein RBD sequence M8).

[0175] According to the present invention there is provided an isolated polypeptide comprising an amino acid sequence according to SEQ ID NO: 35 (designed S protein RBD sequence M9).

[0176] According to the present invention there is provided an isolated polypeptide comprising an amino acid sequence according to SEQ ID NO: 36 (designed S protein RBD sequence M10).

[0177] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 34 (M8), or an amino acid sequence having at least 99% amino acid identity to the amino acid sequence of SEQ ID NO: 34 over its entire length.

[0178] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 34 (M8) or an amino acid sequence having at least 99% amino acid identity to the amino acid sequence of SEQ ID NO: 34 over its entire length comprises at least one or all of the following amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11: 13Q, 25Q, 54T.

[0179] The present invention also provides an isolated polypeptide comprising a coronavirus S protein RBD domain having at least one of the following amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11: 13Q, 25Q, 54T, 203N.

[0180] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 35 (M9), or an amino acid sequence having at least 70% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 35.

[0181] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 35 (M9) or an amino acid sequence having at least 70% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 35 comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 9.1 below. [Table 9-1-1] [Table 9-1-2] [Table 9-1-3]

[0182] Optionally, the isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 35 (M9) or an amino acid sequence having at least 70% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 35 comprises at least one or both of the following amino acid residues: 54T, 203N, at positions corresponding to the amino acid residue positions of SEQ ID NO: 11.

[0183] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 36 (M10), or an amino acid sequence having at least 69% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 36.

[0184] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 36 (M10) or an amino acid sequence having at least 69% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 36 comprises at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 9.2 below. [Table 9-2-1] [Table 9-2-2] [Table 9-2-3]

[0185] Optionally, the isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 36 (M10) or an amino acid sequence having at least 69% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 36 comprises at least one or all of the following amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11: 13Q, 25Q, 54T.

[0186] The effect of glycosylation of the RBD protein appears to be important. We found that M7 and wild-type SARS2 RBD DNA (which appears to result in the expression of glycosylated RBD protein) are superior to recombinant SARS2 RBD protein (non-glycosylated or low-density glycosylated) in inducing a neutralizing response to SARS2. Example 28 below describes mass spectrometry data obtained to examine the glycosylation of SARS-CoV-2 (SARS2) RBD protein in supernatants from HEK cells transfected with pEVAC plasmids encoding the SARS-CoV-2 RBD sequence compared to recombinant SARS-CoV-2 RBD protein (see Figures 21 and 22). The results concluded that there were two major glycosylated forms of the protein obtained from the supernatant compared to the purified (recombinant) protein. The purified protein was either non-glycosylated or low-density glycosylated. This difference in glycosylation is thought to be important because the glycosylation sites surround the epitope region and are conserved among most sarbecoviruses. These glycosylation sites are also important for interaction with some of the antibodies.

[0187] Optionally, a polypeptide of the invention comprising the amino acid sequence of a designed coronavirus spike (S) protein (full-length, truncated, or RBD) comprises at least one glycosylation site in the RBD sequence.

[0188] Optionally, a polypeptide of the invention comprising the amino acid sequence of a designed coronavirus spike (S) protein (full-length, truncated, or RBD) comprises at least two glycosylation sites in the RBD sequence.

[0189] Optionally, a polypeptide of the invention comprising the amino acid sequence of a designed coronavirus spike (S) protein (full-length, truncated, or RBD) comprises at least three glycosylation sites in the RBD sequence.

[0190] Optionally, a polypeptide of the invention comprising the amino acid sequence of a designed coronavirus spike (S) protein (full-length, truncated, or RBD) comprises a glycosylation site located within the last 10 amino acids of the RBD sequence, preferably at a residue position corresponding to residue 203 of the RBD sequence.

[0191] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SARS2 RBD with a glycosylation site located within the last 10 amino acids of the SARS2 RBD sequence, preferably at a residue position corresponding to residue 203 of the RBD sequence.

[0192] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SARS2 RBD having a glycosylation site located within the epitope region of monoclonal antibody CR3022 (the epitope region of mAb CR3022 is shown in Figure 54B).

[0193] The present inventors also found that immunization of mice with wild-type SARS1 S protein or RBD protein, or wild-type SARS2 S protein or RBD protein, induced antibodies that bind to the SARS2 RBD.

[0194] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:5.

[0195] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:11.

[0196] The traditional method for generating cross-reactive antigens is to generate consensus sequences based on natural diversity. The antigen sequences encoded by the nucleic acid sequences of the present invention described herein take into account sampling bias and coevolution between sites. The result is a realistic molecule that induces an immune response against a range of viruses. As a further improvement, the inventors enriched the antigen sequences for known and predicted epitopes. The inventors developed an algorithm for selecting epitope combinations that maximize population protection against a range of target viruses. This algorithm identifies conserved epitopes while maintaining redundancy, ensuring that the selected epitopes are bound by a range of common MHC alleles.

[0197] To avoid disease enhancement, we modify antigens to delete regions associated with immunopathology, often referred to as antibody-dependent enhancement (ADE) and / or complement- or virus-induced proinflammatory responses. To validate these modifications, we developed assays to screen for such ADE-like effects. A modified assay from Yip et al. (Yip et al. "Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus," Virol J. 2014;11:82; Jaume et al. "Anti-Severe Acute Respiratory Syndrome Coronavirus Spike Antibodies Trigger Infection of Human Immune Cells via a pH- and Cysteine ​​Protease-Independent FcγR Pathway," Journal of Virology, October 2011, pp. 10582-10597) can be used to identify non-neutralizing antibodies against non-RBD sites of the S protein that allow SARS-CoV-1 to enter non-ACE2 expressing immune cells bearing Fc-γ-RII.

[0198] After antigens are designed, the DNA sequences encoding them are optimized for expression in mammalian cells. In this DNA form, multiple synthetic genes for the target antigens are inserted into DNA plasmid vectors (e.g., pEVAC, see Figure 3), which are used for both in vitro and in vivo immunoscreening.

[0199] Designed coronavirus full-length S protein sequences to protect against COVID-19 variants Multiple SARS-CoV-2 variants are circulating globally, most notably, several new variants emerged in the fall of 2020.

[0200] A new variant of SARS-CoV-2 (known as 20I / 501Y.V1, VOC202012 / 01, or B.1.1.7) with multiple mutations emerged in the United Kingdom (UK). This variant has since been detected in numerous countries around the world, including the United States (US). In January 2021, the UK reported evidence suggesting the B.1.1.7 variant may be associated with an increased risk of death compared with other variants, although more testing was needed to confirm this finding. This variant was reported in the US at the end of December 2020.

[0201] In South Africa, another variant of SARS-CoV-2 (known as 20H / 501Y.V2 or B.1.351) emerged independently of B.1.1.7. This variant shares several mutations with B.1.1.7. Cases attributed to this variant have been detected in multiple countries outside of South Africa. This variant was reported in the US in late January 2021.

[0202] A variant of SARS-CoV-2 (known as P-1) has emerged in Brazil and was first identified in four travelers from Brazil who were tested during routine screening at Haneda Airport outside Tokyo, Japan. This variant has 17 unique mutations, including three in the receptor-binding domain of the spike protein. This variant was detected in the US in late January 2021.

[0203] Scientists are working to learn more about these variants to better understand how easily they may be transmitted and the effectiveness of currently licensed vaccines against them. New information about the virological, epidemiological, and clinical characteristics of these variants is emerging rapidly.

[0204] As described in more detail below in Example 30, the inventors designed a new full-length S protein sequence ("VOC chimera," or COV_S_T2_29) for use as a COVID-19 vaccine insert to protect against variants B.1.1.7, P.1, and B.1.351. The amino acid sequence of the designed full-length S protein sequence is shown below and in Example 30. >COV_S_T2_29 (VOC chimera) (SEQ ID NO: 53) [ka]

[0205] An alignment of this sequence with the SARS2 reference sequence (EPI_ISL_402130 (Wuhan strain) (SEQ ID NO: 52)) is shown in Example 30 below.

[0206] The amino acid differences between the SARS2 reference sequence (SEQ ID NO: 52) and the designed sequence COV_S_T2_29 (SEQ ID NO: 53) are shown in Table 9.3 below. [Table 9-3-1]

[0207] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:53.

[0208] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:53, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:53.

[0209] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 53, comprises at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.4 below. [Table 9-4]

[0210] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:53, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:53, comprises at least five of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.4.

[0211] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 53, comprises at least 10 of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.4.

[0212] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:53, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:53, comprises at least one or all of the amino acid residues P at position 986 and P at position 987 at positions corresponding to the amino acid residue positions of SEQ ID NO:52, and amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.5 below. [Table 9-5]

[0213] The present invention also provides an isolated polypeptide comprising a coronavirus S protein having at least one or all of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.4 above.

[0214] An isolated polypeptide of the present invention comprising at least one amino acid residue or deletion at a position corresponding to an amino acid residue position of SEQ ID NO:52, as shown in Table 9.4 above, comprises at least five amino acid residues or deletions at positions corresponding to an amino acid residue position of SEQ ID NO:52, as shown in Table 9.4 above.

[0215] An isolated polypeptide of the present invention comprising at least one amino acid residue or deletion at a position corresponding to an amino acid residue position of SEQ ID NO:52, as shown in Table 9.4 above, comprises at least 10 of the amino acid residues or deletions at a position corresponding to an amino acid residue position of SEQ ID NO:52, as shown in Table 9.4 above.

[0216] Optionally, the coronavirus S protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:52.

[0217] Optionally, an isolated polypeptide of the invention comprising at least one of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52 as shown in Table 9.4 above comprises amino acid residue P at position 986 and amino acid residue P at position 987, corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.5 above, and at least one or all of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52.

[0218] As described in more detail in Example 37 below, the inventors designed a new full-length S protein, COV_S_T2_29 (COV_S_T2_29+Q498R; SEQ ID NO: 87), with an arginine residue at position 498 of SEQ ID NO: 52, corresponding to position 495 of SEQ ID NO: 53 (COV_S_T2_29). The designed construct is effective for use as a COVID-19 vaccine insert for protection against variants B.1.617.2, P.1, B.1.351, and BA.1, as described in the Examples. The amino acid sequence of the designed full-length S protein sequence is shown below and in Example 37. [ka]

[0219] The amino acid differences between the SARS2 reference sequence (SEQ ID NO: 52) and the designed sequence COV_S_T2_29+Q498R (SEQ ID NO: 87) are shown in Table 9.6 below. [Table 9-6]

[0220] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:87.

[0221] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:87, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:87.

[0222] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 87, comprises at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.7 below. [Table 9-7]

[0223] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:87, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:87, comprises at least five of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.7.

[0224] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:87, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:87, comprises at least 10 of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.7.

[0225] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:87, or an amino acid sequence having at least 99% amino acid identity thereto over its entire length, comprises at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, such as amino acid residue P at position 986 and amino acid residue P at position 987, as well as amino acid residue positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.8 below. [Table 9-8]

[0226] The present invention also provides an isolated polypeptide comprising a coronavirus S protein having at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.8 above.

[0227] The designed constructs are effective for use as COVID-19 vaccine inserts for protection against variants B.1.617.2, P.1, B.1.351, and BA.1 (delta, gamma, beta, and omicron BA.1, respectively), as described in the Examples. Also, as described in Example 37, the designed constructs generated at least two-fold better neutralizing responses against beta, gamma, and omicron after three doses of DNA vaccination compared to WTdER (Figure 50C). Neutralizing antibody titers against delta challenge were lower than WTdER (Figure 50C) before MVA boost.

[0228] As described in more detail in Example 37 below, the inventors also designed a new full-length S protein COV_S_T2_29+Q498R (COV_S_T2_29+Q498R+dER; SEQ ID NO: 88) with a 19 amino acid C-terminal truncation (dER). The designed construct is effective for use as a COVID-19 vaccine insert for protection against variants B.1.617.2, P.1, B.1.351, and BA.1, as described in the Examples. The amino acid sequence of the designed full-length S protein sequence is shown below and in Example 37. [ka] [ka]

[0229] The amino acid differences between the SARS2 reference sequence (SEQ ID NO: 52) and the designed sequence COV_S_T2_29+Q498R+dER (SEQ ID NO: 88) are shown in Table 9.9 below. [Table 9-9]

[0230] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:88.

[0231] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:88, or an amino acid sequence having at least 98% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:88.

[0232] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:88, or an amino acid sequence having at least 98% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:88, comprises at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.10 below. [Table 9-10]

[0233] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:88, or an amino acid sequence having at least 98% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:88, comprises at least five of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.10.

[0234] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:88, or an amino acid sequence having at least 98% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:88, comprises at least 10 of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.10.

[0235] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:88, or an amino acid sequence having at least 98% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:88, comprises at least 15 of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.10.

[0236] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:88, or an amino acid sequence having at least 98% amino acid identity thereto over its entire length, comprises at least one or all of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, such as amino acid residue P at position 986 and amino acid residue P at position 987, as well as amino acid residue positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.11 below. [Table 9-11]

[0237] The present invention also provides an isolated polypeptide comprising a coronavirus S protein having at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.11 above.

[0238] The designed constructs are effective for use as COVID-19 vaccine inserts for protection against variants B.1.617.2, P.1, B.1.351, and BA.1 (delta, gamma, beta, and omicron, respectively), as described in the Examples. Also, as described in Example 37, the designed constructs generated at least two-fold better neutralizing responses against beta, gamma, and omicron after three doses of DNA vaccination compared to WTdER (Figure 50C). Neutralizing antibody titers against both the ancestral sequence and delta were comparable for the T2_29+Q+dER design to WTdER (Figure 50C).

[0239] Closed, engineered coronovirus S protein sequences to protect against COVID-19 variants and predict future variants The majority of SARS-CoV-2 vaccines currently in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on the virion as a prefusion trimer in which the receptor-binding domain (RBD) is stochastically open or closed. Neutralizing antibodies active against both the open and closed conformations have been described. The long-term success of vaccination strategies depends on inducing antibodies that provide long-lasting, broad-spectrum immunity against evolving and diversifying SARS-CoV-2 strains while avoiding the risk of antibody-dependent enhancement observed with other coronavirus vaccines.

[0240] Carnell et al. ("SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses"; https: / / doi.org / 10.1101 / 2021.01.14.426695, posted January 14, 2021) evaluated the outcome of immunization in a mouse model using an S protein trimer arrested in the closed state to prevent exposure of the receptor-binding site and thus interaction with the receptor. The authors compared this with a range of other modified S protein constructs, including representative examples used in current vaccines. The authors found that all trimeric S proteins induced long-lived and strong neutralizing antibody responses, as well as T cell responses. Notably, the protein-binding characteristics of sera induced by the closed spike differed from those induced by the standard S protein construct. The closed S protein induced a stronger neutralizing response than expected based on the extent to which it inhibited the interaction between the RBD and ACE2. The authors conclude that these findings suggest that closed spikes mobilize a different but equally potent virus-inhibitory immune response than open spikes, and that this likely involves neutralizing antibodies directed against conformational epitopes present in the closed conformation.

[0241] The inventors have realized that amino acid changes in the designed S protein sequences disclosed herein (particularly SEQ ID NO: 53 described in Example 30) can optionally be present in the designed S protein to stall in a closed state, thereby further improving the antibody response of the designed sequence. In particular, the use of such structural constraints may reduce immunodominance to critical regions and broaden the antibody response to focus on other, or less immunodominant, sites.

[0242] Example 31 below describes optional additional amino acid changes that can be made to the designed S protein sequence to enable it to form a closed structure.

[0243] Optionally, the designed S protein sequences of the invention may contain cysteine ​​residues at positions corresponding to positions 413 and 987 of the full-length S protein sequence, for example, G413C and V987C.

[0244] For example, a designed S protein sequence of the invention may comprise the following amino acid sequence (SEQ ID NO:54) (with cysteine ​​residues at positions 410 and 984, which correspond to positions 413 and 987, respectively, of SEQ ID NO:52). [ka] [ka]

[0245] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:54.

[0246] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:54, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:54.

[0247] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 54, comprises at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.4 below. [Table 9-12]

[0248] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 54, comprises at least five of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.4.

[0249] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 54, comprises at least 10 of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.4.

[0250] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 54, comprises at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.5 below.

[0251] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:54, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:54, comprises at least one, or all, of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, including amino acid residue P at position 986, as shown in Table 9.5 below. [Table 9-13]

[0252] The present invention also provides an isolated polypeptide comprising a coronavirus S protein comprising cysteine ​​amino acid residues at positions corresponding to positions 413 and 987 of SEQ ID NO:52, and at least one or all of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO:52, as shown in Table 9.5.

[0253] Optionally, an isolated polypeptide of the invention comprising cysteine ​​amino acid residues at positions corresponding to positions 413 and 987 of SEQ ID NO:52, and at least one of amino acid residues or deletions at positions corresponding to amino acid residue positions of SEQ ID NO:52, as shown in Table 9.5 above, also comprises at least five of amino acid residues or deletions at positions corresponding to amino acid residue positions of SEQ ID NO:52, as shown in Table 9.5.

[0254] Optionally, an isolated polypeptide of the invention comprising cysteine ​​amino acid residues at positions corresponding to positions 413 and 987 of SEQ ID NO:52, and at least one of amino acid residues or deletions at positions corresponding to amino acid residue positions of SEQ ID NO:52, as shown in Table 9.5 above, comprises at least 10 of amino acid residues or deletions at positions corresponding to amino acid residue positions of SEQ ID NO:52, as shown in Table 9.5.

[0255] Optionally, an isolated polypeptide of the invention comprising cysteine ​​amino acid residues at positions corresponding to positions 413 and 987 of SEQ ID NO:52, and at least one of amino acid residues or deletions at positions corresponding to amino acid residue positions of SEQ ID NO:52, as shown in Table 9.5 above, comprises an amino acid residue P at position 986.

[0256] The present inventors have also realized that, in accordance with Carnell et al. (supra), any SARS-CoV-2 spike protein can be modified to include cysteine ​​residues at positions corresponding to 413 and 987 of SEQ ID NO: 52 to enable the formation of a spike protein that is stalled in a closed state, thereby eliciting a stronger neutralizing response compared to the corresponding unmodified protein. For example, Jeong et al. (https: / / virological.org / t / assemblies-of-putative-sars-cov2-spike-encoding-mrna-sequences-for-vaccines-bnt-162b2-and-mrna-1273 / 663-version 0.2Beta 03 / 30 / 21) recently reported experimental sequence information for the RNA components of the early Moderna (https: / / pubmed.ncbi.nlm.nih.gov / 32756549 / ) and PfiZer / BioNTech (https: / / pubmed.ncbi.nlm.nih.gov / 33301246 / ) COVID-19 vaccines, confirming the working assembly of the former and the previously reported sequence information for the latter RNA (see sequences provided in Figures 1 and 2 of the document). The spike protein encoded by such a sequence may be modified to include cysteine ​​residues at positions corresponding to positions 413 and 987 of SEQ ID NO:52.

[0257] The present invention also provides an isolated polypeptide comprising a coronavirus S protein comprising cysteine ​​amino acid residues at positions corresponding to positions 413 and 987 of SEQ ID NO:52.

[0258] Optionally, the coronavirus S protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:52.

[0259] SARS-CoV-2 is continually evolving, with more infectious mutations spreading rapidly. Zahradnik et al., 2021 ("SARS-CoV-2 RBD in vitro evolution follows contagious mutation spread, yet generates an able infection inhibitor"; doi: https: / / doi.org / 10.1101 / 2021.01.06.425392, published January 29, 2021) recently reported that using in vitro evolution to affinity-maturate the receptor-binding domain (RBD) of the spike protein against ACE2, the more infectious mutations S477N, E484K, and N501Y were initially selected, explaining the convergent evolution of the "European" (20E-EU1), "UK" (501.V1), "South African" (501.V2), and "Brazilian" variants (501.V3). The authors report that in vitro evolution of a 600-fold increase in binding provides guidelines for potentially new evolved mutations with even higher infectivity, for example, Q498R is epistatic to N501Y.

[0260] The inventors have appreciated that the designed S protein sequences (RBD, truncated, or full-length) disclosed herein (and particularly in the sections above titled "Designed Coronavirus Full-Length S Protein Sequences to Protect Against COVID-19 Variants," and "Designed Coronovirus S Protein Sequences in a Closed State to Protect Against COVID-19 Variants and Anticipate Future Variants," and Examples 30 and 31 below) can also optionally include amino acid substitutions at one or more residue positions where future COVID-19 variants are predicted to mutate due to a vaccine escape response, for example, at one or more (or all) of positions 446, 452, 477, and 498 (e.g., G446R, S477N, Q498R, particularly Q498R).

[0261] Optionally, the isolated polypeptide of the invention comprises amino acid changes at one or more (or all) of the following positions (corresponding to the amino acid residue positions of SEQ ID NO: 52): 446, 452, 477, and 498 (e.g., G446R, S477N, Q498R, particularly Q498R).

[0262] Optionally, the isolated polypeptide of the invention comprises amino acid changes at positions Q498R and N501Y (corresponding to the amino acid residue positions of SEQ ID NO: 52).

[0263] Designed coronavirus envelope (E) protein sequences The inventors have also generated novel amino acid sequences for coronavirus envelope (E) proteins. Figure 6 shows the amino acid sequence of the SARS envelope (E) protein (SEQ ID NO: 21) and indicates key features of the sequence. As described in Example 10 below, Figure 7 shows a multiple sequence alignment of coronavirus E protein sequences comparing the sequences of isolates NL63 and 229E (α-coronaviruses) and HKU1, MERS, SARS, and SARS2 (β-coronaviruses). The alignment shows that the C-terminus of the E protein for the SARS2 and SARS sequences (β-coronaviruses of the subgenus Sarbeco) contains a deletion compared to the other sequences, and that the SARS2 E protein sequence contains a deletion and an arginine (positively charged) amino acid residue compared to the SARS sequence.

[0264] The novel amino acid sequences of the coronavirus E protein are designated COV_E_T2_1 (designed sarbecovirus sequence) (SEQ ID NO: 22) and COV_E_T2_2 (designed SARS2 sequence) (SEQ ID NO: 23). [ka]

[0265] Alignment of the SARS2 reference E protein sequence in Figure 7 with these designed sequences highlights that there are four amino acid differences between the SARS2 reference E protein sequence and the COV_E_T2_1 designed sequence (SEQ ID NO: 22) and two amino acid differences between the SARS2 reference E protein sequence and the COV_E_T2_2 designed sequence (SEQ ID NO: 23). [ka]

[0266] The C-terminal sequence of the COV_E_T2_2 sequence is identical to the SARS2 reference sequence. Because the C-terminus of the E protein is one of the identified epitopes of the E protein, the amino acid deletion and substitution with an arginine residue present in the SARS2 reference sequence (compared to the SARS reference sequence in Figure 6) was retained in the COV_E_T2_2 designed sequence. Amino acid differences at other positions were optimized to maximize the induction of an immune response that recognizes all sarbecoviruses.

[0267] The amino acid differences are summarized in the table below. [Table 10-1]

[0268] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:22.

[0269] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:22, comprises one or both of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:22, as shown in the table below. [Table 10-2]

[0270] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:22, comprises any, at least two, at least three, or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:22, as shown in the table below. [Table 10-3]

[0271] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:22.

[0272] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:23 or an amino acid sequence having at least 98%, or 99%, amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:23.

[0273] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:23, or an amino acid sequence having at least 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:23, comprises one or both of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:23, as shown in the table below. [Table 10-4]

[0274] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:23.

[0275] Also provided in accordance with the present invention is an isolated polypeptide comprising a coronavirus E protein having one or both of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 10-5]

[0276] The present invention also provides an isolated polypeptide comprising a coronavirus E protein having any, at least two, at least three, or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 10-6]

[0277] Optionally, an isolated polypeptide of the invention comprising a coronavirus E protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:21.

[0278] The alignment above residue 36 of the SARS2 reference sequence is shown as a V, but is actually an A (as correctly shown in Figure 7 and SEQ ID NO: 21). The alignment of SEQ ID NO: 21 with the designed sequences highlights that there are three amino acid differences between the alternative SARS2 reference E protein sequence and the COV_E_T2_1 designed sequence (SEQ ID NO: 22), and one amino acid difference between the SARS2 reference E protein sequence and the COV_E_T2_2 designed sequence (SEQ ID NO: 23). [ka]

[0279] The amino acid differences are summarized in the table below. [Table 10-7]

[0280] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 22 (COV_E_T2_1), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 22.

[0281] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:22, comprises amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:22, as shown in the table below. [Table 10-8]

[0282] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:22, comprises any, at least two, or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:22, as shown in the table below. [Table 10-9]

[0283] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:23, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:23.

[0284] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:23, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:23, comprises amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:23, as shown in the table below. [Table 10-10]

[0285] Also provided in accordance with the present invention is an isolated polypeptide comprising a coronavirus E protein having amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 10-11]

[0286] The present invention also provides an isolated polypeptide comprising a coronavirus E protein having any, at least two, or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 10-12]

[0287] Optionally, an isolated polypeptide of the invention comprising a coronavirus E protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:21.

[0288] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:21.

[0289] The SARS-CoV envelope (E) gene encodes a 76-amino acid transmembrane protein with ion channel (IC) activity, a key function in virus-host interactions. Infection of mice with viruses lacking or exhibiting E protein IC activity revealed activation of the inflammasome pathway, and the exacerbated inflammatory response induced by SARS-CoV was reduced in infection with the ion channel-deficient virus (Nieto-Torres et al., 2014, Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis. PLoS Pathog 10(5):e1004077).

[0290] The inventors have generated new E protein designs CoV_E_T2_3, CoV_E_T2_4, and CoV_E_T2_5, which correspond to the new designs of SARS2 reference (SEQ ID NO: 41), CoV_E_T2_1 (SEQ ID NO: 22), and CoV_E_T2_2 (SEQ ID NO: 23), respectively (see Example 10). These new designs have the point mutation N15A, which suppresses ion channel activity but does not affect structural stability. Nieto-Torres et al., supra, discuss this mutation and the toxic and inflammatory effects of SARS E on host cells.

[0291] The amino acid sequence of the SARS2 envelope protein reference (SEQ ID NO: 41) is as follows: [ka] The amino acid sequences of the new E protein designs are shown below and in Example 25. [ka] [ka]

[0292] An alignment of the E protein design with the SARS2 E protein reference sequence is shown below. [ka]

[0293] The amino acid differences between the SARS2 reference sequence (SEQ ID NO: 41) and the designed sequence are shown in the table below (with differences from the reference sequence highlighted in bold): [Table 10-13]

[0294] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:36.

[0295] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:37.

[0296] In accordance with the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:38.

[0297] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 42 (COV_E_T2_3), or an amino acid sequence having at least 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 42.

[0298] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 42 (COV_E_T2_3) or an amino acid sequence having at least 99% amino acid identity to the amino acid sequence of SEQ ID NO: 42 over its entire length comprises an amino acid residue A at a position corresponding to amino acid residue position 15 of SEQ ID NO: 41.

[0299] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:42.

[0300] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 43 (COV_E_T2_4), or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 43.

[0301] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 43 (COV_E_T2_4), or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 43 over its entire length, comprises at least one or all of the following amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 41: 15A, 55T, 69Q, 70G.

[0302] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:43.

[0303] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 44 (COV_E_T2_5), or an amino acid sequence having at least 98% or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 44.

[0304] Optionally, a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 44 (COV_E_T2_5), or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 44 over its entire length, comprises at least one or all of the following amino acid residues, 15A, 55T, at positions corresponding to the amino acid residue positions of SEQ ID NO: 41.

[0305] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:44.

[0306] The present invention also provides an isolated polypeptide comprising a coronavirus E protein having at least one of the following amino acid residues: 15A, 55T, 69Q, 70G at positions corresponding to the amino acid residue positions of SEQ ID NO:41.

[0307] Optionally, an isolated polypeptide of the invention comprising a coronavirus E protein comprises the following amino acid residues at positions corresponding to amino acid residue positions in SEQ ID NO:41: 15A, 55T.

[0308] Optionally, an isolated polypeptide of the invention comprising a coronavirus E protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:21.

[0309] Designed coronavirus membrane (M) protein sequences Applicants have also generated a novel amino acid sequence for the coronavirus membrane (M) protein. COV_M_T2_1 Sarbecovirus root ancestor (SEQ ID NO: 24), The COV_M_T2_2 epitope was created by optimizing the version of the SARS2 clade ancestor Node88b (removing D4), adding B cell epitopes from the start and end of SARS 2, and then adding T cell epitopes while observing co-evolutionary site constraints (SEQ ID NO: 25).

[0310] The amino acid sequences of these designed sequences are as follows: [ka]

[0311] Figure 8 shows an alignment of the SARS2 reference M protein sequence (SEQ ID NO: 26) with the designed sequence, as described below in Example 11. The alignment shown in Figure 8 highlights the amino acid differences between the SARS2 reference M protein sequence and the COV_M_T2_1 and COV_M_T2_2 designed sequences, as shown in the table below. [Table 11-1]

[0312] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:24 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24.

[0313] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24, comprises at least one of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table below. [Table 11-2]

[0314] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24, comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in Table 11.2.

[0315] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24, comprises all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in Table 11.2.

[0316] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24, comprises at least one of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table below. [Table 11-3]

[0317] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24, comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in Table 11.3.

[0318] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24, comprises at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in Table 11.3.

[0319] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24, comprises at least 15 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in Table 11.3.

[0320] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24, comprises all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in Table 11.3.

[0321] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:24.

[0322] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25.

[0323] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25, comprises at least one of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:25, as shown in the table below. [Table 11-4]

[0324] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25, comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:25, as shown in Table 11.4.

[0325] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25, comprises all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:25, as shown in Table 11.4.

[0326] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25, comprises at least one of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:25, as shown in the table below. [Table 11-5]

[0327] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25, comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:25, as shown in Table 11.5.

[0328] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25, comprises at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:25, as shown in Table 11.5.

[0329] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25, comprises all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:25, as shown in Table 11.5.

[0330] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:25.

[0331] The present invention also provides an isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 11-6]

[0332] The present invention also provides an isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 11-7]

[0333] The present invention also provides an isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 11-8]

[0334] The present invention also provides an isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 11-9]

[0335] Optionally, an isolated polypeptide of the invention comprising a coronavirus M protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:26.

[0336] We further created new M protein designs (COV_M_T2_3, COV_M_T2_4, COV_M_T2_5) in which we deleted the first and second transmembrane domains of the membrane protein to suppress interaction with the S protein. · The string-like constructs with S, M, and E showed higher-order aggregates. · Inhibition of the interaction between S and M can reduce aggregation. · An M-del construct (Cov_M_T2_(3-5)) designed to suppress interaction with S.

[0337] Figure 20 shows a diagram of the M protein. Interactions between the M, E, and N proteins are important for virus assembly. The M protein also binds to the nucleocapsid, and this interaction promotes the completion of virion assembly. These interactions have been mapped to the C-terminus of the endodomain of the M protein and the C-terminal domain of the N protein. In Figure 20, *demonstrated the identification of immunodominant epitopes on the membrane protein of severe acute respiratory syndrome-associated coronovirus, ** shows the mapping of coronavirus membrane protein domains involved in interactions with the spike protein.

[0338] The amino acid sequence of the new M protein design is shown below. [ka]

[0339] Sequence alignments of the new M protein designs (COV_M_T2_3, COV_M_T2_4, COV_M_T2_5) with previous M protein designs (COV_M_T1_1, COV_M_T2_1, COV_M_T2_2) are shown below. [ka]

[0340] The amino acid differences between the SARS2 M protein reference sequence and the designed sequence are shown in the table below (with differences from the reference sequence highlighted in bold). [Table 11-10]

[0341] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:48 or an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:48.

[0342] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:48, or an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:48, comprises a deletion of amino acid residues at positions corresponding to positions 20-75 of SEQ ID NO:26.

[0343] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:48, or an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:48, comprises an amino acid residue G at a position corresponding to amino acid residue position 204 of SEQ ID NO:26.

[0344] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:48.

[0345] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:49 or an amino acid sequence having at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:49.

[0346] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:49, comprises deletions of amino acid residues at positions corresponding to positions 20-75 of SEQ ID NO:26.

[0347] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:49, comprises at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table below. [Table 11-11]

[0348] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:49, comprises at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table below. [Table 11-12]

[0349] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:49.

[0350] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:50 or an amino acid sequence having at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:50.

[0351] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:50, or an amino acid sequence having at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:50, comprises a deletion of amino acid residues at positions corresponding to positions 20-75 of SEQ ID NO:26.

[0352] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:50, or an amino acid sequence having at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:50, comprises at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table below. [Table 11-13]

[0353] Optionally, an isolated polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:50, or an amino acid sequence having at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:50, comprises at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table below. [Table 11-14]

[0354] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:50.

[0355] The present invention also provides an isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 11-15]

[0356] The present invention also provides an isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 11-16]

[0357] The present invention also provides an isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions shown in the table below. [Table 11-17]

[0358] Optionally, an isolated polypeptide of the invention comprising a coronavirus M protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:26.

[0359] Designed coronavirus nucleoprotein (N) sequence We generated new N protein designs, COV_N_T2_1 (SEQ ID NO: 46) and COV_N_T2_2 (SEQ ID NO: 47). The amino acid sequences of these designs are shown below and in Example 15. Sequence COV_N_T2_2 was designed using a methodology and algorithm that selected predicted epitopes based on conservation across sarbecoviruses (while minimizing redundancy), including frequency and number of MHC alleles, limited by the quality of the predicted epitopes, and a small number of user-specified weights. >YP_009724397.2 / 1-419 Nucleocapsid phosphoprotein [SARS-CoV-2] (Reference Sequence) (SEQ ID NO: 45) [ka]

[0360] An alignment of the N protein design with the SARS2 N protein reference sequence is shown below. [ka]

[0361] The amino acid differences between the SARS2 reference sequence and the designed sequences are shown below in Table 12.1 (differences from the reference sequence are highlighted in bold, differences common to all designed sequences are underlined). [Table 12-1-1] [Table 12-1-2]

[0362] Positions 415 and 416 in the SARS2 N protein reference residue position column are italicized because they are not residues in the reference sequence but contain insertions in the N_T2_1 and N_T2_2 sequences.

[0363] The amino acid changes common to both designed sequences are summarized in the table below. [Table 12-2]

[0364] Optional additional changes are summarized in the table below. [Table 12-3]

[0365] Additional variations in alternative options are summarized in the table below. [Table 12-4]

[0366] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 46 (COV_N_T2_1), or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 46.

[0367] Optionally, polypeptides of the invention, including isolated polypeptides comprising the amino acid sequence of SEQ ID NO:46 or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:46, further comprise at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 12.2 above.

[0368] Optionally, polypeptides of the invention, including isolated polypeptides comprising the amino acid sequence of SEQ ID NO:46 or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:46, further comprise at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 12.3 above.

[0369] According to the present invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 46 (COV_N_T2_1).

[0370] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 47 (COV_N_T2_2), or an amino acid sequence having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 47.

[0371] Optionally, polypeptides of the invention, including isolated polypeptides comprising the amino acid sequence of SEQ ID NO:47 or an amino acid sequence having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:47, further comprise at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 12.2 above.

[0372] Optionally, polypeptides of the invention, including isolated polypeptides comprising the amino acid sequence of SEQ ID NO:47 or an amino acid sequence having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:47, further comprise at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in Table 12.4 above.

[0373] The present invention also provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 47 (COV_N_T2_2).

[0374] Also provided in accordance with the present invention is an isolated polypeptide comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above.

[0375] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least five amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above.

[0376] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least 10 amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above.

[0377] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least 15 amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above.

[0378] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.3 above.

[0379] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.3 above.

[0380] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.3 above.

[0381] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.4 above.

[0382] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.4 above.

[0383] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, also comprises at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.4 above.

[0384] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein having at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.2 above, comprises at least 15 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:45, as shown in Table 12.4 above.

[0385] Optionally, an isolated polypeptide of the invention comprising a coronavirus N protein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:45.

[0386] The polypeptides of the invention are particularly advantageous because they are capable of eliciting a broadly neutralizing immune response against several different types of coronaviruses, in particular several different types of β-coronaviruses. The polypeptides of the invention are particularly advantageous because they are capable of eliciting a broadly neutralizing immune response against several different types of coronaviruses, in particular several different types of β-coronaviruses. The polypeptides of the invention are particularly advantageous because they are capable of eliciting a broadly neutralizing immune response against several different types of coronaviruses, in particular several different types of β-coronaviruses. The polypeptides of the invention are particularly advantageous because they are capable of eliciting a broadly neutralizing immune response against several different types of coronaviruses, in particular several different types of β-coronaviruses. The polypeptides of the invention are particularly advantageous because they are capable of eliciting a broadly neutralizing immune response against several different types of coronaviruses, in particular several different types of β-coronaviruses. The polypeptides of the invention are Polypeptides of the invention comprising an amino acid sequence of at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity (an amino acid sequence having at least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity) are also advantageous because they lack non-neutralizing epitopes that may lead to viral immune evasion and disease progression due to ADE (or an ADE-like pro-inflammatory response).

[0387] Similarly, the amino acid sequence of a newly designed coronavirus E protein (e.g., the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length) or the amino acid sequence of a coronavirus M protein (e.g., the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length) can be used. Polypeptides of the invention comprising an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity thereto over their entire length, or the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity thereto over their entire length, are advantageous because they lack non-neutralizing epitopes that may result in viral immune evasion and disease progression due to ADE (or an ADE-like pro-inflammatory response).

[0388] The polypeptide of the present invention may contain one or more conservative amino acid substitutions. Conservative amino acid substitutions are substitutions that, when made, do not significantly disrupt the properties of the original polypeptide, i.e., the structure and, in particular, function of the protein are preserved and are not significantly changed by such substitutions. Examples of conservative substitutions are shown below. Original residue Conservative substitution Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn;Gln Ile Leu, Val Leu Ile;Val Lys Arg;Gln; Met Leu;Ile Phe Met;Leu;Tyr Ser Thr Thr Ser Trp Tyr Tyr;Trp;Phe Val Ile;Leu

[0389] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone, e.g., sheet or helix conformation, in the area of ​​the substitution, (b) the charge or hydrophobicity of the target site molecule, or (c) the bulk of the side chains.

[0390] Generally, substitutions expected to produce the greatest changes in protein properties are non-conservative, such as (a) a hydrophilic residue, e.g., serine or threonine, is substituted for (or is substituted by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine, or alanine; (b) a cysteine ​​or proline is substituted for (or is substituted by) any other residue; (c) a residue having an electrically positive side chain, e.g., lysine, arginine, or histidine, is substituted for (or is substituted by) an electrically negative residue, e.g., glutamate or aspartate; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or is substituted by) one having no side chain, e.g., glycine.

[0391] The term "broadly neutralizing immune response" is used herein to mean an immune response elicited in a subject sufficient to inhibit (i.e., reduce), neutralize, or prevent infection and / or progression of an infection with a virus within the coronavirus family. Optionally, a broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of an infection with two or more types of β-coronaviruses (e.g., SARS-CoV and SARS-CoV-2). Optionally, a broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of an infection with two or more types of β-coronaviruses within the same β-coronavirus lineage (e.g., two or more types of β-coronaviruses within the subgenus Sarbecovirus, such as SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1). Optionally, the broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection with different β-coronavirus lineages, e.g., coronaviruses of type B (e.g., SARS-CoV and SARS-CoV-2) and type C (e.g., MERS-CoV). Optionally, the broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection with most or all different β-coronaviruses. Optionally, the broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection with most or all different viruses of the coronavirus family. Optionally, the broadly neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection with two or more types of β-coronavirus SARS-CoV-2 variants of interest (VOCs), e.g., alpha, beta, gamma, delta, and omicron SARS-CoV-2 VOCs.

[0392] An immune response can be a humoral and / or cellular immune response. A cellular immune response is the response of cells of the immune system, such as B cells, T cells, macrophages, or polymorphonuclear cells, to a stimulus, such as an antigen or a vaccine. An immune response can include any cell of the body that is involved in a host defense response, including, for example, epithelial cells that secrete interferons or cytokines. An immune response includes, but is not limited to, an innate immune response or inflammation.

[0393] Optionally, the polypeptides of the present invention induce a protective immune response. A protective immune response refers to an immune response that protects a subject from infection or disease (i.e., prevents infection or prevents the onset of a disease associated with the infection). Methods for measuring immune responses are well known in the art and include, for example, measuring lymphocyte (such as B or T cell) proliferation and / or activity, cytokine or chemokine secretion, inflammation, or antibody production.

[0394] Optionally, the polypeptides of the invention are capable of inducing the production of an antibody and / or T cell response in a human or non-human animal to which the polypeptide is administered (either as a polypeptide or, for example, expressed from an administered nucleic acid expression vector).

[0395] Optionally, the polypeptide of the present invention is a glycosylated polypeptide.

[0396] nucleic acid molecule The present invention also provides an isolated nucleic acid molecule encoding a polypeptide of the present invention, or its complement.

[0397] Also provided by the present invention is an isolated nucleic acid molecule, or a complement thereof, comprising a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to a nucleic acid molecule of the present invention encoding a polypeptide of the invention.

[0398] Optionally, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence of SEQ ID NO: 18, 16 or 14, or a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO: 18, 16 or 14, or a complement thereof.

[0399] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 33, 34, 35, or 36.

[0400] Optionally, the nucleotide sequence encoding the polypeptide comprising the amino acid sequence of SEQ ID NO: 33, 34, 35, or 36 comprises the nucleotide sequence of SEQ ID NO: 37, 38, 39, or 40, respectively.

[0401] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 34 (M8), or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 34.

[0402] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide comprising a coronavirus S protein RBD domain having at least one of the following amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11: 13Q, 25Q, 54T, 203N.

[0403] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 35 (M9), or an amino acid sequence having at least 70% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 35.

[0404] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 36 (M10), or an amino acid sequence having at least 69% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 36.

[0405] The inventors have found that immunization of mice with nucleic acid (particularly DNA) encoding a SARS2 truncated S protein induces the production of antibodies capable of binding to the SARS2 spike protein (see Example 17, Figure 10).

[0406] According to the present invention, there is provided an isolated nucleic acid molecule encoding a truncated SARS2 S protein (CoV_T2_3) of the amino acid sequence of SEQ ID NO:9.

[0407] Optionally, the isolated nucleic acid molecule encoding the SARS2 truncated S protein (CoV_T2_3) of the amino acid sequence of SEQ ID NO:9 comprises the nucleotide sequence of SEQ ID NO:10.

[0408] The inventors also found that immunization of mice with nucleic acids (particularly DNA) encoding the SARS2 S protein RBD induced the production of antibodies capable of neutralizing SARS2 pseudotyped viruses (see Example 18, Figure 11).

[0409] The inventors also found that M7 and wild-type SARS2 RBD DNA (which would result in expression of a glycosylated RBD protein) were superior to recombinant SARS2 RBD protein (non-glycosylated or low-density glycosylated) in inducing a neutralizing response to SARS2.

[0410] According to the present invention, there is provided an isolated nucleic acid molecule encoding the SARS2S protein RBD (CoV_T2_6) having the amino acid sequence of SEQ ID NO: 11.

[0411] Optionally, the isolated nucleic acid molecule encoding the SARS2S protein RBD (CoV_T2_6) of the amino acid sequence of SEQ ID NO: 11 comprises the nucleotide sequence of SEQ ID NO: 12.

[0412] The present inventors have also found that nucleic acids (particularly DNA) encoding the designed M7 SARS2 S protein RBD have particularly advantageous effects. In particular, the present inventors have found that: Immunization of mice with a DNA vaccine containing nucleic acid encoding M7 SARS2 RBD (SEQ ID NO: 33) induced an immune response with stronger binding to SARS2 RBD than to wild-type SARS2 RBD (see Example 20 and Figure 14). Immunization of mice with a DNA vaccine encoding the M7 SARS2 RBD (SEQ ID NO: 33) elicits a neutralizing immune response more rapidly than a DNA vaccine encoding the wild-type SARS2 RBD (see Example 21 and Figure 15). Immunization of mice with a DNA vaccine encoding the M7 SARS2 RBD (SEQ ID NO: 33) induced a higher neutralizing response than a DNA vaccine encoding the wild-type SARS2 RBD in serum collected from bleeds at week 1 and week 2 (see Example 22 and Figures 16 and 17); The supernatant containing M7 SARS2 RBD effectively competed with three ACE2-binding viruses for ACE2 cell entry (see Example 23 and Figure 18). T cell responses were induced by a DNA vaccine encoding the M7 SARS2 RBD (SEQ ID NO: 33) that were reactive to peptides from an RBD peptide pool but not to the full-length RBD or medium (see Example 24 and Figure 19).

[0413] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:37.

[0414] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 78 (nucleic acid encoding COV_S_T2_13).

[0415] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 79 (nucleic acid encoding COV_S_T2_14).

[0416] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 80 (nucleic acid encoding COV_S_T2_15).

[0417] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 81 (a nucleic acid encoding COV_S_T2_16).

[0418] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 82 (a nucleic acid encoding COV_S_T2_17).

[0419] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 83 (nucleic acid encoding COV_S_T2_18).

[0420] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 84 (nucleic acid encoding COV_S_T2_19).

[0421] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 85 (nucleic acid encoding COV_S_T2_20).

[0422] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 86 (T2_17+pEVAC expression vector).

[0423] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 92 (CoV_S_T2_17+tPA signal sequence).

[0424] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 93 (CoV_S_T2_17+tPA signal sequence).

[0425] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 94 (pURVAC_T2_17+tPA).

[0426] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 95 (pURVAC_CoV_S_T2_29+Q498R+dER).

[0427] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 97 (pMVA Trans TK mH5 T2_17+tPA).

[0428] The present invention also provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 98 (pMVA Trans TK mH5 T2_29+Q498R+dER).

[0429] Sequence identity The similarity between amino acid sequences or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is often measured in terms of the percentage of identity (or similarity or homology), and the higher the percentage, the more similar the two sequences are. Homologs or variants of a given gene or protein have a relatively high degree of sequence identity when aligned using standard methods. Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids' Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994. NCBI Basic Local Alignment Search Tool (BLAST TM ) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0430] Sequence identity between nucleic acid or amino acid sequences can be determined by comparing the alignment of sequences. If the equivalent position in the compared sequences is occupied by the same nucleotide or amino acid, the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at the position shared by the compared sequences. When comparing sequences, optimal alignment may require gaps to be introduced into one or more of the sequences to account for possible insertions and deletions in the sequence. Sequence comparison methods may use gap penalties so that, for the same number of identical molecules in the compared sequences, a sequence alignment with as few gaps as possible, reflecting a higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculating the maximum percent identity involves generating an optimal alignment, taking into account gap penalties.

[0431] Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sectors, including, for example, MatGat (Campanella et al., 2003, BMC Bioinformatics 4:29; program available at http: / / bitincka.com / ledion / matgat), Gap (Needleman & Wunsch, 1970, J. Mol. Biol. 48:443-453), FASTA (Altschul et al., 1990, J. Mol. Biol. 215:403-410; program available at http: / / www.ebi.ac.uk / fasta), Clustal W2.0 and X2.0 (Larkin et al., 2007, Bioinformatics 23:2947-2948; program available at http: / / www.ebi.ac.uk / tools / clustalw2), and EMBOSS Pairwise Alignment Algorithms. (Needleman & Wunsch, 1970, supra; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp. 1-44, Addison Wesley; programs available at http: / / www.ebi.ac.uk / tools / emboss / align). All programs can be run using the default parameters.

[0432] For example, sequence comparison can be performed using the "needle" method of the Emboss Pairwite alignment algorithm, which determines the optimal alignment of two sequences (including gaps), providing a percentage identity score when considered over their entire length. Default parameters for amino acid sequence comparison ("Protein molecule" option) can be: Gap Extend penalty: 0.5, Gap Open penalty: 10.0, Matrix: Blosum62.

[0433] Sequence comparison may be performed over the entire length of the reference sequence.

[0434] Corresponding position The sequences described herein include reference to amino acid sequences that contain amino acid residues at positions that correspond to amino acid residue positions in another sequence. Such corresponding positions may be identified, for example, from alignment of the sequences using the sequence alignment methods described herein or other sequence alignment methods known to those of skill in the art.

[0435] vector The present invention also provides a vector comprising a nucleic acid molecule of the present invention.

[0436] The present invention also provides a vector comprising a nucleic acid molecule encoding a polypeptide of the present invention.

[0437] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17.

[0438] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 15.

[0439] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 13.

[0440] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 27 (COV_S_T2_13), or an amino acid sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 27.

[0441] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 28 (COV_S_T2_14), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 28.

[0442] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 29 (COV_S_T2_15), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 29.

[0443] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 30 (COV_S_T2_16), or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 30.

[0444] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31.

[0445] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32.

[0446] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:33.

[0447] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 34.

[0448] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:22.

[0449] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:23, or an amino acid sequence having at least 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:23.

[0450] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 42 (COV_E_T2_3), or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 42.

[0451] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 43 (COV_E_T2_4), or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 43.

[0452] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 44 (COV_E_T2_5), or an amino acid sequence having at least 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 44.

[0453] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24.

[0454] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25.

[0455] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 46 (COV_N_T2_1), or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 46.

[0456] Optionally, a vector of the invention comprises a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 47 (COV_N_T2_2), or an amino acid sequence having at least 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 47.

[0457] Optionally, the vector of the invention further comprises a promoter operably linked to the nucleic acid.

[0458] Optionally, the promoter is for expression of a polypeptide encoded by the nucleic acid in a mammalian cell.

[0459] Optionally, the promoter is for expression of the polypeptide encoded by the nucleic acid in yeast or insect cells.

[0460] Optionally, a vector of the invention comprises two or more nucleic acid molecules encoding different polypeptides of the invention. Advantageously, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and / or a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and / or a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention.

[0461] Optionally, a vector of the invention comprises two or more nucleic acid molecules encoding different polypeptides of the invention. Advantageously, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and / or a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and / or a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention and / or a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0462] Optionally, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention.

[0463] Optionally, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention.

[0464] Optionally, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0465] Optionally, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention.

[0466] Optionally, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0467] Optionally, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention.

[0468] Optionally, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0469] Optionally, the vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention, and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0470] Optionally, a vector of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention, and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0471] Optionally, the vector of the invention comprises: a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length.

[0472] Optionally, the vector of the invention comprises: a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO:24 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO:24 over its entire length; or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO:25 over its entire length.

[0473] Optionally, the vector of the invention comprises: a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length; a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:24 over its entire length; or a nucleic acid sequence encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:25 over its entire length.

[0474] Optionally, the vector of the invention comprises: a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length; a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO:24 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO:24 over its entire length; or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO:25 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO:25 over its entire length.

[0475] Optionally, the vector of the invention further comprises, for each nucleic acid molecule of the vector that encodes a polypeptide, a separate promoter operably linked to that nucleic acid molecule.

[0476] Optionally, the, or each promoter, is for expression of a polypeptide encoded by the nucleic acid molecule in a mammalian cell.

[0477] Optionally, the, or each promoter, is for expression of a polypeptide encoded by the nucleic acid molecule in a yeast or insect cell.

[0478] Optionally, the vector is a vaccine vector.

[0479] Optionally, the vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector.

[0480] The nucleic acid molecules of the present invention can include DNA or RNA molecules. For embodiments in which the nucleic acid includes an RNA molecule, it is understood that the nucleic acid sequence of the nucleic acid is the same as that set forth in the respective SEQ ID NO: or its complement, but with each "T" nucleotide replaced with a "U".

[0481] For embodiments in which the nucleic acid molecule comprises an RNA molecule, it is understood that the molecule can comprise an RNA sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to or identical to any of SEQ ID NOs: 18, 16, or 14, or a complement thereof, in which each "T" nucleotide is replaced with a "U".

[0482] For example, when an RNA vaccine vector comprising a nucleic acid of the invention is provided, it is understood that the nucleic acid sequence of the nucleic acid of the invention is an RNA sequence and therefore may comprise, for example, an RNA sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or identical to, any of SEQ ID NOs: 18, 16, or 14, and in which each "T" nucleotide is replaced with "U" or its complement.

[0483] Viral vaccine vectors use live viruses to deliver nucleic acids (e.g., DNA or RNA) into human or non-human animal cells. The nucleic acids contained in the viruses encode one or more antigens, which, when expressed in infected human or non-human animal cells, elicit an immune response. Both humoral and cell-mediated immune responses can be induced by viral vaccine vectors. Viral vaccine vectors combine many of the positive qualities of nucleic acid vaccines with those of live attenuated vaccines. Like nucleic acid vaccines, viral vaccine vectors deliver nucleic acids to host cells for the production of antigenic proteins that can be tailored to stimulate a range of immune responses, including antibodies, T helper cells (CD4+ T cells), and cytotoxic T lymphocyte (CTL, CD8+ T cell)-mediated immunity. Unlike nucleic acid vaccines, viral vaccine vectors, like live attenuated vaccines, have the ability to actively invade and replicate in host cells, further activating the immune system much like adjuvants. Thus, viral vaccine vectors generally comprise live, attenuated viruses genetically engineered to carry nucleic acids (e.g., DNA or RNA) encoding protein antigens from unrelated organisms. Viral vaccine vectors can generally generate stronger immune responses than nucleic acid vaccines, but for some diseases, viral vectors are used in combination with other vaccine technologies in a strategy called heterologous prime-boost. In this system, one vaccine is administered as a priming step, followed by vaccination with an alternative vaccine as a booster. The heterologous prime-boost strategy aims to provide a stronger overall immune response. Viral vaccine vectors can be used as both prime and boost vaccines as part of this strategy. Viral vaccine vectors are reviewed by Ura et al., 2014 (Vaccines 2014, 2, 624-641) and Choi and Chang, 2013 (Clinical and Experimental Vaccine Research 2013; 2:97-105).

[0484] Optionally, the viral vaccine vector is based on a viral delivery vector, e.g., a Poxvirus (e.g., Modified Vaccinia Ankara (MVA), NYVAC, AVIPOX), herpesvirus (e.g., HSV of any host species, CMV, adenovirus), morbillivirus (e.g., measles), alphavirus (e.g., SFV, Sendai), flavivirus (e.g., yellow fever), or rhabdovirus (e.g., VSV)-based viral delivery vector, a bacterial delivery vector (e.g., Salmonella, E. coli), an RNA expression vector, or a DNA expression vector.

[0485] Adenoviruses are by far the most widely used and cutting-edge viral vectors developed for SARS2 vaccines. They are non-enveloped double-stranded DNA (dsDNA) viruses capable of packaging foreign genes up to 7.5 kb. Nearly all SARS2 adenovirus-based vaccines are engineered for expression of the SARS2 S protein or RBD subunit. Recombinant adenovirus vectors are widely used due to their high transduction efficiency, high levels of transgene expression, and broad viral tropism. These vaccines are highly cell-specific, highly efficient in gene transduction, and efficient in inducing immune responses. Adenovirus vaccines are effective in eliciting and priming T cells, resulting in long-term and high-level antigen protein expression and therefore long-lasting protection. The AZD1222 (AstraZeneca) vaccine construct contains a recombinant adenovirus vector vaccine encoding the SARS2 S protein. The recombinant adenovirus genome contains the SARS2 S gene in the E1 locus.

[0486] Optionally, the vaccine of the invention (optionally a nucleic acid or polypeptide of the invention) is administered as part of a heterologous prime-boost regimen, for example using a heterologous DNA prime / MVA boost regimen.

[0487] Optionally, a method according to the invention for inducing an immune response against coronavirus in a subject or a method for immunizing a subject against coronavirus comprises administering a nucleic acid of the invention, a vector of the invention, or a pharmaceutical composition of the invention, wherein the nucleic acid, vector, or pharmaceutical composition is administered as part of a heterologous prime-boost regimen.

[0488] Optionally, a heterologous prime-boost regimen involves priming with a DNA vector of the invention followed by boosting with an MVA vector of the invention.

[0489] Optionally, the DNA prime comprises administration of a DNA vaccine vector comprising a nucleic acid molecule of the invention, and the MVA boost comprises administration of an MVA vector comprising a nucleic acid molecule of the invention, and optionally, the nucleic acid molecule of the DNA vaccine vector encodes the same amino acid sequence as the nucleic acid molecule of the invention of the MVA vector.

[0490] For example, a nucleic acid molecule (optionally a DNA molecule) encoding the designed S protein RBD sequence M7 polypeptide (SEQ ID NO: 33) of the present invention can be administered as part of a prime-boost vaccination using an MVA boost. As shown in Example 38 below, the heterologous DNA prime / MVA boost M7 regimen induced higher, broadly neutralizing, and long-lasting antibodies against the variant of interest.

[0491] In a further example, a nucleic acid molecule (or, optionally, a DNA molecule) encoding a designed S protein sequence T2_29 polypeptide of the invention (SEQ ID NO: 88-COV_S_T2_29+Q498R+dER; COV_S_T2_29+Q498R-SEQ ID NO: 87; or COV_S_T2_29-SEQ ID NO: 53) can be administered as part of a heterologous prime-boost vaccination using an MVA boost. As shown in Example 37 below, priming with a DNA vector containing DNA encoding the amino acid sequence of SEQ ID NO: 53, 87, or 88, followed by a boost with an MVA vector containing a nucleic acid encoding the amino acid sequence of SEQ ID NO: 88, induced broad neutralizing responses against all VOCs tested, and at least two-fold better neutralizing responses against alpha, beta, gamma, and omicron VOCs, compared to WT dER after three doses of DNA vaccine.

[0492] In a further example, a nucleic acid molecule (optionally a DNA molecule) encoding the designed S protein sequence T2_17 polypeptide of the present invention (SEQ ID NO: 31) may be administered as part of a heterologous prime-boost vaccination using an MVA boost with an MVA vector comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 31.

[0493] Optionally, priming with a DNA vector of the present invention can involve one, two, or three administrations of the DNA vector prior to an MVA boost.

[0494] The MVA boost can be administered at least 1 day, at least 1 week, or at least 2, 3, 4, 5, 6, or 7 weeks after the final administration of the DNA vector.

[0495] The present invention also provides a kit comprising a DNA vaccine vector comprising a nucleic acid molecule of the present invention, and an MVA vector comprising a nucleic acid molecule of the present invention, wherein, if necessary, the nucleic acid molecule of the present invention in the DNA vaccine vector encodes the same amino acid sequence as the nucleic acid molecule of the present invention in the MVA vector.

[0496] Optionally, the nucleic acid molecule of the present invention in the DNA vaccine vector encodes the designed S protein sequence T2_29 polypeptide of the present invention (SEQ ID NO: 88-COV_S_T2_29+Q498R+dER; COV_S_T2_29+Q498R-SEQ ID NO: 87; or COV_S_T2_29-SEQ ID NO: 53), and the nucleic acid molecule of the present invention in the MVA vector encodes the amino acid sequence of SEQ ID NO: 88.

[0497] Optionally, the nucleic acid molecule of the invention in a DNA vaccine vector encodes the amino acid sequence of SEQ ID NO:33, and the nucleic acid molecule of the invention in an MVA vector encodes the amino acid sequence of SEQ ID NO:33.

[0498] Optionally, the nucleic acid molecule of the present invention in a DNA vaccine vector encodes the amino acid sequence of SEQ ID NO:31, and the nucleic acid molecule of the present invention in an MVA vector encodes the amino acid sequence of SEQ ID NO:31.

[0499] Optionally, the nucleic acid expression vector is a nucleic acid expression vector and a viral pseudotype vector.

[0500] Optionally, the nucleic acid expression vector is a vaccine vector.

[0501] Optionally, the nucleic acid expression vector comprises, in the 5' to 3' direction, a promoter, a splice donor site (SD), a splice acceptor site (SA), and a terminator signal, with a multiple cloning site located between the splice acceptor site and the terminator signal.

[0502] Optionally, the promoter comprises a CMV immediate early 1 enhancer / promoter (CMV-IE-E / P) and / or the terminator signal comprises the terminator signal of the bovine growth hormone gene (Tbgh) lacking a KpnI restriction endonuclease site.

[0503] Optionally, the nucleic acid expression vector further comprises an origin of replication and a nucleic acid encoding resistance to an antibiotic. Optionally, the origin of replication comprises a pUC-plasmid origin of replication and / or the nucleic acid encodes resistance to kanamycin.

[0504] Optionally, the vector is a pEVAC-based expression vector.

[0505] Optionally, the nucleic acid expression vector comprises the nucleic acid sequence of SEQ ID NO: 20 (pEVAC). The pEVAC vector has proven to be a highly versatile expression vector for generating viral pseudotypes and for direct DNA vaccination of animals and humans. The pEVAC expression vector is described in more detail in Example 8 below. Figure 3 shows the plasmid map of pEVAC.

[0506] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0507] The polynucleotides (or nucleic acids) of the present invention may comprise DNA molecules.

[0508] The or each polynucleotide (or nucleic acid) of the pharmaceutical composition, combined preparation or vector of the invention may comprise a DNA molecule.

[0509] The vector of the present invention may be a DNA vector.

[0510] The or each vector of the pharmaceutical composition or combined preparation of the invention may be a DNA vector.

[0511] The polynucleotide (or nucleic acid) of the present invention, or the polynucleotide (or nucleic acid) of the pharmaceutical composition, combined preparation, or vector of the present invention may be provided as part of a DNA vaccine.

[0512] The present invention also provides a DNA vaccine comprising a polynucleotide (or nucleic acid) of the present invention, a vector of the present invention, or a pharmaceutical composition or combined preparation of the present invention comprising one or more polynucleotides (or nucleic acids), wherein the or each polynucleotide (or nucleic acid) is a DNA molecule.

[0513] Optionally, the or each vaccine vector is an RNA vaccine vector.

[0514] Polynucleotides (or nucleic acids) of the present invention can include RNA molecules.

[0515] The or each polynucleotide (or nucleic acid) of the pharmaceutical composition, combined preparation or vector of the invention may comprise an RNA molecule.

[0516] The vector of the present invention may be an RNA vector.

[0517] The or each vector of the pharmaceutical composition or combined preparation of the invention may be an RNA vector.

[0518] The polynucleotide (or nucleic acid) of the present invention, or the polynucleotide (or nucleic acid) of the pharmaceutical composition, combined preparation, or vector of the present invention may be provided as part of an RNA vaccine.

[0519] The present invention also provides an RNA vaccine comprising a polynucleotide (or nucleic acid) of the present invention, a vector of the present invention, or a pharmaceutical composition or combined preparation of the present invention comprising one or more polynucleotides (or nucleic acids), wherein the or each polynucleotide (or nucleic acid) is an RNA molecule.

[0520] The polynucleotides (or nucleic acids) of the present invention may include mRNA molecules.

[0521] The or each polynucleotide (or nucleic acid) of the pharmaceutical composition, combined preparation or vector of the invention may comprise an mRNA molecule.

[0522] The vector of the present invention may be an mRNA vector.

[0523] Optionally, the or each vaccine vector is an mRNA vaccine vector.

[0524] The or each vector of the pharmaceutical composition or combined preparation of the present invention may be an mRNA vector.

[0525] The polynucleotide (or nucleic acid) of the present invention, or the polynucleotide (or nucleic acid) of the pharmaceutical composition, combined preparation, or vector of the present invention may be provided as part of an mRNA vaccine.

[0526] The present invention also provides an mRNA vaccine comprising a polynucleotide (or nucleic acid) of the present invention, a vector of the present invention, or a pharmaceutical composition or combined preparation of the present invention comprising one or more polynucleotides (or nucleic acids), wherein the, or each polynucleotide (or nucleic acid) comprises an mRNA molecule.

[0527] Messenger RNA (mRNA) vaccines are a new form of vaccine (reviewed recently in Pardi et al., Nature Reviews Drug Discovery Volume 17, pages 261-279 (2018); Wang et al., Molecular Cancer (2021) 20:33: mRNA Vaccine: A Potential Therapeutic Strategy). The first mRNA vaccines approved for use were BNT162b2 (Pfizer) and mRNA-1273 (Moderna) during the COVID-19 pandemic. mRNA vaccines have the unique feature of promoting antigen expression transiently (typically for several days). Expression of exogenous antigens is controlled by the lifespan of the encoding mRNA, which is regulated by cellular degradation pathways. While this transient nature of protein expression necessitates repeated administration for the treatment of genetic diseases and cancer, it would be extremely useful for vaccines if prime or prime-boost vaccination were sufficient to generate highly specific adaptive immunity without any exposure to infectious disease.

[0528] mRNA-based vaccines induce an immune response after synthetic mRNA encoding viral antigens is transfected into human cells. The cytosolic mRNA molecules are then translated into specific viral antigens by the host's own cellular machinery. These antigens can then be presented on the cell surface where they can be recognized by immune cells, triggering an immune response.

[0529] The structural elements of vaccine vector mRNA molecules are similar to those of natural mRNAs, including a 5' cap, a 5' untranslated region (UTR), a coding region (e.g., containing an open reading frame encoding a polypeptide of the invention), a 3' UTR, and a poly(A) tail. The 5' UTR (also known as a leader sequence, transcript leader, or leader RNA) is a region of an mRNA immediately upstream of the start codon. This region is important for regulating translation of the transcript. In many organisms, the 5' UTR forms complex secondary structures to regulate translation. The 5' UTR begins at the transcription start site and ends one nucleotide (nt) before the start sequence (usually AUG) of the coding region. In eukaryotes, the length of the 5' UTR tends to be anywhere from 100 to several thousand nucleotides. The different sizes are likely due to the regulatory complexities of eukaryotes, where the 5' UTR similarly supports a larger preinitiation complex than the one that needs to form to initiate translation. Eukaryotic 5' UTRs contain a Kozak consensus sequence (ACC). AUG (start codon underlined), which includes the start codon AUG. An extended Kozak sequence can be used: GCCACC AUG (The start codon is underlined).

[0530] Two main types of RNA are currently being investigated as vaccines: non-replicating mRNA and self-amplifying RNA derived from viruses. Both types of vaccines share a common structure in the mRNA construct, but self-amplifying RNA vaccines contain additional sequences in the coding region for RNA replication, including an RNA-dependent RNA polymerase.

[0531] The BNT162b2 vaccine construct contains lipid nanoparticles (LNPs) encapsulating mRNA molecules encoding the trimerized full-length SARS2 S protein with a PP mutation (residue positions 986-987). The mRNA is encapsulated in 80 nm ionizable cationic lipid nanoparticles. The mRNA-1273 vaccine construct is also based on the LNP vector, but the synthetic mRNA encapsulated within the lipid construct encodes the full-length SARS2 S protein.

[0532] US Patent No. 10,702,600 B1 (ModernaTX) describes betacoronavirus mRNA vaccines, including suitable LNPs for use in such vaccines.

[0533] The nucleic acid vaccines (eg, mRNA) of the present invention may be formulated in lipid nanoparticles.

[0534] mRNA vaccines have several advantages over traditional vaccines containing inactivated (or live-attenuated) disease-causing organisms. First, mRNA-based vaccines can be developed rapidly due to their design flexibility and ability to mimic the antigenic structure and expression of constructs as seen during natural infection. While mRNA vaccines can be developed within days or months based on sequencing information from the target virus, traditional vaccines often take years and require a deep understanding of the target virus to make the vaccine effective and safe. Second, these novel vaccines can be produced rapidly. Due to the high yields from in vitro transcription reactions, mRNA production can be rapid, inexpensive, and scalable. Third, vaccine risks are low. mRNA does not contain infectious viral elements that pose the risk of infection and insertional mutagenesis. Because mRNA is a minimally immunogenic gene vector, it also circumvents anti-vector immunity and allows for repeated vaccine administration. A challenge for the effective application of mRNA vaccines lies in cytosolic delivery. mRNA isolates are rapidly degraded by extracellular RNases and cannot penetrate the cell membrane and be transcribed in the cytosol. However, efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm. Numerous delivery methods have been developed to date, including lipid-, polymer-, or peptide-based delivery, virus-like replicon particles, cationic nanoemulsions, naked mRNA, and dendritic cell-based delivery (reviewed in Wang et al., supra). Decationized lipid nanoparticle (LNP) delivery is the most attractive and commonly used mRNA vaccine delivery tool.

[0535] Exogenous mRNA can be highly immunostimulatory. Single-stranded RNA (ssRNA) molecules are considered pathogen-associated molecular patterns (PAMPs) and are recognized by various Toll-like receptors (TLRs), which induce pro-inflammatory responses. While strong cellular and humoral immune responses are desirable in response to vaccination, innate immune responses elicited by exogenous mRNA can cause unwanted side effects in subjects. U-rich sequences in mRNA are key elements for activating TLRs (Wang et al., supra). Furthermore, enzymatically synthesized mRNA preparations contain double-stranded RNA (dsRNA) contaminants as aberrant products of the in vitro transcription (IVT) process. dsRNA is a potent PAMP and induces downstream responses that result in the inhibition of translation and degradation of cellular mRNA and ribosomal RNA (Pardi et al., supra). Thus, mRNA can suppress antigen expression and thus reduce vaccine efficacy.

[0536] Research over the past decade has shown that the immunostimulatory effects of mRNA can be shaped by purifying IVT mRNA, introducing modified nucleosides, conjugating mRNA to various carrier molecules (Pardi et al., supra), adding poly(A) tails, or optimizing mRNA with GC-rich sequences (Wang et al., supra). Chemical modification of uridine is a common approach to minimize the immunogenicity of foreign mRNA. Incorporation of pseudouridine (ψ) and N1-methylpseudouridine (m1ψ) into IVT mRNA prevents TLR activation and other innate immune sensors, thus reducing proinflammatory signaling in response to exogenous mRNA. Such nucleoside modifications may also suppress the recognition of dsRNA species (Pardi et al., supra) and reduce innate immunity to detect exogenous mRNA translation (Hou et al. Nature Reviews Materials, 2021, https: / / doi.org / 10.1038 / s41578-021-00358-0).

[0537] Other chemical modifications of nucleosides include, but are not limited to, 5-methylcytidine (m5C), 5-methyluridine (m5U), N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), and 5-methoxyuridine (5moU) (Wang et al., supra).

[0538] The IVT mRNA molecules used in the mRNA-1273 and BNT162b2 COVID-19 vaccines were prepared by replacing uridine with m1ψ, and their sequences were optimized to encode a stabilized pre-fusion spike protein with two critical proline substitutions (Hou et al., supra). However, CureVac's mRNA vaccine candidate, CVnCoV, uses unmodified nucleosides and relies on a combination of mRNA sequence changes to enable immune evasion without affecting the expressed protein. First, CVnCoV has a higher GC content (63%) than both competing vaccines (BNT162b2 has 56%) and the original SARS-CoV-2 virus itself (37%). Second, the vaccine contains a C-rich motif that binds to poly(C)-binding proteins, enhancing both mRNA stability and expression. Further modifications of CVnCoV include a histone stem-loop sequence and a poly(A) tail to enhance mRNA longevity and translation (Hubert, B., 2021. The CureVac Vaccine, and a brief tour through some of the wonders of nature. URL https: / / berthub.eu / articles / posts / curevac-vaccine-and-wonders-of-biology / . (Accessed September 15, 2021)). However, the vaccine had disappointing results from Phase III clinical trials, which experts argue depended on the decision not to incorporate chemically modified nucleosides into the mRNA sequence. Nevertheless, CureVac and Acuitas Therapeutics delivered erythropoietin (EPO)-encoding mRNA with a GC-rich codon to pigs using lipid nanoparticles (LNPs). These results demonstrated that EPO-associated responses were elicited without immunogenicity (Wang et al., supra), suggesting that there is still room for unmodified mRNA nucleoside-based vaccines.

[0539] The polynucleotides (or nucleic acids) of the present invention may include mRNA molecules.

[0540] The or each polynucleotide (or nucleic acid) of the pharmaceutical composition, combined preparation or vector of the invention may comprise an mRNA molecule.

[0541] The vector of the present invention may be an mRNA vector.

[0542] The or each vector of the pharmaceutical composition or combined preparation of the present invention may be an mRNA vector.

[0543] The polynucleotide (or nucleic acid) of the present invention, or the polynucleotide (or nucleic acid) of the pharmaceutical composition, combined preparation, or vector of the present invention may be provided as part of an mRNA vaccine.

[0544] The present invention also provides an mRNA vaccine comprising a polynucleotide (or nucleic acid) of the present invention, a vector of the present invention, or a pharmaceutical composition or combined preparation of the present invention comprising one or more polynucleotides (or nucleic acids), wherein the or each polynucleotide (or nucleic acid) comprises an mRNA molecule.

[0545] The RNA or mRNA of a polynucleotide (or nucleic acid) of the invention, or of a polynucleotide (or nucleic acid) of a pharmaceutical composition, combined preparation, vector, or vaccine of the invention, may be produced by in vitro transcription (IVT).

[0546] A polynucleotide (or nucleic acid) of the invention, or of a pharmaceutical composition, combined preparation, vector, or vaccine of the invention, may comprise one or more modified nucleosides.

[0547] One or more modified nucleosides may be present in the DNA or RNA of a polynucleotide (or nucleic acid) of the invention, or of a polynucleotide (or nucleic acid) of a pharmaceutical composition, combined preparation, vector, or vaccine of the invention.

[0548] Optionally, the at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, the chemical modification is N1-ethylpseudouridine.

[0549] For example, the polynucleotides (or nucleic acids) of the invention, or the RNA or mRNA of the polynucleotides (or nucleic acids) of the pharmaceutical compositions, combined preparations, vectors, or vaccines of the invention, may contain one or more of the following modified nucleosides: pseudouridine (ψ); N1-methylpseudouridine (m1ψ) 5-methylcytidine (m5C) 5-methyluridine (m5U) N1-methyladenosine (m1A) N6-methyladenosine (m6A) 2-thiouridine (s2U) 5-Methoxyuridine (5moU) may include one or more of:

[0550] In some embodiments, 100% of the uracils in the open reading frame have a chemical modification. In some embodiments, the chemical modification is at the 5-position of the uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, 100% of the uracils in the open reading frame have N1-methylpseudouridine at the 5-position of the uracil.

[0551] Polynucleotides (or nucleic acids) can contain from about 1% to about 100% modified nucleotides (or nucleosides) (either relative to the total nucleotide content or relative to one or more types of nucleotides (or nucleosides), i.e., any one or more of A, G, U, or C), or any percentage in between (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, It may contain 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). Any remaining percentage is accounted for by unmodified A, G, U, or C occurrences.

[0552] Optionally, the polynucleotides (or nucleic acids) of the invention, or the polynucleotides (or nucleic acids) of the pharmaceutical compositions, combined preparations, vectors, or vaccines of the invention, comprise RNA molecules in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that set forth in the respective SEQ ID NO: or its complement, but with each "U" replaced by mψ.

[0553] Optionally, the polynucleotides (or nucleic acids) of the invention, or the polynucleotides (or nucleic acids) of the pharmaceutical compositions, combination preparations, vectors, or vaccines of the invention, comprise mRNA molecules in which the nucleic acid sequence of the polynucleotide is the same as that set forth in the respective SEQ ID NO: or its complement, but with each "U" replaced by mψ.

[0554] Optionally, a polynucleotide (or nucleic acid) of the invention, or a polynucleotide (or nucleic acid) of a pharmaceutical composition, combination preparation, vector, or vaccine of the invention, comprises an RNA molecule in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that set forth in the respective SEQ ID NO: or its complement, but in which at least 50% of the "U's" are replaced by mlψ. The remaining "U's" may be all unmodified or may include unmodified nucleosides and one or more other modified nucleosides.

[0555] Optionally, the polynucleotides (or nucleic acids) of the invention, or the polynucleotides (or nucleic acids) of the pharmaceutical compositions, combination preparations, vectors, or vaccines of the invention, comprise mRNA molecules in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that set forth in the respective SEQ ID NO: or its complement, but in which at least 50% of the "U's" are replaced by mlψ. The remaining "U's" may be all unmodified or may include unmodified nucleosides and one or more other modified nucleosides.

[0556] Optionally, a polynucleotide (or nucleic acid) of the invention, or a polynucleotide (or nucleic acid) of a pharmaceutical composition, combination preparation, vector, or vaccine of the invention, comprises an RNA molecule in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that set forth in the respective SEQ ID NO: or its complement, but in which at least 90% of the "U"s are replaced by mlψ. The remaining "U"s may be all unmodified or may include unmodified nucleosides and one or more other modified nucleosides.

[0557] Optionally, a polynucleotide (or nucleic acid) of the invention, or a polynucleotide (or nucleic acid) of a pharmaceutical composition, combination preparation, vector, or vaccine of the invention, comprises an mRNA molecule in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that set forth in the respective SEQ ID NO: or its complement, but in which at least 90% of the "U"s are replaced by mlψ. The remaining "U"s may be all unmodified or may include unmodified nucleosides and one or more other modified nucleosides.

[0558] The mRNA vaccines of the present invention may be co-administered with immunological adjuvants, such as MF59 (Novartis), TriMix, RNActive (CureVac AG), RNAdjuvant (reviewed again in Wang et al., supra).

[0559] When mRNA vaccines encoding different polypeptides of the invention are used in accordance with the invention, each different polypeptide of the invention (e.g., a designed coronavirus S protein of the invention (full-length, truncated, or RBD) and / or a designed coronavirus E protein of the invention and / or a designed coronavirus M protein of the invention and / or a designed coronavirus N protein of the invention) is preferably encoded as part of a separate mRNA vaccine vector.

[0560] Thus, in a preferred embodiment, each vector of the pharmaceutical composition or combined preparation of the present invention is an mRNA vaccine vector.

[0561] The present invention also provides an isolated cell comprising or transfected with a vector of the invention.

[0562] The present invention also provides a fusion protein comprising a polypeptide of the present invention.

[0563] Pharmaceutical Composition The present invention also provides a pharmaceutical composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent.

[0564] Optionally, a pharmaceutical composition of the invention comprises two or more different polypeptides of the invention.

[0565] Advantageously, the pharmaceutical composition of the present invention comprises a designed coronavirus S protein (full-length, truncated, or RBD) of the present invention, and / or a designed coronavirus E protein of the present invention and / or a designed coronavirus M protein of the present invention.

[0566] Advantageously, the pharmaceutical composition of the present invention comprises a designed coronavirus S protein (full-length, truncated, or RBD) of the present invention, and / or a designed coronavirus E protein of the present invention, and / or a designed coronavirus M protein of the present invention and / or a designed coronavirus N protein of the present invention.

[0567] Optionally, the pharmaceutical compositions of the invention comprise an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention and an engineered coronavirus E protein of the invention.

[0568] Optionally, the pharmaceutical compositions of the invention comprise an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention and an engineered coronavirus M protein of the invention.

[0569] Optionally, a pharmaceutical composition of the invention comprises an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention and an engineered coronavirus N protein of the invention.

[0570] Optionally, the pharmaceutical composition of the invention comprises a designed coronavirus E protein of the invention and a designed coronavirus M protein of the invention.

[0571] Optionally, the pharmaceutical composition of the invention comprises a designed coronavirus E protein of the invention and a designed coronavirus N protein of the invention.

[0572] Optionally, the pharmaceutical compositions of the invention comprise a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, a designed coronavirus E protein of the invention, and a designed coronavirus M protein of the invention.

[0573] Optionally, the pharmaceutical composition of the invention comprises a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, a designed coronavirus E protein of the invention, and a designed coronavirus N protein of the invention.

[0574] Optionally, the pharmaceutical composition of the invention comprises a designed coronavirus E protein of the invention, a designed coronavirus M protein of the invention, and a designed coronavirus N protein of the invention.

[0575] Optionally, the pharmaceutical composition of the present invention comprises: a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; A polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length.

[0576] Optionally, the pharmaceutical composition of the present invention comprises: a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; A polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0577] Optionally, the pharmaceutical composition of the present invention comprises: a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length; A polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0578] Optionally, the pharmaceutical composition of the present invention comprises: a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length; A polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0579] The present invention also provides a pharmaceutical composition comprising a nucleic acid of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent.

[0580] Optionally, a pharmaceutical composition of the invention comprises two or more nucleic acid molecules of the invention encoding different polypeptides of the invention.

[0581] Advantageously, the pharmaceutical composition of the present invention comprises a nucleic acid molecule of the present invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the present invention, and / or a nucleic acid molecule of the present invention encoding a designed coronavirus E protein of the present invention, and / or a nucleic acid molecule of the present invention encoding a designed coronavirus M protein of the present invention.

[0582] Advantageously, the pharmaceutical composition of the present invention comprises a nucleic acid molecule of the present invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the present invention, and / or a nucleic acid molecule of the present invention encoding a designed coronavirus E protein of the present invention, and / or a nucleic acid molecule of the present invention encoding a designed coronavirus M protein of the present invention and / or a nucleic acid molecule of the present invention encoding a designed coronavirus N protein of the present invention.

[0583] Optionally, a pharmaceutical composition of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention.

[0584] Optionally, a pharmaceutical composition of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention.

[0585] Optionally, a pharmaceutical composition of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0586] Optionally, a pharmaceutical composition of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention.

[0587] Optionally, a pharmaceutical composition of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0588] Optionally, the pharmaceutical compositions of the invention comprise a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention.

[0589] Optionally, a pharmaceutical composition of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0590] Optionally, the pharmaceutical composition of the invention comprises a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention, and a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention.

[0591] Optionally, the pharmaceutical composition of the present invention comprises: a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length.

[0592] Optionally, the pharmaceutical composition of the present invention comprises: a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:24 over its entire length; or a nucleic acid sequence encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:25 over its entire length.

[0593] Optionally, the pharmaceutical composition of the present invention comprises: a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length; a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:24 over its entire length; or a nucleic acid sequence encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:25 over its entire length.

[0594] Optionally, the pharmaceutical composition of the present invention comprises: a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length; a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:24 over its entire length; or a nucleic acid sequence encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO:25 over its entire length.

[0595] The present invention also provides a pharmaceutical composition comprising a vector of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent.

[0596] Optionally, the pharmaceutical composition of the present invention further comprises an adjuvant to enhance the immune response in the subject to the polypeptide of the composition or to a polypeptide encoded by the nucleic acid.

[0597] Optionally, the pharmaceutical composition of the present invention further comprises an adjuvant to enhance the immune response in the subject to the polypeptide of the composition or to a polypeptide encoded by the nucleic acid.

[0598] The present invention also provides pseudotyped viruses comprising the polypeptides of the present invention.

[0599] Combination Preparations The term "combination preparation," as used herein, refers to a "kit of parts" in the sense that combination components (i) and (ii), or (i), (ii) and (iii), or (i), (ii), (iii) and (iv), as defined herein, can be administered independently or by using distinct amounts of combination components (i) and (ii), or (i), (ii) and (iii), or different fixed combinations of (i), (ii), (iii) and (iv). The components can be administered simultaneously or sequentially. When the components are administered sequentially, preferably, the time interval between administrations is selected so that the therapeutic effect of the combined use of the components is greater than the effect that would be obtained by using only any one of the combination components (i) and (ii), or (i), (ii) and (iii), or (i), (ii), (iii) and (iv).

[0600] The components of the combination preparation may be present in one combined unit dosage form, or as a first unit dosage form of component (i) and a separate second unit dosage form of component (ii), or as a first unit dosage form of component (i), a separate second unit dosage form of component (ii), and a separate third unit dosage form of component (iii), or as a first unit dosage form of component (i), a separate second unit dosage form of component (ii), a separate third unit dosage form of component (iii), and a separate third unit dosage form of component (iv). The ratio of the total amount of combination component (i) to combination component (ii), or combination component (i) to combination component (ii) and combination component (iii), or combination component (i) to combination component (ii), combination component (iii) and combination component (iv) administered in a combination preparation may vary, for example, to address the needs of a patient subpopulation being treated, or the needs of a single patient, which may be due, for example, to the particular disease, age, sex, or weight of the patient.

[0601] Preferably, at least one beneficial effect, such as an enhancement of the effect of component (i), or an enhancement of the effect of component (ii), or a mutual enhancement of the effects of the combined components (i) and (ii), or an enhancement of the effect of component (i), or an enhancement of the effect of component (ii), or an enhancement of the effect of component (iii), or a mutual enhancement of the effects of the combined components (i), (ii) and (iii), or an enhancement of the effect of component (i), or an enhancement of the effect of component (ii), or an enhancement of the effect of component (iii), or an enhancement of the effect of component (iv), or a combination. There is a mutual enhancement of the effects of components (i), (ii), (iii) and (iv), e.g., a greater than additive effect, an additive beneficial effect, lower side effects, lower toxicity or combined therapeutic effect compared to the effective dosage of one or both of the combination components (i) and (ii), or (i), (ii) and (iii), or (i), (ii), (iii) and (iv), highly preferably a synergistic effect of the combination components (i) and (ii), or (i), (ii) and (iii), or (i), (ii), (iii) and (iv).

[0602] The combined preparation of the present invention may be provided as a pharmaceutical combined preparation for administration to a mammal, preferably a human. Component (i) may be provided, if necessary, together with a pharmaceutically acceptable carrier, excipient, or diluent, and / or component (ii) may be provided, if necessary, together with a pharmaceutically acceptable carrier, excipient, or diluent, or component (i) may be provided, if necessary, together with a pharmaceutically acceptable carrier, excipient, or diluent, and / or component (ii) may be provided, if necessary, together with a pharmaceutically acceptable carrier, excipient, or diluent, and / or component (iii) may be provided, if necessary, together with a pharmaceutically acceptable carrier, excipient, or diluent. Component (i) may be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, if desired, and / or component (ii) may be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, if desired, and / or component (iii) may be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, if desired, and / or component (iv) may be provided together with a pharmaceutically acceptable carrier, excipient, or diluent, if desired.

[0603] According to the present invention, i) an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention, and / or ii) an engineered coronavirus E protein of the invention, and / or iii) an engineered coronavirus M protein of the invention, and / or iv) The engineered coronavirus N protein of the present invention A combined preparation is provided, comprising:

[0604] According to the present invention, i) an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention, and / or ii) an engineered coronavirus E protein of the invention, and / or iii) Engineered Coronavirus M Proteins of the Invention A combined preparation is provided, comprising:

[0605] According to the present invention, i) an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention; ii) the engineered coronavirus E proteins of the invention; iii) an engineered coronavirus M protein of the invention, and iv) The engineered coronavirus N protein of the present invention A combined preparation is provided, comprising:

[0606] According to the present invention, i) an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) Designed Coronavirus E Proteins of the Invention A combined preparation is provided, comprising:

[0607] According to the present invention, i) an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) Engineered Coronavirus M Proteins of the Invention A combined preparation is provided, comprising:

[0608] According to the present invention, i) an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) The engineered coronavirus N proteins of the present invention A combined preparation is provided, comprising:

[0609] According to the present invention, i) an engineered coronavirus E protein of the invention, and ii) Engineered Coronavirus M Proteins of the Invention A combined preparation is provided, comprising:

[0610] According to the present invention, i) an engineered coronavirus E protein of the invention, and ii) The engineered coronavirus N proteins of the present invention A combined preparation is provided, comprising:

[0611] According to the present invention, i) an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) an engineered coronavirus E protein of the invention, and iii) Engineered Coronavirus M Proteins of the Invention A combined preparation is provided, comprising:

[0612] According to the present invention, i) an engineered coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) an engineered coronavirus E protein of the invention, and iii) The engineered coronavirus N proteins of the present invention A combined preparation is provided, comprising:

[0613] According to the present invention, i) an engineered coronavirus E protein of the invention, and ii) an engineered coronavirus M protein of the invention, and iii) The engineered coronavirus N proteins of the present invention A combined preparation is provided, comprising:

[0614] Optionally, the combined preparation of the present invention comprises: i) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; ii) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence which has at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence which has at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length.

[0615] Optionally, the combined preparation of the present invention comprises: i) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; ii) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence which has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence which has at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0616] Optionally, the combined preparation of the present invention comprises: i) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length, ii) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence which has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence which has at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0617] Optionally, the combined preparation of the present invention comprises: i) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; ii) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length, iii) a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence which has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence which has at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0618] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and / or ii) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and / or iii) A nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention. A combined preparation is provided, comprising:

[0619] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and / or ii) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and / or iii) a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention, and / or iv) A nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention. A combined preparation is provided, comprising:

[0620] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention; ii) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention; iii) a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention, and iv) A nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention. A combined preparation is provided, comprising:

[0621] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention A combined preparation is provided, comprising:

[0622] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention A combined preparation is provided, comprising:

[0623] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention A combined preparation is provided, comprising:

[0624] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and ii) a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention A combined preparation is provided, comprising:

[0625] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and ii) a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention A combined preparation is provided, comprising:

[0626] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and iii) A nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention. A combined preparation is provided, comprising:

[0627] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus S protein (full-length, truncated, or RBD) of the invention, and ii) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and iii) a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention A combined preparation is provided, comprising:

[0628] According to the present invention, i) a nucleic acid molecule of the invention encoding a designed coronavirus E protein of the invention, and ii) a nucleic acid molecule of the invention encoding a designed coronavirus M protein of the invention, and iii) a nucleic acid molecule of the invention encoding a designed coronavirus N protein of the invention A combined preparation is provided, comprising:

[0629] Optionally, the combined preparation of the present invention comprises: i) a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; ii) a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence which has at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence which has at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length.

[0630] Optionally, the combined preparation of the present invention comprises: i) a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; ii) a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence which has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence which has at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0631] Optionally, the combined preparation of the present invention comprises: i) a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length; ii) a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence which has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence which has at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0632] Optionally, the combined preparation of the present invention comprises: i) a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17; ii) a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 22 over its entire length, or a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 23 over its entire length; iii) a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence which has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 24 over its entire length, or a nucleic acid molecule encoding a polypeptide of the invention comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence which has at least 95%, 96%, 97%, 98% or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 25 over its entire length.

[0633] Each different nucleic acid molecule of the combined preparation of the invention may be provided as part of a separate vector.

[0634] The present invention also provides a combined preparation comprising a vector of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent.

[0635] Optionally, the combined preparation of the present invention further comprises an adjuvant to enhance the immune response in the subject to the polypeptide of the composition or the polypeptide encoded by the nucleic acid.

[0636] Optionally, the combined preparation of the present invention further comprises an adjuvant to enhance the immune response in the subject to the polypeptide of the composition or the polypeptide encoded by the nucleic acid.

[0637] string Embodiments of the invention in which different polypeptides of the invention are encoded as part of the same polynucleotide (or nucleic acid) or provided in the same polypeptide (i.e., as "strings" of different subunits, e.g., the S protein RBD, and / or the E protein, and / or the M protein, and / or the N protein) are particularly advantageous because the use of such "strings" as part of a vaccine requires testing of only a single product containing the "string" for safety and efficacy, rather than testing each different subunit individually. This dramatically reduces the time and cost of vaccine development compared to individual subunits. In some embodiments, combinations of different strings (polynucleotides and / or polypeptides), or combinations of one or more strings and one or more single subunits (polypeptides or encoded subunits) may be used.

[0638] Strategies for multigene co-expression include the introduction of multiple vectors, the use of multiple promoters in a single vector, fusion proteins, intergenic protein cleavage sites, internal ribosome entry sites (IRES), and "self-cleaving" 2A peptides. Multicistronic vectors based on IRES nucleotide sequences and self-cleaving 2A peptides are reviewed in Shaimardanova et al. (Pharmaceutics 2019, 11, 580; doi:10.3390 / pharmaceutics11110580).

[0639] vaccine Vaccines can be provided, for example, as nucleic acid vaccines, as separate polynucleotides encoding each of the different subunits (for administration together or separately), or as a single polynucleotide encoding all of the subunits, grouped together in a string. The separate polynucleotides can be administered together as a mixture (e.g., as a pharmaceutical composition comprising the separate polynucleotides), or co-administered or sequentially administered in any order (in which case the separate polynucleotides can be provided as a combined preparation for co-administration or sequential administration). Nucleic acid vaccines can be provided as DNA, RNA, or mRNA vaccines. The generation and application of multicistronic constructs (e.g., when the subunits are provided in a string as a single polynucleotide) is reviewed in Shaimardanova et al. (Pharmaceutics 2019, 11, 580; doi:10.3390 / pharmaceutics11110580).

[0640] The vaccine constructs of the invention can also be provided, for example, as separate polypeptides each containing a different designed subunit, or as a single polypeptide containing all of the subunits, grouped together in a string. The separate polypeptides can be administered together as a mixture (e.g., as a pharmaceutical composition containing the separate polypeptides), or co-administered or administered sequentially in any order (in which case the separate polypeptides can be provided as a combined preparation for co-administration or sequential administration).

[0641] Treatment and Use The present invention also provides a method for inducing an immune response against coronavirus in a subject, the method comprising administering to the subject an effective amount of a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0642] The present invention also provides a method for immunizing a subject against coronavirus, the method comprising administering to the subject an effective amount of a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention.

[0643] An effective amount is an amount that generates an antigen-specific immune response in a subject.

[0644] The present invention further provides the polypeptide of the present invention, the nucleic acid of the present invention, the vector of the present invention, or the pharmaceutical composition for use as a pharmaceutical.

[0645] The present invention further provides a polypeptide of the present invention, a nucleic acid of the present invention, a vector of the present invention, or a pharmaceutical composition for use in the prevention, treatment, or amelioration of coronavirus infection.

[0646] The present invention also provides use of the polypeptide of the present invention, the nucleic acid of the present invention, the vector of the present invention, or the pharmaceutical composition in the manufacture of a medicament for the prevention, treatment, or amelioration of coronavirus infection.

[0647] Optionally, the coronavirus is a β-coronavirus.

[0648] Optionally, the β-coronavirus is a type B or type C β-coronavirus.

[0649] Optionally, the β-coronavirus is a type B β-coronavirus.

[0650] Optionally, the type B beta-coronavirus is SARS-CoV or SARS-CoV-2.

[0651] Optionally, the type C beta-coronavirus is MERS-CoV.

[0652] Optionally, the immune response is directed against more than one type B beta-coronavirus.

[0653] Optionally, the immune response is directed against SARS-1 and SARS-2 beta-coronaviruses.

[0654] Optionally, the immune response is directed against SARS-1 and MERS beta-coronaviruses.

[0655] Optionally, the immune response is directed against SARS-2 and MERS beta-coronaviruses.

[0656] Optionally, the immune response is directed against SARS-1, SARS-2, and MERS beta-coronaviruses.

[0657] Optionally, the beta-coronavirus is a variant of interest (VOC).

[0658] Where appropriate, the beta-coronavirus is the SARS-CoV-2 VOC.

[0659] Where appropriate, the beta-coronavirus is the SARS-CoV-2 lineage B1.248 (Brazilian P1 lineage) VOC.

[0660] Where appropriate, the beta-coronavirus is the SARS-CoV-2 lineage B1.351 (South Africa) VOC.

[0661] Optionally, the beta-coronavirus is a SARS-CoV-2 beta, gamma, or delta coronavirus.

[0662] Where appropriate, the beta-coronavirus is SARS-CoV-2 beta-coronavirus.

[0663] Where appropriate, the beta-coronavirus is the SARS-CoV-2 gamma coronavirus.

[0664] Where appropriate, the beta-coronavirus is the SARS-CoV-2 delta coronavirus.

[0665] Where appropriate, the beta-coronavirus is the SARS-CoV-2 alpha-coronavirus.

[0666] Where appropriate, the beta-coronavirus is the SARS-CoV-2 Omicron VOC.

[0667] Where appropriate, the beta-coronavirus is SARS-CoV-2 Omicron BA.1.

[0668] Optionally, the beta-coronavirus is SARS-CoV-2 Omicron BA.2.

[0669] Whether an immune response has been induced against a beta-coronavirus can be readily determined using methods well known to those skilled in the art, for example, the pseudotype neutralization assay described in any of the Examples below can be used.

[0670] Administration Any suitable route of administration can be used. Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal, intranasal, inhalation, or oral. Parenteral administration, such as subcutaneous, intravenous, or intramuscular administration, is generally achieved by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type described above. Administration can be systemic or local. Routes for systemic administration generally include, for example, transdermal, oral, and parenteral routes, such as subcutaneous, intravenous, intramuscular, intraarterial, intradermal, and intraperitoneal injections and / or intranasal routes. Routes for local administration generally include, for example, topical administration routes, as well as intradermal, transdermal, subcutaneous, or intramuscular injections, or intralesional, intracranial, intrapulmonary, intracardiac, and sublingual injections.

[0671] The composition can be administered in any suitable manner, such as a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered and by the particular method used to administer the composition. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases.

[0672] Some of the compositions may be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanol amines.

[0673] Administration can be achieved by single or multiple doses. In the context of the present disclosure, the dose administered to a subject should be sufficient to induce a beneficial therapeutic response in the subject over time, or to inhibit or prevent infection. The required dose will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the infection being treated, the specific composition being used, and its mode of administration. The appropriate dose can be determined by one skilled in the art using only routine experimentation.

[0674] The present disclosure includes methods comprising administering an RNA vaccine, an mRNA vaccine, or a DNA vaccine to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, etc.

[0675] RNA or DNA is usually formulated into dosage unit form for ease of administration and uniformity of dosage.However, it is understood that the total daily amount of RNA can be determined by attending physician within the scope of sound medical judgment.The specific therapeutically effective, prophylactically effective or suitable imaging dose level for any specific patient depends on a variety of factors, including the disorder to be treated and the severity of the disorder, the activity of the specific compound used, the specific composition used, the age, weight, general condition, sex and diet of the patient, the timing of administration, the route of administration and the excretion rate of the specific compound used, the duration of treatment, the drugs used in combination with or simultaneously with the specific compound used, and similar factors well known in the medical field.

[0676] Effective amounts of RNA or DNA provided herein can be as low as 20 pg, for example, administered as a single dose or as two 10 pg doses. In some embodiments, an effective amount is 20 μg to 300 μg or a total dose of 25 μg to 300 μg. For example, an effective amount can be a total dose of 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, or 300 μg. In some embodiments, the effective amount is a 20 μg total dose. In some embodiments, the effective amount is a 25 pg total dose. In some embodiments, the effective amount is a 50 μg total dose. In some embodiments, the effective amount is a 75 μg total dose. In some embodiments, the effective amount is a 100 μg total dose. In some embodiments, the effective amount is a 150 μg total dose. In some embodiments, the effective amount is a 200 μg total dose. In some embodiments, the effective amount is a 250 pg total dose. In some embodiments, the effective amount is a 300 μg total dose.

[0677] The RNA or DNA described herein can be formulated into dosage forms described herein, for example, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0678] If desired, the RNA (eg, mRNA) or DNA vaccine is formulated in an amount effective to generate an antigen-specific immune response in the subject.

[0679] In some embodiments, the effective amount is a total dose of 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a 25 μg dose administered to a subject a total of two times. In some embodiments, the effective amount is a 100 μg dose administered to a subject a total of two times. In some embodiments, the effective amount is a 400 μg dose administered to a subject a total of two times. In some embodiments, the effective amount is a 500 μg dose administered to a subject a total of two times.

[0680] If desired, a dosage of 10 μg / kg to 400 μg / kg of nucleic acid vaccine is administered to the subject. In some embodiments, the dosage of RNA or DNA polynucleotide (or nucleic acid) is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25 μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100 μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-150 μg, 80-200 μg, 80-300 μg, 80-350 μg, 80-400 μg, 80-450 μg, 80-500 μg, 80-600 μg, 80-800 μg, 80-1000 μg, 80-1200 μg, 80-1500 μg, 80-1500 μg, 80-2000 μg, 80-3500 μg, 80-4500 μg, 80-5000 μg, 80-60 ... The dose is 0 to 200 μg, 100 to 200 μg, 120 to 250 μg, 150 to 250 μg, 180 to 280 μg, 200 to 300 μg, 50 to 300 μg, 80 to 300 μg, 100 to 300 μg, 40 to 300 μg, 50 to 350 μg, 100 to 350 μg, 200 to 350 μg, 300 to 350 μg, 320 to 400 μg, 40 to 380 μg, 40 to 100 μg, 100 to 400 μg, 200 to 400 μg, or 300 to 400 μg. In some embodiments, the nucleic acid vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the nucleic acid vaccine is administered to the subject on day zero. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day 21.

[0681] Pharmaceutically acceptable carrier Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile, and the formulation is suitable for the mode of administration. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, or powder. The composition can be formulated as a suppository using traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Any common pharmaceutical carrier, such as sterile saline solution or sesame oil, can be used. The vehicle can also contain conventional pharmaceutical auxiliary materials, such as pharmaceutically acceptable salts for adjusting osmotic pressure, buffers, preservatives, etc. Other vehicles that can be used in the compositions and methods provided herein are normal saline and sesame oil.

[0682] In some embodiments, the composition comprises a pharmaceutically acceptable carrier and / or an adjuvant. For example, the adjuvant can be alum, Freund's complete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (such as a CpG oligonucleotide).

[0683] Pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. See Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA , 15th Edition (1975) describes compositions and formulations suitable for the pharmaceutical delivery of one or more therapeutic compositions, such as one or more influenza vaccines, as well as additional agents.

[0684] The nature of the carrier will generally depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solution, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sodium monolaurate.

[0685] Optionally, the polypeptides, nucleic acids or compositions of the invention are administered intramuscularly.

[0686] Optionally, the polypeptides, nucleic acids or compositions of the invention may be administered intramuscularly, intradermally, subcutaneously, by needle or gene gun or by electroporation.

[0687] Diagnostic methods The present invention also provides a method for diagnosing whether a subject has a coronavirus infection, comprising determining whether a polypeptide of the present invention binds to an antibody produced by the subject.

[0688] Optionally, the method is an in vitro method.

[0689] Optionally, the antibody is in a biological sample obtained from the subject, or in a sample derived from a biological sample obtained from the subject.

[0690] "Biological sample" encompasses a wide variety of sample types obtained from an individual and can be used in diagnostic or monitoring assays. The definition includes blood and other liquid samples of biological origin, solid tissue samples, such as biopsy specimens, or tissue cultures or cells derived therefrom, and their progeny. The definition also includes samples that have been manipulated in any way after their procurement, for example, by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses clinical samples, including cells in culture, cell supernatants, cell lysates, serum, plasma, body fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions such as plasma and serum, and the like. The term "biological sample" also includes solid tissue samples, tissue culture samples, and cell samples.

[0691] Optionally, the biological sample is selected from the group consisting of blood, serum, plasma, urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, a solid tissue sample, a tissue culture sample, and a cell sample.

[0692] Optionally, the biological sample is a blood or serum sample.

[0693] Suitable methods for determining whether a polypeptide of the present invention binds to antibodies produced by a subject are well known to those of skill in the art, including, for example, ELISA, luminex, legendplex.

[0694] The diagnostic methods of the present invention can be used to determine the stage (severity) of a coronavirus infection. The diagnostic methods of the present invention can be used to monitor the progression of a coronavirus infection in a subject. The diagnostic methods of the present invention can be used to determine a subject's response to a therapeutic regimen for treating a coronavirus infection.

[0695] The diagnostic methods of the present invention generally involve (a) determining the amount of an antibody (or antibodies) that binds to a polypeptide of the present invention in a biological sample obtained from a subject, and (b) comparing the amount of the antibody (or antibodies) in the biological sample with a reference, standard, or normal control value indicating the amount of the antibody (or antibodies) in a normal control subject. A significant difference between the amount of the antibody (or antibodies) in the biological sample and the normal control value indicates that the individual has a coronavirus infection. In some embodiments, the determining step involves contacting the biological sample with a polypeptide of the present invention and quantifying binding of the polypeptide to the antibody (or antibodies) present in the sample.

[0696] Various aspects of the present invention are defined in the following numbered paragraphs. 1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 17. 2. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:15, or an amino acid sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:15. 3. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:13, or an amino acid sequence having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:13. 4. The isolated polypeptide of any preceding paragraph, comprising at least one of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 17, as shown in the table below. [Table 13-1] [Table 13-2] 5. The isolated polypeptide of any preceding clause, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 17, as set out in the table below. [Table 14-1] [Table 14-2] 6. The isolated polypeptide of paragraph 5, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 17, as shown in the table below. [Table 15-1] [Table 15-2] 7. The isolated polypeptide of paragraph 5, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 17, as shown in the table below. [Table 16-1] [Table 16-2] 8. The polypeptide of any preceding clause, comprising the amino acid sequence of SEQ ID NO: 17. 9. An isolated polypeptide comprising a coronavirus S protein RBD domain having any or all of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 17] 10. The isolated polypeptide of paragraph 9, comprising at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table. 11. An isolated polypeptide comprising a coronavirus S protein RBD domain having any or all of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 18-1] [Table 18-2] 12. An isolated polypeptide comprising a coronavirus S protein RBD domain having any or all of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 19-1] [Table 19-2] 13. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 27 (COV_S_T2_13), or an amino acid sequence having at least 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 27. 14. The polypeptide of paragraph 13, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 20] 15. The polypeptide of paragraph 13 or 14, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 21] 16. The isolated polypeptide according to any one of items 13 to 15, comprising the amino acid sequence of SEQ ID NO: 27 (COV_S_T2_13). 17. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 28 (COV_S_T2_14), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 28. 18. The polypeptide of paragraph 17, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 22] 19. The polypeptide of paragraph 17 or 18, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 23] 20. The polypeptide according to any one of items 17 to 19, comprising at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 24] 21. The polypeptide according to any one of items 17 to 20, comprising the amino acid sequence of SEQ ID NO: 28 (COV_S_T2_14). 22. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 29 (COV_S_T2_15), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 29. 23. The polypeptide of paragraph 22, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 25] 24. The polypeptide of paragraph 22 or 23, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 26] 25. The polypeptide of any one of items 22 to 24, comprising at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 27] 26. The isolated polypeptide of any one of items 22 to 25, comprising the amino acid sequence of SEQ ID NO: 29 (COV_S_T2_15). 27. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 30 (COV_S_T2_16), or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 30. 28. The polypeptide of paragraph 27, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 28] 29. The isolated polypeptide of paragraph 27 or 28, comprising the amino acid sequence of SEQ ID NO: 30 (COV_S_T2_16). 30. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 31. 31. The polypeptide of paragraph 30, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 29] 32. The polypeptide of paragraph 30 or 31, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 30] 33. The polypeptide of any of items 30 to 32, comprising at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 31] 34. The polypeptide of any of items 30 to 33, comprising at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 32] 35. The isolated polypeptide of any one of items 30 to 34, comprising the amino acid sequence of SEQ ID NO: 31 (COV_S_T2_17). 36. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18), or an amino acid sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 32. 37. The polypeptide of paragraph 36, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 33] 38. The polypeptide of paragraph 36 or 37, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions in SEQ ID NO: 11, as shown in the table below. [Table 34] 39. The polypeptide of any of items 36 to 38, comprising at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 35] 40. The polypeptide of any of items 36 to 39, comprising at least one or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 36] 41. The isolated polypeptide of any of items 36 to 40, comprising the amino acid sequence of SEQ ID NO: 32 (COV_S_T2_18). 42. An isolated polypeptide comprising a coronavirus S protein RBD domain having at least 1, at least 5, at least 10, at least 15, or all of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11, as shown in the table below. [Table 37] 43. The isolated polypeptide of paragraph 42, further comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 38] 44. The isolated polypeptide of paragraph 42 or 43, further comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 39] 45. The isolated polypeptide of any of paragraphs 42 to 44, further comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 40] 46. ​​The isolated polypeptide of any of paragraphs 42 to 45, further comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 11, as shown in the table below. [Table 41] 47. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 33. 48. An isolated polypeptide comprising the amino acid sequence of SARS2 RBD having a glycosylation site located within the last 10 amino acids of the SARS2 RBD sequence, preferably at residue position 203. 49. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 34. 50. The polypeptide of paragraph 49, comprising at least one or all of the following amino acid residues at positions corresponding to amino acid residue positions in SEQ ID NO:11: 13Q, 25Q, 54T. 51. An isolated polypeptide comprising a coronavirus S protein RBD domain having at least one of the following amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:11: 13Q, 25Q, 54T, 203N. 52. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 35 (M9), or an amino acid sequence having at least 70% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 35. 53. The polypeptide of paragraph 52, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 42-1] [Table 42-2] 54. The polypeptide of paragraph 52 or 53.54, comprising at least one of the following amino acid residues, 54T, 203N, or both, at positions corresponding to amino acid residue positions in SEQ ID NO:11. 55. The polypeptide according to any one of items 52 to 54, comprising the amino acid sequence of SEQ ID NO: 35 (M9). 56. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 36 (M10), or an amino acid sequence having at least 69% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 36. 57. The polypeptide of paragraph 56, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 43-1] [Table 43-2] 58. The polypeptide of paragraph 56 or 578, comprising at least one or all of the following amino acid residues at positions corresponding to amino acid residue positions in SEQ ID NO:11: 13Q, 25Q, 54T. 59. The polypeptide according to any one of items 56 to 58, comprising the amino acid sequence of SEQ ID NO: 36 (M10). 60. The polypeptide of any preceding clause, comprising at least one glycosylation site within the amino acid sequence of the receptor binding domain (RBD). 61. The polypeptide of any preceding clause, comprising a glycosylation site located within the last 10 amino acids of the amino acid sequence of the RBD, preferably at a residue position corresponding to residue position 203 of the RBD sequence. 62. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:22, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:22. 63. The isolated polypeptide of paragraph 62, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 22, as shown in the table below. [Table 44] 64. The isolated polypeptide of paragraph 63, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 22, as shown in the table below. [Table 45] 65. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 23. 66. The isolated polypeptide of paragraph 65, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 23, as shown in the table below. [Table 46] 67. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 42 (COV_E_T2_3), or an amino acid sequence having at least 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 42. 68. The polypeptide of paragraph 67, comprising an amino acid residue A at a position corresponding to amino acid residue position 15 of SEQ ID NO:41. 69. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 43 (COV_E_T2_4), or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 43. 70. The polypeptide of paragraph 69, comprising at least one or all of the following amino acid residues: 15A, 55T, 69Q, 70G at positions corresponding to amino acid residue positions in SEQ ID NO:41. 71. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 44 (COV_E_T2_5), or an amino acid sequence having at least 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 44. 72. The polypeptide of paragraph 71, comprising at least one or all of the following amino acid residues: 15A, 55T at positions corresponding to amino acid residue positions in SEQ ID NO:41. 73. An isolated polypeptide comprising a coronavirus E protein having at least one of the following amino acid residues: 15A, 55T, 69Q, 70G at positions corresponding to the amino acid residue positions of SEQ ID NO:41. 74. The isolated polypeptide of paragraph 73, comprising at least one or all of the following amino acid residues at positions corresponding to amino acid residue positions in SEQ ID NO:41: 15A, 55T. 75. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:24. 76. The isolated polypeptide of paragraph 75, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 26, as shown in the table below. [Table 47] 77. The isolated polypeptide of paragraph 75, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 26, as shown in the table below. [Table 48] 78. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:25. 79. The isolated polypeptide of paragraph 78, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 25, as shown in the table below. [Table 49] 80. The isolated polypeptide of paragraph 78, comprising amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 25, as shown in the table below. [Table 50] 81. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:48, or an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:48. 82. The polypeptide of paragraph 81, comprising a deletion of amino acid residues at positions corresponding to positions 20 to 75 of SEQ ID NO:26. 83. The polypeptide of paragraph 81 or 82, comprising an amino acid residue G at a position corresponding to amino acid residue position 204 of SEQ ID NO:26. 84. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:49, or an amino acid sequence having at least 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:49. 85. The polypeptide of paragraph 84, comprising a deletion of amino acid residues at positions corresponding to positions 20 to 75 of SEQ ID NO:26. 86. The polypeptide of paragraph 84 or 85, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions in SEQ ID NO: 26, as shown in the table below. [Table 51] 87. The polypeptide of paragraph 86, comprising at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table. 88. The polypeptide of paragraph 84 or 85, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions in SEQ ID NO: 26, as shown in the table below. [Table 52] 89. The polypeptide of paragraph 88, comprising at least 5, at least 10, or at least 15 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table. 90. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:50, or an amino acid sequence having at least 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO:50. 91. The polypeptide of paragraph 90, comprising a deletion of amino acid residues at positions corresponding to positions 20 to 75 of SEQ ID NO:26. 92. The polypeptide of paragraph 90 or 91, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions in SEQ ID NO: 26, as shown in the table below. [Table 53] 93. The polypeptide of paragraph 92, comprising at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table. 94. The polypeptide of paragraph 90 or 91, comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions in SEQ ID NO: 26, as shown in the table below. [Table 54] 95. The polypeptide of paragraph 94, comprising at least 5 or at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 26, as shown in the table. 96. An isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 55] 97. The polypeptide of paragraph 96, comprising at least five of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table. 98. An isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 56] 99. The polypeptide of paragraph 98, comprising at least 5, at least 10, or at least 15 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 26, as shown in the table. 100. An isolated polypeptide comprising a coronavirus M protein having any or all of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table below. [Table 57] 101. The polypeptide of paragraph 100, comprising at least 5 or at least 10 of the amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO:26, as shown in the table. 102. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 46 (COV_N_T2_1), or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity over its entire length to the amino acid sequence of SEQ ID NO: 46. 103. The polypeptide of paragraph 102, further comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue positions as set forth in Table 12.2 above. 104. The polypeptide of paragraph 103, comprising at least 5, at least 10, or at least 15 of the amino acid residues at positions corresponding to the amino acid residue positions as shown in the table. 105. The polypeptide of any of paragraphs 102 to 104, further comprising at least one, or all, of the amino acid residues at positions corresponding to the amino acid residue pos...

Claims

1. An isolated polypeptide comprising: a) the amino acid sequence of SEQ ID NO: 88 (COV_S_T2_29+Q498R+dER), or an amino acid sequence having at least 98% or 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 88 (COV_S_T2_29+Q498R+dER), and optionally including at least one or all of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.11 below. Table 80 or b) the amino acid sequence of SEQ ID NO: 87 (COV_S_T2_29+Q498R), or an amino acid sequence having at least 99% amino acid identity over its entire length with the amino acid sequence of SEQ ID NO: 87 (COV_S_T2_29+Q498R), and optionally including at least one or all of the amino acid residues or deletions at positions corresponding to the amino acid residue positions of SEQ ID NO: 52, as shown in Table 9.8 below. Table 82 1. An isolated polypeptide comprising:

2. 2. The polypeptide of claim 1, comprising an R amino acid residue at a position corresponding to amino acid residue position 498 of SEQ ID NO:

52.

3. 3. The polypeptide of claim 1, comprising a deletion of amino acid residues at positions corresponding to amino acid residue positions 1255 to 1273 of SEQ ID NO:

52.

4. 2. The polypeptide of claim 1, comprising an amino acid residue P at a position corresponding to amino acid residue position 986 of SEQ ID NO:52, and an amino acid residue P at a position corresponding to amino acid residue position 987 of SEQ ID NO:

52.

5. 10. An isolated nucleic acid molecule encoding the polypeptide of claim 1, or its complement.

6. or encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:87, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:90 or a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical over its entire length to the nucleotide sequence of SEQ ID NO:90; or 6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:88, and wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:91 or a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of SEQ ID NO:91 over its entire length.

7. A vector comprising the nucleic acid molecule of claim 5.

8. 8. The vector of claim 7, further comprising a promoter operably linked to said nucleic acid, preferably said promoter for expression of the polypeptide encoded by said nucleic acid in a mammalian cell.

9. 8. The vector of claim 7, which is a vaccine vector, preferably a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, a DNA vaccine vector, an mRNA vaccine vector, a pURVac vector, preferably the pURVac vector comprises a nucleic acid molecule encoding a polypeptide of claim 1, the vector comprising the nucleotide sequence of SEQ ID NO: 95, or a modified vaccinia virus Ankara (MVA) vector, preferably the MVA vector comprises a nucleic acid molecule encoding a polypeptide of claim 1, the vector comprising the nucleotide sequence of SEQ ID NO:

98.

10. An isolated cell comprising the vector of claim 7.

11. A fusion protein or pseudotyped virus comprising the polypeptide of claim 1.

12. A pharmaceutical composition comprising the polypeptide of claim 1, the nucleic acid molecule of claim 5, or the vector of claim 7, and a pharmaceutically acceptable carrier, excipient, or diluent, optionally further comprising an adjuvant for enhancing the immune response in a subject to the polypeptide or a polypeptide encoded by the nucleic acid of the composition.

13. A pharmaceutical composition comprising the nucleic acid of claim 5, the vector of claim 7, or the polypeptide of claim 1, the nucleic acid of claim 5, or the vector of claim 7, wherein the nucleic acid comprises one or more modified nucleosides, optionally wherein the one or more modified nucleosides comprise a 1-methylpseudouridine modification, and optionally wherein at least 80% of the uridines in the open reading frame are modified, and a pharmaceutically acceptable carrier, excipient, or diluent, optionally further comprising an adjuvant of the pharmaceutical composition for enhancing the immune response in a subject to the polypeptide or a polypeptide encoded by the nucleic acid.

14. 14. The nucleic acid, vector, or pharmaceutical composition of claim 13, wherein the nucleic acid comprises messenger RNA (mRNA).

15. A composition comprising the polypeptide of claim 1, the nucleic acid of claim 5, or the vector of claim 7, for use as a pharmaceutical, or a pharmaceutical composition comprising the polypeptide of claim 1, the nucleic acid of claim 5, or the vector of claim 7, together with a pharmaceutically acceptable carrier, excipient, or diluent, optionally further comprising an adjuvant for enhancing the immune response in a subject to the polypeptide or a polypeptide encoded by the nucleic acid of the pharmaceutical composition.

16. 10. A composition comprising the polypeptide of claim 1, the nucleic acid of claim 5, or the vector of claim 7, for use in the prevention, treatment, or amelioration of a coronavirus infection, wherein optionally the coronavirus is a beta-coronavirus, optionally the β-coronavirus is a type B beta-coronavirus, preferably the type B β-coronavirus is SARS-CoV or SARS-CoV-2, optionally the beta-coronavirus is a SARS-CoV-2 VOC, optionally the beta-coronavirus is a SARS-CoV-2 beta, gamma, delta, or omicron VOC; or a pharmaceutical composition comprising the polypeptide of claim 1, the nucleic acid of claim 5, or the vector of claim 7, and a pharmaceutically acceptable carrier, excipient, or diluent, optionally further comprising an adjuvant for enhancing an immune response in a subject to the polypeptide or the polypeptide encoded by the nucleic acid of the pharmaceutical composition.

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