Coronavirus vaccine that induces broad-spectrum immunity against variants

Abstract

Designed coronavirus polypeptide sequences and their use as vaccines against viruses of the coronavirus family are described. The designed sequences include the designed coronavirus spike (S) protein and its fragments, including CoV_S_T2_35 (SEQ ID NO: 1), CoV_S_T2_36 (SEQ ID NO: 2), and Omicron_Vaccine (SEQ ID NO: 3). Variants of CoV_S_T2_35 (Deome), including CoV_S_T3_1 (SEQ ID NO: 31) and CoV_S_T3_2 (SEQ ID NO: 32), and their fragments, are also described. Nucleic acid molecules encoding 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 polynucleotides, polypeptides, vectors, cells, fusion proteins, pharmaceutical compositions, complex 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 include zoonotic diseases such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Viruses in this family generally cause mild, self-regulated respiratory infections in immune individuals, but they can also cause severe, fatal illnesses characterized by fever, extreme fatigue, difficulty breathing, anoxia, and pneumonia. CoVs are transmitted through close contact via respiratory droplets from infected individuals, 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 positive-sense RNA genome, ranging in length from 25 to 31 kilobases (Siddell SG1995, The Coronaviridae), with the largest genomes so far being found in RNA viruses. The Coronaviridae family is subtyped into four genera—α, β, γ, and δ coronaviruses—based on phylogenetic clustering, and each genus is further subdivided into clusters depending on the virus strain. For example, within the β-CoV genus (group 2CoV), four lineages (a, b, c, and d) are commonly recognized. • Lineage A (Embecovirus subgenus) includes HCoV-OC43 and HCoV-HKU1 (various species). · Lineage B (Sarbecovirus) includes SARSr-CoV (including all strains such as SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1). · Lineage C (Merbecovirus) includes Tylonycteris bat coronavirus HKU4 (BtCoV-HKU4), Pipistrellus bat coronavirus HKU5 (BtCoV-HKU5), and MERS-CoV (various species). · Lineage D (Novelbetacoronavirus) includes Rousettus bat coronavirus HKU9 (BtCoV-HKU9).

[0004] The fifth subgenus, Hibecovirus, which is a type of bat CoV, is also recognized.

[0005] CoV virions are spherical and have characteristic club-shaped spike protrusions that emanate from the surface of the virion. The virion contains 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-CoV also contain a fifth structural protein, hemagglutinin-esterase (HE), which enhances cell entry via the S protein and virus spread through the mucosa via its acetyl esterase activity. The homotrimers of the S glycoprotein form distinctive spike structures on the surface of the virus. These trimers are class I fusion proteins that mediate virus binding to the host receptor 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) (the exact positioning of the RBD varies depending on the viral strain), and S2 forms the stem of the spike molecule.

[0006] Figure 1 shows the structure of the SARS S protein. 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 modelling analysis depicts the origin and infectivity of 2019-nCoV, a new coronavirus which caused a pneumonia outbreak in Wuhan, China. 2020). The figure shows the S domain, which includes the S1 and S2 domains involved in receptor binding and membrane fusion, respectively.

[0007] The full length of SARS-CoV-2 S is 1273 amino acids, consisting of a signal peptide located at the N-terminus (amino acids 1-13), an S1 subunit (residues 14-685), and an S2 subunit (residues 686-1273). The last two regions are involved in receptor binding and membrane fusion, respectively. SARS2 binds to the human angiotensin-converting enzyme 2 (ACE2) receptor for viral attachment and entry. The S1 subunit contains an N-terminal domain (residues 14-305) and a receptor-binding domain (RBD, residues 319-541). The S2 subunit consists of a fusion peptide (FP) (788-806 residues), heptapeptide repeat 1 (HR1) (912-984 residues), HR2 (1163-1213 residues), the TM domain (1213-1237 residues), and the cytoplasmic domain (1237-1273 residues). The S protein trimer visually forms a characteristic bulbous coronal halo surrounding the virus particle. Based on the structure of the coronavirus S protein monomer, the S1 and S2 subunits form the bulbous head and stalk regions. The structure of the SARS-CoV-2 trimer S protein has been determined at the atomic level by cryo-electron microscopy / X-ray crystallography, revealing the different conformations of the open and closed S RBD domain and their corresponding functions.

[0008] Because viral RNA polymerase lacks the proofreading ability of DNA polymerase, RNA viruses generally have a much higher mutation rate compared to DNA viruses (with the exception of CoV, which has ExoN, nsp14 as its proofreading system). This is one reason why viruses can be transmitted from their natural host reservoirs to other species and from human to human, and why it is difficult to create effective vaccines to prevent diseases caused by RNA viruses. In most cases, current vaccine candidates against RNA viruses are limited by the viral strain used as the vaccine insert, which is often selected based on the availability of wild-type strains rather than informed design. Technical challenges in developing vaccines for enveloped RNA viruses include: i) viral variability in wild-type isolate glycoproteins (GPs) limits the range of protection as a vaccine antigen; ii) the selection of vaccine antigens expressed by vaccine inserts is highly experimental, with immunogen selection being slow and trial-and-error; and iii) the time required to develop new vaccine candidates in the event of an evolved or unexpected viral outbreak, which can delay vaccine development.

[0009] Prior to 2002, CoVs were thought to cause only mild respiratory problems and were endemic in human populations, causing 15-30% of respiratory infections annually. Since their initial discovery in the 1960s, the CoV family has expanded on a large scale, causing numerous pandemics in both humans and animals. Towards the end of 2019, a new CoV, SARS-CoV-2, group 2b β-CoV, emerged. The pandemic began in Wuhan, China, in the latter half of 2019. As the virus spread to more than 25 countries within a month of its emergence, the WHO declared a global public health emergency on January 30, 2020. The number of SARS-CoV cases increased exponentially in many countries around the world. Efforts were made to stop the spread of the virus, which led to a decrease in the number of infection cases and deaths caused by the virus. However, multiple waves of the disease have occurred in many countries, resulting in global figures of over 555 million confirmed cases and over 6 million confirmed deaths (according to the WHO, as of July 14, 2022).

[0010] Since the first human infection with SARS-CoV-2 was described in December 2019, more than 37 vaccines have been approved for human use, and many more are under 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 adenovirus vector. Two of the vaccines currently in use worldwide, BNT162b2 (a BioNTech vaccine manufactured by Pfizer) and mRNA-1273 (manufactured by Moderna), are based on delivering mRNA encoding a pre-fusion-stabilized form of the S protein derived from SARS-CoV-2, isolated in the early stages of the outbreak in Wuhan, China, via lipid nanoparticles. All of these vaccines demonstrated efficacy exceeding 94% in preventing COVID-19 in Phase III clinical trials conducted in multiple countries in the latter half of 2020 (Polack et al., C4591001 Clinical Trial Group (2020). Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N.Engl.J.Med. 383, 2603-2615; Baden et al., COVE Study Group (2021). Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N.Engl.J.Med. 384, 403-416).However, the emergence of newly circulating variants has raised serious concerns about the effectiveness of current vaccines, particularly in countries where the outbreak 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 rapidly become globally dominant after its emergence was D614G. In the UK, it was known as B.1.1.7(VOC-202012 / 01 or 501Y.V1, or alpha A new strain called B.1.1.7 (also known as B.1.1.7) rapidly emerged. B.1.1.7 contains D614G and N501Y in the ACE2 receptor-binding domain (RBD), three amino acid deletions and seven missense mutations in the spike, and has been reported to be more infectious than the variant with only the D614G mutation. Regarding the transmission of SARS-CoV-2 between humans and Danish mink, a variant called mink cluster 5 or B.1.1.298 has also been reported, which contains two amino acid deletions in the RBD and four missense mutations, including Y453F. Another variant that emerged in California is called B.1.429, which contains four missense mutations in the S protein, one of which is a single L452R RBD mutation. Novel variants arising from the B.1.1.28 strain, first described in Brazil and Japan, are P.2 (with three spike missense mutations) and P.1 ( gamma Also known as strain P.1, it contains the E484K mutation and has 12 spike missense mutations. P.1 also contains the K417T and N501Y mutations in the RBD. These strains spread rapidly but are no longer detected in the EU / EEA, or are detected at very low levels.

[0012] Past concerns stemmed from multiple strains of the B.1.351 lineage, which were first reported in South Africa and have since spread globally. betaThis was the emergence of the strain (also known as 501Y.V2). This line contains three RBD mutations, K417N, E484K, and N501Y, in addition to several mutations outside of RBD. Subsequently, B.1.617.2( delta Mutants have emerged and are becoming more transmissible. This mutant, 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) mutant has emerged, which contains 30 mutations in the S protein, 15 of which are in the RBD, and has been shown to cause significant humoral immune evasion and high transmissibility. Since then, many sub-mutants of Omicron have emerged, including BA.2, BA.3, BA.4, and BA.5. Some of these sub-mutants also include a sub-mutant containing BA.2.12.1. BA.5 mutates to BQ.1 and BQ.1.1, and BA.2 mutates to XBB1.5, which can bind more strongly to ACE-2 than its predecessor. Figure 2 provides details of the variants of interest (VOIs) circulating globally as of April 3, 2023 (https: / / www.ecdc.europa.eu / en / covid-19 / variants-concerns). At this point, there are no variants of concern for epidemic risk, as many omicron subvariants have been reduced by the European Centre for Disease Prevention and Control (ECDC).

[0013] BA.1, BA.2, and BA.3 share 12 RBD mutations, namely G339D, S373P, S375F, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y505H. The BA.2 sublineage includes four additional mutations compared to BA.1, including S371F, T376A, D405N, and R408S, and lacks S371L, G446S, and G496S, which are present in BA.1. BA.3 has distinct mutations S371F, D405N, and G446S. Importantly, new omicron variants continue to emerge. BA.2 variants contain the same RBD sequence as BA.2 but have further substitutions at L452 and F486, namely BA.2.12.1 (L452Q), BA.2.13 (L452M), BA.4, and BA.5 (L452R+F486V), all of which exhibit a higher transmission advantage than BA.2, BA.4, and BA.5. The study suggests that antibodies induced by a three-dose vaccination (CoronaVac) are less effective than those of the original Omicron strain in neutralizing BA.4 and BA.5. Furthermore, subjects with hybrid immunity from vaccination and previous infection with BA.1 produce antibodies that attempt to neutralize BA.4 and BA.5 (Cao, Y et al. Nature (2022). DOI: https: / / www.nature.com / articles / s41586-022-04980-y). In fact, infection with BA.1 induces a relatively narrow neutralizing antibody response, which appears to leave subjects vulnerable to BA.4 and BA.5 infections. This is thought to be due to the L452R and F486V S protein mutations in the BA.4 and BA.5 variants. Therefore, it is clear that the population is at risk of infection by newly emerging SARS2 variants and subvariants that SARS2 cannot be effectively neutralized.

[0014] The emergence of novel variants that appear to evade the immune response has prompted vaccine manufacturers to develop boosters for these spike variants. However, the continued emergence of such VOCs during the ongoing COVID-19 pandemic, and the constant threat of new zoonotic transmission of coronaviruses from animals to humans, highlight the need for next-generation vaccines with broader and more robust protection against ACE-2-conjugated salvecovirus. In particular, there is a need to protect against more coronavirus variants and subvariants than current vaccines, and to provide stronger protection against those variants and subvariants. Specifically, there is a need to provide improved vaccines that induce a broader neutralizing immune response against the omicron virus of coronaviruses and the omicron virus of neonatal coronaviruses. There is also a need for vaccines with stronger neutralizing capabilities against such viruses. Furthermore, there is a need for vaccines that effectively counter vaccine evasion by new SARS-CoV-2 variants.

[0015] Therefore, there is a need to provide an improved coronavirus vaccine that induces antibodies that broadly neutralize SARS-CoV-2 variants, particularly those of current and recent concern. delta There is a need to provide an effective vaccine that induces a broad-spectrum neutralizing immune response to protect against the strain and several Omicron strains.

[0016] Furthermore, there is a need to provide a vaccine that effectively counters vaccine evasion by new SARS-CoV-2 variants.

[0017] Designed SARS-CoV-2 spike (S) protein sequence The SARS-CoV-2 S protein plays a crucial role in ACE-2 receptor recognition and the fusion of the viral envelope with the host cell membrane. A comparison of 303,250 human SARS-CoV-2 spike protein sequences with the reference protein sequence Wuhan-Hu-1 showed that approximately 96.5% of spike protein sequences had mutated by February 2022, since the outbreak of the COVID-19 pandemic disease was first reported in December 2019 (Guruprasad. Current Research in Structural Biology. Vol 4, 2022; 41-50). A total of 1,269,629 mutations were detected, corresponding to 1,229 different mutation sites within the spike protein, which contains 1,273 amino acid residues. Therefore, approximately 3.5% of human SARS-CoV-2 spike protein sequences remained unchanged over the past two years. This figure is expected to decrease further since the document disclosing the comparison was published at the beginning of 2022. The S protein is the major antigenic component of the structural protein of SARS-CoV-2. nAbs, which are involved in inducing the host immune response, target the S protein and can induce protective immunity against viral infection.

[0018] As mentioned above, while vaccines against the SARS2 S protein exist, these vaccines cannot induce a broad-spectrum neutralizing immune response to previous SARS2 variants, as well as to recently emerged SARS2 variants, including Omicron BA.4 and BA.5. Therefore, there is a need to provide an improved vaccine that induces a broader neutralizing immune response against the SARS2 virus, particularly the recently emerged SARS2 variants and newly emerging SARS2 variants. [Prior art documents] [Non-patent literature]

[0019] [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-Patent Document 2] 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 [Non-Patent 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 [Non-Patent Document 4] 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 [Non-Patent Document 5] Garcia-Beltran et al.,2021,Cell 184,2372-2383:Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity [Non-Patent Document 6] Cao,Y et al.Nature(2022).DOI:https: / / www.nature.com / articles / s41586-022-04980-y [Non-Patent Document 7] Guruprasad.Current Research in Structural Biology.Vol 4,2022;41-50 [Overview of the project]

[0020] The applicant has recently appeared delta We designed amino acid sequences that induce a broad-spectrum neutralizing immune response against key SARS2 strains, including substrains of Omicron. These designs are referred to herein as CoV_S_T2_35 and CoV_S_T2_36. Such polypeptides and the nucleic acid sequences that encode them were used in Wuhan, alpha , beta , gamma , delta It is particularly advantageous because it induces broad-spectrum neutralizing antibody responses to a panel of coronaviruses, including Omicron BA.1, BA.2, BA.2.12, and BA.4 / 5 pseudoviruses. The polypeptide and the encoding nucleic acid sequence also induce broad-spectrum neutralizing antibody responses to Omicron BA.2.75, BA.2.75.2, BA.2.3.20, BQ.1.1, XBB, and XBB.1.5 pseudoviruses. In particular, CoV_S_T2_25 induces broad-spectrum neutralizing antibody responses to Omicron BA.2.75, BA.2.75.2, BA.2.3.20, BQ.1.1, XBB, and XBB.1.5 pseudoviruses. CoV_S_T2_26 induces broad-spectrum neutralizing antibody responses to Omicron BA.2.75 and BA.2.3.20 pseudoviruses.

[0021] The applicant has also identified modifications to the amino acid sequence for better expression of the polypeptide of the present invention. The applicant has further identified amino acid residues involved in increasing the stability of the polypeptide of the present invention. [Modes for carrying out the invention]

[0022] Based on the Omicron BA.1 consensus sequence, the applicant designed a further polypeptide sequence, Omicron_Vaccine, which includes amino acid modifications that the applicant identified as important for the stability and expression of the vaccine polypeptide.

[0023] The amino acid sequence of the S protein according to the present invention is described below.

[0024] CoV_S_T2_35(Deom)(Sequence ID 1) According to the present invention, an isolated polypeptide is provided that contains the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an amino acid having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length.

[0025] >CoV_S_T2_35(Deom)(Sequence ID 1) Amino acid sequence: MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGK QGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL ALHRSYLTPGDSSSGWTAGAAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCT LKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCV ADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYN YKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNG VAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFN FNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGT NTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYEC DIPIGAGICASYQTHTNSRGSASSVASQSIIAYTMSLGAENSVAYNNNSIAIPTNFTISV TTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVF AQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFVKQYGDCLG DIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQM AYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTL VKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASA NLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAIC HDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQ PELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE LGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCCSCLKGCCSCGSCC

[0026] Figure 3a shows the amino acid sequence alignment of CoV_S_T2_35(deome)(SEQ ID NO: 1) with the reference sequences of coronavirus S protein S sequences 4-13 and the previously designed vaccine sequences 14-29. CoV_S_T2_35 is a coronavirus delta It is phylogenetically most similar to the mutant, and therefore, delta C is shown as the reference sequence for comparison. The figure shows the alignment of the aforementioned sequences at the position where CoV_S_T2_35 contains the novel amino acid residue, as will be discussed below. Sequence IDs 5-13 are the consensus sequences of the wild-type (WT) strain of coronavirus. CoV_S_T2_35 (deome) (Sequence ID 1) and delta Table 1 below shows the amino acid differences from the CS protein reference sequence (SEQ ID NO: 8).

[0027] [Table 1-1]

[0028] Amino acid residues involved in stabilizing polypeptide design are shown in bold. Among these residues, novel amino acid residues are shown in bold and underlined. Deletions of amino acids corresponding to residues 1253-1271 of Sequence ID No. 8 are indicated as "deletion". Deletions of these residues promote the expression of the design. The residue positions shown in the table are: delta This corresponds to the residue positions in both the C and CoV_S_T2_35 sequences.

[0029] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes at least one amino acid residue of SEQ ID NO: 1 at a position corresponding to the amino acid residue position of SEQ ID NO: 8, as shown in Table 1.

[0030] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes at least five amino acid residues of SEQ ID NO: 1 at positions corresponding to the amino acid residue positions of SEQ ID NO: 8, as shown in Table 1.

[0031] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes at least 10 amino acid residues of SEQ ID NO: 1 at positions corresponding to the amino acid residue positions of SEQ ID NO: 8, as shown in Table 1.

[0032] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes at least 15 amino acid residues of SEQ ID NO: 1 at positions corresponding to the amino acid residue positions of SEQ ID NO: 4, as shown in Table 1.

[0033] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes at least 20 amino acid residues of SEQ ID NO: 1 at positions corresponding to the amino acid residue positions of SEQ ID NO: 8, as shown in Table 1.

[0034] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes an amino acid residue of SEQ ID NO: 1 at a position corresponding to the amino acid residue position of SEQ ID NO: 8, as shown in Table 1.

[0035] CoV_S_T2_35(deom)(sequence number 1) and new delta Table 3 below shows the amino acid differences from the CS protein reference sequence (SEQ ID NO: 8). The residue positions shown in the table below correspond to: delta This corresponds to the residue positions of both C and CoV_S_T2_35.

[0036] [Table 3-1]

[0037] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes at least one amino acid residue of SEQ ID NO: 1 at a position corresponding to the amino acid residue position of SEQ ID NO: 8, as shown in Table 3.

[0038] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes an amino acid residue of SEQ ID NO: 1 at a position corresponding to the amino acid residue position of SEQ ID NO: 8, as shown in Table 3.

[0039] The applicant has recognized that polypeptides of the present invention, including the above-described residue changes, have better stability than polypeptides containing wild-type residues at these positions.

[0040] CoV_S_T2_35(deom)(sequence number 1) and present in at least one reference sequence or previously designed (DIOS) sequence, delta Table 2 below shows the amino acid differences from the CS protein reference sequence (SEQ ID NO: 8). Generally, these amino acid differences are wild-type. These mutations have been found in coronavirus sequences, and their incorporation into vaccine design ensures that variants, including those involving amino acid changes, are captured by the vaccine design.

[0041] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or amino acids having at least 97%, 98%, or 99% amino acid identity across the entire length of the amino acid sequence of SEQ ID NO: 1, as shown in Table 2. Street The amino acid residues of sequence number 1 , at the position corresponding to the amino acid residue position of SEQ ID NO: 8 include.

[0042] [Table 2-1]

[0043] The applicant states that such polypeptides are used in Wuhan, alpha , beta , gamma , delta , and one or more We found that the polypeptides induce broad-spectrum neutralizing immune responses to a panel of SARS-CoV-2 pseudoviruses, including Omicron substrains BA.1, BA.2, BA.2.12.1, and BA.4 / 5. The polypeptides also induce broad-spectrum neutralizing antibody responses to Omicron BA.2.75, BA.2.75.2, BA.2.3.20, BA.2.12, BQ.1.1, XBB, and XBB.1.5 pseudoviruses. In particular, CoV_S_T2_25 induces broad-spectrum neutralizing antibody responses to Omicron BA.2.75, BA.2.75.2, BA.2.3.20, BQ.1.1, XBB, and XBB.1.5 pseudoviruses. CoV_S_T2_26 induces broad-spectrum neutralizing antibody responses to Omicron BA.2.75 and BA.2.3.20 pseudoviruses.

[0044] BA.4 and BA.5 were the dominant SARS-CoV-2 strains prevalent in many countries around the world recently (as of April 2023).

[0045] Optionally, the polypeptide of the present invention comprises amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35) or the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes the following amino acid residues at positions corresponding to residues 984 and 985 of SEQ ID NO: 8. ·984:P, and 985:P.

[0046] Advantageously, the proline residues at positions 984 and 985 increase the stability of the vaccine design.

[0047] Optionally, the polypeptide of the present invention comprises amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35) or the amino acid sequence of SEQ ID NO: 1 over its entire length, and includes the following amino acid residues at positions corresponding to residues 680, 681, 683, 984, and 985 of SEQ ID NO: 8. ·680:G, ·681:S, ·683:S, ·984:P, and 985:P.

[0048] The applicant has recognized that polypeptides of the present invention, including the above-described residue changes, have better stability than polypeptides containing wild-type residues at these positions.

[0049] Optionally, the polypeptide of the present invention contains amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35) over its entire length, and does not contain the residue KFDEDDSEPVLKGVKLHYT at the positions corresponding to amino acid residue positions 1253-1271 of SEQ ID NO: 8.

[0050] The applicant recognized that the deletion of 19 amino acid residues from the C-terminus of the SARS-CoV-2 S protein at positions corresponding to residue positions 1253-1271 of Sequence ID No. 8 (endoplasmic reticulum (ER) signal sequence) improves the expression of the designed amino acid sequence.

[0051] According to the present invention, an isolated polypeptide containing the CoV_S_T2_35(deome) amino acid sequence (SEQ ID NO: 1) is provided.

[0052] According to the present invention, as shown in Table 3 below Of the amino acid residues At least one or all at the position corresponding to the amino acid residue position of SEQ ID NO: 8 Isolated polypeptides containing the coronavirus S protein are also provided.

[0053] [Table 3-2]

[0054] Optionally, an isolated polypeptide containing coronavirus S protein is provided, as shown in Table 3, which includes 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 across the entire length of the amino acid sequence of SEQ ID NO: 8, and which includes at least one or all amino acid residues at positions corresponding to the amino acid residue positions of SEQ ID NO: 8.

[0055] Optionally, the isolated polypeptide containing the coronavirus S protein according to the present invention has the following amino acid residues at the positions corresponding to residues 984 and 985 of SEQ ID NO: 8 : ·984:P, and ·985:P including .

[0056] Optionally, the isolated polypeptide containing the coronavirus S protein according to the present invention may have the following amino acid residues at the positions corresponding to residues 680, 681, 683, 984, and 985 of SEQ ID NO: 8 : ·680:G, ·681:S, ·683:S, ·984:P, and ·985:P including .

[0057] The following options are optional and can be selected from Table 2 below. Street The amino acid residues of sequence number 1 , at the position corresponding to the amino acid residue position of SEQ ID NO: 8 An isolated polypeptide containing the coronavirus S protein is provided.

[0058] [Table 2-2]

[0059] The following options are optional and can be selected from Table 1 below. Street The amino acid residues of sequence number 1 , at the position corresponding to the amino acid residue position of SEQ ID NO: 8 An isolated polypeptide containing the coronavirus S protein is provided.

[0060] [Table 1-2]

[0061] Optionally, the coronavirus S protein according to the present invention does not contain the residue KFDEDDSEPVLKGVKLHYT at the positions corresponding to amino acid residue positions 1253-1271 of SEQ ID NO: 8.

[0062] CoV_S_T2_35 scaffold sequence The inventors also designed a scaffold S protein polypeptide sequence (SEQ ID NO: 30) based on CoV_S_T2_35(deome) (SEQ ID NO: 1), which includes a constant region and variable amino acid residues of the sequence. The variable amino acid residues can be modified to provide different antigens that induce a neutralizing immune response to new SARS-CoV-2 variants (and / or future SARS-CoV-2 variants) as new variants arise.

[0063] Sequence ID 30 below shows the scaffold S protein sequence, which provides the amino acid sequence of the constant region of the scaffold, with each variable amino acid residue represented by X (indicated by an underline in the following sequence).

[0064] [ka]

[0065] In this sequence, X can be any amino acid residue.

[0066] CoV_S_T2_35(deome)(SEQ ID NO: 1) is an example of a polypeptide sequence covered by a scaffold sequence. Other examples of polypeptide sequences covered by scaffold sequences are shown below.

[0067] COV_S_T3_1(Deom_v2)(Sequence ID 31): MFVFLVLLPL VSSQCVNLRT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS 60 NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV 120 NNATNVVIKV CEFQFCNDPF LDVYYHKNNK SWMESGVYSS ANNCTFEYVS QPFLMDLEGK 180 QGNFKNLREF VFKNIDGYFK IYSKHTPINL VRDLPQGFSA LEPLVDLPIG INITRFQTLL 240 ALHRSYLTPG DSSSGWTAGA AAYYVGYLQP RTFLLKYNEN GTITDAVDCA LDPLSETKCT 300 LKSFTVEKGI YQTSNFRVQP TESIVRFPNI TNLCPFDEVF NATRFASVYA WNRKRISNCV 360 ADYSVLYNFA PFFAFKCYGV SPTKLNDLCF TNVYADSFVI RGNEVSQIAP GQTGNIADYN 420 YKLPDDFTGC VIAWNSNKLD SKVGGNYNYR YRLFRKSKLK PFERDISTEI YQAGNKPCNG 480 VAGPNCYFPL QSYGFRPTYG VGHQPYRVVV LSFELLHAPA TVCGPKKSTN LVKNKCVNFN 540 FNGLKGTGVL TESNKKFLPF QQFGRDIADT TDAVRDPQTL EILDITPCSF GGVSVITPGT 600 NTSNQVAVLY QGVNCTEVPV AIHADQLTPT WRVYSTGSNV FQTRAGCLIG AEHVNNSYEC 660 DIPIGAGICA SYQTHTNSRG SASSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTISV 720 TTEILPVSMT KTSVDCTMYI CGDSTECSNL LLQYGSFCTQ LNRALTGIAV EQDKNTQEVF 780 AQVKQIYKTP PIKDFGGFNF SQILPDPSKP SKRSFIEDLL FNKVTLADAG FVKQYGDCLG 840 DIAARDLICA QKFNGLTVLP PLLTDEMIAQ YTSALLAGTI TSGWTFGAGA ALQIPFAMQM 900 AYRFNGIGVT QNVLYENQKL IANQFNSAIG KIQDSLSSTA SALGKLQNVV NQNAQALNTL 960 VKQLSSNFGA ISSVLNDILS RLDPPEAEVQ IDRLITGRLQ SLQTYVTQQL IRAAEIRASA 1020 NLAATKMSEC VLGQSKRVDF CGKGYHLMSF PQSAPHGVVF LHVTYVPAQE KNFTTAPAIC 1080 HDGKAHFPRE GVFVSNGTHW FVTQRNFYEP QIITTDNTFV SGNCDVVIGI VNNTVYDPLQ 1140 PELDSFKEEL DKYFKNHTSP DVDLGDISGI NASVVNIQKE IDRLNEVAKN LNESLIDLQE 1200 LGKYEQYIKW PWYIWLGFIA GLIAIVMVTI MLCCMTSCCS CLKGCCSCGS CC 1252

[0068] COV_S_T3_2(Deom_v3)(SEQ ID NO: 32): MFVFLVLLPL VSSQCVNLRT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS 60 NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV 120 NNATNVVIKV CEFQFCNDPF LDVYYHKNNK SWMESGVYSS ANNCTFEYVS QPFLMDLEGK 180 QGNFKNLREF VFKNIDGYFK IYSKHTPINL VRDLPQGFSA LEPLVDLPIG INITRFQTLL 240 ALHRSYLTPG DSSSGWTAGA AAYYVGYLQP RTFLLKYNEN GTITDAVDCA LDPLSETKCT 300 LKSFTVEKGI YQTSNFRVQP TESIVRFPNI TNLCPFHEVF NATTFASVYA WNRKRISNCV 360 ADYSVIYNFA PFFAFKCYGV SPTKLNDLCF TNVYADSFVI RGNEVSQIAP GQTGNIADYN 420 YKLPDDFTGC VIAWNSNKLD SKPSGNYNYL YRLFRKSKLK PFERDISTEI YQAGNKPCNG 480 VAGPNCYSPL QSYGFRPTYG VGHQPYRVVV LSFELLHAPA TVCGPKKSTN LVKNKCVNFN 540 FNGLKGTGVL TESNKKFLPF QQFGRDIADT TDAVRDPQTL EILDITPCSF GGVSVITPGT 600 NTSNQVAVLY QGVNCTEVPV AIHADQLTPT WRVYSTGSNV FQTRAGCLIG AEHVNNSYEC 660 DIPIGAGICA SYQTHTNSRG SASSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTISV 720 TTEILPVSMT KTSVDCTMYI CGDSTECSNL LLQYGSFCTQ LNRALTGIAV EQDKNTQEVF 780 AQVKQIYKTP PIKDFGGFNF SQILPDPSKP SKRSFIEDLL FNKVTLADAG FVKQYGDCLG 840 DIAARDLICA QKFNGLTVLP PLLTDEMIAQ YTSALLAGTI TSGWTFGAGA ALQIPFAMQM 900 AYRFNGIGVT QNVLYENQKL IANQFNSAIG KIQDSLSSTA SALGKLQNVV NQNAQALNTL 960 VKQLSSNFGA ISSVLNDILS RLDPPEAEVQ IDRLITGRLQ SLQTYVTQQL IRAAEIRASA 1020 NLAATKMSEC VLGQSKRVDF CGKGYHLMSF PQSAPHGVVF LHVTYVPAQE KNFTTAPAIC 1080 HDGKAHFPRE GVFVSNGTHW FVTQRNFYEP QIITTDNTFV SGNCDVVIGI VNNTVYDPLQ 1140 PELDSFKEEL DKYFKNHTSP DVDLGDISGI NASVVNIQKE IDRLNEVAKN LNESLIDLQE 1200 LGKYEQYIKW PWYIWLGFIA GLIAIVMVTI MLCCMTSCCS ​​CLKGCCSCGS CC 1252

[0069] Figure 16 shows the amino acid sequence alignment of CoV_S_T2_35(deome) (SEQ ID NO: 1), COV_S_T3_1(deome_v2) (SEQ ID NO: 31), and COV_S_T3_2(deome_v3) (SEQ ID NO: 32). Differences between sequences are indicated by residues enclosed in squares.

[0070] The following table lists the residues at variable positions in the amino acid sequences of CoV_S_T2_35(deome) (SEQ ID NO: 1), CoV_S_T3_1(deome_v2) (SEQ ID NO: 31), and CoV_S_T3_2(deome_v3) (SEQ ID NO: 32).

[0071] [Table 8]

[0072] According to the present invention, an isolated polypeptide is provided which contains the amino acid sequence of SEQ ID NO: 30 (CoV_S_T2_35 scaffold sequence), where X at amino acid residue positions 337, 344, 366, 371, 373, 374, 403, 406, 443, 444, 450, 458, 484, 488, 491, and 494 is any amino acid residue.

[0073] The RBD portion of Sequence ID No. 30 consists of residues 317-530 (shown below as Sequence ID No. 33). RVQPTESIVR FPNITNLCPF XEVFNATXFA SVYAWNRKRI SNCVADYSVX YNSAXFXXFK 60 CYGVSPTKLN DLCFTNVYAD SFVIRGXEVX QIAPGQTGNI ADYNYKLPDD FTGCVIAWNS 120 NKLDSKXXGN YNYXYRLFRK SXLKPFERDI STEIYQAGNK PCNGVAGXNC YXPLXSYXFR 180 PTYGVGHQPY RVVVLSFELL HAPATVCGPK KSTN 214

[0074] According to the present invention, an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 33 (the RBD portion of the CoV_S_T2_35 scaffold sequence (SEQ ID NO: 30)) is also provided, where X at the amino acid residue positions corresponding to amino acid residue positions 337, 344, 366, 371, 373, 374, 403, 406, 443, 444, 450, 458, 484, 488, 491, and 494 of SEQ ID NO: 30 is any amino acid residue.

[0075] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains amino acid residues G, D, or H at the amino acid residue position corresponding to position 337 of SEQ ID NO: 30.

[0076] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains an amino acid residue R or T at the amino acid residue position corresponding to position 344 of SEQ ID NO: 30.

[0077] Optionally, an isolated polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 30 or 33 includes amino acid residue L or I at the amino acid residue position corresponding to position 366 of SEQ ID NO: 30.

[0078] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains an amino acid residue with amino acid residue S or P at the amino acid residue position corresponding to position 371 of SEQ ID NO: 30.

[0079] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains amino acid residue S or F at the amino acid residue position corresponding to position 373 of SEQ ID NO: 30.

[0080] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains an amino acid residue T or A at the amino acid residue position corresponding to position 374 of SEQ ID NO: 30.

[0081] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains amino acid residue D or N at the amino acid residue position corresponding to position 403 of SEQ ID NO: 30.

[0082] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains an amino acid residue R or S at the amino acid residue position corresponding to position 406 of SEQ ID NO: 30.

[0083] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains amino acid residue V or P at the amino acid residue position corresponding to position 443 of SEQ ID NO: 30.

[0084] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains an amino acid residue S or G at the amino acid residue position corresponding to position 444 of SEQ ID NO: 30.

[0085] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains an amino acid residue L or R at the amino acid residue position corresponding to position 450 of SEQ ID NO: 30.

[0086] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains amino acid residue N or K at the amino acid residue position corresponding to position 458 of SEQ ID NO: 30.

[0087] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains amino acid residue F or P at the amino acid residue position corresponding to position 484 of SEQ ID NO: 30.

[0088] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains amino acid residue F or S at the amino acid residue position corresponding to position 488 of SEQ ID NO: 30.

[0089] Optionally, an isolated polypeptide of the present invention comprising the amino acid sequence of SEQ ID NO: 30 or 33 includes an amino acid residue R or Q at the amino acid residue position corresponding to position 491 of SEQ ID NO: 30.

[0090] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 or 33 contains amino acid residue S or G at the amino acid residue position corresponding to position 494 of SEQ ID NO: 30.

[0091] Optionally, the isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 also contains the amino acid sequence of SEQ ID NO: 31.

[0092] Optionally, the isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 30 also contains the amino acid sequence of SEQ ID NO: 32.

[0093] The RBD portion of sequence number 1 (CoV_S_T2_35(deom)) is shown below as sequence number 34. RVQPTESIVR FPNITNLCPF GEVFNATRFA SVYAWNRKRI SNCVADYSVL YNSASFSTFK 60 CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGNI ADYNYKLPDD FTGCVIAWNS 120 NKLDSKVSGN YNYLYRLFRK SNLKPFERDI STEIYQAGNK PCNGVAGFNC YFPLRSYSFR 180 PTYGVGHQPY RVVVLSFELL HAPATVCGPK KSTN 214

[0094] The RBD portion of sequence number 31 (COV_S_T3_1(Deom_v2)(Sequence Number 31)) is shown below as sequence number 35. RVQPTESIVR FPNITNLCPF DEVFNATRFA SVYAWNRKRI SNCVADYSVL YNFAPFFAFK 60 CYGVSPTKLN DLCFTNVYAD SFVIRGNEVS QIAPGQTGNI ADYNYKLPDD FTGCVIAWNS 120 NKLDSKVGGN YNYRYRLFRK SKLKPFERDI STEIYQAGNK PCNGVAGPNC YFPLQSYGFR 180 PTYGVGHQPY RVVVLSFELL HAPATVCGPK KSTN 214

[0095] The RBD portion of sequence number 32 (COV_S_T3_2(Deom_v3)(Sequence Number 32)) is shown below as sequence number 36. RVQPTESIVR FPNITNLCPF HEVFNATTFA SVYAWNRKRI SNCVADYSVI YNFAPFFAFK 60 CYGVSPTKLN DLCFTNVYAD SFVIRGNEVS QIAPGQTGNI ADYNYKLPDD FTGCVIAWNS 120 NKLDSKPSGN YNYLYRLFRK SKLKPFERDI STEIYQAGNK PCNGVAGPNC YSPLQSYGFR 180 PTYGVGHQPY RVVVLSFELL HAPATVCGPK KSTN 214

[0096] Optionally, the isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 33 also contains the amino acid sequence of SEQ ID NO: 34.

[0097] Optionally, the isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 33 also contains the amino acid sequence of SEQ ID NO: 35.

[0098] Optionally, an isolated polypeptide of the present invention containing the amino acid sequence of SEQ ID NO: 33 may also contain the amino acid sequence of SEQ ID NO: 36.

[0099] The RBD portion of SEQ ID NOs: 1, 30, 31, or 32 may be provided without further SARS-CoV-2 S protein sequence, or as part of a longer polypeptide having a SARS-CoV-2 S protein sequence, for example, as part of a full-length SARS-CoV-2 S protein. The S protein sequence may be a sequence derived from the S protein disclosed herein, or another S protein sequence. For example, an S protein containing the RBD of SEQ ID NOs: 33, 34, 35, or 36 may be provided over its entire length. delta The CS protein (SEQ ID NO: 8) may have an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with the amino acid sequence of the CS protein (SEQ ID NO: 8). Alternatively, the S protein containing RBD of SEQ ID NO: 33, 34, 35, or 36 may have an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with the amino acid sequence of the BA.1C S protein (SEQ ID NO: 9) throughout its entire length.

[0100] CoV_S_T2_36(Omid)(Sequence ID 2) According to the present invention, an isolated polypeptide is provided that contains the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or an amino acid having at least 97%, 98%, or 99% amino acid identity across the entire length of the amino acid sequence of SEQ ID NO: 2.

[0101] >CoV_S_T2_36(Omid)(Sequence ID 2) Amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNN ATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN FKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA DYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGV EGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN TSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECD IPIGAGICASYQTQTKSHGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMA YRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLV KQLSSKFGAISSVLNDIFSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC

[0102] Figure 3b shows the alignment of the amino acid sequence of CoV_S_T2_36(omido) (SEQ ID NO: 9) with the coronavirus S protein reference sequences SEQ ID NOs. 4-13 and the previously designed vaccine sequences SEQ ID NOs. 14-29. CoV_S_T2_36 is phylogenetically most similar to the Omicron BA.1 variant of coronavirus, and therefore BA.1 is shown as the comparative reference sequence. The figure shows the alignment of the aforementioned sequences at the position where CoV_S_T2_36 contains novel amino acid residues, as will be discussed below. SEQ ID NOs. 5-13 are the consensus sequences of the wild-type (WT) strain of coronavirus. Table 4 below shows the amino acid differences between CoV_S_T2_36(omido) (SEQ ID NO: 2) and the Omicron BA.1C S protein reference sequence (SEQ ID NO: 9).

[0103] [Table 4-1]

[0104] Amino acid residues involved in stabilizing polypeptide design are shown in bold. Of these residues, novel amino acid residues in CoV_S_T2_36 are shown in bold and underlined. Deletions of amino acids corresponding to residues 1252-1270 of Sequence ID No. 8 are indicated as "deletion". Deletions of these residues promote the expression of the designed polypeptide sequence. The residue positions shown in the table correspond to residue positions in both the BA.1C and CoV_S_T2_36 sequences.

[0105] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and includes at least one or all of the amino acid residues of SEQ ID NO: 2 at the positions corresponding to the amino acid residue positions of SEQ ID NO: 9, as shown in Table 4.

[0106] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and includes at least five amino acid residues of SEQ ID NO: 2 at positions corresponding to the amino acid residue positions of SEQ ID NO: 9, as shown in Table 4.

[0107] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and includes at least 10 amino acid residues of SEQ ID NO: 2 at positions corresponding to the amino acid residue positions of SEQ ID NO: 9, as shown in Table 4.

[0108] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and includes at least 15 amino acid residues of SEQ ID NO: 2 at positions corresponding to the amino acid residue positions of SEQ ID NO: 9, as shown in Table 4.

[0109] Optionally, the polypeptide of the present invention contains amino acids having at least 97%, 98%, or 99% amino acid identity across the entire length of the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), and contains at least 25 amino acid residues of SEQ ID NO: 2 at positions corresponding to the amino acid residue positions of SEQ ID NO: 9, as shown in Table 4.

[0110] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and includes at least 25 amino acid residues of SEQ ID NO: 2 at positions corresponding to the amino acid residue positions of SEQ ID NO: 9, as shown in Table 4.

[0111] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and includes an amino acid residue of SEQ ID NO: 2 at a position corresponding to the amino acid residue position of SEQ ID NO: 9, as shown in Table 4.

[0112] Table 6 below shows the amino acid differences between CoV_S_T2_36(omido) (SEQ ID NO: 2) and the novel Omicron BA.1C S protein reference sequence (SEQ ID NO: 9) used in the design of the CoV-S-T2_36(omido) vaccine. As explained above, these residue changes stabilize the designed sequence of the S protein.

[0113] [Table 6-1]

[0114] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity across the entire length of the amino acid sequence of SEQ ID NO: 2, and includes at least one amino acid residue of SEQ ID NO: 2 at a position corresponding to the amino acid residue position of SEQ ID NO: 9, as shown in Table 6.

[0115] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and includes an amino acid residue of SEQ ID NO: 2 at a position corresponding to the amino acid residue position of SEQ ID NO: 9, as shown in Table 6.

[0116] The applicant has recognized that polypeptides of the present invention, including the above-described residue changes, have better stability than polypeptides containing wild-type residues at these positions.

[0117] Table 5 below shows the amino acid differences between CoV_S_T2_36(omido) (SEQ ID NO: 2) and the omicron BA.1C S protein reference sequence (SEQ ID NO: 9), in which amino acid differences exist in at least one reference sequence or a previously designed (DIOS) sequence. Generally, these amino acid differences are found in wild-type coronavirus sequences, and their incorporation into vaccine design ensures that variants containing amino acid changes are captured by the vaccine design.

[0118] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and includes an amino acid residue of SEQ ID NO: 2 at a position corresponding to the amino acid residue position of SEQ ID NO: 9, as shown in Table 5.

[0119] [Table 5-1]

[0120] The applicant states that such polypeptides are used in Wuhan, alpha , beta , gamma , deltaWe found that the polypeptides induce broad-spectrum neutralizing immune responses to a diverse panel of SARS-CoV-2 pseudoviruses, including Omicron substrains BA.1, BA.2, BA.2.12.1, and BA.4 / 5. The polypeptides also induce broad-spectrum neutralizing antibody responses to Omicron BA.2.75, BA.2.75.2, BA.2.3.20, BQ.1.1, XBB, and XBB.1.5 pseudoviruses. In particular, CoV_S_T2_25 induces broad-spectrum neutralizing antibody responses to Omicron BA.2.75, BA.2.75.2, BA.2.3.20, BQ.1.1, XBB, and XBB.1.5 pseudoviruses. CoV_S_T2_26 induces broad-spectrum neutralizing antibody responses to Omicron BA.2.75 and BA.2.3.20 pseudoviruses. As described above, BA.4 and BA.5 are currently the dominant SARS-CoV-2 strains circulating in many countries around the world (as of April 2023).

[0121] Optionally, the polypeptide of the present invention comprises amino acids having at least 97%, 98%, or 99% amino acid identity across the entire length of the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), and the following amino acid residues at the positions corresponding to residues 983 and 984 of SEQ ID NO: 9 : ·983:P, and ·984:P including .

[0122] Advantageously, the proline residues at positions 983 and 984 increase the stability of the vaccine design.

[0123] Optionally, the polypeptide of the present invention comprises amino acids having at least 97%, 98%, or 99% amino acid identity across the entire length of the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), and the following amino acid residues at the positions corresponding to residues 679, 680, 682, 983, and 984 of SEQ ID NO: 9 : ·679:G, ·680:S, ·682:S, ·983:P, and ·984:P including .

[0124] The applicant has recognized that polypeptides of the present invention, including the above-described residue changes, have better stability than polypeptides containing wild-type residues at these positions.

[0125] Optionally, the polypeptide of the present invention contains amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36) over its entire length, and does not contain the residue KFDEDDSEPVLKGVKLHYT at the positions corresponding to amino acid residue positions 1252-1270 of SEQ ID NO: 9.

[0126] The applicant recognized that the deletion of 19 amino acid residues from the C-terminus of the SARS-CoV-2 S protein at positions corresponding to residue positions 1252-1270 of Sequence ID No. 9 (ER signal sequence) improves the expression of the designed polypeptide sequence.

[0127] According to the present invention, an isolated polypeptide containing the CoV_S_T2_36(omido) amino acid sequence (SEQ ID NO: 2) is provided.

[0128] According to the present invention, as shown in Table 6 below The amino acid residue or deletion as described above at least one Alternatively, place all of them in positions corresponding to the amino acid residue positions of SEQ ID NO: 9 An isolated polypeptide containing the coronavirus S protein is provided.

[0129] [Table 6-2]

[0130] Optionally, isolated polypeptides containing coronavirus S protein are provided, which include 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 across the entire length of SEQ ID NO: 9, as shown in Table 6. Among the amino acid residues or deletions at least one Alternatively, place all of them in positions corresponding to the amino acid residue positions of SEQ ID NO: 9 include.

[0131] Optionally, the isolated polypeptide according to the present invention containing coronavirus S protein has the following amino acid residues at the positions corresponding to residues 984 and 985 of SEQ ID NO: 9 : ·983:P, and ·984:P including .

[0132] Optionally, the isolated polypeptide according to the present invention containing coronavirus S protein has the following amino acid residues at positions corresponding to residues 680, 681, 683, 984, and 985 of SEQ ID NO: 9 : ·679:G, ·680:S, ·682:S, ·983:P, and ·984:P including .

[0133] Optionally, 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 across the entire length of the amino acid sequence of SEQ ID NO: 9, as shown in Table 5 below. Street The amino acid residues of SEQ ID NO: 2 At the position corresponding to the amino acid residue position of SEQ ID NO: 9 An isolated polypeptide containing the coronavirus S protein is provided.

[0134] [Table 5-2]

[0135] Optionally, 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 across the entire length of the amino acid sequence of SEQ ID NO: 9, as shown in Table 4 below. Street The amino acid residues of SEQ ID NO: 2 At the position corresponding to the amino acid residue position of SEQ ID NO: 9 An isolated polypeptide containing the coronavirus S protein is provided.

[0136] [Table 4-2]

[0137] According to the present invention, an isolated polypeptide is also provided that comprises a coronavirus S protein having 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 across the entire length of the amino acid sequence of SEQ ID NO: 9, and that does not contain the residue KFDEDDSEPVLKGVKLHYT at the position corresponding to amino acid residue positions 1252-1270 of SEQ ID NO: 9.

[0138] Omicron Vaccine (Sequence ID 3) According to the present invention, an isolated polypeptide is also provided which contains the amino acid sequence of SEQ ID NO: 3 (Omicron_Vaccine), or an amino acid having at least 99% amino acid identity over the entire length of the amino acid sequence of SEQ ID NO: 3.

[0139] >Omicron_Vaccine (SEQ ID NO: 3) Amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNN ATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN FKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVA DYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNY KLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGV AGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN TSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECD IPIGAGICASYQTQTKSHGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMA YRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLV KQLSSKFGAISSVLNDIFSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCSCGSCC

[0140] Omicron_Vaccine (SEQ ID NO: 3) is phylogenetically most similar to Omicron BA.1C among the reference sequences. Omicron_Vaccine differs from BA.1 by a double proline mutation at positions 983 and 984 of BA.1C (incorporated to assess the effect of such mutations on vaccine stability) and by a deletion of the ER signaling sequence to assess the effect on protein expression. The amino acid differences between Omicron_Vaccine (SEQ ID NO: 3) and the Omicron BA.1C S protein reference sequence (SEQ ID NO: 9) are shown in Table 7 below.

[0141] [Table 7-1]

[0142] Amino acid residues involved in stabilizing polypeptide design are shown in bold. Deletions of amino acids corresponding to residues 1252-1270 of SEQ ID NO: 9 are indicated as “Deletion”. Deletions of these residues promote the expression of the designed polypeptide sequence. The residue positions shown in the table correspond to residue positions in both the BA.1C and Omicron_Vaccine sequences.

[0143] Optionally, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 3 (Omicron_Vaccine), or amino acids having at least 99% amino acid identity across the entire length of the amino acid sequence of SEQ ID NO: 3, and is presented as follows: 7( (The following is a copy without indicating any missing parts.) As shown At least one or both of the amino acid residues of SEQ ID NO: 3 At the position corresponding to the amino acid residue position of SEQ ID NO: 9 include.

[0144] [Table 7-2]

[0145] The applicant has recognized that polypeptides of the present invention, including the above-described residue changes, have better stability than polypeptides containing wild-type residues at these positions.

[0146] Optionally, the polypeptide of the present invention contains the amino acid sequence of SEQ ID NO: 3 (Omicron_Vaccine), or amino acids having at least 99% amino acid identity with the amino acid sequence of SEQ ID NO: 3 over its entire length, and does not contain the residue KFDEDDSEPVLKGVKLHYT at the position corresponding to amino acid residue positions 1252-1270 of SEQ ID NO: 9.

[0147] The applicant has recognized that deletion of residues at positions 1252-1270 of SEQ ID NO: 9 (endoplasmic reticulum signal sequence) from the polypeptide of the present invention results in better polypeptide expression than polypeptides containing such residues.

[0148] According to the present invention, an isolated polypeptide containing the omicron_vaccine amino acid sequence (SEQ ID NO: 3) is provided.

[0149] The applicant believes that such polypeptides are deltaWe found that it induces a broad-spectrum neutralizing immune response to a panel of SARS-CoV-2 pseudoviruses, including Omicron substrains BA.1, BA.2, BA.2.12.1, and BA.4 / 5. As mentioned above, BA.4 and BA.5 are currently the dominant SARS-CoV-2 strains circulating in many countries around the world (as of April 2023).

[0150] The polypeptide of the present invention is one or more This may include conserved amino acid substitutions. Conservative amino acid substitutions are those that, when made, do little to interfere with the properties of the original polypeptide; that is, the protein's structure, especially its function, is preserved and not significantly altered by such substitutions. Examples of conserved substitutions are shown below.

[0151] [Table 9]

[0152] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the substitution region, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0153] Generally, substitutions that are expected to result in the greatest change in protein properties are non-conservative and include, for example, (a) a hydrophilic residue, e.g., serine or threonine, substituting (or being substituted by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine, or alanine; (b) cysteine ​​or proline substituting (or being substituted by) any other residue; (c) a residue with an electrically positive side chain, e.g., lysine, arginine, or histidine, substituting (or being substituted by) an electrically negative residue, e.g., glutamate or aspartate; or (d) a residue with a bulky side chain, e.g., phenylalanine, substituting (or being substituted by) one without a side chain, e.g., glycine.

[0154] In this specification, the term “broad neutralizing immune response” is used to mean an immune response induced in a subject that is sufficient to inhibit (i.e., mitigate), neutralize, or prevent infection and / or progression of infection by multiple strains and / or variants of SARS-CoV-2. Preferably, the broad neutralizing immune response is one of the Omicron strains of SARS-CoV-2. or more Sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by variants of SARS-CoV-2. Optionally, a broad-spectrum neutralizing immune response is effective against SARS-CoV-2. delta One of the stocks or more Sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by variants of SARS-CoV-2. Optionally, a broad-spectrum neutralizing immune response is effective against SARS-CoV-2. alpha One of the stocks or more Sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by variants of SARS-CoV-2. Optionally, a broad-spectrum neutralizing immune response is effective against SARS-CoV-2. beta One of the stocks or more Sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by variants of SARS-CoV-2. Optionally, a broad-spectrum neutralizing immune response is effective against SARS-CoV-2. gamma One of the stocks or more Sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by variants of SARS-CoV-2. Optionally, a broad-spectrum neutralizing immune response is effective against one of the Wuhan strains of SARS-CoV-2. or more The broad-spectrum neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by variants of the virus.Optionally, the broad-spectrum neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by most or all different virus strains of the SARS-CoV-2 virus.Optionally, the broad-spectrum neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by multiple types of β-coronavirus SARS-CoV-2 variants of concern (VOCs) or variants of interest (VOIs), e.g., multiple alpha , beta , gamma , delta Omicron is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection among multiple types of SARS-CoV-2 virus.

[0155] Selectively, broad-spectrum neutralizing immune responses are sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by viruses belonging to the coronavirus family. Selectively, broad-spectrum neutralizing immune responses involve multiple types of β - Sufficient to inhibit, neutralize, or prevent infection and / or progression of coronaviruses (e.g., SARS-CoV and SARS-CoV-2). Optionally, a broad-spectrum neutralizing immune response is also effective. - Multiple types of beta belonging to the coronavirus lineage - Coronaviruses (e.g., SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1, etc., multiple types of β-viruses belonging to the subgenus Salvecovirus) - Sufficient to inhibit, neutralize, or prevent infection and / or progression of coronavirus infection. Optionally, broad-spectrum neutralizing immune responses are effective against different β strains such as lineage B (e.g., SARS-CoV, SARS-CoV-2) and lineage C (e.g., MERS-CoV). - Sufficient to inhibit, neutralize, or prevent infection and / or progression of coronaviruses of the coronavirus lineage. Optionally, a broad-spectrum neutralizing immune response is effective against almost all different β- - Sufficient to inhibit, neutralize, or prevent infection and / or progression of coronavirus infection. Optionally, broad-spectrum neutralizing immune responses are sufficient to inhibit, neutralize, or prevent infection and / or progression of infection with most or all different viruses of the coronavirus family.

[0156] Immune responses can be humoral and / or cellular immune responses. Cellular immune responses are the responses of cells in the immune system, such as B cells, T cells, macrophages, or polymorphonuclear cells, to stimuli such as antigens or vaccines. Immune responses can include any cells in the body involved in the host defense response, such as epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate immune responses or inflammation.

[0157] Optionally, the polypeptide of the invention induces a protective immune response. A protective immune response refers to an immune response that protects a subject from infection or disease (i.e., prevents infection or the onset of a disease associated with infection). Methods for measuring an immune response are well known in the art and include, for example, measuring the proliferation and / or activation of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, or antibody production.

[0158] Optionally, the polypeptide of the invention can induce antibody production and / or a 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).

[0159] Optionally, the polypeptide of the invention is a glycosylated polypeptide.

[0160] Nucleic acid molecule According to the invention, there is provided an isolated nucleic acid molecule encoding a polypeptide according to the invention, or its complement.

[0161] According to the invention, there is also provided an isolated nucleic acid molecule or its complement comprising 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 nucleic acid molecule of the invention encoding the polypeptide of the invention over its entire length.

[0162] According to the invention, there is provided an isolated nucleic acid molecule or its complement comprising a nucleotide sequence encoding an isolated polypeptide having the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity to the amino acid sequence of SEQ ID NO: 1 over its entire length.

[0163] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 1, or a complement thereof.

[0164] Optionally, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity over the full length of the amino acid sequence of SEQ ID NO: 2, or a complement thereof.

[0165] According to the present invention, there is provided an isolated polynucleotide molecule comprising a nucleotide sequence encoding an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a complement thereof. [[ID=I0]]

[0166] [[ID=II]] According to the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (Omicron_vaccine), or an amino acid sequence having 99% amino acid identity over the full length of the amino acid sequence of SEQ ID NO: 3, or a complement thereof.

[0167] According to the present invention, there is provided an isolated polynucleotide molecule comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, or a complement thereof.

[0168] mRNA molecule It has been recognized that the mRNA immunogen encoding the coronavirus spike protein according to the present invention provides advantageous immunogenic properties (e.g., increased antibody response and / or expanded breadth of immune response).

[0169] According to the present invention, there is also provided an isolated mRNA encoding a polypeptide comprising the amino acid sequence of the coronavirus spike protein CoV_S_T2_35 (SEQ ID NO: 1).

[0170] According to the present invention, isolated mRNA encoding a polypeptide containing the amino acid sequence (SEQ ID NO: 2) of the coronavirus spike protein CoV_S_T2_36 is also provided.

[0171] According to the present invention, isolated mRNA encoding a polypeptide containing the amino acid sequence (SEQ ID NO: 3) of the coronavirus spike protein omicron_vaccine is also provided.

[0172] According to the present invention, an isolated mRNA molecule containing an RNA sequence encoding the polypeptide according to the present invention or its complement is provided.

[0173] The present invention also provides an isolated mRNA molecule or its complement containing an RNA sequence that is identical to the mRNA molecule of the present invention encoding the polypeptide of the present invention by 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% over its entire length.

[0174] According to the present invention, an isolated mRNA molecule or its complement is provided, which comprises an RNA sequence encoding an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length.

[0175] Optionally, the RNA sequence may include the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an isolated polypeptide encoding an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length, as shown in Table 3 below. Street The amino acid residues of sequence number 1 At the position corresponding to the amino acid residue position of SEQ ID NO: 8 Isolated mRNA molecules containing the mRNA are provided.

[0176] [Table 3-3]

[0177] Optionally, an RNA sequence encoding an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity over the entire length thereof with the amino acid sequence of SEQ ID NO: 1, and having the following amino acid residues at positions corresponding to residues 984 and 985 of SEQ ID NO: 8 : · 984: P, and · 985: P Isolated mRNA molecules containing the above are provided. .

[0178] Optionally, an RNA sequence encoding an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity over the entire length thereof with the amino acid sequence of SEQ ID NO: 1, and having the following amino acid residues at positions corresponding to residues 680, 681, 683, 984, and 985 of SEQ ID NO: 8 : · 680: G, · 681: S, · 683: S, · 984: P, and · 985: P Isolated mRNA molecules containing the above are provided. .

[0179] Optionally, provided is an isolated mRNA molecule comprising an RNA sequence encoding an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an amino acid sequence having at least 97%, 98% or 99% amino acid identity over the entire length thereof with the amino acid sequence of SEQ ID NO: 1, and not containing the residue KFDEDDSEPVLKGVKLHYT at the position corresponding to amino acid residue positions 1253 - 1271 of SEQ ID NO: 8.

[0180] According to the present invention, as shown in Table 3 below The amino acid residues at least one, or all at the position corresponding to the amino acid residue position of SEQ ID NO: 8An isolated mRNA molecule is provided that contains an RNA sequence encoding an isolated polypeptide containing the coronavirus S protein.

[0181] [Table 3-4]

[0182] An isolated mRNA molecule or its complement is provided, optionally containing an RNA sequence encoding an isolated polypeptide that includes the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length.

[0183] Optionally, the RNA sequence may include the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or an isolated polypeptide encoding an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 across its entire length, as shown in Table 6 below. Street The amino acid residues of SEQ ID NO: 2 At the position corresponding to the amino acid residue position of SEQ ID NO: 9 Isolated mRNA molecules containing the mRNA are provided.

[0184] [Table 6-3]

[0185] Optionally, an RNA sequence encoding an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 across its entire length, with the following amino acid residues at the positions corresponding to residues 983 and 984 of SEQ ID NO: 9 : ·983:P, and ·984:P Isolated mRNA molecules containing the above are provided. .

[0186] Optionally, an RNA sequence encoding an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 across its entire length, wherein the following amino acid residues are present at the positions corresponding to residues 679, 680, 682, 983, and 984 of SEQ ID NO: 9. : ·679:G, ·680:S, ·682:S, ·983:P, and ·984:P Isolated mRNA molecules containing the above are provided. .

[0187] An isolated mRNA molecule is provided that optionally includes an RNA sequence encoding an isolated polypeptide that comprises the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length, and does not contain the residue KFDEDDSEPVLKGVKLHYT at the positions corresponding to amino acid residue positions 1252-1270 of SEQ ID NO: 9.

[0188] According to the present invention, as shown in Table 6 below Among the amino acid residues as described above At least one, or all of them at the position corresponding to the amino acid residue position of SEQ ID NO: 9 An isolated mRNA molecule is provided that contains an RNA sequence encoding an isolated polypeptide containing the coronavirus S protein.

[0189] [Table 6-4]

[0190] According to the present invention, an isolated mRNA molecule or its complement is provided, which comprises an RNA sequence encoding an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 3 (Omicron_Vaccine), or an amino acid sequence having 99% amino acid identity with the amino acid sequence of SEQ ID NO: 3 over its entire length.

[0191] Optionally, an RNA sequence encoding an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 3 (Omicron_Vaccine) or the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence having at least 99% amino acid identity across its entire length, with the following amino acid residues at the positions corresponding to residues 983 and 984 of SEQ ID NO: 9 : ·983:P, and ·984:P Isolated mRNA molecules containing the above are provided. .

[0192] The inventors have found that immunization of mice with nucleic acids (particularly mRNA) encoding the SARS2 S protein design of the present invention induces the production of antibodies that can bind to and neutralize coronavirus pseudoviruses expressing the spike protein (see Example 3 / Figures 6 and 7). In particular, immunization of mice with mRNA vaccines containing nucleic acids encoding the CoV_S_T2_35(deome) mRNA vaccine design (SEQ ID NO: 1) of the present invention was found to be effective in Wuhan, alpha , beta , gamma , delta Furthermore, Omicron BA.1, BA.2, BA.2.12.1, and BA.4 / 5 induced broad-spectrum neutralizing antibody responses against SARS-CoV-2 (Figures 6B and 6C, Figure 7). Similarly, immunization of mice with mRNA vaccines containing nucleic acids encoding the CoV_S_T2_36(omido)mRNA vaccine design of the present invention (SEQ ID NO: 2) induced broad-spectrum neutralizing antibody responses against Omicron strains BA.1, BA.2, BA.2.12.1, and BA.4 / 5 substrains, as well as SARS-CoV-2 from Wuhan. alpha , beta , gamma , and delta This induced a broad-spectrum neutralizing antibody response against the strain (Figures 6B and 6C, Figure 7). Immunization of mice with an mRNA vaccine containing nucleic acids encoding the Omicron Vaccine (Deome) design of the present invention (SEQ ID NO: 3) was effective against SARS-CoV-2. beta , gamma BA.1, BA.2, BA.4 / 5, and delta This induced a broad-spectrum neutralizing antibody response against the strain.

[0193] Sequence identity The similarity between amino acid sequences or nucleic acid sequences is expressed in terms of similarity between sequences, otherwise it is called sequence identity. Sequence identity is often measured in terms of a percentage of identity (or similarity or homology), with a higher percentage indicating greater similarity between the two sequences. Homologs or variants of a given gene or protein exhibit 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 cited 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; and Altschul et al., Nature It is described in Genet. 6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLAST®) (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.

[0194] Sequence identity between nucleic acid sequences or amino acid sequences can be determined by comparing their alignments. If equivalent positions in the comparison sequences are occupied by the same nucleotide or amino acid, the molecules are identical at those positions. Scoring alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at positions shared by the comparison sequences. When comparing sequences, the optimal alignment is one that takes into account possible insertions and deletions in the sequences. or more It may be necessary to introduce gaps into the sequence. The sequence comparison method may use a gap penalty, and when the same number of identical molecules in the sequences are compared, a sequence alignment with as few gaps as possible reflects a higher relevance between the two sequences being compared and achieves a higher score than one with many gaps. Calculating the maximum identity percentage involves generating the optimal alignment, taking the gap penalty into account.

[0195] Suitable computer programs for performing sequence comparisons are widely available in the commercial and public sectors. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4:29, program available from http: / / bitincka.com / ledion / matgat), Gap (Needleman & Wunsch, 1970, J.Mol.Biol.48:443-453), FASTA (Altschul et al., 1990, J.Mol.Biol.215:403-410, program available from http: / / www.ebi.ac.uk / fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23:2947-2948, program available from http: / / www.ebi.ac.uk / tools / clustalw2), and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch). References include Wunsch, 1970, cited above; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley (the program is available from http: / / www.ebi.ac.uk / tools / emboss / align). All programs can be run using default parameters.

[0196] For example, sequence comparison can be performed using the "needle" method of EMBOSS Pairwise Alignment Algorithms, which determines the optimal alignment (including gaps) when considering the two sequences over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparison ("Protein Molecule" option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, and Matrix: Blosum 62.

[0197] Sequence comparisons can be performed over the entire length of the reference sequence.

[0198] Corresponding position The sequences described herein include references to amino acid sequences that contain amino acid residues at “positions corresponding to amino acid residue positions” of another sequence. Such corresponding positions may be identified, for example, by sequence alignment using the sequence alignment methods described herein, or by other sequence alignment methods known to those skilled in the art.

[0199] Vectors and vaccines According to the present invention, a vector comprising a nucleic acid molecule encoding the polypeptide of the present invention is also provided.

[0200] Optionally, the vector of the present invention comprises a nucleic acid molecule encoding the polypeptide of the present invention, which includes the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length.

[0201] Optionally, the vector of the present invention includes a polynucleotide molecule or its complement containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0202] Optionally, the vector of the present invention comprises a nucleic acid molecule encoding the polypeptide of the present invention, which comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length.

[0203] Optionally, the vector of the present invention includes a polynucleotide molecule or its complement containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0204] Optionally, the vector of the present invention includes a nucleic acid molecule encoding the polypeptide of the present invention, which comprises the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 3 and having at least 99% amino acid identity over its entire length.

[0205] Optionally, the vector of the present invention comprises a nucleic acid molecule encoding the polypeptide of the present invention, which includes the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 99% amino acid identity with the amino acid sequence of SEQ ID NO: 3 over its entire length.

[0206] Optionally, the vector of the present invention includes a polynucleotide molecule or its complement containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3.

[0207] Optionally, the vector of the present invention includes the nucleic acid molecule of the present invention that encodes the coronavirus S protein of the present invention.

[0208] Optionally, the vector of the present invention further comprises a promoter operably linked to a nucleic acid.

[0209] Optionally, the promoter is for the expression of nucleic acid-encoded polypeptides in mammalian cells.

[0210] Optionally, the promoter is for the expression of nucleic acid-encoded polypeptides in yeast or insect cells.

[0211] The vector is of optional choice and is a vaccine vector.

[0212] The vector can be optionally a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector.

[0213] The nucleic acid molecule of the present invention may include DNA or RNA molecules. In embodiments in which the nucleic acid includes RNA molecules, it will be noted that the nucleic acid sequence of the nucleic acid is the same as or a complement thereof as described in the respective sequence numbers, but each "T" nucleotide is replaced with "U".

[0214] In embodiments in which the nucleic acid molecule includes an RNA molecule, it will be recognized that the molecule may include an RNA sequence or its complement 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, a polynucleotide sequence encoding any of the polypeptide sequences of Sequence ID No. 1, 2, or 3, in which each "T" nucleotide is replaced with "U".

[0215] For example, if an RNA vaccine vector containing the nucleic acid of the present invention is provided, the nucleic acid sequence of the nucleic acid of the present invention is an RNA sequence, and is therefore recognized as potentially containing an RNA nucleic acid sequence or its complement, which 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: 1, 2, or 3, in which each "T" nucleotide is replaced by "U".

[0216] Viral vaccine vectors use live viruses to deliver nucleic acids (e.g., DNA or RNA) to human or non-human animal cells. A single nucleic acid is contained within the virus. or more Viral vaccine vectors encode antigens that, when expressed in infected human or non-human animal cells, trigger 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. Similar to nucleic acid vaccines, viral vaccine vectors carry nucleic acids to host cells for the production of antigenic proteins that can be tuned to stimulate a broad immune response, including antibody, T helper cell (CD4+ T cell), and cytotoxic T lymphocyte (CTL, CD8+ T cell) mediated immunity. Unlike nucleic acid vaccines, viral vaccine vectors also have the ability to actively enter and replicate in host cells, like live attenuated vaccines, and further activate the immune system like an adjuvant. Therefore, viral vaccine vectors generally contain live attenuated viruses engineered to carry nucleic acids (e.g., DNA or RNA) encoding protein antigens from an unrelated organism. Viral vaccine vectors can generally produce a stronger immune response than nucleic acid vaccines, but in some diseases, viral vectors are used in combination with other vaccine technologies in a strategy called heterologous prime-boost. In this system, one vaccine is given as a priming step, followed by vaccination with a surrogate vaccine as a booster. The heterologous prime-boost strategy aims to produce a stronger overall immune response. Viral vaccine vectors can be used as both prime and booster vaccines as part of this strategy. Viral vaccine vectors have been outlined by Ura et al., 2014 (Vaccines 2014, 2, 624-641) and Choi and Chang, 2013 (Clinical and Experimental Vaccine Research 2013; 2: 97-105).

[0217] Optionally, the viral vaccine vector is a viral delivery vector, e.g., poxvirus (e.g., Modified Vaccinia Ankara (MVA), NYVAC, AVIPOX), herpesvirus (e.g., HSV, CMV, adenovirus of any host species), morbillivirus (e.g., measles), alpha These are based on viral delivery vectors (e.g., SFV, Sendai), flaviviruses (e.g., yellow fever), or rhabdoviruses (e.g., VSV), bacterial delivery vectors (e.g., Salmonella, E. coli), RNA expression vectors, or DNA expression vectors. Optionally, viral vaccine vectors are pURVac vaccine vectors, derivatives of DNA vaccine vectors.

[0218] Adenoviruses are the most widely used and advanced viral vectors developed for SARS-2 vaccines. They are non-enveloped double-stranded DNA (dsDNA) viruses with the ability to package foreign genes up to 7.5 kb. Almost all SARS-2 adenovirus-based vaccines are engineered to express the SARS-2 S protein or RBD subunit. Recombinant adenovirus vectors are widely used due to their high transduction efficiency, high levels of transgene expression, and broad virtropy. These vaccines exhibit high cell specificity, high gene transduction efficiency, and high efficiency in inducing immune responses. Adenovirus vaccines are effective in inducing and priming T cells, resulting in prolonged and high levels of antigen protein expression, and therefore, long-lasting protection. The AZD1222 (AstraZeneca) vaccine construct contains a recombinant adenovirus vector vaccine encoding the SARS-2 S protein. The recombinant adenovirus genome contains the SARS-2 S gene at the E1 locus.

[0219] Optionally, the vaccine of the present invention (optionally, the nucleic acid or polypeptide of the present invention) may be administered as part of a heterologous prime-boost regimen, for example, using a heterologous DNA prime / MVA boost regimen.

[0220] Optionally, a method according to the present invention for inducing an immune response to coronavirus in a subject, or for immunizing a subject to coronavirus, comprises administering the nucleic acid, vector, or pharmaceutical composition of the present invention, wherein the nucleic acid, vector, or pharmaceutical composition is administered as part of a heterologous prime boost regimen.

[0221] Optionally, the heterologous prime-boost regimen includes priming with the DNA vector of the present invention, followed by a boost with the MVA vector of the present invention.

[0222] Optionally, DNA Prime comprises administration of a DNA vaccine vector containing the nucleic acid molecule of the present invention, and MVA Boost comprises administration of an MVA vector containing the nucleic acid molecule of the present invention. Optionally, 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. Optionally, the nucleic acid molecule of the present invention in the DNA vaccine vector encodes a different amino acid sequence than the nucleic acid molecule of the present invention in the MVA vector.

[0223] For example, nucleic acid molecules (optionally DNA molecules) encoding the designed S protein sequence T2_29 polypeptide (SEQ ID NO: 29, COV_S_T2_29+Q498R+dER), (COV_S_T2_29+Q498R-SEQ ID NO: 28), or (COV_S_T2_29-SEQ ID NO: 27) of the present invention can be administered as part of a heterologous prime-boost vaccine using MVA boosting. As shown in Example 3 below, priming with a DNA vector containing DNA encoding the amino acid sequence of SEQ ID NO: 27, 28, or 29, followed by a boost with an MVA vector containing nucleic acid encoding the amino acid sequence of SEQ ID NO: 29, induced a broad neutralization response for all VOCs tested, and after three doses of the DNA vaccine, compared to WTdER, beta , gamma , delta It also induced a neutralization response that was at least twice as good against omicron VOCs.

[0224] Optionally, priming with the DNA vector of the present invention may include one, two, or three doses of the DNA vector prior to MVA boosting.

[0225] The MVA boost may be administered at least one day, at least one week, or at least two, three, four, five, six, or seven weeks after the final dose of the DNA vector.

[0226] According to the present invention, a kit is also provided comprising a DNA vaccine vector containing the nucleic acid molecule of the present invention and an MVA vector containing the nucleic acid molecule of the present invention, wherein the nucleic acid molecule of the present invention in the DNA vaccine vector optionally encodes the same amino acid sequence as the nucleic acid molecule of the present invention in the MVA vector.

[0227] Optionally, the nucleic acid molecule of the present invention in a DNA vaccine vector encodes the designed S protein sequence T2_29 polypeptide (SEQ ID NO: 29-COV_S_T2_29+Q498R+dER, COV_S_T2_29+Q498R-SEQ ID NO: 28, or COV_S_T2_29-SEQ ID NO: 27), and the nucleic acid molecule of the present invention in an MVA vector encodes the amino acid sequence of SEQ ID NO: 29.

[0228] Optionally, nucleic acid expression vectors include nucleic acid expression vectors and viral pseudotype vectors.

[0229] Optionally, the nucleic acid expression vector is a vaccine vector.

[0230] Optionally, the nucleic acid expression vector includes a promoter, a splice donor (SD), a splice acceptor (SA), and a terminator signal in the 5' to 3' direction, with multiple cloning sites located between the splice acceptor and the terminator signal.

[0231] Optionally, the promoter may include the CMV earliest 1 enhancer / promoter (CMV-IE-E / P), and / or the terminator signal may include the terminator signal of the bovine growth hormone gene (Tbgh) lacking the KpnI restriction endonuclease site.

[0232] Optionally, the nucleic acid expression vector further comprises an origin of replication and a nucleic acid encoding resistance to antibiotics. Optionally, the origin of replication comprises a pUC-plasmid origin of replication, and / or the nucleic acid encodes resistance to kanamycin.

[0233] Optionally, the vector is a pEVAC-based expression vector.

[0234] pEVAC vectors have proven to be highly versatile expression vectors for generating viral pseudotypes and for direct DNA vaccination in animals and humans.

[0235] Optionally, the vector is a pURVAC vector.

[0236] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0237] The polynucleotide (or nucleic acid) of the present invention may include DNA molecules.

[0238] The polynucleotide (or nucleic acid) or each polynucleotide (or nucleic acid) of the pharmaceutical composition or vector of the present invention may contain DNA molecules.

[0239] The vector of the present invention may be a DNA vector.

[0240] The vectors in the pharmaceutical compositions of the present invention, or each vector, may be DNA vectors.

[0241] The polynucleotide (or nucleic acid) of the present invention, or the polynucleotide (or nucleic acid) of the pharmaceutical composition or vector of the present invention, may be provided as part of a DNA vaccine.

[0242] According to the present invention, the polynucleotide (or nucleic acid) of the present invention, the vector of the present invention, or one or more A DNA vaccine comprising the pharmaceutical composition of the present invention, which contains a polynucleotide (or nucleic acid), is also provided, wherein the polynucleotide (or nucleic acid) or each polynucleotide (or nucleic acid) is a DNA molecule.

[0243] Optionally, the vector or each vector is an RNA vaccine vector.

[0244] The polynucleotide (or nucleic acid) of the present invention may include RNA molecules.

[0245] The polynucleotide (or nucleic acid) or each polynucleotide (or nucleic acid) of the pharmaceutical composition or vector of the present invention may include RNA molecules.

[0246] The vectors in the pharmaceutical compositions or complex preparations of the present invention, or each individual vector, may be RNA vectors.

[0247] The polynucleotide (or nucleic acid) of the present invention, or the polynucleotide (or nucleic acid) of the pharmaceutical composition or vector of the present invention, may be provided as part of an RNA vaccine.

[0248] According to the present invention, the polynucleotide (or nucleic acid) of the present invention, the vector of the present invention, or one or moreAn RNA vaccine comprising the pharmaceutical composition of the present invention, which contains a polynucleotide (or nucleic acid), is also provided, wherein the polynucleotide (or nucleic acid) or each polynucleotide (or nucleic acid) is an RNA molecule.

[0249] The polynucleotide (or nucleic acid) of the present invention may include mRNA molecules.

[0250] The polynucleotide (or nucleic acid) of the pharmaceutical composition or vector of the present invention, or each polynucleotide (or nucleic acid), may contain mRNA molecules.

[0251] The vector of the present invention may be mRNA.

[0252] Optionally, the vaccine vector or each vaccine vector is an mRNA vaccine vector.

[0253] The vectors of the pharmaceutical compositions of the present invention, or each vector, may be mRNA vectors.

[0254] The polynucleotide (or nucleic acid) of the present invention, or the mRNA molecule, or the polynucleotide (or nucleic acid) of the pharmaceutical composition or vector of the present invention, may be provided as part of an mRNA vaccine.

[0255] According to the present invention, an mRNA vaccine is also provided which includes the mRNA of the present invention encapsulated in lipid nanoparticles (LNPs) or the mRNA vaccine vector of the present invention.

[0256] According to the present invention, the polynucleotide (or nucleic acid) of the present invention, the vector of the present invention, or one or more An mRNA vaccine is also provided, comprising the pharmaceutical composition of the present invention, which comprises a polynucleotide (or nucleic acid), wherein the polynucleotide (or nucleic acid) or each polynucleotide (or nucleic acid) comprises an mRNA molecule.

[0257] Messenger RNA (mRNA) vaccines are a new form of vaccine (recently outlined in Pardi et al., Nature Reviews Drug Discovery Volume 17, pages 261-279 (2018), and Wang et al., Molecular Cancer (2021) 20:33: mRNA vaccine: a potential therapeutic strategy). The first mRNA vaccines approved for use during the COVID-19 pandemic were BNT162b2 (Pfizer) and mRNA-1273 (Moderna). mRNA vaccines have the unique characteristic of temporarily (typically for several days) boosting antigen expression. The expression of exogenous antigens is controlled by the lifespan of the mRNA encoding it, which is regulated by cytodegradation pathways. This transient nature of protein expression is extremely beneficial for vaccines where prime or prime-boost vaccination is sufficient to develop highly specific adaptive immunity without exposure to the infectious disease, although repeated doses are required for the treatment of hereditary diseases and cancers.

[0258] mRNA-based vaccines induce an immune response after synthetic mRNA encoding viral antigens is transfected into human cells. The cytoplasmic mRNA molecules are then translated into specific viral antigens by the host's own cellular mechanisms. These antigens are then present on the cell surface, where they can be recognized by immune cells and trigger an immune response.

[0259] The structural elements of the vaccine vector mRNA molecule are similar to those of natural mRNA and include a 5' cap, a 5' untranslated region (UTR), a coding region (e.g., including an open reading frame encoding the polypeptide of the present invention), a 3' UTR, and a poly(A) tail. The 5' UTR (also known as the leader sequence, transcript leader, or leader RNA) is the region of mRNA immediately upstream of the start codon. This region is crucial for regulating transcript translation. In many organisms, the 5' UTR forms a complex secondary structure for regulating translation. The 5' UTR begins at the transcription start site and ends with a single nucleotide (nt) preceding the start sequence of the coding region (usually AUG). In eukaryotes, the length of the 5' UTR tends to range from 100 to several thousand nucleotides. These length differences are likely due to the complexity of the eukaryotic regulation borne by the 5' UTR and the larger pre-initiation complex that must be formed to initiate translation. The 5'UTR of eukaryotes is a Kosack consensus sequence containing the start codon AUG.

[0260] [ka] (The underlined part is the start codon) contains an extended Kozak sequence,

[0261] [ka] (The underlined part is the start codon) can be used.

[0262] The 5' and 3' UTR elements surrounding the coding sequence strongly influence mRNA stability and translation, and both are important considerations for vaccines. These regulatory sequences originate from viral or eukaryotic genes and can significantly increase the half-life and expression of therapeutic mRNA. For example, the 5' UTR of the mRNA of the present invention may include a Kosack consensus sequence or an extended Kosack sequence along with the mRNA start codon. Optionally, the 5' UTR of the mRNA of the present invention may include the sequence GGAGACGCCACC immediately upstream of the start codon sequence.

[0263] Efficient protein production from mRNA requires a 5' cap structure. Various versions of the 5' cap can be added during or after the transcription reaction using vaccinia virus capping enzymes, or by incorporating synthetic caps or anti-reverse cap analogs (see Pardi et al., cited above). Anti-reverse cap analogs (ARCA) are cap analogs used in vitro during transcription to produce capped transcripts. ARCA are modified to ensure incorporation in the forward direction only. Anti-reverse cap analogs (ARCA) have a 3'OH group (m 7 It is a modified cap analog in which the part (close to G) is replaced with -OCH3.

[0264] [ka] Conventional cap analogues: R=H, m 7 G(5')pppG, ARCA: R=CH3, 3'-0-Me-m 7 G(5')pppG

[0265] Due to this substitution, RNA polymerase can initiate transcription using only the remaining hydroxyl group, thereby forcing ARCA into the forward direction. As a result, unlike transcripts synthesized using conventional cap analogues, 100% of transcripts synthesized with ARCA at the 5' end are translatable, resulting in a strong translational stimulus.

[0266] Poly(A) tails also play an important regulatory role in mRNA translation and stability. Therefore, poly(A) of the optimal length must be added to mRNA either directly from the coding DNA template or using poly(A) polymerase (see Pardi et al., op. cit.). The appropriate length of the poly(A) tail is, for example, poly(A) 120 )

[0267] Codon usage frequency further influences protein translation. Replacing rare codons in mRNA with frequently used synonymous codons for which the corresponding tRNA is abundant in the cytoplasm is a common technique for increasing protein production. G enrichment:C content constitutes another form of sequence optimization, which has been shown to increase steady-state mRNA levels in vitro and protein expression in vivo (see Pardi et al., op. cit.).

[0268] Two main types of RNA, non-replicating mRNA and virus-derived self-amplified RNA, are currently being studied as vaccines. Both types of vaccines share a common structure in their mRNA constructs, but self-amplified RNA vaccines contain additional sequences in the coding region for RNA replication, including RNA-dependent RNA polymerase.

[0269] The BNT162b2 vaccine construct contains a lipid nanoparticle (LNP)-encapsulated mRNA molecule encoding a trimerized full-length SARS2 S protein with a PP mutation (residue positions 986-987). The mRNA is encapsulated within 80 nm ionizable cationic lipid nanoparticles. The mRNA-1273 vaccine construct is also LNP vector-based, but the synthetic mRNA encapsulated within the lipid construct encodes the full-length SARS2 S protein.

[0270] U.S. Patent No. 10,702,600(B1) (ModernaTX) contains LNPs suitable for use in such vaccines. beta This document describes coronavirus mRNA vaccines.

[0271] The nucleic acid vaccine (e.g., mRNA) of the present invention can be formulated in lipid nanoparticles.

[0272] mRNA vaccines offer several advantages over conventional vaccines containing inactivated (or bio-attenuated) pathogens. Firstly, mRNA-based vaccines can be developed rapidly due to their design flexibility and the ability of constructs to mimic the structure and expression of antigens observed in natural infection processes. mRNA vaccines can be developed within days to months based on the target virus's sequence information, whereas conventional vaccines often take several years to develop, requiring a deep understanding of the target virus to create an effective and safe vaccine. Secondly, these novel vaccines can be manufactured rapidly. Due to high yields from in vitro transcription reactions, mRNA generation can be rapid, inexpensive, and scalable. Thirdly, vaccine-related risks are low. mRNA does not contain infectious viral elements that pose a risk of infection or insertional mutation. Because mRNA is a gene vector with minimal immunogenicity, anti-vector immunization is also avoided, allowing for repeated administration of the vaccine. A challenge for the effective application of mRNA vaccines lies in cytoplasmic delivery. mRNA isolates are rapidly degraded by extracellular RNases and are not susceptible to transcribing across the cell membrane and being transcribed in the cytoplasm. However, by formulating mRNA into a carrier molecule, efficient in vivo delivery can be achieved, enabling rapid uptake and expression into the cytoplasm. To date, numerous delivery methods have been developed, including lipid, polymer, or peptide-based delivery, virus-like replicon particles, cationic nanoemulsions, naked mRNA, and dendritic cell-based delivery (each outlined in Wang et al., cited above). Delivery using divalent cationic lipid nanoparticles (LNPs) is the most promising and commonly used method for delivering mRNA vaccines.

[0273] Exogenous mRNA can be highly immunostimulant. Single-stranded RNA (ssRNA) molecules are considered pathogen-associated molecular patterns (PAMPs) and are recognized by various Toll-like receptors (TLRs) that trigger pro-inflammatory responses. While a strong cellular and humoral immune response in response to vaccination is desirable, the innate immune response induced by exogenous mRNA can cause undesirable side effects in the target population. U-rich sequences in mRNA are important components for activating TLRs (Wang et al., op. cit.). Furthermore, enzymatically synthesized mRNA preparations contain double-stranded RNA (dsRNA) contaminants as abnormal products of the in vitro transcription (IVT) process. dsRNA is a potent PAMP and induces downstream responses that lead to translational inhibition and degradation of intracellular mRNA and ribosomal RNA (Pardi et al., op. cit.). Therefore, mRNA can suppress antigen expression and thus reduce the effectiveness of the vaccine.

[0274] Studies over the past decade have shown that the immunostimulatory effects of mRNA can be induced by purification of IVT mRNA, introduction of modified nucleosides, complexation of mRNA with various carrier molecules (Pardi et al., op. cit.), addition of poly(A) tails, or optimization of mRNA with GC-rich sequences (Wang et al., op. cit.). Uridine chemical modification is a common technique for minimizing the immunogenicity of exogenous mRNA. Incorporation of pseudouridine (Ψ) and N1-methylpseudridine (m1Ψ) into IVT mRNA interferes with TLR activation and other innate immune sensors, thus reducing pro-inflammatory signaling in response to exogenous mRNA. Such nucleoside modifications also suppress the recognition of dsRNA species (Pardi et al., cited above) and may reduce the innate immune sensing of exogenous mRNA translation (Hou et al. Nature Reviews Materials, 2021, https: / / doi.org / 10.1038 / s41578-021-00358-0).

[0275] Other nucleoside chemical modifications include, but are not limited to, 5-methylcytidine (m5C), 5-methyluridine (m5U), N1-methyladenosine (m1A), N6-methyladenosine (m6A), 2-thiouridine (s2U), and 5-methoxyuridine (5moU) (Wang et al., cited above).

[0276] The IVT mRNA molecules used in the mRNA-1273 and BNT162b2 COVID-19 vaccines were prepared by substituting uridine with m1Ψ, and their sequences were optimized to encode a stabilized pre-fusion spike protein with two central proline substitutions (Hou et al., op. cit.). However, CureVac's mRNA vaccine candidate, CVnCoV, uses an unmodified nucleoside and relies on a combination of mRNA sequence mutations to enable immune evasion without affecting the expressed protein. Firstly, CVnCoV has a higher GC content (63%) than rival vaccines (BNT162b2 has 56%) and the original SARS-CoV-2 virus itself (37%). Secondly, the vaccine contains a C-rich motif that binds to poly(C)-binding proteins, enhancing both mRNA stability and expression. Further modifications to CVnCoV include the inclusion of histone stem-loop sequences and poly(A) tails to enhance mRNA lifespan 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 15.09.21)). However, the vaccine showed disappointing results in Phase III clinical trials, which experts argue are due to the decision not to incorporate chemically modified nucleosides into the mRNA sequence. Nevertheless, CureVac and Acuitas Therapeutics delivered GC codon-rich mRNA-encoding erythropoietin (EPO) to pigs using lipid nanoparticles (LNPs). The results showed that an EPO-related response was induced without immunogenicity (Wang et al.). (al., cited above), this suggests that there is still room for improvement in unmodified mRNA nucleoside-based vaccines.

[0277] The RNA or mRNA of the polynucleotide (or nucleic acid) of the present invention, or the RNA or mRNA of the polynucleotide (or nucleic acid) of the pharmaceutical composition, complex preparation, vector, or vaccine of the present invention, can be generated by in vitro transcription (IVT).

[0278] Optionally, the IVT mRNA of the present invention includes a polyadenylated (poly(A)) tail downstream of the open reading frame (ORF) encoding the polypeptide.

[0279] The polynucleotide (or nucleic acid) or mRNA molecule of the present invention, or the polynucleotide (or nucleic acid) or mRNA molecule of the pharmaceutical composition, vector, or vaccine of the present invention, is one or more It may contain modified nucleosides.

[0280] 1 or more The modified nucleoside may be present in the DNA or RNA of the polynucleotide (or nucleic acid) of the present invention, or in the DNA or RNA of the polynucleotide (or nucleic acid) of the pharmaceutical composition, vector, or vaccine of the present invention.

[0281] Optionally, at least one chemical modification is selected from pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deazapseudridine, 2-thio-1-methylpseudridine, 2-thio-5-azauridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-azauridine, dihydropseudridine, 5-methoxyuridine, and 2'-O-methyluridine, 5-methylcytosine, N1-methyladenosine, and N6-methyladenosine. In some embodiments, the chemical modification is located at the 5-position of uracil. In some embodiments, the chemical modification is N1-methylpseuduridine. In some embodiments, the chemical modification is N1-ethylpseuduridine.

[0282] For example, the RNA or mRNA of the polynucleotide (or nucleic acid) of the present invention, or the RNA or mRNA of the polynucleotide (or nucleic acid) of the pharmaceutical composition, vector, or vaccine of the present invention, may have the following modified nucleosides : Pseudouridine (Ψ), N1-methylpseudridine (m1Ψ) 5-methylcytidine (m5C) 5-methyluridine (m5U) N1-Methyladenosine (m1A) N6-methyladenosine (m6A) 2-thiouridine (s2U) 5-Methoxyuridine (5 moU) It may include one or more of these.

[0283] In some embodiments, 100% of the uracil in the open reading frame has a chemical modification. In some embodiments, the chemical modification is at position 5 of uracil. In some embodiments, the chemical modification is N1-methylpseudouridine. In some embodiments, 100% of the uracil in the open reading frame has N1-methylpseudouridine at position 5 of uracil.

[0284] Polynucleotides (or nucleic acids) consist of approximately 1% to 100% modified nucleotides (or nucleosides) (relative to the total nucleotide content, or one of the nucleotides (or nucleosides)). or more The type, i.e., one of A, G, U, or C or more (relative to) or any intervening percentage (for example, 1%~20%, 1%~25%, 1%~50%, 1%~60%, 1%~70%, 1%~80%, 1%~90%, 1%~95%, 10%~20%, 10%~25%, 10%~50%, 10%~60%, 10%~70%, 10%~80%, 10%~90%, 10%~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%) It may contain (~70%, 20%~80%, 20%~90%, 20%~95%, 20%~100%, 50%~60%, 50%~70%, 50%~80%, 50%~90%, 50%~95%, 50%~100%, 70%~80%, 70%~90%, 70%~95%, 70%~100%, 80%~90%, 80%~95%, 80%~100%, 90%~95%, 90%~100%, and 95%~100%). The remaining percentage is made up of unmodified A, G, U, or C.

[0285] Optionally, at least 50% of the uridine in the ORF is modified.

[0286] Optionally, at least 50% of the uridine in the ORF is modified with m1ψ.

[0287] Optionally, the polynucleotide (or nucleic acid) or mRNA molecule of the present invention, or the polynucleotide (or nucleic acid) or mRNA molecule of the pharmaceutical composition, vector, or vaccine of the present invention, includes an RNA molecule or its complement in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that described in the respective sequence number, but each "U" is replaced with m1ψ.

[0288] Optionally, the polynucleotide (or nucleic acid) or mRNA molecule of the present invention, or the polynucleotide (or nucleic acid) or mRNA molecule of the pharmaceutical composition, vector, or vaccine of the present invention, includes an mRNA molecule or its complement in which the nucleic acid sequence of the polynucleotide is the same as that described in the respective sequence number, but each "U" is replaced with m1ψ.

[0289] Optionally, the polynucleotide (or nucleic acid) or mRNA molecule of the present invention, or the polynucleotide (or nucleic acid) or mRNA molecule of the pharmaceutical composition, vector, or vaccine of the present invention, comprises an RNA molecule or its complement in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that described in the respective sequence number, but at least 50% of the "U"s are replaced with m1ψ. The remaining "U"s may all be unmodified, or one unmodified nucleoside and one or more It may also contain other modified nucleosides.

[0290] Optionally, the polynucleotide (or nucleic acid) or mRNA molecule of the present invention, or the polynucleotide (or nucleic acid) or mRNA molecule of the pharmaceutical composition, vector, or vaccine of the present invention, comprises an mRNA molecule or its complement in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that described in the respective sequence number, but at least 50% of the "U"s are replaced with m1ψ. The remaining "U"s may all be unmodified, or one unmodified nucleoside and one or more It may also contain other modified nucleosides.

[0291] Optionally, the polynucleotide (or nucleic acid) or mRNA molecule of the present invention, or the polynucleotide (or nucleic acid) or mRNA molecule of the pharmaceutical composition, vector, or vaccine of the present invention, comprises an RNA molecule or its complement in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that described in the respective sequence number, but at least 90% of the "U"s are replaced with m1ψ. The remaining "U"s may all be unmodified, or one unmodified nucleoside and one or more It may also contain other modified nucleosides.

[0292] Optionally, the polynucleotide (or nucleic acid) or mRNA molecule of the present invention, or the polynucleotide (or nucleic acid) or mRNA molecule of the pharmaceutical composition, vector, or vaccine of the present invention, comprises an mRNA molecule or its complement in which the nucleic acid sequence of the polynucleotide (or nucleic acid) is the same as that described in the respective sequence number, but at least 90% of the "U"s are replaced with m1ψ. The remaining "U"s may all be unmodified, or one unmodified nucleoside and one or more It may also contain other modified nucleosides.

[0293] The mRNA vaccine of the present invention may be administered simultaneously with an immunological adjuvant, such as MF59 (Novartis), TriMix, RNActive (CureVac AG), or RNAdjuvant (also outlined in Wang et al., cited above).

[0294] According to the present invention, isolated cells containing or transfected with the vector of the present invention are also provided.

[0295] According to the present invention, a fusion protein containing the polypeptide of the present invention is also provided.

[0296] According to the present invention, a pharmaceutical composition comprising the mRNA of the present invention, the mRNA vaccine vector of the present invention, or the mRNA vaccine of the present invention, and a pharmaceutically acceptable carrier, excipient, or diluent is further provided.

[0297] According to the present invention, mRNA, mRNA vaccine vectors, mRNA vaccines, or pharmaceutical compositions of the present invention are also provided for use as pharmaceuticals.

[0298] According to the present invention, mRNA, mRNA vaccine vectors, mRNA vaccines, or pharmaceutical compositions of the present invention are further provided for use in the prevention, treatment, or improvement of coronavirus infection.

[0299] The present invention also provides for the use of the mRNA of the present invention, the mRNA vaccine vector of the present invention, the mRNA vaccine of the present invention, or the pharmaceutical composition of the present invention in the manufacture of pharmaceuticals for the prevention, treatment, or improvement of coronavirus infection.

[0300] The present invention also provides a method for inducing an immune response to coronavirus in a subject, comprising administering to the subject an effective amount of the mRNA of the present invention, the mRNA vaccine vector of the present invention, the mRNA vaccine of the present invention, or the pharmaceutical composition of the present invention.

[0301] The present invention also provides a method for immunizing a subject against coronavirus, comprising administering to the subject an effective amount of the mRNA of the present invention, the mRNA vaccine vector of the present invention, the mRNA vaccine of the present invention, or the pharmaceutical composition of the present invention.

[0302] Optionally, the method of the present invention includes administering the mRNA of the present invention, the mRNA vaccine vector of the present invention, the mRNA vaccine of the present invention, or the pharmaceutical composition of the present invention as part of a prime boost regimen.

[0303] If optional, the coronavirus is beta - It's the coronavirus.

[0304] Optional, beta - Coronavirus is of lineage B or lineage C beta - It's the coronavirus.

[0305] Optional, beta - Coronavirus is of lineage B beta - It's the coronavirus.

[0306] Optionally, system B beta - The coronavirus is either SARS-CoV or SARS-CoV-2.

[0307] Optionally, system C beta - The coronavirus is MERS-CoV.

[0308] Optional, beta - The coronavirus is a volatile organic compound (VOC).

[0309] Optional, beta- The coronavirus is the target variant (VOI).

[0310] Optional, beta - The coronavirus is SARS-CoV-2 VOC.

[0311] Optional, beta - The coronavirus is SARS-CoV-2 beta , gamma , delta Alternatively, it is an omicron VOC.

[0312] Optional, beta - The coronavirus is SARS-CoV-2 omicron VOC.

[0313] Optional, beta - The coronavirus is SARS-CoV-2 omicron BA.1.

[0314] Optional, beta - The coronavirus is SARS-CoV-2 omicron BA.2.

[0315] Optional, beta- The coronavirus is SARS-CoV-2 Omicron BA.2.12.

[0316] Optional, beta- The coronavirus is SARS-CoV-2 omicron BA.4.

[0317] Optional, beta- The coronavirus is SARS-CoV-2 omicron BA.5.

[0318] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBB.

[0319] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBB.1.5.

[0320] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BA.2.12.1.

[0321] Optional, beta- The coronavirus is SARS-CoV-2 Omicron BA.2.75.2.

[0322] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BQ.1.1.

[0323] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BA.2.3.20.

[0324] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BA.2.75.

[0325] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BF.7.

[0326] Optional, beta- The coronavirus is SARS-CoV-2 Omicron XBB.1.19.1.

[0327] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBC.

[0328] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BQ.1.12.

[0329] Optional, beta - The coronavirus is SARS-CoV-2 Omicron CH.1.1.1.

[0330] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBB.1.9.1.

[0331] The subjects are selected voluntarily, and the subjects are human.

[0332] Pharmaceutical composition The present invention also provides a pharmaceutical composition comprising the isolated polypeptide of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent.

[0333] Optionally, the pharmaceutical composition of the present invention comprises two or more different isolated polypeptides of the present invention.

[0334] According to the present invention, a pharmaceutical composition comprising the nucleic acid of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent is also provided.

[0335] Optionally, the pharmaceutical composition of the present invention comprises two or more nucleic acid molecules of the present invention that encode different polypeptides of the present invention.

[0336] According to the present invention, a pharmaceutical composition comprising the mRNA molecule of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent is also provided.

[0337] Optionally, the pharmaceutical composition of the present invention comprises two or more mRNA molecules of the present invention that encode different polypeptides of the present invention.

[0338] According to the present invention, a pharmaceutical composition comprising the vector of the present invention and a pharmaceutically acceptable carrier, excipient, or diluent is also provided.

[0339] Optionally, the pharmaceutical composition of the present invention further comprises an adjuvant for enhancing the immune response in a target to a polypeptide encoded by the polypeptide of the composition or a nucleic acid.

[0340] Optionally, the pharmaceutical composition of the present invention further comprises an adjuvant for enhancing the immune response in a target to a polypeptide encoded by the polypeptide of the composition or a nucleic acid.

[0341] According to the present invention, a pseudovirus containing the polypeptide of the present invention is also provided.

[0342] Treatment and Usage The present invention also provides a method for inducing an immune response to coronavirus in a subject, which comprises administering to the subject an effective amount of the polypeptide, nucleic acid, mRNA, vector, pharmaceutical composition, or vaccine of the present invention.

[0343] Furthermore, the present invention also provides a method for immunizing a subject against coronavirus, which comprises administering to the subject an effective amount of the polypeptide, nucleic acid, mRNA, vector, vaccine, or pharmaceutical composition of the present invention.

[0344] The effective dose is the amount that elicits an antigen-specific immune response in the target population.

[0345] The present invention further provides polypeptides, nucleic acids, mRNAs, vectors, vaccines, or pharmaceutical compositions of the present invention for use as pharmaceuticals.

[0346] The present invention further provides polypeptides, nucleic acids, mRNAs, vectors, vaccines, or pharmaceutical compositions of the present invention for use in the prevention, treatment, or improvement of coronavirus infection.

[0347] The present invention also provides for the use of the polypeptide, nucleic acid, mRNA, vector, vaccine, or pharmaceutical composition of the present invention in the manufacture of pharmaceuticals for the prevention, treatment, or improvement of coronavirus infection.

[0348] By choice, the coronavirus is a β-coronavirus.

[0349] The β-coronavirus, chosen at random, is either a β-coronavirus of lineage B or lineage C.

[0350] The β-coronavirus, selected by arbitrary selection, is a β-coronavirus of lineage B.

[0351] Optionally, the β-coronavirus of lineage B is either SARS-CoV or SARS-CoV-2.

[0352] By arbitrary selection, the β-coronavirus of lineage C is MERS-CoV.

[0353] In an optional selection, the immune response was observed in multiple strains of B. beta - Induced by the coronavirus

[0354] Optionally, the immune response to SARS-1 and SARS-2 beta - Induced by the coronavirus

[0355] In optional selection, the immune response was observed in SARS-1 and MERS. beta - Induced by the coronavirus

[0356] In optional selection, the immune response was observed in SARS-2 and MERS. beta - Induced by the coronavirus

[0357] Optionally, the immune response to SARS-1, SARS-2, and MERS beta - Coronavirus It is guided towards S.

[0358] Optional, beta - The coronavirus is a volatile organic compound (VOC).

[0359] Optional, beta - The coronavirus is SARS-CoV-2 VOC.

[0360] Optional, beta - The coronavirus is SARS-CoV-2 lineage B1.248 (Brazil P1 lineage) VOC.

[0361] Optional, beta - The coronavirus is SARS-CoV-2 strain B1.351 (South Africa) VOC.

[0362] Optional, beta - The coronavirus is SARS-CoV-2 beta , gamma or delta It is a VOC (Volatile Organic Compound).

[0363] Optional, beta - The coronavirus is SARS-CoV-2 beta It is a VOC (Volatile Organic Compound).

[0364] Optional, beta - The coronavirus is SARS-CoV-2 gamma It is a VOC (Volatile Organic Compound).

[0365] Optional, beta - The coronavirus is SARS-CoV-2 delta It is a VOC (Volatile Organic Compound).

[0366] Optional, beta - The coronavirus is SARS-CoV-2 alpha It is a VOC (Volatile Organic Compound).

[0367] Optional, beta - The coronavirus is SARS-CoV-2 omicron VOC.

[0368] Optional, beta - The coronavirus is SARS-CoV-2 omicron BA.1.

[0369] Optional, beta - The coronavirus is SARS-CoV-2 omicron BA.2.

[0370] Optional, beta- The coronavirus is SARS-CoV-2 Omicron BA.2.12. (Optional selection) beta- The coronavirus is SARS-CoV-2 omicron BA.4.

[0371] Optional, beta- The coronavirus is SARS-CoV-2 omicron BA.5.

[0372] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBB.

[0373] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBB.1.5.

[0374] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BA.2.12.1.

[0375] Optional, beta- The coronavirus is SARS-CoV-2 Omicron BA.2.75.2.

[0376] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BQ.1.1.

[0377] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BA.2.3.20.

[0378] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BA.2.75.

[0379] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BF.7.

[0380] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBB.1.19.1.

[0381] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBC.

[0382] Optional, beta - The coronavirus is SARS-CoV-2 Omicron BQ.1.12.

[0383] Optional, beta - The coronavirus is SARS-CoV-2 Omicron CH.1.1.1.

[0384] Optional, beta - The coronavirus is SARS-CoV-2 Omicron XBB.1.9.1.

[0385] Using methods well known to those skilled in the art, beta - It is possible to easily determine whether an immune response has been induced against the coronavirus. For example, a pseudo-neutralization assay described in one of the following examples can be used.

[0386] Administration Any suitable route of administration can be used. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, vaginal, rectal, nasal, inhalation, or oral. Parenteral administration, such as subcutaneous, intravenous, or intramuscular administration, is generally achieved by injection. Injectable preparations can be prepared in conventional forms, either as a liquid solution or suspension, a solid suitable for dissolving or suspending in liquid before injection, or as an emulsion. Injectable solutions and suspensions can be prepared from the aforementioned types of sterile powders, granules, and tablets. Administration may be systemic or local. Routes for systemic administration generally include transdermal, oral, and parenteral routes, including, for example, subcutaneous, intravenous, intramuscular, intra-arterial, intradermal, and intraperitoneal injection and / or intranasal administration routes. Common local administration routes include For example, this includes not only topical administration routes, but also intradermal, transdermal, subcutaneous or intramuscular injections, or intra-lesional, intracranial, intrapulmonary, intracardiac and sublingual injections.

[0387] In the case of lipid nanoparticles, the route of administration is often determined by the properties of the nanoparticles and their therapeutic applicability. After intravenous (IV) administration, many lipid nanoparticles can accumulate in the liver. The liver is inherently capable of producing secretory proteins, and therefore, IV administration of lipid nanoparticle-mRNA preparations can be used to produce proteins deficient in hereditary metabolic and hematological disorders, or to produce antibodies that neutralize pathogens or target cancer cells. These applications require protein translation without stimulating an immune response, which may limit the efficiency of repeated administration. However, IV administration can also lead to the accumulation of lipid nanoparticles in multiple lymph nodes throughout the body, which can increase the immune response to mRNA vaccines. For example, IV administration of mRNA vaccines compared to local injection It has been shown to induce a stronger antigen-specific cytotoxic T cell response. Broad distribution of mRNA vaccines in the body can lead to systemic adverse effects; therefore, it may be necessary to develop lipid nanoparticles that enable targeted delivery of mRNA vaccines to tissues with abundant immune cells.

[0388] Local administration routes are also being investigated for mRNA therapeutics. Local administration aims to achieve local therapeutic effects; for example, local injection of lipid nanoparticle-mRNA preparations allows for the supplementation of therapeutic proteins in specific tissues such as the heart, eyes, and brain. Furthermore, lipid nanoparticle-mRNA preparations can be administered to the lungs by inhalation.

[0389] Local administration of mRNA vaccines can also prime a systemic response. For example, intradermal (id), intramuscular (im), and subcutaneous (sc) injections are commonly used for vaccination because resident and recruited antigen-presenting cells (APCs) are present in the skin and muscles and can internalize and process mRNA-encoded antigens. Furthermore, the blood vessels and lymphatic vessels in these tissues help APCs and mRNA vaccines stimulate T-cell immunity, particularly in the excretory lymph nodes. In fact, both intramuscular and intradermal administration of lipid nanoparticle-mRNA vaccines have resulted in robust immune responses at well-tolerated doses in human trials. Vaccination can also be performed by intranasal administration because APCs in peripheral lymph nodes can readily endocytose administered lipid nanoparticle-mRNA preparations.

[0390] mRNA vaccines delivered by lipid nanoparticles may contain cationic lipids and / or ionizable lipids (see Overview: Lipid Nanoparticles for mRNA Delivery, Nature Reviews Materials, 6 1078-1094, 2021). In addition to cationic or ionizable lipids, lipid nanoparticle-mRNA formulations typically contain other lipid components such as phospholipids (e.g., phosphatidylcholine and phosphatidylethanolamine), cholesterol, or polyethylene glycol (PEG)-functionalized lipids (PEG-lipids). These lipids can improve nanoparticle properties such as particle stability, delivery efficacy, tolerability, and biodistribution.

[0391] The compositions of the present invention can be administered by any suitable method, along with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is determined in part by the specific composition to be administered and by the specific 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, alcohol / aqueous solutions, emulsions, or suspensions, and include saline and buffering media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose, and sodium chloride, lactated ringer's, or fixative oil. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as ringer's dextrose-based ones), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0392] Some of the compositions may, in some cases, be administered as pharmaceutically acceptable acid or base-added salts formed by the reaction of 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 the reaction of inorganic bases such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases such as mono-, di-, trialkyl and arylamines and substituted ethanolamines.

[0393] The administration can be achieved by a single or multiple dose. In the context of this 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 depending on the subject's species, age, weight, and overall condition, the severity of the infection being treated, the specific composition used, and the mode of administration. The appropriate dose can be determined by those skilled in the art using only routine experiments.

[0394] This disclosure includes methods for administering mRNA vaccines or DNA vaccines to subjects in need. The exact amount required will vary depending on the subject, depending on the species, age and overall condition, disease severity, specific composition, mode of administration, mode of activity, etc.

[0395] mRNA or DNA is usually formulated in unit dosage forms to ensure ease of administration and uniformity of dosage. However, it should be understood that the total daily dose of mRNA or DNA can be determined by the attending physician within the bounds of appropriate medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient depends on a variety of factors well known in the medical field, including the disorder being treated and its severity; the activity of the specific compound used; the specific composition used; the patient's age, weight, overall health, sex, and diet; the timing of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; and drugs used in combination with or concurrently with the specific compound used.

[0396] The effective dose of mRNA or DNA provided herein is at least 20 pg and can be administered, for example, as a single dose or as two doses of 10 pg each. In some embodiments, the effective dose is a total dose of 20 μg to 300 μg or 25 μg to 300 μg. For example, the effective dose may be a total dose of 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 250 μg, or 300 μg. In some embodiments, the effective dose is a total dose of 20 μg. In some embodiments, the effective dose is a total dose of 25 pg. In some embodiments, the effective dose is a total dose of 50 μg. In some embodiments, the effective dose is a total dose of 75 μg. In some embodiments, the effective dose is a total dose of 100 μg. In some embodiments, the effective dose is a total dose of 150 μg. In some embodiments, the effective dose is a total dose of 200 μg. In some embodiments, the effective dose is a total dose of 250 pg. In some embodiments, the effective dose is a total dose of 300 μg.

[0397] The mRNA or DNA described herein can be formulated into the dosage forms described herein, for example, intranasal, intratracheal, or for injection (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0398] Optionally, mRNA or DNA vaccines are formulated in amounts effective to elicit an antigen-specific immune response in the target population.

[0399] In some embodiments, the effective dose is a total dose of 25 μg to 1000 μg, or 50 μg to 1000 μg. In some embodiments, the effective dose is a total dose of 100 μg. In some embodiments, the effective dose is administered to the subject in a total of two doses of 25 μg. In some embodiments, the effective dose is administered to the subject in a total of two doses of 100 μg. In some embodiments, the effective dose is administered to the subject in a total of two doses of 400 μg. In some embodiments, the effective dose is administered to the subject in a total of two doses of 500 μg.

[0400] Nucleic acid vaccines are administered to the target population at a dose of 10 μg / kg to 400 μg / kg, at the discretion of the patient. In some embodiments, the dose of mRNA 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, 80-200 μg The single dose is 100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg, 80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg, 300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or 300-400 μg. 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 0. In some embodiments, a second dose of the nucleic acid vaccine is administered to the subject on day 21.

[0401] In some embodiments, the mRNA vaccine is administered to the subject by intradermal or intramuscular injection. In some embodiments, the mRNA vaccine is administered to the subject on day 0. In some embodiments, a second dose of the mRNA vaccine is administered to the subject on day 21.

[0402] In a strategy called "prime-boost," a first dose of the mRNA vaccine is administered as a priming step, followed by a second dose as a booster. The prime-boost strategy aims to elicit a stronger overall immune response. The booster may be administered at least one day, at least one week, or at least two, three, four, five, six, or seven weeks after the primer, or at least two, three, four, five, or six months after the primer. For example, the booster may be administered at least three weeks after the primer.

[0403] Pharmacologically acceptable carriers Pharmaceutically acceptable carriers include, but are not limited to, physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carriers and compositions may be sterile, and the formulations may be suitable for various administration modes. Compositions may also contain small amounts of wetting or emulsifying agents, or pH buffers. Compositions may be liquid solutions, suspensions, emulsions, tablets, pills, capsules, sustained-release formulations, or powders. Compositions may be formulated as suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Either sterile physiological saline solution or common pharmaceutical carriers such as sesame oil may be used. The medium may also contain conventional pharmaceutical adjuncts, such as pharmaceutically acceptable salts for adjusting osmotic pressure, buffering agents, preservatives, etc. Other media that can be used in the compositions and methods provided herein include ordinary physiological saline and sesame oil.

[0404] In some embodiments, the composition comprises a pharmaceutically acceptable carrier and / or adjuvant. For example, the adjuvant may be alum, Freund's complete adjuvant, a biological adjuvant, or an immunostimulatory oligonucleotide (such as a CpG oligonucleotide).

[0405] The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional ones. (EW Martin, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton) PA The 15th Edition (1975) includes one or more Therapeutic compositions and compositions and formulations suitable for the delivery of additional pharmaceuticals are described.

[0406] Generally, the properties of the carrier depend on the specific mode of administration used. For example, parenteral formulations typically contain an injectable fluid as a vehicle, which includes pharmaceutically and physiologically acceptable fluids such as water, saline, equilibrium salt solutions, aqueous dextrose, and glycerol. In solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic auxiliary substances such as humectants or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sodium monolaurate.

[0407] Optionally, the polypeptide, nucleic acid, composition, or mRNA vaccine of the present invention may be administered intramuscularly.

[0408] Optionally, the polypeptide, nucleic acid, composition, or mRNA vaccine of the present invention may be administered by inhalation.

[0409] Optionally, the polypeptide, nucleic acid, composition, or mRNA vaccine of the present invention may be administered intramuscularly, intradermally, subcutaneously using a needle or gene gun, or by electroporation.

[0410] Sequence identity The similarity between amino acid sequences or nucleic acid sequences is expressed in terms of similarity between sequences, otherwise it is called sequence identity. Sequence identity is often measured in terms of a percentage of identity (or similarity or homology), with a higher percentage indicating greater similarity between the two sequences. Homologs or variants of a given gene or protein exhibit 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 cited 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; and Altschul et al., Nature It is described in Genet. 6:119-129, 1994. The NCBI Basic Local Alignment Search Tool (BLAST®) (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.

[0411] Sequence identity between nucleic acid sequences or amino acid sequences can be determined by comparing their alignments. If equivalent positions in the comparison sequences are occupied by the same nucleotide or amino acid, the molecules are identical at those positions. Scoring alignment as a percentage of identity is a function of the number of identical nucleotides or amino acids at positions shared by the comparison sequences. When comparing sequences, the optimal alignment takes into account possible insertions and deletions in the sequences. One or more of the array It may be necessary to introduce gaps. The sequence comparison method may use a gap penalty, and when the same number of identical molecules in the sequences are compared, a sequence alignment with as few gaps as possible reflects a higher relevance between the two sequences being compared and achieves a higher score than one with many gaps. Calculating the maximum identity percentage involves generating an optimal alignment that takes the gap penalty into account.

[0412] Suitable computer programs for performing sequence comparisons are widely available in the commercial and public sectors. Examples include MatGat (Campanella et al., 2003, BMC Bioinformatics 4:29, program available from http: / / bitincka.com / ledion / matgat), Gap (Needleman & Wunsch, 1970, J.Mol.Biol.48:443-453), FASTA (Altschul et al., 1990, J.Mol.Biol.215:403-410, program available from http: / / www.ebi.ac.uk / fasta), Clustal W 2.0 and X 2.0 (Larkin et al., 2007, Bioinformatics 23:2947-2948, program available from http: / / www.ebi.ac.uk / tools / clustalw2), and EMBOSS Pairwise Alignment Algorithms (Needleman & Wunsch). References include Wunsch, 1970, cited above; Kruskal, 1983, In: Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Sankoff & Kruskal (eds), pp 1-44, Addison Wesley (the program is available from http: / / www.ebi.ac.uk / tools / emboss / align). All programs can be run using default parameters.

[0413] For example, sequence comparison can be performed using the "needle" method of EMBOSS Pairwise Alignment Algorithms, which determines the optimal alignment (including gaps) when considering the two sequences over their entire length and provides a percentage identity score. Default parameters for amino acid sequence comparison ("Protein Molecule" option) may be Gap Extend penalty: 0.5, Gap Open penalty: 10.0, and Matrix: Blosum 62.

[0414] Sequence comparisons can be performed over the entire length of the reference sequence.

[0415] Conservative amino acid substitutions The mRNA-encoded polypeptide of the present invention is one or more This may include conserved amino acid substitutions. Conservative amino acid substitutions are those that, when made, do little to interfere with the properties of the original polypeptide; that is, the protein's structure, especially its function, is preserved and not significantly altered by such substitutions. Examples of conserved substitutions are shown below.

[0416] [Table 10]

[0417] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the substitution region, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

[0418] Generally, substitutions that are expected to result in the greatest change in protein properties are non-conservative and include, for example, (a) a hydrophilic residue, e.g., serine or threonine, substituting (or being substituted by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine, or alanine; (b) cysteine ​​or proline substituting (or being substituted by) any other residue; (c) a residue with an electrically positive side chain, e.g., lysine, arginine, or histidine, substituting (or being substituted by) an electrically negative residue, e.g., glutamate or aspartate; or (d) a residue with a bulky side chain, e.g., phenylalanine, substituting (or being substituted by) one without a side chain, e.g., glycine.

[0419] Broad-spectrum neutralizing immune response In this specification, the term “broad neutralizing immune response” is used to mean an immune response induced in a subject that is sufficient to inhibit (i.e., mitigate), neutralize, or prevent infection and / or progression of infection by a virus within the coronavirus family. Optionally, a broad neutralizing immune response may be multiple types of β - Sufficient to inhibit, neutralize, or prevent infection and / or progression of coronaviruses (e.g., SARS-CoV and SARS-CoV-2). Optionally, a broad-spectrum neutralizing immune response is also effective. - Multiple types of β within the coronavirus lineage - Coronaviruses (e.g., SARS-CoV, SARS-CoV-2, and Bat SL-CoV-WIV1, among others, are multiple types of β within the subgenus Salvecovirus) - Sufficient to inhibit, neutralize, or prevent infection and / or progression of coronavirus infection. Optionally, broad-spectrum neutralizing immune responses are effective against different β strains such as lineage B (e.g., SARS-CoV, SARS-CoV-2) and lineage C (e.g., MERS-CoV). - Sufficient to inhibit, neutralize, or prevent infection and / or progression of coronaviruses of the coronavirus lineage. Optionally, a broad-spectrum neutralizing immune response is effective against almost all different β- -Sufficient to inhibit, neutralize, or prevent infection and / or progression of coronavirus infection. Optionally, broad-spectrum neutralizing immune responses are sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by most or all different viruses of the coronavirus family. Optionally, broad-spectrum neutralizing immune responses are, beta , gamma , delta The broad-spectrum neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by most or all volatile organic compounds (VOCs) of SARS-CoV-2, including Omicron (BA.1). Optionally, the broad-spectrum neutralizing immune response is effective against SAR-CoV, WIV16, RaTG13, SARS-CoV-2, and SARS-CoV-2. beta SARS-CoV-2 gamma SARS-CoV-2 delta The broad-spectrum neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by SARS-CoV-2 omicrons (BA.1, BA.2, BA.2.12.1, BA.4, BA.5, XBB 1.5). Optionally, the broad-spectrum neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by SARS-CoV-2 omicrons BA.2.75, BA.2.75.2, BA.2.3.20, BQ.1.1, BA.2.12, BA.4 / 5, and XBB. Optionally, the broad-spectrum neutralizing immune response is sufficient to inhibit, neutralize, or prevent infection and / or progression of infection by SARS-CoV-2 omicrons BA.2.75 and BA.2.3.20.

[0420] Immune responses can be humoral and / or cellular immune responses. Cellular immune responses are the responses of cells in the immune system, such as B cells, T cells, macrophages, or polymorphonuclear cells, to stimuli such as antigens or vaccines. Immune responses can include any cells in the body involved in the host defense response, such as epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate immune responses or inflammation.

[0421] Optionally, polypeptides encoded by the mRNA of the present invention induce a protective immune response. A protective immune response refers to an immune response that protects an object from infection or disease (i.e., prevents infection or the development of an infection-related disease). Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and / or activity of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, or antibody production.

[0422] Optionally, the polypeptides of the present invention induce a protective immune response. A protective immune response refers to an immune response that protects an object from infection or disease (i.e., prevents infection or the development of an infection-related disease). Methods for measuring immune responses are well known in the art and include, for example, measuring the proliferation and / or activity of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, or antibody production.

[0423] Optionally, the polypeptides of the present invention can induce the production of antibodies and / or T cell responses in humans or non-human animals to which the polypeptide has been administered (either as polypeptides or, for example, expressed from an administered nucleic acid expression vector).

[0424] Optionally, the polypeptide of the present invention is a glycosylated polypeptide.

[0425] Optionally, the mRNA-encoded polypeptide of the present invention can induce antibody production and / or T cell responses in humans or non-human animals (e.g., those expressed from administered mRNA vaccines) to which the mRNA has been administered. Embodiments of the present invention are described below merely as examples with reference to the accompanying drawings. [Brief explanation of the drawing]

[0426] [Figure 1]The structure of the SARS S protein is shown. 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 far more diverse than the highly conserved C-terminal region (Dong et al, Genomic and protein structure modelling analysis depicts the origin and infectivity of 2019-nCoV, a new coronavirus which caused a pneumonia outbreak in Wuhan, China. 2020). The figure shows the S domain, including the S1 and S2 domains, which are involved in receptor binding and cell membrane fusion, respectively. [Figure 2] Details of the largest VOI circulating globally as of April 3, 2023 are shown. In the table, x = includes its sublineage (BN, CH, and others). Omicron-Omicron recombinants XBF and XBK, which share the same spike as BA.2.75, are monitored under the BA.2.75 lineage. Y = W152R, F157L, I210V, G257S, D339H, G446S, N460K, Q493 (reverted) Z = XBB and sublineages, excluding XBB.1.5-like lines. Recombinant lines of BJ.1 (BA.2.10.1.1) and BM.1.1.1 (BA.2.75.3.1.1.1) A = Monitors umbrellas of SARS-CoV-2 lines that have similar spike protein profiles and are characterized by specific sets of mutations (S:Q183E, S:F486P, and S:F490S). This umbrella includes, for example, systems XBB.1.5, XBB.1.9.1*, XBB.1.9.2*, and XBB.1.16. [Figure 3] Figure 3a shows the alignment of the amino acid sequences of CoV_S_T2_35, CoV_S_T2_36, and Omicron_Vaccine with reference sequences SEQ ID NOs. 4-29. Figure 3b shows the alignment of CoV_S_T2_35 at the position where the designed sequence contains a novel amino acid residue, compared to Delta C. Figure 3b shows the alignment of CoV_S_T2_36 at the position where the residue contains a novel amino acid residue, compared to BA.1C. [Figure 4] This image shows the surface representation of the extravirion region of the SARS-CoV-2 spike protein. The three subunits are colored pale yellow (center subunit), pale blue (left subunit), and gray (right subunit). Mutations reported in different variants are colored red (shown as shaded dots in the variant representation). Mutations introduced into the spike vaccine antigen are colored orange in T2_29, T2_35, and T2_36 (shown as shaded dots in the design antigen representation). [Figure 5] The immunization and blood sampling schedule (A), neutralization data for each blood sampling (B), and neutralization data for guinea pigs primed with WTdER and T2_29, T2_29+Q, and T2_29+Q+dER design DNA sequences (C) and boosted with T2_29+Q+dER design MVA sequence (D) are shown. The box plots are color-coded according to the primer vaccine, with WTdER, T2_29, T2_29+Q, and T2_29+Q+dER in that order from left to right for each challenged PV. [Figure 6] The neutralization data for mice primed (B) and boosted (C) with WTdER, ancestral, and T2_35 and T2_36 mRNA design sequences are shown. The box plots are color-coded according to the vaccine used, and for each challenged PV, from left to right, are PBS, T2_35, T2_36, BA.1, and ancestral. [Figure 7] This shows the final blood neutralization data for coronavirus challenge in mice. The box plots are color-coded according to the mRNA vaccine used, and for each challenge PV, from left to right are PBS, T2_35, T2_36, BA.1, and ancestral. [Figure 8] This shows distance-based phylogenetic trees for wild-type coronavirus and the designed sequence, generated using observed distances. [Figure 9] Figure 5a shows the VOC RBD-binding antibody levels in guinea pigs at blood sampling 4 of the schedule, as indicated by ELISA. [Figure 10]This chart shows the neutralizing titers of guinea pigs immunized with the DNA T2-29 vaccine. Data points for the WT vaccine appear on the left for each coronavirus pseudovirus, while data points for the combined T2-29 vaccine appear on the right for each coronavirus pseudotype. [Figure 11] We show neutralization data from guinea pigs immunized with a designed T2_29 DNA construct and then boosted with MVA T2_29+Q+dER. [Figure 12] This shows neutralization data from mice immunized with T2_35 and T2_36 mRNA vaccines, and presents a statistical comparison between COV-S-T2_36 and BA.1. [Figure 13] This shows neutralization data from mice immunized with T2_35 and T2_36 mRNA vaccines, along with statistical comparisons and IC50 neutralization values ​​for all vaccine groups. [Figure 14] This shows neutralization data from mice immunized with T2_35 and T2_36 mRNA vaccines, along with statistical comparisons and IC50 neutralization values ​​for all vaccine groups. [Figure 15] This report shows neutralization data for mice immunized with T2_35 and T2_36 mRNA vaccines, as well as Wuhan and BA.1 spike vaccines, against SARS-CoV-1 and SARS-like pseudoviruses. Data are based on statistical differences between immunized and untreated animals. Mann-Whitney U test (white = ns, light gray = *p<0.05, dark gray = **p<0.01). [Figure 16-1] This shows the amino acid sequence alignment of CoV_S_T2_35(deome) (SEQ ID NO: 1), COV_S_T3_1(deome_v2) (SEQ ID NO: 31), and COV_S_T3_2(deome_v3) (SEQ ID NO: 32). Differences between sequences are indicated by residues enclosed in squares. [Figure 16-2] This shows the amino acid sequence alignment of CoV_S_T2_35(deome) (SEQ ID NO: 1), COV_S_T3_1(deome_v2) (SEQ ID NO: 31), and COV_S_T3_2(deome_v3) (SEQ ID NO: 32). Differences between sequences are indicated by residues enclosed in squares. [Figure 16-3]This shows the amino acid sequence alignment of CoV_S_T2_35(deome) (SEQ ID NO: 1), COV_S_T3_1(deome_v2) (SEQ ID NO: 31), and COV_S_T3_2(deome_v3) (SEQ ID NO: 32). Differences between sequences are indicated by residues enclosed in squares. [Figure 17-1] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-2] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-3] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-4] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-5] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-6]The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-7] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-8] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-9] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-10] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-11] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-12]The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-13] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 17-14] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with delta C representing the alignment reference sequence. [Figure 18-1] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-2] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-3] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-4]The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-5] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-6] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-7] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-8] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-9] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-10]The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-11] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-12] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-13] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 18-14] The amino acid sequence alignment of the designed S proteins CoV_S_T2_35(deome) (SEQ ID NO: 1) and CoV_S_T2_36(omid) (SEQ ID NO: 2) with previously designed S protein sequences, RBD sequences, and reference sequences is shown, with BA.1 being the alignment reference sequence. [Figure 19]This shows the in-silico designs of the vaccine antigens T2_32 (also known as CoV_T2_29+Q+dER), T2_35, and T2_36. It also shows the surface representation of the extravirion region of the SARS-CoV-2 spike protein. The three subunits are colored pale yellow (center subunit), pale blue (left subunit), and gray (right subunit). Mutations reported in different variants are colored red (shown as shaded dots on the variant representation). Mutations introduced into the spike vaccine antigens are colored orange in T2_32, T2_35, and T2_36 (shown as shaded dots on the designed antigens). [Figure 20] This shows the immunogenicity testing of T2_32 (also known as CoV_T2_29+Q+dER) in guinea pigs. A) Blood sampling and immunization schedule in guinea pigs (DNA prime, followed by MVA boost). B) Distribution of neutralizing titers against Wu-Hu-1 pseudotype in immunization with WTdER. The x-axis represents the number of blood samples, and the y-axis represents the log10 (IC50) value. C) Distribution of neutralizing titers of blood samples 4 against Wu-Hu-1 and VOCs-beta, gamma, delta, BA.1, BA.2, XBB, and XBB.1.5. The x-axis represents the pseudovirus tested for neutralization, and the y-axis represents the log10 (IC50) value. The box plots are color-coded according to vaccine, and for each PV tested, WTdER and T2_32 are shown from left to right. D) Distribution of neutralizing titers of blood sample 6 against Wu-Hu-1 and VOCs-beta, gamma, delta, BA.1, BA.2, XBB, and XBB.1.5. The x-axis represents the pseudovirus tested for neutralization, and the y-axis represents the log10(IC50) value. The box plots are color-coded according to the vaccine, with WTdER and T2_32 in order from left to right for each tested PV. The Mann-Whitney U test is used as a statistical significance test for all plots (p-values: *≦0.05, **<0.01, ***≦0.001). Distributions that are not statistically significant are not labeled in the plots. [Figure 21]This shows the immunogenicity studies of T2_35 and T2_36 in mice. A) Blood collection and immunization schedule for T2_35 and T2_36 mRNA vaccines in mice. B) Distribution of neutralization titers of final blood collections for Wu-Hu-1 and VOCs. The x-axis represents the pseudovirus tested for neutralization, and the y-axis represents the log10(IC50) value. The box plots are color-coded according to the vaccine, and for each PV tested, from left to right are Vehicle, T2_35, T2_36, BA.1, and Wuhan. The Mann-Whitney U test is used as a statistical significance test for all plots (p-values: *≦0.05, **<0.01, ***≦0.001). Distributions that are not statistically significant are not labeled in the plots. C) Heatmap display of median log10(IC50) values ​​for all vaccines and SARS-CoV-2 variants tested. The darker the color, the higher the log10IC50. All medians less than 1.5 are plotted as zero. [Figure 22] This shows the immunogenicity test of CoV_S_T2_35 (Deome, SEQ ID NO: 1) in mice. Mice were immunized twice with mRNA at week 0 and week 3, and blood samples were collected at 3-week intervals starting from day 0. The data shown uses serum from the final blood collection (6 weeks after boost). The x-axis represents the pseudovirus that underwent neutralization testing, and the y-axis represents the log10 (IC50) value. [Figure 23-1] This study presents immunogenicity studies of optimized coronavirus "superspike" mRNA constructs COV_S_T2_35 (Deome, SEQ ID NO: 1), COV_S_T3_1 (Deome_v2, SEQ ID NO: 31), and COV_S_T3_2 (Deome_v3, SEQ ID NO: 32) in mice. Mice were immunized with two doses of 10 μg of mRNA in 100 μL of vehicle, with a 3-week interval between doses. Blood samples were collected from mice 3 weeks and 6 weeks after the first dose. The final blood sample was collected 9 weeks after the first dose. Serum was challenged with a SARS-CoV-2 lentivirus pseudovirus expressing the VOC spike protein. The x-axis represents the pseudovirus that underwent neutralization testing, and the y-axis represents the log10 (IC50) value. [Figure 23-2]This study presents immunogenicity studies of optimized coronavirus "superspike" mRNA constructs COV_S_T2_35 (Deome, SEQ ID NO: 1), COV_S_T3_1 (Deome_v2, SEQ ID NO: 31), and COV_S_T3_2 (Deome_v3, SEQ ID NO: 32) in mice. Mice were immunized with two doses of 10 μg of mRNA in 100 μL of vehicle, with a 3-week interval between doses. Blood samples were collected from mice 3 weeks and 6 weeks after the first dose. The final blood sample was collected 9 weeks after the first dose. Serum was challenged with a SARS-CoV-2 lentivirus pseudovirus expressing the VOC spike protein. The x-axis represents the pseudovirus that underwent neutralization testing, and the y-axis represents the log10 (IC50) value. [Examples]

[0427] [Table 11]

[0428] Example 1 - Vaccine Sequence This embodiment provides amino acid and nucleic acid sequences of full-length S proteins for embodiments of the present invention known as CoV_S_T2_35 (Deome), CoV_S_T2_36 (Omid), and Omicron_Vaccine.

[0429] >CoV_S_T2_35(Deom)(Sequence ID 1) Amino acid sequence: MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGK QGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL ALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCT LKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCV ADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYN YKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNG VAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFN FNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGT NTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYEC DIPIGAGICASYQTHTNSRGSASSVASQSIIAYTMSLGAENSVAYNNNSIAIPTNFTISV TTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVF AQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFVKQYGDCLG DIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQM AYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTL VKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASA NLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAIC HDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQ PELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE LGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC

[0430] >CoV_S_T2_36(Omid)(Sequence ID 2) Amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNN ATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN FKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA DYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNY KLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGV EGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN TSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECD IPIGAGICASYQTQTKSHGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGITITSGWTFGAGAALQIPFAMQMA YRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLV KQLSSKFGAISSVLNDIFSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCSCGSCC

[0431] >Omicron_Vaccine (SEQ ID NO: 3) Amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNN ATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN FKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVA DYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNY KLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGV AGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN TSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECD IPIGAGICASYQTQTKSHGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGITITSGWTFGAGAALQIPFAMQMA YRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLV KQLSSKFGAISSVLNDIFSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCSCGSCC

[0432] Example 2 Differences in amino acid sequences of influenza Tier 2 (T2) S protein vaccine candidates CoV_S_T2_35, CoV_S_T2_36, and Omicron_Vaccine. This example identifies the differences in amino acid residues in the newly designed sequences CoV_S_T2_35, CoV_S_T2_36, and Omicron_Vaccine compared to previous Tier2(T2)S protein and RBD designs, as well as the wild-type S protein sequence. Figure 3 shows the alignment of the amino acid sequences of CoV_S_T2_35 and CoV-S_T2_36 with the coronavirus S protein reference sequences SEQ ID NOs. 4-13 and the previously designed vaccine sequences SEQ ID NOs. 14-29. Specifically, Figure 3a shows the sequence alignment at the position containing the novel residues in CoV_S_T2_35, and Figure 3b shows the alignment for CoV_S_T2_36. Figures 17 and 18 show the sequence alignment across the entire S protein sequence.

[0433] Table 3 below summarizes the novel amino acid residue changes in the amino acid sequence of the coronavirus S protein of an embodiment of the present invention known as CoV_S_T2_35 (SEQ ID NO: 1). delta The amino acid residues at positions 680, 681, 683, 984, and 985 of CoV_S_T2_35, corresponding to residue positions 680, 681, 683, and 984-985 of C (SEQ ID NO: 8), are novel. In particular, CoV_S_T2_35 includes the RRAR680-684GSAS and KV984-985PP mutations to stabilize the vaccine design. The designed sequence also includes a C-terminal cleavage, where 19 amino acid residues, including the ER signal sequence, delta The carbon molecule is deleted at residue positions 1253–1271. This cleavage improves the surface expression of the antigen.

[0434] Table 6 below summarizes the novel amino acid residue changes in the amino acid sequence of the coronavirus S protein of an embodiment of the present invention known as CoV_S_T2_36 (SEQ ID NO: 2). The amino acid residues at positions 679, 680, 682, 983, and 984 of CoV_S_T2_36 are novel, corresponding to residue positions 679, 680, 682, 983, and 984 of Omicron BA.1 (SEQ ID NO: 9). In particular, CoV_S_T2_36 includes the RRAR679-682GSAS and KV983-984PP mutations to stabilize the vaccine design. The designed sequence also includes a C-terminal cleavage, where 19 amino acid residues, including the ER signaling sequence, are deleted at residue positions 1252-1270 of Omicron BA.1C. This cleavage improves antigen surface expression.

[0435] The Omicron vaccine contains the wild-type Omicron BA.1 sequence with the KV983-984PP mutation to stabilize the vaccine design. The designed sequence also includes a C-terminal truncation, where 19 amino acid residues, including the ER signaling sequence, are deleted at residue positions 1252–1270 of Omicron BA.1C. The designed sequence is used as a direct comparison to the CoV_S_T2_35 and CoV_S_T2_36 vaccine designs.

[0436] [Table 3-5]

[0437] [Table 6-5]

[0438] Amino acid sequence of the SARS2 S protein reference sequence: ·Wuhan_Hu_1 (Sequence number 4) · alpha C (Sequence ID 5) · beta C (Sequence ID 6) · gamma C (Sequence ID 7) · deltaC (Sequence ID 8) Omicron BA.1C (Sequence ID 9) Omicron BA.2C (Sequence ID 10) Omicron BA.2.12.1C (Sequence ID 11) Omicron BA.4C (Sequence ID 12) Omicron BA.5C (Sequence ID 13) The following is shown.

[0439] >NC_045512.2_Wuhan_Hu_1 (Wuhan) (Sequence number 4) Amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSY ECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGITITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDP LQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGVKLHYT

[0440] > alpha C (Sequence ID 5) Amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGL TGTGVLTESNKKFLPFQQFGRDIDDTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCL IGAEHVNNSYECDIPIGAGICASYQTQTNSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPINFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL QYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGL TVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNT LVKQLSSNFGAISSVLNDILARLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHV TYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTHNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASVVNIQKEIDRLNEVANNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0441] > beta C (Sequence ID 6) Amino acid sequence:

[0442] > gamma C (Sequence ID 7) Amino acid sequence:

[0443] > delta C (Sequence ID 8) Amino acid sequence: MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGK QGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLL ALHRSYLTPGDSSSGWTAGAAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCT LKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCV ADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYN YKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNG VEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFN FNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGT NTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYEC DIPIGAGICASYQTQTNSRRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISV TTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVF AQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLG DIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGITITSGWTFGAGAALQIPFAMQM AYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTL VKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASA NLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAIC HDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQ PELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE LGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSE PVLKGVKLHYT

[0444] >Omicron BA.1C (Sequence ID 9) Amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNN ATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN FKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVA DYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNY KLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGV AGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN TSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECD IPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMA YRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLV KQLSSKFGAISSVLNDIFSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEP VLKGVKLHYT

[0445] >Omicron BA.2C (Sequence ID 10) Amino acid sequence: MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVT WFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNA TNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQ GNFKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVA DYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNY KLPDDFTGCVIAWNSNKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGV AGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN TSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECD IPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMA YRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLV KQLSSKFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEP VLKGVKLHYT

[0446] >Omicron BA.2.12.1C (Sequence ID 11) Amino acid sequence: MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVT WFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNA TNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQ GNFKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLA LHRSYLTPGDSSSGWTAGAAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL KSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVA DYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNY KLPDDFTGCVIAWNSNKLDSKVGGNYNYQYRLFRKSNLKPFERDISTEIYQAGNKPCNGV AGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNF NGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTN TSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECD IPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENLVAYSNNSIAIPTNFTISVT TEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFA QVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD IAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGITITSGWTFGAGAALQIPFAMQMA YRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLV KQLSSKFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASAN LAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICH DGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQP ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEP VLKGVKLHYT

[0447] >Omicron BA.4C (Sequence ID 12) Amino acid sequence: MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVT WFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATN VVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN FKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALH RSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS FTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADY SVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKL PDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAG VNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS NQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIP IGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTE ILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQV KQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIA ARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYR FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQ LSSKFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLA ATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG KAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPEL DSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK YEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL KGVKLHYT

[0448] >Omicron BA.5C (Sequence ID 13) Amino acid sequence: MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVT WFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATN VVIKVCEFQFCNDPFLDVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGN FKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLPIGINITRFQTLLALH RSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS FTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADY SVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKL PDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAG VNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG LTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS NQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIP IGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTE ILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQV KQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIA ARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYR FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQ LSSKFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLA ATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDG KAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPEL DSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGK YEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVL KGVKLHYT

[0449] Amino acid sequences of previously designed Tier 2 (T2)S proteins and RBD designs: · CoV_T2_1 (Sequence ID 14) • CoV_T2_8 (Sequence ID 15) · CoV_T2_9 (Sequence ID 16) • CoV_T2_10 (Sequence ID 17) · CoV_T2_11 (Sequence ID 18) · CoV_T2_13 (Sequence ID 19) • CoV_T2_14 (Sequence ID 20) • CoV_T2_15 (Sequence ID 21) · CoV_T2_16 (Sequence ID 22) • CoV_T2_17 (Sequence ID 23) · CoV_T2_18 (Sequence ID 24) • CoV_T2_19 (Sequence ID 25) · CoV_T2_20 (Sequence ID 26) • CoV_T2_29 (Sequence ID 27) • CoV_T2_31 (Sequence ID 28) • CoV_T2_32 (Sequence ID 29) This is disclosed in the sequence listing of this application.

[0450] Example 3 Digitally optimized immune spike vaccines induce a broad neutralization response against concerning variants of SARS-CoV-2.

[0451] overview The successive waves of SARS-CoV-2 variants of concern (VOCs) have increased their ability to evade existing immunity in both vaccinated and infected populations. Currently approved SARS-CoV-2 vaccines have a poor ability to induce or boost neutralizing antibodies against the latest variants. A new vaccine strategy capable of inducing broad protective immunity across VOCs is needed. The evolution of SARS-CoV-2 variants can be reproduced from detailed global surveillance efforts and epidemiological sequence data on VOCs. Using this data, the inventors employed a structure-based approach to computationally generate artificial spike genes designed to induce neutralizing antibody responses across the VOC spectrum. The first study involved the Q498R variant, a variant later acquired by the Omicron lineage VOC, and a VOC variant-informed spike called T2_29, which has multiple versions including C-terminal truncated and Q498R variants. Three DNA immunizations in guinea pigs between C-terminally cleaved ancestral spikes with or without the C-terminal cleavage and Q498R mutation and T2_29 spikes revealed superior immune responses across VOCs with T2_29 and modified T2_29 constructs compared to the C-terminally cleaved ancestral construct. We further boosted all groups with MVA expressing T2_29 with the C-terminal cleavage and Q498R modification. MVA boosting significantly increased the immune response across all groups to all mutants tested. However, delta Since the immune response to omicrons was low compared to other VOCs, the inventors designed second-generation vaccine antigens T2_35 and T2_36. These were administered to mice as mRNA in lipid nanoparticle formulations to target VOCs, particularly delta We also studied the immune response to the newly formed Omicron line variants. Superior broad-spectrum neutralizing ability was observed in both T2_35 and T2_36 compared to mice immunized with the original Wuhan or Omicron BA.1 spike immunogen. These findings demonstrate the potential of a novel, computationally based, structure-informed synthetic spike gene designed to induce a pan-mutant neutralizing immune response.

[0452] Introduction Over the past two years, SARS-CoV-2 has acquired numerous spike mutations with varying degrees of effect on its interaction with the host, as well as its ability to evade pre-existing human immune responses acquired through vaccination and / or infection. In addition to the evolution of SARS-CoV-2 in humans, the virus has been reported to have been transmitted to other mammals, such as certain species of mink, cats, dogs, and deer. Interspecies transmission of SARS-CoV-2, resulting in species-specific variants, provides further dimensions to the rate of evolution, their fitness, and immune-evading characteristics, which could enable future outbreaks of SARS-CoV-2 variants. Since late 2020, alpha , beta , gamma , delta Numerous volatile organic compounds (VOCs) have been reported, starting with the latest variants of the Omicron lineage. The evolution of the spike protein enables immune escape and evasion, influenced by several different selective pressures, including immunopressure. The emergence of adaptive mutations in the S protein can strongly influence host tropism and viral transmission. Faced with a growing immune population, immunological evasion from host immunity acquired during previous infection and / or vaccination is necessary for future variants to acquire advantageous changes that allow them to replicate and transmit within immune human populations.

[0453] With the subsequent emergence of various concerning variants (VOCs), the levels of vaccine-induced neutralizing antibodies induced by the ancestral spike antigen (Wuhan Hu-1 strain) used by all current-generation COVID-19 vaccines have decreased. Of these, deltaOmicron and Omicron subvariants have been reported to have higher transmission rates and immune evasion from both innate and vaccine-acquired immunity. This necessitates an urgent update of current SARS-CoV-2 vaccines that still use ancestral strains. Continued use of ancestral sequence-based vaccines is less effective in promoting a de novo response to novel epitopes of new variants. Leading COVID-19 mRNA vaccine manufacturers have provided better protection against variants of the Omicron lineage by adding the Omicron BA.1 spike antigen as a bivalent vaccine to Wuhan spike-based vaccines, thereby matching the vaccine to the Omicron lineage and administering it as either a monovalent or bivalent vaccine. Matching a vaccine to a specific lineage may be beneficial in providing protection against newly emerging variants from vaccine-matching lineages, but may not provide desirable protection against emerging antigenically different lineages of SARS-CoV-2 or re-emergences of already reported antigenically different lineages of SARS-CoV-2. To circumvent this problem, we have developed a next-generation single-spike-based vaccine antigen that expresses diverse epitopes covering the majority of known VOCs. These novel vaccine antigens, T2_29, T2_35, and T2_36, are derived from the ancestral Wuhan spike. delta It showed considerable neutralization against SARS-CoV-2 pseudotypes expressing the strain, as well as against Omicron BA.1, BA.2, and BA.4 / 5 mutants.

[0454] method Computer-based design of vaccine antigens The consensus sequence was determined using the sequence deposited in the NCBI virus, for each VOC, i.e., alpha , beta , gamma , deltaNext-generation spike-based vaccine antigens were generated for , and omicron BA.1. Each mutation was mapped to a different region of the spike, and clusters of mutations from different structural domains were sequentially combined to generate next-generation spike-based vaccine antigens. The structural integrity of the obtained vaccine antigens was confirmed by generating homology models using Modeller software.

[0455] Plasmid generation and transformation The vaccine design sequences were gene-optimized and adapted to human codon usage using the GeneOptimizer algorithm. These genes were cloned into pEVAC (GeneArt, Germany) via restriction digestion. Plasmids were transformed via heat shock into chemically induced competent E. coli DH5α cells (Invitrogen18265-017). Plasmid DNA was extracted from bacterial cultures transformed via a plasmid mini-kit (Qiagen12125). All plasmids were then quantified using UV spectrophotometric analysis (NanoDrop®, Thermo Scientific).

[0456] Vaccination experiments in guinea pigs Four groups of four 7-week-old female Hartley guinea pigs were purchased from Envigo (Maastricht, Netherlands). The guinea pigs were immunized at two-week intervals by intradermal administration of 200 μg of DNA vaccine containing the antigen gene in a pURVac vector, using a Pharmajet (copyright) instrument, in a total volume of 200 μL into the hind limbs. The animals received three DNA doses via the same route, and then, seven weeks after the first three doses, were boosted with MVA via the intramuscular route at a dose of 1 e7 PFU / dose. Blood was collected from the saphenous vein at two-week intervals.

[0457] Vaccination experiments in mice Five groups of six 8-10 week old female BALB / c mice were purchased from Charles River Laboratories (Kent, UK). The mice were immunized twice at 3-week intervals. The vaccine antigens T2_35 and T2_36, modified ancestral type and modified omicron BA.1 were delivered via mRNA in lipid nanoparticle formulations. A total of 100 μL of PBS containing 10 μg of lipid-encapsulated mRNA was administered intramuscularly to both hind limbs. The untreated mouse group received 100 μL of PBS. Blood samples were collected at 3-week intervals.

[0458] Generation of lentivirus pseudotypes Lentiviral pseudotypes were generated by transient transfection of HEK293T / 17 cells with packaging plasmids p8.91 and pCSFLW and different spike expression plasmids using Fugene-HD transfection reagents. After 48 hours, the supernatant was collected, filtered through 0.45 μm, and titrated onto HEK293T / 17 cells transiently expressing human ACE-2 and TMPRSS2. The target cells used were HEK293T / 17 cells transfected 24 hours prior with 2 μg of huACE-2 and 75 ng of TMPRSS2.

[0459] Pseudotype-based microneutralization assay The pseudotype-based microneutralization assay was performed as previously described. Briefly, serial dilutions of serum, along with SARS-CoV-2 spike-containing lentivirus pseudotypes, were incubated in 96-well leukocyte culture plates at 37°C and 5% CO2 for 1 hour. Next, 1.5 x 10⁴ HEK293T / 17 cells transiently expressing human ACE-2 and TMPRSS2 were added per well and incubated in a humidified incubator at 37°C and 5% CO2 for 48 hours. Bright-Glo (Promega) was then added to each well, and luminescence was read after a 5-minute incubation period. Experimental data points were normalized to 100% and 0% neutralization controls, and nonlinear regression analysis was performed in GraphPad Prism 9 to generate neutralization curves and IC50 values.

[0460] statistical analysis For all comparisons, a two-tailed Mann-Whitney U test was performed using the Python sklearn package. All plots were generated using the Python Matplotlib package.

[0461] result Computer-based design of vaccine antigens The structure of the SARS-CoV-2 spike protein can be antigenically divided into three distinct regions: the N-terminal domain (NTD), the receptor-binding domain (RBD), and the stalk region. The RBD contains most of the experimentally characterized epitopes, followed by the NTD and the stalk. The relevance of these epitopes in protection against SARS-CoV-2 can also be recognized from the observation of multiple mutations in the RBD and NTD in the SARS-CoV-2 VOC. For our next-generation mutant vaccine antigen, we clustered all reported mutations in the VOC. alpha , beta , gamma , delta , and omicron (BA.1) are inserted into the NTD, RBD, and stalk regions. Regarding our bioinformatics analysis, we consider the epitopes in the NTD, RBD, and stalk to be non-synergistic in inducing immune responses, and it is important to note that the immune responses to these domains are independent of each other. After the epitopes were clustered, antigens were generated by sequential combinations of different VOC-specific mutations in the NTD, RBD, and stalk. Only mutations reported in the immunodominant region were considered for design. Based on these combinations, the first-generation spike vaccine antigen T2_29 (Figure 4) was developed. alpha , beta , and gammaThe variants were generated using available data on the variants. The modified T2_29 spike was further modified into three other antigens, namely T2_29+Q and T2_29+Q+dER. The variant Q498R was observed to be prominent in the target variant of the circulating SARS-CoV-2 prior to April 2021, and was incorporated into the T2_29 backbone, giving rise to the T2_29+Q design as a preemptive antigen design for future variants. It is interesting to note that the Q498R variant was later acquired by the Omicron variant in late 2021. A C-terminal deletion version of T2_29+Q was also generated for comparison. The deletion of 19 amino acids from the C-terminus was reported to result in better expression of the spike protein on the cell surface compared to the full-length version, and therefore, a greater presence of the antigen. We also deleted this C-terminal region from the WT ancestral type as a control (hereinafter referred to as WTdER). All of these vaccine antigens have stabilizing double proline mutations, as reported in most current vaccines. Our first-generation antigen T2_29 is delta And, since they were designed before the global pandemic of omicron, the inventors further generated second-generation spike antigens T2_35 and T2_36 (Figure 4) to contain these VOCs. In addition to the stabilizing double proline mutation, the inventors also modified the furin cleavage site RRAR to GSAS and introduced a C-terminal cleavage.

[0462] First-generation spike vaccine antigens delivered by DNA and MVA in guinea pigs Guinea pigs were immunized three times with the antigens T2_29, T2_29+Q, T2_29+Q+dER, and WTdER in DNA vectors, and boosted once with MVA expressing T2_29+Q+dER (Figure 5A). Neutralizing titers were analyzed over time for WTdER against pseudoviruses (PVs) expressing VOC spikes. Neutralizing antibodies peaked in blood sample 4 after three immunizations and in blood sample 6 after MVA boosting (Figure 5B). Neutralizing titers for all VOCs and ancestral sequences were measured for these blood samples (Figures 5C and 5D). The first-generation spike vaccine antigen T2_29 and its modifications, namely T2_29+Q and T2_29+Q+dER, were able to induce a broad neutralizing response against all VOCs tested. T2-29-based antigens, compared to WTdER, showed the following after three doses of DNA vaccine: alpha , beta , gamma , and yielded at least a twice as good a neutralization response against omicron (Figure 5C). Ancestral sequence and delta The neutralizing antibody titers against both T2_29 and T2_29+Q+dER were equivalent to those of WTdER (Figure 5C). However, a lower titer was observed for T2_29+Q before MVA boosting. WTdER resulted in a very weak neutralizing antibody titer against omicron, while all of our vaccine antigens resulted in a strong neutralizing antibody response against omicron. Interestingly, T2_29+Q showed a lower neutralizing titer against omicron compared to T2_29 and T2_29+Q_dER. We believe that the higher neutralizing titer of T2_29+Q_dER compared to T2_29+Q is due to higher construct expression. T2_29 is, delta It is important to note that these do not include many of the mutations reported in omicron variants. delta And because it was designed before the Omicron pandemic. delta Furthermore, despite lacking many of the key mutations reported in omicron neutralization, T2_29 induces a high titer against omicrons. deltaFor this, titers comparable to the wild type were induced. The inventors further boosted all groups of guinea pigs with MVA expressing T2_29+Q+dER. The inventors selected this particular construct because it was antigenically close to Omicron BA.1. Boosting with MVA significantly increased the neutralizing titers of all vaccine antigens (Figure 5D). Most importantly, the neutralizing titer of WTdER against Omicron BA.1 increased threefold when boosted with MVA expressing T2_29+Q+dER. This strongly supports the applicability of these spike antigens to boost the immune response against variants emerging in already vaccinated or infected human populations.

[0463] Next-generation spike vaccine antigen delivered by mRNA in mice T2_29 is different from the wild type. alpha , beta , gamma , and showed excellent response to omicron variants, delta Considering that the titer was low for this, delta And by including the mutations observed in Omicron BA.1, we developed a second-generation version of T2_29. We tested these second-generation modified spike vaccine antigens, T2_35 and T2_36-, in mice. Since Omicron BA.1 has mutations in most of the epitopes reported in NTD and RBD, we generated two sets of designs using different combinations of mutations observed in Omicron to achieve a broader neutralization response to all VOCs. Since currently approved booster vaccines are delivered as mRNA, we immunized mice with mRNA formulated in lipid nanoparticles. A 3-week interval prime-boost regimen was followed (Figure 6A). These second-generation spike antigens, alpha , gamma , beta , delta Furthermore, we were able to induce broad-spectrum neutralization responses to many important omicron / omicron-like lines (Figures 6B and 6C, Figure 7).

[0464] Both T2_35 and T2_36 produced strong neutralizing titers for all VOCs, but differences in titers were observed. For example, immunization with T2_35 produced a large neutralizing titer comparable to immunization with BA.1 vaccine (Omicron vaccine) when challenging the Omicron mutant, but produced a much higher titer for other VOCs than BA.1 vaccine. T2_36 produced a slightly lower neutralizing titer for the Omicron mutant compared to BA.1 vaccine, but a higher neutralizing titer for other VOCs compared to BA.1 vaccine. The difference in neutralizing titers of our designs compared to BA.1 vaccine and the wild type can also be explained by the phylogenetic distance between the designs and all known VOCs (Figure 8). T2_35 is positioned near the Omicron line, while both T2_29 and T2_36 are positioned near the ancestral type and the remaining VOCs. The inventors also further investigated the cross-neutralization range of these mRNA vaccines against related salvecoviruses, namely SARS-1, WIV16, and RaTG13 (Figure 7). Neutralizing titers similar to those of the ancestral SARS-2 antigen were observed for T2_36 against WIV16 and RaTG13, while lower neutralizing titers were observed for T2_35. No neutralization was observed against SARS-1. For related salvecoviruses as well, T2_36 showed higher neutralization against WIV16 and RaTG13 compared to T2_35 due to this closer similarity to the ancestral sequence. These observations validate the inventors' design strategy for designing antigens that yield a broad response against all VOCs.

[0465] Consideration Advances in vaccine technology and genomics have led to the successful development and distribution of vaccines against COVID-19. While these vaccines have successfully controlled the transmission and mortality of COVID-19, the rapid emergence of new SARS-CoV-2 variants has caused concerning trends in infection rates and associated hospitalizations. These variants have been observed to evade the immune response resulting from either natural infection or vaccination, leading to reinfection and re-infection. This has led to further boosts in the immune response by immunizing the population with booster doses of the original vaccine. While boosts result in increased antibody titers, they may still be ineffective against emerging variants. Considering this, many vaccine manufacturers are deploying vaccines with updated antigens, including the latest variants or combinations of the original Wuhan-Hu-1 based antigen with the latest Omicron BA.1 variant. These may provide protection against circulating variants, but may be ineffective against new variants that are phylogenetically distinct from current circulating variants, or against new variants that are phylogenetically similar to known variants. Here, we describe the design of a novel spike-based antigen that incorporates information on the mutations observed across reported VOCs. First, alpha , beta , and gamma We designed spike antigens using mutations observed in [specific location] and validated the immunogenicity of the designs in guinea pigs using a DNA / MVA vaccination regimen. A strong neutralizing titer was observed after three DNA vaccinations. T2_29 is [specifically,] delta It yielded excellent neutralization responses to all tested VOCs except for one, and was comparable to the ancestral Wuhan-Hu-1 antigen. Interestingly, and importantly, by T2_29 delta Furthermore, the induction of an excellent immune response comparable to that of Omicron BA.1 encourages and validates the inventors' rationale that a novel spike antigen containing mutational information across VOCs is a better vaccine antigen against nascent variants compared to naturally occurring variant sequences. The inventors believe that, deltaFurthermore, this rationale was further validated by incorporating mutation information from Omicron BA.1 into the T2_29 design sequence to generate second-generation designs, T2_35 and T2_36. The inventors verified the immunogenicity of these candidates as mRNA in mice. Both candidate T2_35 and T2_36 elicited a strong neutralizing response to all VOCs tested. The broad neutralizing response of the inventors' vaccine candidates in two animal models and across two platforms supports the superiority of these designs regardless of platform and animal model. Overall, from the data presented below, the inventors can conclude that antigenically engineered spike genes can induce superior immune breadth than combinations of the original spike antigen with mutant or chimeric spike antigens.

[0466] Supplementary Information Figure 6 shows the ancestor, Omicron, and delta This shows the final blood neutralization data for the coronavirus challenge in mice.

[0467] Figure 6A shows the blood sampling schedule for mice. Figure 6B shows the ancestral type and VOC-BA.1, BA.2, BA.4 / 5, and delta The distribution of neutralizing titers for blood sample 2 is shown. The x-axis represents the sham virus test for neutralization, and the y-axis represents the log10(IC50) value. The box plots are color-coded according to the vaccine. Figure 6C shows the ancestral type and VOC-BA.1, BA.2, BA.4 / 5, and delta This shows the distribution of neutralizing titers in the final blood sample. The x-axis represents the sham virus test for neutralization, and the y-axis represents the log10(IC50) value. The box plots are color-coded according to the vaccine. The Mann-Whitney U test is used for statistical significance testing in all plots (p-value: * ≤0.05, ** <0.01, *** (≤0.001). Distributions that are not statistically significant are not labeled on the plot.

[0468] Figure 7 shows the terminal bleed neutralization data in mice against the coronavirus challenge. Figure 7A shows the distribution of neutralization titers at terminal bleed against the ancestral type and VOC- alpha , beta , and gamma . The x-axis represents the pseudovirus test for neutralization, and the y-axis represents the log10(IC50) value. The box-and-whisker plots are color-coded according to the vaccine. Figure 7B shows the distribution of neutralization titers at terminal bleed against SARS-1, WIV16, RaTG13, and the ancestral type. The x-axis represents the pseudovirus test for neutralization, and the y-axis represents the log10(IC50) value. The box-and-whisker plots are color-coded according to the vaccine. The Mann-Whitney U test is used as the statistical significance test for all plots (p-value: * ≤0.05, ** <0.01, *** ≤0.001). Distributions that are not statistically significant are not labeled on the plots.

[0469] Example 4 Supplementary Information Background: Current COVID-19 vaccines are based on wild-type spike SARS-CoV-2 derived from the original Wuhan sequence or the Omicron BA.1 spike variant.

[0470] Problem: As new variants continue to emerge, the use of "historical" spike antigens from past waves of SARS-CoV-2 variants reduces the benefit against the prevention of the emergence of new variants. Thus, repeated boosting of the immune response from past immunization or infection ("antigenic原罪") may be less effective in promoting a de novo response to new epitopes and preventing the emergence of new variants.

[0471] Objective 1: To determine whether a single engineered SARS-CoV-2 spike design expressing diverse epitopes can induce neutralization across the SARS-CoV-2 variants of concern (VOCs), and to increase the breadth of protective immunity achievable by novel immunogens for defense against future SARS-CoV-2 variants.

[0472] Objective 2: To demonstrate that these effects can be mediated by different vaccine vector delivery systems (i.e., DNA, mRNA, or MVA).

[0473] First-generation DNA, T2_29 ("Superspike") Study 1a: Neutralizing antibody titers in uninbred guinea pigs after DNA immunization using SARS-CoV-2 spikes designed with VOC mutations.

[0474] Test design: Group 1: Complete spike constructs of SARS-CoV-2 based on the original Wuhan sequence with ER retention signal deletion. Group 2a: T2_29, Group 2b: T2_29+Q, Group 2c: T2_29+Q+dER mutation (all in combination with N501Y)

[0475] result: Figure 9 shows the VOC RBD-binding antibody levels in guinea pig blood sample 4, as indicated by ELISA. The area under the curve (AUC), calculated from the logarithmic dilution curves, is plotted for different vaccine constructs. A-F: Binding to each RBD variant is plotted. The overall signal intensity varied among RBD variants, and therefore comparisons could only be made within a single RBD variant. For each group, four individual values ​​and the mean with a 95% confidence interval were plotted.

[0476] As shown, in blood sample 4, serum from guinea pigs immunized with the WT spike DNA construct showed the second highest level of neutralization against PV with homologous WT spikes (mean IC50). 50 (=1,438). alpha Neutralization against PV was higher than against wild-type (average IC). 50 (=3,844).

[0477] VOC delta (573) beta (94) gamma(46) and when assayed against Omicron BA.1 / BA.2 (15, 26), WTΔER showed a continuous decrease in mean nAb titer and hardly neutralized Omicron. When all three groups of 2 superspikes (2a, 2b, 2c) were compared to the WTΔER immunized group of group 1, beta ( ** (p<0.01), gamma ( ** (p<0.005), Omicron BA.1( * , p<0.05), and Omicron BA.2( * When assayed against p<0.05, the mean IC 50 There is a significant increase in nAb values. T2_29+Q, one of two constructs with an additional Q498R mutation, is T2_29(mean IC 50 Neutralization levels lower than 3,045 (average IC) are omicron BA.1 (average IC) 50 This is shown for =295).

[0478] The RBD for the T2_29 structure is: beta It is identical to the RBD, gamma It has K417N instead of K417T. gamma It is almost identical to Omicron. T2_29 shares three AA mutations with Omicron, and T2_29+Q(+ / -ΔER) further contains Omicron's Q498R, making it the genetically closest construct to Omicron in this study. On the other hand, delta The variant possesses two RBD mutations not found in other VOCs (except T478K in BA.2) or in any of the superspike designs. Therefore, delta RBD is most antigenically distant from the superspike constructs, particularly those containing Q498R.

[0479] Figure 10 shows the distribution of neutralizing titers in guinea pig serum (blood sample 4) against ancestral types and VOCs after DNA immunization using the WT vaccine (WTdER) and the T2_29 vaccine group (2a, 2b, 2c, combined data). The x-axis represents the pseudovirus test for neutralization, and the y-axis represents the IC50 value. The WT vaccine appears on the left for each coronavirus pseudovirus, and the combined T2_29 vaccine appears on the right for each coronavirus pseudotype.

[0480] Discussion: Average IC of combined T2_29 groups (2a, 2b, 2c) for the WTΔER group. 50 The significant difference in values ​​is greater than that of the WTΔER immunized group. beta , gamma This reveals a strong increase in neutralization with respect to omicron. beta and gamma IC 50 The levels are very similar.

[0481] Neither the effect of NTD mutations nor the effect of N417 vs. T417 in PV could be observed. WT and delta The nAb activity of T2_29 group 2 against the mutated pseudovirus remains similar to that of the WTΔER group.

[0482] Adding VOC mutations is not a "zero-sum game" where any gain of neutralization for one variant results in an equal loss for another variant.

[0483] The T2-29 group was compared to the WTΔER immunized group. beta , gamma , and a strong increase in neutralization with respect to omicron is revealed. WT and delta The nAb levels in the T2_29 group for mutant PV remain similar to those in the WTΔER group.

[0484] Study 1b: Neutralizing antibody titers in MVA-boosted DNA-immunized guinea pigs with MVA T2_29+Q+dER.

[0485] Test design: Group 1: DNA delivery WT spike + dER, all boosted by MVA T2_29 + Q + dER. Group 2: DNA delivery groups 2a, 2b, and 2c were all boosted with MVA T2_29+Q+dER.

[0486] Figure 11G outlines the 3× DNA and MVA-boosted immunization and blood sampling schedules for Groups 1 and 2. Guinea pigs were immunized with plasmid DNA on days 0, 14, and 70 (guinea pig icon, PharmaJet instrument shown in green). A fourth immunization with MVA (guinea pig with syringe) was performed on day 113. Blood sampling (blood drop icon) was performed before the start of immunization, 2 and 4 weeks after each immunization, and at the time of euthanasia (final blood sampling).

[0487] result: Figures 11A–11F show immunization with either wild-type (WT) or designed DNA constructs, followed by MVA. This figure shows neutralization data from blood sampling 6 for guinea pigs boosted with T2_29+Q+dER. This figure shows neutralization data for each vaccine construct when challenged with a panel of VOCs. The x-axis represents the sham virus test for neutralization, and the y-axis represents the IC50 value.

[0488] Group 1: Despite heterogeneous boosting by T2_29+Q+ΔER, the nAb levels in the WTΔER group at blood sample 6 were still strongly correlated with those at blood sample 4 (Spearman r=0.83). **** No correlation (p>0.05) was observed between the WTΔER group (6 blood samples) and the T2_29+Q+ΔER group (4 blood samples). In particular, the WT vaccine group given heterologous MVA T2_29+Q+ΔER boost was found to only partially expand mutant neutralization.

[0489] Group 2: As expected, the three Group 2 (2a, 2b, 2c) MVA T2_29+Q+ΔER boosted groups showed a neutralization pattern very similar to blood sample 4. Neutralization of the BA.1 pseudovirus in the T2_29 group was beta and gammaIt did not provide the same level of boost as neutralizing the fake virus.

[0490] Figure 5 shows a summary of the data from this example. In guinea pigs, the spike vaccine antigen T2_29 was delivered by DNA and MVA.

[0491] Figure 5A. Guinea pig blood sampling schedule.

[0492] Figure 5B. Distribution of neutralizing titers in guinea pigs against ancestral viral pseudotypes during immunization with WTdER. The x-axis represents the number of blood samples, and the y-axis represents the log10 (IC50) value.

[0493] Figure 5C. Ancestral type and VOC- beta , gamma , delta Distribution of neutralizing titers of blood sample 4 against BA.1. The x-axis represents the sham virus test for neutralization, and the y-axis represents the log10(IC50) value. The box plots are color-coded according to the vaccine, and for each challenge variant, they appear from left to right in the order of WT dER, T2_29, T2_29+Q, and T2_29+Q+dER.

[0494] Figure 5D. Ancestral type and VOC- beta , gamma , delta Distribution of neutralizing titers of blood sample 6 against BA.1. The x-axis represents the sham virus test for neutralization, and the y-axis represents the log10(IC50) value. The box plots are color-coded according to the vaccine, and the vaccines appear in the same order as in Figure 5C. The Mann-Whitney U test is used as a statistical significance test for all plots (p-value: * ≤0.05, ** <0.01, *** (≤0.001). Distributions that are not statistically significant are not labeled on the plot.

[0495] NextGen digital design for neutralizing broad-spectrum VOCs: mRNA delivery of SARS-CoV-2 spikes Objective: To demonstrate mRNA delivery of digitally immuno-optimized synthetic spike genes compared to WT Wuhan and Omicron BA.1 spikes. All spikes compared had the same proline-stabilizing modification.

[0496] Groups: (Four groups of the same age, each containing 6 Balb-c mice) 1) WT Spikes 2) BA.1 Spikes 3) COV-S-T2_35 4) COV-S-T2_36)

[0497] Methods: Two different digital immuno-optimized synthetic spike genes (COV-S-T2_35 and COV-S-T2_36) were compared to mRNA-delivered WT Wuhan and Omicron BA.1 spikes administered intramuscularly at weeks 0 and 3 in a group of six BALB / c mice.

[0498] Assay: SARS-CoV-2 pseudotype neutralization assay, originally from Wuhan. delta The same tests were performed on Omicron BA.1, BA.2, BA.2.12.1, and BA.4 / BA.5 mutants (Figures 12-14).

[0499] Findings: Three weeks after the second immunization, serum from mice immunized with the Wuhan spike showed a significant titer against Wuhan. delta It had a slightly reduced titer and showed a weak neutralization response to all four omicron variants tested. Similarly, serum from mice immunized with BA.1 sufficiently neutralized BA.1, BA.2, and BA.2.12, but Wuhan, delta Furthermore, it showed reduced neutralization against BA.4 / BA.5 pseudoviruses. In contrast, immunization with COV-S-T2_36 showed a significant neutralization response across all variants tested.

[0500] Figure 12 shows a statistical comparison of COVID-19 (COV-S-T2_36) versus BA.1 spikes, comparing the Log IC50 of the virus between the neutralization and immunization groups.

[0501] Figure 13 shows a statistical comparison between BA.1 and BA.4 / 5 PV, and displays the IC50 neutralization values ​​for all vaccine groups.

[0502] Figure 14 shows the IC50 neutralization values ​​for all vaccine groups.

[0503] Conclusion: 1. Proof of the concept that a single DIOS-generating spike can induce a broad neutralization response on its own. 2. The response showed excellent breadth or response when tested against a single wild-type Wuhan or BA.1 spike antigen proposed for use in human SARS-CoV-2 booster vaccine campaigns. 3. At week 6 (3 weeks after the second administration), the S-T2_36-induced neutralization response to BA.4 / BA.5 was not significantly different, but clear Wuhan / Omicron cleavage was observed for WT antigens. At week 9, the S-T2_35 > S-T2_36 > BA.1 response to BA.4 / BA.5 was 3. Digitally optimized SARS-CoV-2 spike genes may induce a superior immune broadening than combinations of wild-type Wuhan antigen and mutant spike antigen.

[0504] Example 5 T2_35-induced broad-spectrum neutralizing immune response to SARS-CoV-2 variants of concern, including omicron submutants. SARS-CoV-2 and SARS-like pseudoviruses were used as challenge viruses against serum from mice immunized with T2_35, T2_36, Wuhan spike, and BA.1 spike (Figure 15).

[0505] Knowledge The vaccine candidate antigen T2_35 offers a broader range of applicability against SARS-CoV-2 variants of concern, including omicron sub-variants. T2_35 is superior to T2_36 in neutralization across omicron variants and sub-variants (Figure 15).

[0506] The Wuhan and BA.1 spike vaccine antigens generally induce neutralizing antibody responses limited to pre-omicron and post-omicron variants, respectively, while T2_35 shows a broader range across the entire panel.

[0507] Example 6 CoV_S_T2_35 scaffold sequence Sequence ID 30 below shows the scaffold S protein sequence, which contains the amino acid sequence of the scaffold's constant region, with each variable amino acid residue (i.e., an amino acid residue that may change to provide an antigen that induces a neutralizing immune response to novel and / or future SARS-CoV-2 variants) represented by X (underlined in the following sequence).

[0508] [ka]

[0509] Examples of sequences provided herein that are covered by this scaffold sequence are sequence number 1 (CoV_S_T2_35(deome)), sequence number 35 (CoV_S_T3_1(deome_v2)), and sequence number 36 (CoV_S_T3_2(deome_v3)).

[0510] Figure 16 shows the amino acid sequence alignment of CoV_S_T2_35(deome) (SEQ ID NO: 1), COV_S_T3_1(deome_v2) (SEQ ID NO: 31), and COV_S_T3_2(deome_v3) (SEQ ID NO: 32). Differences between sequences are indicated by residues enclosed in squares.

[0511] The following table lists the residues at variable positions in the amino acid sequences of CoV_S_T2_35(deome) (SEQ ID NO: 1), CoV_S_T3_1(deome_v2) (SEQ ID NO: 31), and CoV_S_T3_2(deome_v3) (SEQ ID NO: 32).

[0512] [Table 12]

[0513] The amino acid sequences of sequence numbers 31 (CoV_S_T3_1) (Deome_v2) and 32 (CoV_S_T3_2) (Deome_v3) are shown below.

[0514] COV_S_T3_1(Deom_v2)(Sequence ID 31): MFVFLVLLPL VSSQCVNLRT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS 60 NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV 120 NNATNVVIKV CEFQFCNDPF LDVYYHKNNK SWMESGVYSS ANNCTFEYVS QPFLMDLEGK 180 QGNFKNLREF VFKNIDGYFK IYSKHTPINL VRDLPQGFSA LEPLVDLPIG INITRFQTLL 240 ALHRSYLTPG DSSSGWTAGA AAYYVGYLQP RTFLLKYNEN GTITDAVDCA LDPLSETKCT 300 LKSFTVEKGI YQTSNFRVQP TESIVRFPNI TNLCPFDEVF NATRFASVYA WNRKRISNCV 360 ADYSVLYNFA PFFAFKCYGV SPTKLNDLCF TNVYADSFVI RGNEVSQIAP GQTGNIADYN 420 YKLPDDFTGC VIAWNSNKLD SKVGGNYNYR YRLFRKSKLK PFERDISTEI YQAGNKPCNG 480 VAGPNCYFPL QSYGFRPTYG VGHQPYRVVV LSFELLHAPA TVCGPKKSTN LVKNKCVNFN 540 FNGLKGTGVL TESNKKFLPF QQFGRDIADT TDAVRDPQTL EILDITPCSF GGVSVITPGT 600 NTSNQVAVLY QGVNCTEVPV AIHADQLTPT WRVYSTGSNV FQTRAGCLIG AEHVNNSYEC 660 DIPIGAGICA SYQTHTNSRG SASSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTISV 720 TTEILPVSMT KTSVDCTMYI CGDSTECSNL LLQYGSFCTQ LNRALTGIAV EQDKNTQEVF 780 AQVKQIYKTP PIKDFGGFNF SQILPDPSKP SKRSFIEDLL FNKVTLADAG FVKQYGDCLG 840 DIAARDLICA QKFNGLTVLP PLLTDEMIAQ YTSALLAGTI TSGWTFGAGA ALQIPFAMQM 900 AYRFNGIGVT QNVLYENQKL IANQFNSAIG KIQDSLSSTA SALGKLQNVV NQNAQALNTL 960 VKQLSSNFGA ISSVLNDILS RLDPPEAEVQ IDRLITGRLQ SLQTYVTQQL IRAAEIRASA 1020 NLAATKMSEC VLGQSKRVDF CGKGYHLMSF PQSAPHGVVF LHVTYVPAQE KNFTTAPAIC 1080 HDGKAHFPRE GVFVSNGTHW FVTQRNFYEP QIITTDNTFV SGNCDVVIGI VNNTVYDPLQ 1140 PELDSFKEEL DKYFKNHTSP DVDLGDISGI NASVVNIQKE IDRLNEVAKN LNESLIDLQE 1200 LGKYEQYIKW PWYIWLGFIA GLIAIVMVTI MLCCMTSCCS ​​CLKGCCSCGS CC 1252

[0515] COV_S_T3_2 (Deom_v3) (SEQ ID NO: 32): MFVFLVLLPL VSSQCVNLRT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS 60 NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV 120 NNATNVVIKV CEFQFCNDPF LDVYYHKNNK SWMESGVYSS ANNCTFEYVS QPFLMDLEGK 180 QGNFKNLREF VFKNIDGYFK IYSKHTPINL VRDLPQGFSA LEPLVDLPIG INITRFQTLL 240 ALHRSYLTPG DSSSGWTAGA AAYYVGYLQP RTFLLKYNEN GTITDAVDCA LDPLSETKCT 300 LKSFTVEKGI YQTSNFRVQP TESIVRFPNI TNLCPFHEVF NATTFASVYA WNRKRISNCV 360 ADYSVIYNFA PFFAFKCYGV SPTKLNDLCF TNVYADSFVI RGNEVSQIAP GQTGNIADYN 420 YKLPDDFTGC VIAWNSNKLD SKPSGNYNYL YRLFRKSKLK PFERDISTEI YQAGNKPCNG 480 VAGPNCYSPL QSYGFRPTYG VGHQPYRVVV LSFELLHAPA TVCGPKKSTN LVKNKCVNFN 540 FNGLKGTGVL TESNKKFLPF QQFGRDIADT TDAVRDPQTL EILDITPCSF GGVSVITPGT 600 NTSNQVAVLY QGVNCTEVPV AIHADQLTPT WRVYSTGSNV FQTRAGCLIG AEHVNNSYEC 660 DIPIGAGICA SYQTHTNSRG SASSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTISV 720 TTEILPVSMT KTSVDCTMYI CGDSTECSNL LLQYGSFCTQ LNRALTGIAV EQDKNTQEVF 780 AQVKQIYKTP PIKDFGGFNF SQILPDPSKP SKRSFIEDLL FNKVTLADAG FVKQYGDCLG 840 DIAARDLICA QKFNGLTVLP PLLTDEMIAQ YTSALLAGTI TSGWTFGAGA ALQIPFAMQM 900 AYRFNGIGVT QNVLYENQKL IANQFNSAIG KIQDSLSSTA SALGKLQNVV NQNAQALNTL 960 VKQLSSNFGA ISSVLNDILS RLDPPEAEVQ IDRLITGRLQ SLQTYVTQQL IRAAEIRASA 1020 NLAATKMSEC VLGQSKRVDF CGKGYHLMSF PQSAPHGVVF LHVTYVPAQE KNFTTAPAIC 1080 HDGKAHFPRE GVFVSNGTHW FVTQRNFYEP QIITTDNTFV SGNCDVVIGI VNNTVYDPLQ 1140 PELDSFKEEL DKYFKNHTSP DVDLGDISGI NASVVNIQKE IDRLNEVAKN LNESLIDLQE 1200 LGKYEQYIKW PWYIWLGFIA GLIAIVMVTI MLCCMTSCCS ​​CLKGCCSCGS CC 1252

[0516] Example 7 Digitally optimized immune spike vaccines induce a broad neutralization response to concerning SARS-CoV-2 variants.

[0517] overview Current SARS-CoV-2 vaccines are unable to induce potent neutralizing antibodies against the latest volatile organic compounds (VOCs). Here, we describe three novel computer-in-programmed spike-based antigens capable of inducing neutralizing antibodies across the spectrum of SARS-CoV-2 VOCs. delta The previous (also called T2_32, T2_29+Q+dER) and delta Three sets of antigens were designed using post-sequence data (T2_35 and T2_36). When delivered with DNA prime booster regimen, T2_32 induced a superior neutralizing response against many VOCs compared to spikes derived from the initial Wu-Hu-1 strain. Furthermore, heterologous boosting with attenuated poxvirus MVA alone induced a broader neutralizing immune response than DNA immunization with T2_32 alone. When delivered by mRNA, T2_35 and T2_36 induced superior broad neutralizing ability in mice compared to the omicron BA.1 sequence. These findings demonstrate the potential for computationally induced structural-informed modification of spike-based antigens for inducing panmutant neutralizing immune responses.

[0518] Introduction Since its emergence in late 2019, SARS-CoV-2 has acquired numerous mutations that have varying degrees of effect on its interaction with the host, as well as its ability to evade the existing human immune response acquired through vaccination and / or infection. 1 In addition to the evolution of SARS-CoV-2 in humans, it has been reported that the virus has been transmitted to other mammals, such as certain species of mink, cats, dogs, and deer. 2 Interspecies transmission of SARS-CoV-2 adds another dimension to the rate of evolution, fitness, and immune evasion features, which could enable future outbreaks of SARS-CoV-2 variants. From the latter half of 2020, alpha , beta , gamma , deltaNumerous volatile organic compounds (VOCs), including recent variants of the Omicron lineage, have been reported. Immune evasion and the ability to evade are primarily due to the evolution of the spike protein. The emergence of adaptive mutations in the spike protein can strongly influence host tropism and viral transmission. 1,3 Faced with a growing immune population, immunological evasion from host immunity is a characteristic of SARS-CoV-2 variants that allows them to replicate in immune human populations and spread globally.

[0519] With the subsequent emergence of various concerning variants (VOCs) or sub-variants, the neutralizing activity of antibodies induced by SARS-CoV-2 Wu-Hu-1 strain-based vaccines has decreased. Of these, delta Furthermore, the omicron submutant has been reported to have a higher transmission rate and immune evasion from both innate and vaccine-acquired immunity. 4~6 This necessitated the continuous updating of SARS-CoV-2 vaccines to match the circulating strains. Major COVID-19 mRNA vaccine manufacturers are adapting their vaccines to the Omicron lineage and administering them as either monovalent or bivalent vaccines. 7~9 Over the past year, rapid evolution and divergence of the Omicron lineage have been observed, with multiple strains being dominant in different geographical locations. 10 In situations with multiple dominant strains, tailoring a vaccine to a single strain may not be the optimal approach, and it may not provide partial protection for the population from SARS-CoV-2, a situation already observed in the field of influenza vaccines. 11 .

[0520] Here, the inventors propose that antigens expressing diverse epitopes covering the range of the most dominant VOCs are better alternatives for adapting the vaccine to one of the circulating strains. Using publicly available sequence data, the inventors have identified three antigens, namely, delta The previous (also called T2_32, T2_29+Q_dER) and two delta Post-designed antigens (T2_35 and T2_36) were designed. The T2_32 antigen was compared to a Wu-Hu-1 based spike antigen in guinea pigs, and T2_35 and T2_36 were compared to an omicron BA.1 spike antigen in mice. All three digitally immune-optimized synthetic vaccine (DIOSynVax) spike vaccine antigens showed a broader neutralization profile compared to wild-type antigens. The superior neutralization width against VOCs that emerged post-design highlights the importance of this class of digitally designed antigens for inclusion as next-generation COVID-19 booster vaccine candidates.

[0521] method Computer-based design of vaccine antigens The consensus sequence was determined using sequences deposited in NCBI Virus, and VOCs- alpha , beta , and gamma Generated for the spike protein (February 2021) 12 Using the MAFFT algorithm, Wu-Hu-1, alpha , beta , and gamma Regarding this, we created multiple sequence alignments (MSA). 13 For each position along the length of the multiple alignment, amino acids that did not match Wu-Hu-1 were mapped. Furthermore, IEDB 14 Available epitope information was mapped along the length of the MSA. Each epitope residue and mismatched amino acids were clustered into three structural domains: the N-terminal domain (NTD), the receptor-binding domain (RBD), and the stalk (S2) region. Novel spike-based vaccine antigens were generated by combining clusters of mismatched amino acids from different structural domains and epitope regions. Further mutations, namely K986P 15,16 and V987P 15,16 and Q498R 17 This was incorporated into the final design T2_32.

[0522] Using the same design protocol, consensus delta Using MSAs including Omicron BA.1 (December 2021), two designs, T2_35 and T2_36, were generated. In addition to the double proline mutation, these designs were further stabilized by replacing the furin cleavage site with a GSAS motif. 15 The structural integrity of the obtained vaccine antigen was confirmed by generating a homology model using Modeller software. 18 The endoplasmic reticulum retention (ER) signal was removed from all designs, including the wild-type control.

[0523] Plasmid generation and transformation The vaccine design sequences (T2_32 and Wu-Hu-1) were RNA and codon-optimized for high-level expression in human cells using the GeneOptimizer algorithm. 19 These genes were cloned into pEVAC (GeneArt / Thermofisher, Germany) by restriction digestion. Plasmids were transformed via heat shock in chemically competent E. coli DH5α cells (Invitrogen 18265-017). Plasmid DNA was extracted from bacterial cultures transformed via a plasmid mini-kit (Qiagen 12125). DNA plasmids were purified using an EndoFree Plasmid mega-kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Plasmids were quality-controlled by sequencing and quantified using UV spectrophotometric analysis (NanoDrop®, Thermo Scientific) to assess the absence of endotoxins.

[0524] MVA generation The MVA strain used in this study is MVA-CR19. 20 As described above, recombinant MVA expressing T2_32 was generated. 21In short, pure recombinant virus was obtained by serial plaque purification under agarose overlay, and PCR screening confirmed the absence of parental MVA-CR19 TK-GFP contamination. MVA-CR19 (rMVA) encoding T2_32 was further purified by plaque three times. The resulting recombinant MVA-CR19 T2_32 (MVA T2_32) virus stock was generated in suspended AGE1.CR.pIX cells, continuously purified by ultracentrifugation, and titrated to DF-1 cells. The absence of revertant mutants or identity of parental MVA and T2_32 insertions was confirmed by PCR amplification and Sanger sequencing. Accurate expression of T2_32 was confirmed by Western blotting analysis with the monoclonal antibody CR3022 (Invivogen, Toulouse, France), and cell lysates from HEK293 cells were collected 24 hours after infection with MVA T2_32 (MOI 2).

[0525] Vaccination experiments in guinea pigs Two groups of four 7-week-old female Hartley guinea pigs were purchased from Envigo (Maastricht, Netherlands). The guinea pigs were immunized with 200 μg of DNA vaccine containing the antigen gene in a pEVAC vector at 14-day intervals, and a total of 200 μL was administered intradermally to the hind limbs using a Pharmajet (copyright) instrument. A third dose of DNA was administered 70 days postprime and boosted intramuscularly on 112 days. MVA encoding T2_32 was 2.0E 7 It was administered at PFU / dose. Blood was collected from the saphenous vein at 2-week intervals.

[0526] mRNA synthesis and packaging mRNA sequences encoding vaccine antigens (T2_35, T2_36, and Omicron BA.1) were synthesized by in vitro transcription (IVT) from a linear plasmid DNA template using modified nucleotides to produce partially modified mRNA. After IVT, the mRNA was dephosphorylated and enzymatically polyadenylated. Purification was performed by precipitation, and the formulation was then prepared in water for injection at a concentration of 1 mg / mL. The mRNA was stored at -80°C until LNP encapsulation. Each mRNA was microfluidically mixed with ionized lipids, structural lipids, helper lipids, and polyethylene glycol (PEG) lipids in citrate buffer (pH 4.5) and ethanol, and encapsulated in LNPs by nanoprecipitation. Concentration was then performed by buffer exchange and tangential flow filtration. The mRNA LNPs were filtered through a 0.2 μm membrane and stored at -20°C until use. The formulations were analytically characterized and evaluated as acceptable for in vivo use.

[0527] Vaccination experiments in mice Five groups of six 8-10 week old female BALB / c mice were purchased from Charles River Laboratories (Kent, UK). The mice were immunized twice, 21 days apart. 100 μL of PBS containing 10 μg of lipid-encapsulated mRNA encoding the antigen was administered intramuscularly to both hind limbs. The untreated mouse group received 100 μL of PBS. Blood was collected three weeks after each immunization, and the final blood collection was performed six weeks after the second immunization.

[0528] Generation of lentivirus pseudotypes Lentivirus pseudotypes are obtained using the Fugene-HD (Promega E2311) transfection reagent. 25,26 Using packaging plasmid p8.91 22,23 and pCSFLW 24This was generated by transient transfection of HEK293T / 17 cells with expression plasmids containing different SARS-CoV-2 VOC spikes in the pEVAC backbone. After 48 hours, the supernatant was collected, passed through a 0.45 μm cellulose acetate filter, and titrated onto HEK293T / 17 cells transiently expressing human ACE and TMPRSS2. Target HEK293T / 17 cells were prepared 24 hours prior to T75 tissue culture in a flask. 27,28 The cells were then transfected with 2 μg of pCAGGS153 huACE-2 and 150 ng of pCAGGS-TMPRSS2.

[0529] Pseudotype-based microneutralization assay The pseudotype-based microneutralization assay (pMN) was performed as described above. 29 In short, serial dilutions of serum were incubated with lentiviral pseudotypes containing SARS-CoV-2 and SARS-CoV-2 VOC spikes in 96-well white cell culture plates at 37°C and 5% CO2 for 1 hour. Next, 1.5 × 10⁶ cells transiently expressing human ACE-2 and TMPRSS2 were incubated. 4 Each well was populated with HEK293T / 17 cells, and the plate was incubated in a humidified incubator at 37°C and 5% CO2 for 48 hours. Bright-Glo (Promega) was then added to each well, and luminescence was read after a 5-minute incubation period. Experimental data points were normalized to 100% and 0% using a neutralization control and nonlinear regression analysis performed in GraphPad Prism 9 to generate neutralization curves and IC50 values.

[0530] statistical analysis For each pair, 167 two-tailed Mann-Whitney U tests were performed using the Python sklearn package. 30 All plots were created using the Python Matplotlib and statannotat packages. 31 It was generated using [this method].

[0531] animal research ethics All animal studies were approved by the Home Office under project license P8143424B and by the Animal Welfare Ethical Review Body (AWERB).

[0532] result Computer-based design of antigens The structure of the SARS-CoV-2 spike protein can be divided antigenically into three distinct regions: the N-terminal domain (NTD), the receptor-binding domain (RBD), and the stalk (S2) region. The RBD contains most of the experimentally characterized epitopes, followed by the NTD and stalk. The frequent appearance of multiple mutations in the RBD and NTD of SARS-CoV-2 VOCs suggests that these epitopes are important targets of the protective immune mechanism. Computer-aided antigen design is used for VOC- alpha , beta , and gamma The mutations observed in were mapped onto the spike protein, and then in IEDB 14 We used a database to map these mutations as epitope regions and referenced the literature. 32 This involved classifying the immunodominant epitopes using peptides reported in [the relevant study]. All mutations in the immunodominant region were clustered based on both the VOC in which they were observed and the region in which they corresponded within the spike protein.

[0533] beta and gamma The mutants, with the exception of K417N / T, have the same mutation in the immunodominant region of the RBD (E484K, N501Y). In addition to the RBD region, gamma The strain also has mutations in the reported immunodominant regions of the NTD (L18F, T20N, P26S). gamma Since mutations in the variant were reported in two immunodominant regions, the set of mutations reported in NTDs was considered for design. Based on the clustering of mutations, alpha (69HΔ, 70VΔ, 144YΔ) and gammaThe set of mutations reported in NTDs for the (L18F, T20N, P26S) variants, beta The set of mutations reported in RBD for the (K417N, E484K) variants, alpha For the (P681H) variant, we considered the set of mutations reported in S2, as well as the common sets of mutations N501Y and D614G, for design. Another mutation, Q498R, was also reported by the Schreiber group as the most potent immune-evading and high-affinity mutation using in vitro evolution. 17 This mutation was included in the inventors' design as a pre-emptive step. Interestingly, this mutation was later observed in the Omicron line by the second half of 2021. Furthermore, K986P 15,16 V987P 15,16 The antigen-T2_32 was stabilized by introducing a deletion of 19 amino acids from the C-terminus (dER) (Figure 19). The deletion of 19 amino acids from the C-terminus increased the expression of the spike protein on the cell surface compared to the full-length version. 33 Therefore, it was reported that a higher concentration of the antigen was present. This antigen was evaluated in DNA-MVA prime-boost regimen in guinea pigs.

[0534] T2_32 is delta And designed before the global pandemic of omicron variants. The latter is the maximum number of reported mutations. 5,34 Since it has been reported that the accumulation of leads to avoidance of previous immune responses, the inventors designed another set of antigens as a preemptive measure and tested these designs as mRNA antigens in mice. Omicron is T2_32 and delta Since it has multiple different mutations in multiple immunodominant regions compared to the mutant, the inventors combined the set of skeletal mutations of T2_32 in the following two ways: (a) NTD and S2 regions delta (b) The RBD region is enriched with mutations observed in the mutant, and the NTD and S2 regions are enriched with mutations observed in the Omicron BA.1 mutant, and the RBD region is enriched with mutations observed in the Omicron BA.1 mutant. deltaThe mutant T2_36 was enriched with the mutations observed (Figure 19). This was to ensure that all important immunodominant regions were represented for all important VOCs. In addition to all other stabilizing mutations in T2_32, the furin cleavage sites were knocked out in these two antigens (682RRAR to GSAS). 15 .

[0535] Spike vaccine antigen (T2_32) delivered by DNA and MVA in guinea pigs. Guinea pigs were immunized three times with the antigen T2_32 and the dER version of the Wu-Hu-1 spike in the pEVAC plasmid, and boosted once with MVA.CR19 expressing T2_32 (Figure 20A). Neutralizing titers were analyzed over time for WTdER against pseudoviruses (PVs) expressing VOC spikes. Neutralizing antibodies peaked in blood sample 4 after three immunizations and in blood sample 6 after MVA boosting (Figure 20B). Neutralizing titers against all VOCs and Wu-Hu-1 strains were measured for these blood samples (Figures 20C and 20D). Some recent VOCs were included to test the robustness of T2_32.

[0536] Both antigens were tested after three DNA immunization cycles. beta , gamma , and delta Neutralizing titers were induced against the mutants (Figure 20C). High neutralizing titers against BA.1 and BA.2 were observed only in guinea pigs immunized with T2_32 (Figure 20C). Against the recent mutants XBB and XBB.1.5, only one guinea pig immunized with T2_32 produced a neutralizing titer (Figure 20C). Wu-Hu-1 and delta With the exception of the Wu-Hu-1 strain, T2_32 yielded at least logarithmically higher titers than the WTdER antigen. deltaFor both strains, the titers were comparable for both antigens. Since higher titers were observed in the group immunized with T2_32, we boosted both groups of guinea pigs with MVA expressing the heterologous vector T2_32. MVA has shown promising heterologous boosting for DNA, resulting in increased neutralizing titers. 21 Boosting with MVA expressing T2-32 resulted in at least logarithmically higher neutralizing titers across the entire VOC panel in both guinea pig groups (Figure 20D). Most importantly, significantly higher titers were observed for three of the omicron variants, namely BA.1, BA.2, and XBB, in both guinea pig groups, and similarly, significantly higher titers were observed for XBB.1.5 in the T2_32-primed group. In summary, these results indicate that our T2_32 design is far superior to Wu-Hu-1-based spike antigens and demonstrates its potential for many VOCs.

[0537] Spike vaccine antigens (T2_35 and T2_36) delivered by mRNA in mice. While the T2_32 study was underway, the Omicron variant became globally dominant in human populations. The Omicron variant was reported to exhibit greater resistance to patient-derived serum and most known therapeutic antibodies. 35 As a preemptive measure, the inventors designed two additional antigens, T2_35 and T2_36, and formulated them into mRNA. The dER version of Omicron BA.1 and the Wu-Hu-1 sequence were used as controls in the study. A 3-week prime-boost regimen was followed (Figure 21A). These spike antigens were alpha , beta , gamma , delta Furthermore, we were able to induce broad neutralization responses to many important omicron / omicron-like lines (Figures 21B and 21C).

[0538] Both T2_35 and T2_36 exhibited strong neutralizing titers for all VOCs, but a clear pattern was observed for both T2_35 and T2_36 regarding VOCs before and after the omicron. The former exhibited a higher neutralizing titer for VOCs from BA.1 to the omicron sub-lineage, while T2_36 exhibited a higher neutralizing titer for pre-omicron VOCs. alpha ~ delta The neutralizing antibody against had a higher neutralizing titer (Figures 21B and 21C). This difference in neutralizing titer can be theoretically explained by the set of mutations represented in the design. T2_35 was enriched with the mutation observed in the omicron BA.1 variant in the RBD region, while T2_36 was delta The mutations observed in the mutants are enriched. This data supports immunodominant epitopes in the RBD region rather than in the NTD or S2 region.

[0539] Interestingly, candidate T2_35 produced a neutralizing titer comparable to BA.1, or for all omicron mutants that developed after BA.1. delta T2_36 yielded significantly higher titers than BA.1 against omicron variants. T2_36 yielded lower neutralizing titers against omicron variants compared to BA.1, but significantly induced higher neutralizing antibody titers against the remaining VOCs compared to the BA.1 response. Neutralizing titers for all omicron VOCs were higher for T2_36 compared to Wu-Hu-1 spike-based antigens. To capture the breadth and efficacy of T2_35 and T2_36 compared to Wu-Hu-1 and BA.1 spike-based antigens, we used median log for all pseudoviruses tested in our panel. 10 I C 50 The values ​​were plotted (all medians less than 1.5 were scored as zero) (Figure 21C). T2_35 induced the highest coverage with moderate potency across all VOCs. Furthermore, neutralization data for T2_35 for more recent VOCs are plotted in Figure 22.

[0540] Consideration Advances in genomic tracking of viral epidemics, and the progress witnessed during the COVID-19 pandemic, have enabled real-time evaluation of vaccines to neutralize emerging viral variants. Here, we leverage pathogen genomics tracking to digitally design next-generation COVID-19 vaccine antigens with improved neutralizing ability against rapidly emerging variants. While currently approved vaccines have helped mitigate the severity of COVID-19 disease, continued transmission accompanied by the rapid emergence of new SARS-CoV-2 variants remains a cause for concern in infection rates. The most successful new variants have been observed to evade the immune response induced by either natural infection or vaccination, leading to further infection and reinfection. Boosting the immune response with the original historical spike antigen increases antibody titers, however 36 These are ineffective against future immune-evading mutants such as the XBB and BQ.1 lines. 36~38 Considering these challenges, the WHO recommends matching COVID vaccine antigens to the dominant circulating variant or strain. 39 However, the problem of "seasonal" immunization with viral spike vaccines derived from viral strains that have occurred in the past (even recently) does not prevent, and may even promote, the continuous evolution of vaccine-evading variants.

[0541] Over the three years of SARS-CoV-2 variant evolution, the vaccine composition has been updated twice (BA.1, BA.4 / 5) to match the dominant circulating variant at that time. This is to update the immunity status of the human population in accordance with the evolving situation of SARS-CoV-2 variant emergence. The updated vaccine composition has resulted in increased titers against circulating variants but has proven to have lower titers against emerging VOCs. In the current situation of continuously evolving SARS-CoV-2 variants, it would be more beneficial to have a vaccine antigen that can broaden the range of response to both circulating variants and potential future variants.

[0542] Here, we describe the design of a novel spike-based antigen that incorporates key mutations observed in the evolution of SARS-CoV-2 VOCs. alpha , beta and gamma Using the sequence of the mutant spike protein, we generated the spike design T2_32 and compared its immunogenicity to that of the approved SARS-CoV-2 Wu-Hu-1 strain. T2_32 demonstrated a superior neutralization response to all VOCs tested, including currently circulating mutants.

[0543] Furthermore, boosting animals immunized with the Wu-Hu-1 spike antigen with the T2_32 vaccine antigen significantly increased titers against all VOCs. This observation validates the usefulness of T2_32 as a booster antigen for broadening the range of immune responses to encompass many future variants. The immunogenicity of T2_32 validates our rationale that the novel DIOSynVax spike antigen as a booster vaccine provides greater vaccine efficacy against future VOCs compared to the use of historical SARS-CoV-2 variant spike antigens as a vaccine. This observation is important in the context of using these new designs in populations primed and boosted with first-generation COVID-19 vaccines.

[0544] While the T2-29 study is underway, delta The Omicron lineage emerged and became dominant in the human population. The Omicron lineage was reported to have multiple mutations in both the NTD and RBD regions of the spike protein, allowing it to evade previous existing COVID-19 vaccine immunity. 5,34As a preemptive measure, the inventors designed two additional antigens (T2_35 and T2_36). The RBD of T2_35 and the NTD of T2_36 have the greatest identity with the Omicron BA.1 lineage, while the NTD of T2_35 and the RBD of T2_36 have the greatest identity with the VOCs prior to the emergence of the Omicron lineage. The inventors validated the immunogenicity of these candidates by LNP-formulated mRNA immunization in mice. Both candidate T2_35 and T2_36 elicited a strong neutralization response to all VOCs tested. The post-immunization response with T2_35 was compared to that for the BA.1 spike antigen, and the response to the Omicron lineage was similar, but the neutralization response was delta The titer was significantly higher for the strain. In contrast, post-immunization serum against T2_36 resulted in lower neutralizing titers against the Omicron strain compared to the BA.1 spike antigen, but induced significantly higher neutralizing titers compared to the Wu-Hu-1 spike. The immunogenicity profile of T2_36 against the Omicron strain compared to the Wu-Hu-1 spike antigen supports the role of antibodies produced against NTD / Stork regions in a broader immune response to VOCs. From comparisons of the width and potency induced in mice across the tested VOCs, T2_35 was found to have the best immunological profile, followed by T2_36 and BA.1. These studies support human clinical research to evaluate the immunogenicity of this new class of spike-based antigens as boosters in the background of complex SARS-CoV-2 immunity in human populations.

[0545] In summary, the inventors conclude that antigenically engineered spike antigens have the ability to induce superior immune breadth compared to combinations of historical SARS-CoV-2 spike antigens. Broad neutralization responses with these digitally immuno-optimized synthetic spike vaccine candidates support the clinical evaluation of this new class of COVID-19 vaccine antigens.

[0546] References 1.Carabelli,A.M.et al.SARS-CoV-2 variant biology:immune escape,transmission and fitness.Nat Rev Microbiol 21,162-177(2023)。 2.Tan,C.C.S.et al.Transmission of SARS-CoV-2 from humans to animals and potential host adaptation.Nat Commun 13,2988(2022)。 3.Starr,T.N.et al.Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.Cell 182,1295-1310.e20(2020)。 4.Qu,P.et al.Neutralization of the SARS-CoV-2 Omicron BA.4 / 5 and BA.2.12.1 Subvariants.New England Journal of Medicine 386,2526-2528(2022)。 5.Hu,J.et al.Increased immune escape of the new SARS-CoV-2 variant of concern Omicron.Cell Mol Immunol 19,293-295(2022)。 6.Lewnard,J.A.et al.Association of SARS-CoV-2 BA.4 / BA.5 Omicron lineages with immune escape and clinical outcome.Nat Commun 14,1407(2023)。 7.Chalkias,S.et al.A Bivalent Omicron-Containing Booster Vaccine against Covid-19。 N Engl J Med 387,1279-1291(2022)。 8.Collier,A.Y.et al.Immunogenicity of BA.5 Bivalent mRNA Vaccine Boosters.N Engl J Med 388,565-567(2023)。 9.Offit,P.A.Bivalent Covid-19 Vaccines-A Cautionary Tale.New England Journal of Medicine 388,481-483(2023)。 10.Markov,P.V.et al.The evolution of SARS-CoV-2.Nat Rev Microbiol 21,361-379(2023)。 11.Krammer,F.The human antibody response to influenza A virus infection and vaccination.Nat Rev Immunol 19,383-397(2019)。 12.Brister,J.R.,Ako-Adjei,D.,Bao,Y.& Blinkova,O.NCBI viral genomes resource。 Nucleic Acids Res 43,D571-577(2015)。 13.Katoh,K.,Misawa,K.,Kuma,K.& Miyata,T.MAFFT:a novel method for rapid multiple sequence alignment based on fast Fourier transform.Nucleic Acids Research 30,3059-3066(2002)。 14.Vita,R.et al.The Immune Epitope Database(IEDB):2018 update.Nucleic Acids Res 47,D339-D343(2019)。 15.Amanat,F.et al.Introduction of Two Prolines and Removal of the Polybasic Cleavage Site Lead to Higher Efficacy of a Recombinant Spike-Based SARS-CoV-2 Vaccine in the Mouse Model.mBio 12,e02648-20(2021)。 16.Bos,R.et al.Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses.npj Vaccines 5,1-11(2020)。 17.Zahradnik,J.et al.SARS-CoV-2 variant prediction and antiviral drug design are enabled by RBD in vitro evolution.Nat Microbiol 6,1188-1198(2021)。 18.Sali,A.& Blundell,T.L.Comparative protein modelling by satisfaction of spatial restraints.J Mol Biol 234,779-815(1993)。 19.Raab,D.,Graf,M.,Notka,F.,Schodl,T.& Wagner,R.The GeneOptimizer Algorithm:using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization.Syst Synth Biol 4,215-225(2010)。 20.Jordan,I.et al.A Deleted D 417 eletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara(MVA).Virol Sin 35,212-226(2020)。 21.Carnell,G.W.et al.Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD of SARS-CoV-2 and its variants.Frontiers in Immunology 14,(2023)。 22.Zufferey,R.,Nagy,D.,Mandel,R.J.,Naldini,L.& Trono,D.Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.Nat Biotechnol 15,871-875(1997)。 23.Naldini,L.et al.In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector.http: / / science.sciencemag.org / 。 24.Demaison,C.et al.High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency[correction of imunodeficiency]virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter。 Human gene therapy 13,803-813(2002)。 25.Sampson,A.T.et al.Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.Viruses 13,1579(2021)。 26.Di Genova,C.et al.Production,Titration,Neutralisation,Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)Lentiviral Pseudotypes.Bio Protoc 11,e4236(2021)。 27.Hoffmann,M.et al.SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.Cell 181,271-280.e8(2020)。 28.Bertram,S.et al.Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts。 PLoS ONE 7,1-8(2012)。 29.Carnell,G.,Grehan,K.,Ferrara,F.,Molesti,E.& Temperton,N.J.An Optimised Method for the Production using PEI,Titration and Neutralization of SARS-CoV Spike Luciferase Pseudotypes.Bio-Protocol 7,(2017)。 30.Pedregosa,F.et al.Scikit-learn:Machine Learning in Python.Journal of Machine Learning Research 12,2825-2830(2011)。 31.Hunter,J.D.Matplotlib:A 2D Graphics Environment.Computing in Science Engineering 9,90-95(2007)。 32.Lu,S.et al.The immunodominant and neutralization linear epitopes for SARS-CoV-2.Cell Rep 34,108666(2021)。 33.Ujike,M.,Huang,C.,Shirato,K.,Makino,S.& Taguchi,F.The contribution of the cytoplasmic retrieval signal of severe acute respiratory syndrome coronavirus to intracellular accumulation of S proteins and incorporation of S protein into virus-like particles.J Gen Virol 97,1853-1864(2016)。 34.Fan,Y.et al.SARS-CoV-2 Omicron variant:recent progress and future perspectives。 Sig Transduct Target Ther 7,1-11(2022)。 35.Ju,B.et al.Immune escape by SARS-CoV-2 Omicron variant and structural basis of its effective neutralization by a broad neutralizing human antibody VacW-209.Cell Res 32,491-494(2022)。 36.Miller,J.et al.Substantial Neutralization Escape by SARS-CoV-2 Omicron Variants BQ.1.1 and XBB.1.New England Journal of Medicine 388,662-664(2023)。 37.Uraki,R.et al.Antiviral and bivalent vaccine efficacy against an Omicron XBB.1.5 isolate.The Lancet Infectious Diseases 23,402-403(2023)。 38.Yang,J.et al.Low levels of neutralizing antibodies against XBB Omicron subvariants after BA.5 infection.Sig Transduct Target Ther 8,1-12(2023). 39.466 Statement on the antigen composition of COVID-19 vaccines. https: / / www.who.int / news / item / 18-05-2023-statement-on-the-antigen-composition-of-covid-19-vaccines.

[0547] Example 8 Next-generation SuperSpike family vaccine antigens induce a broad neutralization response to concerning SARS-CoV-2 variants. We developed the next-generation SuperSpike design sequences, COV_S_T3_1(Deome_v2)(SEQ ID NO: 31) and COV_S_T3_2(Deome_v3)(SEQ ID NO: 32). The next-generation sequences are covered by scaffold SEQ ID NO: 30. The amino acid residues of COV_S_T3_1(Deome_v2) and COV_S_T3_2(Deome_v3) at the corresponding variable positions of scaffold sequence SEQ ID NO: 30, as well as the first-generation designed sequence COV_S_T2_35(Deome), are shown in Example 6.

[0548] Figure 23 shows neutralization data using mouse antiserum produced by immunization with the mRNA platform-based "SuperSpike" vaccine constructs and controls against a panel of SARS-CoV-2 lentivirus pseudoviruses expressing the VOC spike protein. Mice were immunized with two doses of 10 μg of mRNA in 100 μL of vehicle, with a 3-week interval between doses. Blood samples were collected from mice 3 weeks and 6 weeks after the first dose. The final blood sample was collected 9 weeks after the first dose. The next-generation "SuperSpike" constructs COV_S_T3_1 (Deome_v2, SEQ ID NO: 31) and COV_S_T3_2 (Deome_v3, SEQ ID NO: 32) induced neutralizing antibody responses across a diverse omicron PV panel, showing a clear improvement over the first-generation SuperSpike COV_S_T2_35 (Deome, SEQ ID NO: 1) across this panel. In certain embodiments, for example, the following are provided: (Item 1) An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length. (Item 2) Table 1 below: [Table 1-3] The polypeptide described in item 1, wherein at least one of the amino acid residues of SEQ ID NO: 1, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 8. (Item 3) Table 3 below: [Table 3-6] The polypeptide according to item 1 or 2, wherein the amino acid residue of SEQ ID NO: 1, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 8. (Item 4) Table 2 below: [Table 2-3] A polypeptide as described in any of the preceding items, which contains the amino acid residues of SEQ ID NO: 1 as shown in the image, at the positions corresponding to the amino acid residue positions of SEQ ID NO: 8. (Item 5) The following amino acid residues are located at the positions corresponding to residues 984 and 985 of SEQ ID NO: 8: ·984:P, and ·985:P A polypeptide, including any of the preceding items. (Item 6) The following amino acid residues are located at the positions corresponding to residues 680, 681, 683, 984, and 985 of SEQ ID NO: 8: ·680:G、 ·681:S、 ·683:S、 ·984:P, and ·985:P A polypeptide, including any of the preceding items. (Item 7) Table 1 below: Table 1-4 A polypeptide as described in any of the preceding items, which contains the amino acid residues of SEQ ID NO: 1 as shown in the image, at the positions corresponding to the amino acid residue positions of SEQ ID NO: 8. (Item 8) A polypeptide as described in any of the preceding entries, which does not contain the residue KFDEDDSEPVLKGVKLHYT at the amino acid residue positions 1253-1271 of SEQ ID NO: 8. (Item 9) A polypeptide containing the amino acid sequence CoV_S_T2_35(deome) (SEQ ID NO: 1) as described in any of the preceding entries. (Item 10) Table 3 below: Table 3-7 An isolated polypeptide comprising a coronavirus S protein having at least one or all of the amino acid residues shown in the image at the positions corresponding to the amino acid residue positions in SEQ ID NO: 8. (Item 11) The polypeptide according to item 10, wherein 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 with the amino acid sequence of SEQ ID NO: 8 over its entire length. (Item 12) The following amino acid residues are located at the positions corresponding to residues 983 and 984 of SEQ ID NO: ·984:P, and ·985:P A polypeptide, including those listed in item 10 or 11. (Item 13) Table 2 below: Table 2-4 A polypeptide according to any of items 10 to 12, wherein the amino acid residue of SEQ ID NO: 1, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 8. (Item 14) Table 1 below: Table 1-5 A polypeptide according to any of items 10 to 13, wherein the amino acid residue of SEQ ID NO: 1, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 8. (Item 15) A polypeptide as described in any of items 10 to 14, which does not contain the residue KFDEDDSEPVLKGVKLHYT at the amino acid residue positions 1253 to 1271 of SEQ ID NO: 8. (Item 16) An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length. (Item 17) Table 4 below: Table 4-3 The polypeptide according to item 16, wherein at least one of the amino acid residues of SEQ ID NO: 2, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 9. (Item 18) Table 6 below: Table 6-6 The polypeptide according to item 16 or 17, comprising the amino acid residue of SEQ ID NO: 2 as shown, at the position corresponding to the amino acid residue position of SEQ ID NO: 9. (Item 19) Table 5 below: Table 5-3 A polypeptide according to any of items 16 to 18, wherein the amino acid residue of SEQ ID NO: 2, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 9. (Item 20) The following amino acid residues are located at the positions corresponding to residues 983 and 984 of SEQ ID NO: 9: ·983:P, and ·984:P polypeptides listed in any of items 16-19, including (Item 21) The following amino acid residues are located at the positions corresponding to residues 679, 680, 682, 983, and 984 of SEQ ID NO: 9: ·679:G、 ·680:S、 ·682:S、 ·983:P, and ·984:P Polypeptides listed in any of items 16-20, including (Item 22) Table 4 below: Table 4-4 A polypeptide according to any of items 16 to 21, wherein the amino acid residue of SEQ ID NO: 2, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 9. (Item 23) A polypeptide as described in any of items 16-22, which does not contain the residue KFDEDDSEPVLKGVKLHYT at the amino acid residue positions 1252-1270 of SEQ ID NO: 9. (Item 24) A polypeptide containing the CoV_S_T2_36(omido) amino acid sequence (SEQ ID NO: 2), as described in any of items 16-23. (Item 25) Table 6 below: Table 6-7 An isolated polypeptide comprising a coronavirus S protein having at least one or all of the amino acid residues shown in the image at positions corresponding to the amino acid residue positions of SEQ ID NO: 9. (Item 26) The polypeptide according to item 25, wherein 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 with the amino acid sequence of SEQ ID NO: 9 over its entire length. (Item 27) The following amino acid residues are located at the positions corresponding to residues 983 and 984 of SEQ ID NO: 9: ·983:P, and ·984:P Polypeptides as described in item 25 or 26, including those listed in item 25 or 26. (Item 28) Table 5 below: Table 5-4 A polypeptide according to any of items 25 to 27, wherein the amino acid residue of SEQ ID NO: 2, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 9. (Item 29) Table 4 below: Table 4-5 A polypeptide according to any of items 25 to 28, wherein the amino acid residue of SEQ ID NO: 2, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 9. (Item 30) A polypeptide as described in any of items 25-29, which does not contain the residue KFDEDDSEPVLKGVKLHYT at the position corresponding to amino acid residue positions 1252-1270 of SEQ ID NO: 9. (Item 31) An isolated polypeptide containing the amino acid sequence of SEQ ID NO: 3 (Omicron_Vaccine), or amino acids having at least 99% amino acid identity across the entire length of the amino acid sequence of SEQ ID NO: 3. (Item 32) Table 7 below: Table 7-3 The polypeptide according to item 31, wherein at least one of the amino acid residues of SEQ ID NO: 3, as shown, is located at the position corresponding to the amino acid residue position of SEQ ID NO: 9. (Item 33) The following amino acid residues are located at the positions corresponding to residues 983 and 984 of SEQ ID NO: 9: ·983:P, and ·984:P A polypeptide as described in item 31 or 32, including the polypeptides described in item 31 or 32. (Item 34) A polypeptide as described in any of items 31 to 33, which does not contain the residue KFDEDDSEPVLKGVKLHYT at the amino acid residue positions 1252 to 1270 of SEQ ID NO: 9. (Item 35) An isolated polypeptide described in any of items 31-34, containing the amino acid sequence of Sequence ID No. 3 (Omicron_Vaccine). (Item 36) An isolated polypeptide containing the amino acid sequence of SEQ ID NO: 30 (CoV_S_T2_35 scaffold sequence), wherein X at amino acid residue positions 337, 344, 366, 371, 373, 374, 403, 406, 443, 444, 450, 458, 484, 488, 491, and 494 is any amino acid residue. (Item 37) An isolated polypeptide containing the amino acid sequence of SEQ ID NO: 33 (the RBD portion of the CoV_S_T2_35 scaffold sequence (SEQ ID NO: 30)), wherein X at the amino acid residue positions corresponding to amino acid residue positions 337, 344, 366, 371, 373, 374, 403, 406, 443, 444, 450, 458, 484, 488, 491, and 494 of SEQ ID NO: 30 is any amino acid residue. (Item 38) An isolated polypeptide according to item 36 or 37, comprising amino acid residue G, D, or H at the amino acid residue position corresponding to position 337 of SEQ ID NO: 30. (Item 39) An isolated polypeptide according to any of items 36-38, comprising an amino acid residue R or T at the amino acid residue position corresponding to position 344 of SEQ ID NO: 30. (Item 40) An isolated polypeptide according to any of items 36 to 39, comprising an amino acid residue L or I at the amino acid residue position corresponding to position 366 of SEQ ID NO: 30. (Item 41) An isolated polypeptide according to any of items 36-40, comprising an amino acid residue S or P at the amino acid residue position corresponding to position 371 of SEQ ID NO: 30. (Item 42) An isolated polypeptide according to any of items 36 to 41, comprising an amino acid residue S or F at the amino acid residue position corresponding to position 373 of SEQ ID NO: 30. (Item 43) An isolated polypeptide according to any of items 36 to 42, comprising an amino acid residue T or A at the amino acid residue position corresponding to position 374 of SEQ ID NO: 30. (Item 44) An isolated polypeptide according to any of items 36 to 43, comprising an amino acid residue D or N at the amino acid residue position corresponding to position 403 of SEQ ID NO: 30. (Item 45) An isolated polypeptide according to any of items 36 to 44, comprising an amino acid residue R or S at the amino acid residue position corresponding to position 406 of SEQ ID NO: 30. (Item 46) An isolated polypeptide according to any of items 36 to 45, comprising amino acid residue V or P at the amino acid residue position corresponding to position 443 of SEQ ID NO: 30. (Item 47) An isolated polypeptide according to any of items 36 to 46, comprising an amino acid residue S or G at the amino acid residue position corresponding to position 444 of SEQ ID NO: 30. (Item 48) An isolated polypeptide according to any of items 36 to 47, comprising an amino acid residue L or R at the amino acid residue position corresponding to position 450 of SEQ ID NO: 30. (Item 49) An isolated polypeptide according to any of items 36 to 48, which contains an amino acid residue N or K at the amino acid residue position corresponding to position 458 of SEQ ID NO: 30. (Item 50) An isolated polypeptide according to any of items 36 to 49, comprising amino acid residue F or P at the amino acid residue position corresponding to position 484 of SEQ ID NO: 30. (Item 51) An isolated polypeptide according to any of items 36 to 50, comprising an amino acid residue F or S at the amino acid residue position corresponding to position 488 of SEQ ID NO: 30. (Item 52) An isolated polypeptide according to any of items 36 to 51, comprising an amino acid residue R or Q at the amino acid residue position corresponding to position 491 of SEQ ID NO: 30. (Item 53) An isolated polypeptide according to any of items 36 to 52, comprising an amino acid residue S or G at the amino acid residue position corresponding to position 494 of SEQ ID NO: 30. (Item 54) An isolated polypeptide described in any of items 36-53, containing the amino acid sequence of SEQ ID NO: 31. (Item 55) An isolated polypeptide described in any of items 36-53, containing the amino acid sequence of SEQ ID NO: 32. (Item 56) An isolated polypeptide described in any of items 36-53, containing the amino acid sequence of SEQ ID NO: 34. (Item 57) An isolated polypeptide described in any of items 36-53, containing the amino acid sequence of SEQ ID NO: 35. (Item 58) An isolated polypeptide described in any of items 36-53, containing the amino acid sequence of SEQ ID NO: 36. (Item 59) An isolated nucleic acid molecule encoding a polypeptide described in any of items 1 to 58, or its complement. (Item 60) A vector containing the nucleic acid molecule described in item 59. (Item 61) A vector as described in item 60, comprising a nucleic acid molecule encoding a polypeptide as described in any of items 1-15. (Item 62) A vector as described in item 60, comprising a nucleic acid molecule encoding a polypeptide as described in any of items 16-30. (Item 63) A vector as described in item 60, comprising a nucleic acid molecule encoding a polypeptide as described in any of items 31-35. (Item 64) A vector according to any one of items 60 to 63, further comprising a promoter operably linked to the nucleic acid. (Item 65) The vector according to item 64, wherein the promoter is for the expression of a polypeptide encoded by the nucleic acid in mammalian cells. (Item 66) The vector according to item 64, wherein the promoter is for the expression of a polypeptide encoded by the nucleic acid in yeast or insect cells. (Item 67) A vaccine vector, as described in any of items 60-65. (Item 68) A vector as described in item 67, which is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector. (Item 69) The mRNA vaccine vector, as described in item 67. (Item 70) A DNA vaccine vector, as described in item 67. (Item 71) A modified vaccinia virus ankara (MVA) vector, as described in item 67, 68, or 70. (Item 72) The pURVac vector, as described in item 67, 68, or 70. (Item 73) Isolated cells containing a vector described in any of items 60-72. (Item 74) A fusion protein containing a polypeptide listed in any of items 1-58. (Item 75) A pharmaceutical composition comprising a polypeptide described in any of items 1 to 58 and a pharmaceutically acceptable carrier, excipient, or diluent. (Item 76) A pharmaceutical composition according to item 75, comprising a polypeptide described in any of items 1 to 15. (Item 77) A pharmaceutical composition according to item 75, comprising a polypeptide described in any of items 16 to 30. (Item 78) A pharmaceutical composition according to item 75, comprising a polypeptide described in any of items 31 to 35. (Item 79) A pharmaceutical composition comprising a nucleic acid molecule as described in item 59 and a pharmaceutically acceptable carrier, excipient, or diluent. (Item 80) A pharmaceutical composition according to item 79, comprising a nucleic acid molecule encoding a polypeptide described in any of items 1 to 15. (Item 81) A pharmaceutical composition according to item 79, comprising a nucleic acid molecule encoding a polypeptide described in any of items 16 to 30. (Item 82) A pharmaceutical composition according to item 79, comprising a nucleic acid molecule encoding a polypeptide described in any of items 31 to 35. (Item 83) A pharmaceutical composition comprising a vector described in any of items 60 to 72, and a pharmaceutically acceptable carrier, excipient, or diluent. (Item 84) A pharmaceutical composition according to any one of items 75 to 83, further comprising an adjuvant for enhancing the immune response of the composition to the polypeptide or to the polypeptide encoded by the nucleic acid in a target. (Item 85) A pharmaceutical composition according to any one of items 79 to 82, wherein the nucleic acid molecule is provided by a vector. (Item 86) The pharmaceutical composition according to item 85, wherein the vector is a vaccine vector. (Item 87) The pharmaceutical composition according to item 86, wherein the vaccine vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, an mRNA vaccine vector, or a DNA vaccine vector. (Item 88) The pharmaceutical composition according to item 87, wherein the vaccine vector is a DNA vaccine vector. (Item 89) The pharmaceutical composition according to item 88, wherein the DNA vaccine vector is a pURVac vector. (Item 90) The pharmaceutical composition according to item 87, wherein the viral vaccine vector is a modified vaccinia virus ankara (MVA) vector. (Item 91) The pharmaceutical composition according to item 87, wherein the vaccine vector is an mRNA vaccine vector. (Item 92) Nucleic acids as described in item 59, comprising one or more modified nucleosides. (Item 93) The vector according to any one of items 60 to 69, wherein the nucleic acid of the vector comprises one or more modified nucleosides. (Item 94) The pharmaceutical composition according to any one of items 79 to 82, wherein the nucleic acid of the composition comprises one or more modified nucleosides. (Item 95) The nucleic acid described in item 92, the vector described in item 93, or the pharmaceutical composition described in item 94, wherein the nucleic acid comprises messenger RNA (mRNA). (Item 96) A nucleic acid according to item 92 or 95, a vector according to item 93 or 95, or a pharmaceutical composition according to item 94 or 95, wherein one or more of the modified nucleosides include a 1-methylpseudolidine modification. (Item 97) A nucleic acid according to any one of items 92, 95, or 96, a vector according to any one of items 93, 95, or 96, or a pharmaceutical composition according to any one of items 94 to 96, wherein at least 80% of the uridine in the open reading frame is modified. (Item 98) A pseudovirus containing a polypeptide listed in any of items 1 through 58. (Item 99) A method for inducing an immune response to coronavirus in a subject, comprising administering to the subject an effective amount of a polypeptide described in any of items 1 to 58, a nucleic acid described in any of items 59, 92, or 95 to 97, a vector described in any of items 60 to 72, 93, or 95 to 97, or a pharmaceutical composition described in any of items 75 to 91 or 94 to 97. (Item 100) A method for immunizing a subject against coronavirus, comprising administering to the subject an effective amount of a polypeptide described in any of items 1 to 58, a nucleic acid described in any of items 59, 92, or 95 to 97, a vector described in any of items 60 to 72, 93, or 95 to 97, or a pharmaceutical composition described in any of items 75 to 91 or 94 to 97. (Item 101) A polypeptide as described in any of items 1 to 58, a nucleic acid as described in any of items 59, 92, or 95 to 97, a vector as described in any of items 60 to 72, 93, or 95 to 97, or a pharmaceutical composition as described in any of items 75 to 91 or 94 to 97, for use as a pharmaceutical. (Item 102) A polypeptide according to any of items 1 to 58, a nucleic acid according to any of items 59, 92, or 95 to 97, a vector according to any of items 60 to 72, 93, or 95 to 97, or a pharmaceutical composition according to any of items 75 to 91, or 94 to 97, for use in the prevention, treatment, or improvement of coronavirus infection. (Item 103) Use of a polypeptide described in any of items 1 to 58, a nucleic acid described in any of items 59, 92, or 95 to 97, a vector described in any of items 60 to 72, 93, or 95 to 97, or a pharmaceutical composition described in any of items 75 to 91 or 94 to 97, in the manufacture of a pharmaceutical for the prevention, treatment, or improvement of coronavirus infection. (Item 104) The method described in item 99 or 100, wherein the coronavirus is a beta-coronavirus, a polypeptide, nucleic acid, vector or pharmaceutical composition for use as described in item 102, or the use as described in item 103. (Item 105) A polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or the method or use described in item 104, wherein the beta-coronavirus is a beta-coronavirus of lineage B or lineage C. (Item 106) A polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or the method or use described in item 104, wherein the β-coronavirus is a beta-coronavirus of lineage B. (Item 107) The β-coronavirus of lineage B is SARS-CoV or SARS-CoV-2, and the method or use described in item 105 or 106, or a polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or use. (Item 108) The method, or use, of the aforementioned lineage C beta-coronavirus, wherein MERS-CoV is present, a polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or use, as described in item 105. (Item 109) Polypeptides, nucleic acids, vectors, or pharmaceutical compositions for use, or by the method or use described in item 104, wherein the aforementioned beta-coronavirus is a volatile organic compound (VOC). (Item 110) The method, or use of a polypeptide, nucleic acid, vector, or pharmaceutical composition, or use, as described in item 104, wherein the beta-coronavirus is SARS-CoV-2 VOC. (Item 111) Polypeptides, nucleic acids, vectors, or pharmaceutical compositions for use or by the method described in item 104, wherein the beta-coronavirus is SARS-CoV-2 beta, gamma, delta, or omicron VOC. (Item 112) A method for diagnosing whether a subject has coronavirus infection, comprising determining whether a polypeptide described in any of items 1 to 58 is bound by an antibody produced by the subject. (Item 113) The method according to item 112, wherein the antibody is present in a biological sample obtained from the subject, or in a sample derived from a biological sample obtained from the subject. (Item 114) The method according to item 113, wherein the biological sample is a serum sample. (Item 115) Polypeptides, nucleic acids, vectors, or pharmaceutical compositions for use or the method described in item 104, or for use, wherein the beta-coronavirus is the Omicron BA.1, BA.2, BA.2.75, 2.75.2, BA.2.3.20, BA.4, BA.5, BQ.1.1, XBB, XBB.1.5, BF.7, XBB.1.19.1, XBC, BQ.1.12, CH.1.1.1, or XBB.1.9.1 virus.

Claims

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (CoV_S_T2_35), or an amino acid sequence having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 1 over its entire length.

2. As shown in Table 1 below, at the position corresponding to the amino acid residue position of SEQ ID NO: 8, at least one of the amino acid residues of SEQ ID NO: 1 is included. Table 1-3 The polypeptide according to claim 1.

3. As shown in Table 3 below, the amino acid residue of SEQ ID NO: 1 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

8. Table 3-6 The polypeptide according to claim 1 or 2.

4. As shown in Table 2 below, the amino acid residue of SEQ ID NO: 1 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

8. Table 2-3 The polypeptide according to any one of claims 1 to 3.

5. The following amino acid residues are present at the positions corresponding to residues 984 and 985 of SEQ ID NO: 8 984: P, and ・985:P The polypeptide according to any one of claims 1 to 4.

6. The following amino acid residues are included at the positions corresponding to residues 680, 681, 683, 984, and 985 of SEQ ID NO: 8, ・680: G, ・681: S, ・683: S, 984: P, and ・985:P The polypeptide according to any one of claims 1 to 4.

7. As shown in Table 1 below, the amino acid residue of SEQ ID NO: 1 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

8. Table 1-4 The polypeptide according to claim 1.

8. The polypeptide according to any one of claims 1 to 7, wherein the residue KFDEDDSEPVLKGVKLHYT is not present at the positions corresponding to amino acid residue positions 1253 to 1271 of SEQ ID NO:

8.

9. A polypeptide according to any one of claims 1 to 8, comprising the CoV_S_T2_35 (deome) amino acid sequence (SEQ ID NO: 1).

10. As shown in Table 3 below, the coronavirus S protein contains at least one or all of the amino acid residues at the position corresponding to the amino acid residue position of SEQ ID NO:

8. Table 3-7 The polypeptide according to claim 1 or 2.

11. The polypeptide according to claim 10, wherein 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 with the amino acid sequence of Sequence ID No. 8 over its entire length.

12. The positions corresponding to residues 983 and 984 of SEQ ID NO: 8 contain the following amino acid residues: 984: P, and ・985:P The polypeptide according to claim 10 or 11.

13. As shown in Table 2 below, the amino acid residue of SEQ ID NO: 1 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

8. Table 2-4 The polypeptide according to any one of claims 10 to 12.

14. As shown in Table 1 below, the amino acid residue of SEQ ID NO: 1 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

8. Table 1-5 The polypeptide according to any one of claims 10 to 13.

15. The polypeptide according to any one of claims 10 to 14, wherein the residue KFDEDDSEPVLKGVKLHYT is not present at the positions corresponding to amino acid residue positions 1253 to 1271 of SEQ ID NO:

8.

16. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (CoV_S_T2_36), or amino acids having at least 97%, 98%, or 99% amino acid identity with the amino acid sequence of SEQ ID NO: 2 over its entire length.

17. As shown in Table 4 below, at the position corresponding to the amino acid residue position of SEQ ID NO: 9, at least one of the amino acid residues of SEQ ID NO: 2 is included. Table 4-3 The polypeptide according to claim 16.

18. As shown in Table 6 below, the amino acid residue of SEQ ID NO: 2 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

9. Table 6-6 The polypeptide according to claim 16 or 17.

19. As shown in Table 5 below, the amino acid residue of SEQ ID NO: 2 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

9. Table 5-3 The polypeptide according to any one of claims 16 to 18.

20. The positions corresponding to residues 983 and 984 of Sequence ID No. 9 contain the following amino acid residues: 983: P, and ・984:P Polypeptide according to any one of claims 16 to 19

21. The following amino acid residues are present at the positions corresponding to residues 679, 680, 682, 983, and 984 of SEQ ID NO: 9, ・679: G, ・680: S, ・682: S, 983: P, and ・984:P Polypeptide according to any one of claims 16 to 20

22. As shown in Table 4 below, the amino acid residue of SEQ ID NO: 2 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

9. Table 4-4 The polypeptide according to any one of claims 16 to 21.

23. The polypeptide according to any one of claims 16 to 22, wherein the residue KFDEDDSEPVLKGVKLHYT is not present at the positions corresponding to amino acid residue positions 1252 to 1270 of SEQ ID NO:

9.

24. A polypeptide according to any one of claims 16 to 23, comprising the CoV_S_T2_36 (omido) amino acid sequence (SEQ ID NO: 2).

25. As shown in Table 6 below, the coronavirus S protein contains at least one or all of the amino acid residues at the position corresponding to the amino acid residue position of SEQ ID NO:

9. Table 6-7 Isolated polypeptide.

26. The polypeptide according to claim 25, wherein 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 with the amino acid sequence of Sequence ID No. 9 over its entire length.

27. The positions corresponding to residues 983 and 984 of Sequence ID No. 9 contain the following amino acid residues: 983: P, and ・984:P The polypeptide according to claim 25 or 26.

28. As shown in Table 5 below, the amino acid residue of SEQ ID NO: 2 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

9. Table 5-4 The polypeptide according to any one of claims 25 to 27.

29. As shown in Table 4 below, the amino acid residue of SEQ ID NO: 2 is located at the position corresponding to the amino acid residue position of SEQ ID NO:

9. Table 4-5 The polypeptide according to any one of claims 25 to 28.

30. The polypeptide according to any one of claims 25 to 29, wherein the residue KFDEDDSEPVLKGVKLHYT is not present at the positions corresponding to amino acid residue positions 1252 to 1270 of SEQ ID NO:

9.

31. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 3 (Omicron_Vaccine), or an amino acid having at least 99% amino acid identity with the amino acid sequence of SEQ ID NO: 3 over its entire length.

32. As shown in Table 7 below, at the position corresponding to the amino acid residue position of SEQ ID NO: 9, at least one of the amino acid residues of SEQ ID NO: 3 is included. Table 7-3 The polypeptide according to claim 31.

33. The positions corresponding to residues 983 and 984 of Sequence ID No. 9 contain the following amino acid residues: 983: P, and ・984:P Polypeptide according to claim 31 or 32

34. The polypeptide according to any one of claims 31 to 33, wherein the residue KFDEDDSEPVLKGVKLHYT is not present at the positions corresponding to amino acid residue positions 1252 to 1270 of SEQ ID NO:

9.

35. An isolated polypeptide according to any one of claims 31 to 34, comprising the amino acid sequence of SEQ ID NO: 3 (Omicron_Vaccine).

36. An isolated polypeptide containing the amino acid sequence of Sequence ID No. 30 (CoV_S_T2_35 scaffold sequence), wherein X at amino acid residue positions 337, 344, 366, 371, 373, 374, 403, 406, 443, 444, 450, 458, 484, 488, 491, and 494 is any amino acid residue.

37. An isolated polypeptide containing the amino acid sequence of SEQ ID NO: 33 (the RBD portion of the CoV_S_T2_35 scaffold sequence (SEQ ID NO: 30)), wherein X at the amino acid residue positions corresponding to amino acid residue positions 337, 344, 366, 371, 373, 374, 403, 406, 443, 444, 450, 458, 484, 488, 491, and 494 of SEQ ID NO: 30 is any amino acid residue.

38. The isolated polypeptide according to claim 36 or 37, comprising amino acid residue G, D, or H at the amino acid residue position corresponding to position 337 of SEQ ID NO:

30.

39. The isolated polypeptide according to any one of claims 36 to 38, comprising an amino acid residue R or T at the amino acid residue position corresponding to position 344 of SEQ ID NO:

30.

40. The isolated polypeptide according to any one of claims 36 to 39, wherein the amino acid residue L or I is located at the amino acid residue position corresponding to position 366 of SEQ ID NO:

30.

41. The isolated polypeptide according to any one of claims 36 to 40, comprising an amino acid residue S or P at the amino acid residue position corresponding to position 371 of SEQ ID NO:

30.

42. The isolated polypeptide according to any one of claims 36 to 41, comprising an amino acid residue S or F at the amino acid residue position corresponding to position 373 of SEQ ID NO:

30.

43. The isolated polypeptide according to any one of claims 36 to 42, wherein the amino acid residue T or A is located at the amino acid residue position corresponding to position 374 of SEQ ID NO:

30.

44. The isolated polypeptide according to any one of claims 36 to 43, wherein the amino acid residue D or N is located at the amino acid residue position corresponding to position 403 of SEQ ID NO:

30.

45. The isolated polypeptide according to any one of claims 36 to 44, comprising an amino acid residue R or S at the amino acid residue position corresponding to position 406 of SEQ ID NO:

30.

46. The isolated polypeptide according to any one of claims 36 to 45, comprising an amino acid residue V or P at the amino acid residue position corresponding to position 443 of SEQ ID NO:

30.

47. The isolated polypeptide according to any one of claims 36 to 46, comprising an amino acid residue S or G at the amino acid residue position corresponding to position 444 of SEQ ID NO:

30.

48. The isolated polypeptide according to any one of claims 36 to 47, comprising an amino acid residue L or R at the amino acid residue position corresponding to position 450 of SEQ ID NO:

30.

49. The isolated polypeptide according to any one of claims 36 to 48, comprising an amino acid residue N or K at the amino acid residue position corresponding to position 458 of SEQ ID NO:

30.

50. The isolated polypeptide according to any one of claims 36 to 49, comprising an amino acid residue F or P at the amino acid residue position corresponding to position 484 of SEQ ID NO:

30.

51. The isolated polypeptide according to any one of claims 36 to 50, comprising an amino acid residue F or S at the amino acid residue position corresponding to position 488 of SEQ ID NO:

30.

52. The isolated polypeptide according to any one of claims 36 to 51, comprising an amino acid residue R or Q at the amino acid residue position corresponding to position 491 of SEQ ID NO:

30.

53. The isolated polypeptide according to any one of claims 36 to 52, comprising an amino acid residue S or G at the amino acid residue position corresponding to position 494 of SEQ ID NO:

30.

54. An isolated polypeptide according to any one of claims 36 to 53, comprising the amino acid sequence of SEQ ID NO:

31.

55. An isolated polypeptide according to any one of claims 36 to 53, comprising the amino acid sequence of SEQ ID NO:

32.

56. An isolated polypeptide according to any one of claims 36 to 53, comprising the amino acid sequence of SEQ ID NO:

34.

57. An isolated polypeptide according to any one of claims 36 to 53, comprising the amino acid sequence of SEQ ID NO:

35.

58. An isolated polypeptide according to any one of claims 36 to 53, comprising the amino acid sequence of SEQ ID NO:

36.

59. An isolated nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 58, or a complement thereof.

60. A vector comprising the nucleic acid molecule described in claim 59.

61. The vector according to claim 60, comprising a nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 15.

62. The vector according to claim 60, comprising a nucleic acid molecule encoding a polypeptide according to any one of claims 16 to 30.

63. The vector according to claim 60, comprising a nucleic acid molecule encoding a polypeptide according to any one of claims 31 to 35.

64. The vector according to any one of claims 60 to 63, further comprising a promoter operably coupled to the nucleic acid.

65. The vector according to claim 64, wherein the promoter is for the expression of a polypeptide encoded by the nucleic acid in mammalian cells.

66. The vector according to claim 64, wherein the promoter is for the expression of a polypeptide encoded by the nucleic acid in yeast or insect cells.

67. A vaccine vector, as described in any one of claims 60 to 65.

68. The vector according to claim 67, which is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, or a DNA vaccine vector.

69. The vector according to claim 67, which is an mRNA vaccine vector.

70. The vector according to claim 67, which is a DNA vaccine vector.

71. The vector according to claim 67, 68, or 70, which is a modified vaccinia virus ankara (MVA) vector.

72. The vector according to claim 67, 68, or 70, which is a pURVac vector.

73. Isolated cells comprising the vector according to any one of claims 60 to 72.

74. A fusion protein comprising the polypeptide described in any one of claims 1 to 58.

75. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 58 and a pharmaceutically acceptable carrier, excipient, or diluent.

76. The pharmaceutical composition according to claim 75, comprising the polypeptide described in any one of claims 1 to 15.

77. The pharmaceutical composition according to claim 75, comprising the polypeptide described in any one of claims 16 to 30.

78. The pharmaceutical composition according to claim 75, comprising the polypeptide described in any one of claims 31 to 35.

79. A pharmaceutical composition comprising a nucleic acid molecule according to claim 59 and a pharmaceutically acceptable carrier, excipient, or diluent.

80. The pharmaceutical composition according to claim 79, comprising a nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 15.

81. The pharmaceutical composition according to claim 79, comprising a nucleic acid molecule encoding a polypeptide according to any one of claims 16 to 30.

82. The pharmaceutical composition according to claim 79, comprising a nucleic acid molecule encoding a polypeptide according to any one of claims 31 to 35.

83. A pharmaceutical composition comprising a vector according to any one of claims 60 to 72 and a pharmaceutically acceptable carrier, excipient, or diluent.

84. The pharmaceutical composition according to any one of claims 75 to 83, further comprising an adjuvant for enhancing the immune response in a target to the polypeptide or the polypeptide encoded by the nucleic acid of the composition.

85. The pharmaceutical composition according to any one of claims 79 to 82, wherein the nucleic acid molecule is provided by a vector.

86. The pharmaceutical composition according to claim 85, wherein the vector is a vaccine vector.

87. The pharmaceutical composition according to claim 86, wherein the vaccine vector is a viral vaccine vector, a bacterial vaccine vector, an RNA vaccine vector, an mRNA vaccine vector, or a DNA vaccine vector.

88. The pharmaceutical composition according to claim 87, wherein the vaccine vector is a DNA vaccine vector.

89. The pharmaceutical composition according to claim 88, wherein the DNA vaccine vector is a pURVac vector.

90. The pharmaceutical composition according to claim 87, wherein the viral vaccine vector is a modified vaccinia virus ankara (MVA) vector.

91. The pharmaceutical composition according to claim 87, wherein the vaccine vector is an mRNA vaccine vector.

92. The nucleic acid according to claim 59, comprising one or more modified nucleosides.

93. The vector according to any one of claims 60 to 69, wherein the nucleic acid of the vector comprises one or more modified nucleosides.

94. The pharmaceutical composition according to any one of claims 79 to 82, wherein the nucleic acid of the composition comprises one or more modified nucleosides.

95. The nucleic acid according to claim 92, the vector according to claim 93, or the pharmaceutical composition according to claim 94, wherein the nucleic acid comprises messenger RNA (mRNA).

96. The nucleic acid according to claim 92 or 95, the vector according to claim 93 or 95, or the pharmaceutical composition according to claim 94 or 95, wherein one or more of the modified nucleosides include a 1-methylpseudolidine modification.

97. A nucleic acid according to any one of claims 92, 95, or 96, a vector according to any one of claims 93, 95, or 96, or a pharmaceutical composition according to any one of claims 94 to 96, wherein at least 80% of the uridine in the open reading frame is modified.

98. A pseudovirus comprising the polypeptide described in any one of claims 1 to 58.

99. A method for inducing an immune response to coronavirus in a subject, comprising administering to the subject an effective amount of a polypeptide according to any one of claims 1 to 58, a nucleic acid according to any one of claims 59, 92, or 95 to 97, a vector according to any one of claims 60 to 72, 93, or 95 to 97, or a pharmaceutical composition according to any one of claims 75 to 91 or 94 to 97.

100. A method for immunizing a subject against coronavirus, comprising administering to the subject an effective amount of a polypeptide according to any one of claims 1 to 58, a nucleic acid according to any one of claims 59, 92, or 95 to 97, a vector according to any one of claims 60 to 72, 93, or 95 to 97, or a pharmaceutical composition according to any one of claims 75 to 91 or 94 to 97.

101. A polypeptide according to any one of claims 1 to 58, a nucleic acid according to any one of claims 59, 92, or 95 to 97, a vector according to any one of claims 60 to 72, 93, or 95 to 97, or a pharmaceutical composition according to any one of claims 75 to 91 or 94 to 97, for use as a pharmaceutical.

102. A polypeptide according to any one of claims 1 to 58, a nucleic acid according to any one of claims 59, 92, or 95 to 97, a vector according to any one of claims 60 to 72, 93, or 95 to 97, or a pharmaceutical composition according to any one of claims 75 to 91 or 94 to 97, for use in the prevention, treatment, or improvement of coronavirus infection.

103. Use of a polypeptide according to any one of claims 1 to 58, a nucleic acid according to any one of claims 59, 92, or 95 to 97, a vector according to any one of claims 60 to 72, 93, or 95 to 97, or a pharmaceutical composition according to any one of claims 75 to 91 or 94 to 97, in the manufacture of a pharmaceutical for the prevention, treatment, or improvement of coronavirus infection.

104. The method according to claim 99 or 100, wherein the coronavirus is a β-coronavirus, a polypeptide, nucleic acid, vector, or pharmaceutical composition for use according to claim 102, or the use according to claim 103.

105. The method according to claim 104, or a polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or use, wherein the β-coronavirus is a β-coronavirus of lineage B or lineage C.

106. The method according to claim 104, or a polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or use, wherein the β-coronavirus is a β-coronavirus of lineage B.

107. A polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or a method or use according to claim 105 or 106, wherein the β-coronavirus of lineage B is SARS-CoV or SARS-CoV-2.

108. The method according to claim 105, or a polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or use, wherein the β-coronavirus of lineage C is MERS-CoV.

109. A polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or a method or use according to claim 104, wherein the β-coronavirus is a variant of concern (VOC).

110. The method according to claim 104, or a polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or use, wherein the β-coronavirus is SARS-CoV-2 VOC.

111. Polypeptides, nucleic acids, vectors, or pharmaceutical compositions for use or the method or use described in claim 104, wherein the β-coronavirus is SARS-CoV-2β, γ, Δ, or omicron VOC.

112. A method for diagnosing whether a subject has coronavirus infection, comprising determining whether a polypeptide according to any one of claims 1 to 58 binds to an antibody produced by the subject.

113. The method according to claim 112, wherein the antibody is present in a biological sample obtained from the subject, or in a sample derived from a biological sample obtained from the subject.

114. The method according to claim 113, wherein the biological sample is a serum sample.

115. The method according to claim 104, or a polypeptide, nucleic acid, vector, or pharmaceutical composition for use, or use, wherein the β-coronavirus is Omicron BA. 1, BA. 2, BA. 2.75, 2.75.2, BA. 2.3.20, BA. 4, BA. 5, BQ. 1.1, XBB, XBB. 1.5, BF. 7, XBB. 1.19.1, XBC, BQ. 1.12, CH. 1.1.1, or XBB. 1.9.1 virus.

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