Coronavirus spike protein variants

Abstract

Provided are coronavirus spike protein variants for use as antigens for vaccines for coronavirus infections.　The present invention provides coronavirus spike protein variants comprising (a) a receptor binding domain (RBD) formed from the amino acid sequence set forth in SEQ ID NO: 1, (b) an RBD formed from an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 1, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to RBD formed from the amino acid sequence set forth in SEQ ID NO: 1, or (c) an RBD formed from an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to RBD formed from the amino acid sequence set forth in SEQ ID NO: 1.

Description

Coronavirus spike protein variants

[0001] The present invention relates to a coronavirus spike protein variant suitable as an antigen for a novel coronavirus vaccine (also referred to as the "(coronavirus) spike protein variant of the present invention"), or an immunogenic composition containing the spike protein variant (also referred to as the "immunogenic composition of the present invention").

[0002] SARS-CoV-1 (severe acute respiratory syndrome related coronavirus, sometimes referred to as "SARS-1") is an RNA virus belonging to the Coronaviridae family of the Nidovirales order, which causes severe acute respiratory syndrome (SARS). SARS first emerged in November 2002 and caused SARS epidemics from 2002 to 2004. SARS-CoV-2 is also an RNA virus belonging to the Coronaviridae family of the Nidovirales order, which causes acute respiratory symptoms. Since its outbreak in 2019, the infection caused by SARS-CoV-2 (COVID-19) has spread to countries around the world, causing a global pandemic. The infection caused by SARS-CoV-2 has been named COVID-19.

[0003] BNT162b2 (BioNTech and Pfizer) and mRNA-1273 (Moderna) are mRNA vaccines for the prevention of COVID-19 and have demonstrated high efficacy in preventing COVID-19 (Non-Patent Documents 1 and 2).

[0004] All of the above initial mRNA vaccines encoded the spike protein of the original strain, but the Omicron strain, which has a high immune evasion ability, emerged, and a bivalent vaccine was developed to counter the Omicron strain by adding the spike protein of the Omicron strain (Non-Patent Document 3).

[0005] Variants (mutant strains) that evade coronavirus vaccines and immunity caused by coronavirus infection have since been reported (Non-Patent Document 4).

[0006] Furthermore, SARS-CoV-1 and SARS-CoV-2 are thought to be zoonotic infectious diseases of animal origin, and coronaviruses closely related to SARS-CoV-2 have been isolated from bats and other animals (Non-Patent Document 5). There are concerns that pandemics caused by new coronaviruses will continue to occur in the future.

[0007] Non-Patent Document 6 demonstrates that mRNA vaccines encoding various chimeric spikes, in which the RBD, NTD, and S2 of the spike protein are sequences of SARS-CoV-1, SARS-CoV-2, etc., have broad cross-reactivity. Non-Patent Document 7 discloses a spike protein in which the RBD of the Delta strain is inserted into the N-terminal side of the RBD of the Omicron strain (BA.1 strain) spike protein, aiming to create a vaccine antigen with broad cross-reactivity against various variants of concern (VOCs). Non-Patent Document 8 provides a review of universal vaccines against SARS-CoV-2 variants, and Figure 3 in this document discloses spike proteins with various designs. Non-Patent Document 9 discloses nanoparticles carrying the RBDs of eight sarbecoviruses, including SARS-CoV-2 and WIV-1, with the aim of achieving broad spectrum coverage. However, they are trying to achieve broad spectrum by inducing antibodies against conserved regions rather than inducing antibodies against epitopes with different sequences in each RBD. Non-Patent Document 10 discloses three types of antigens in which the receptor-binding motif (RBM) of the RBD is that of SARS-CoV-2 and the portions other than the RBM region are those of other strains such as SARS-CoV-1, WIV-1, and SARS-CoV-2.

[0008] However, none of the above documents describes the coronavirus spike protein mutant of the present invention.

[0009] N Engl J Med. 2021 Nov 4;385(19):1761-1773. N Engl J Med. 2021 Feb 4;384(5):403-416. Lancet Infect Dis. 2023 Aug 2; S1473-3099(23)00373-0. Nat Rev Microbiol. 2023 Mar; 21(3):162-177. Cell. 2021 Aug 19;184(17):4380-4391. Science. 2021 Aug 27;373(6558):991-998. NPJ Vaccines. 2022 Dec 19;7(1):167. Mol Ther Nucleic Acids. 2022 Dec 13;30:465-476. Science. 2022 Aug 5;377(6606):eabq0839. Cell Rep. 2022 Mar 22;38(12):110561.

[0010] An object of the present invention is to provide an antigen or the like that has immunogenicity against betacoronaviruses including sarbecoviruses and is suitable for vaccines to prevent coronavirus infections, including novel coronavirus disease (COVID-19).

[0011] The present inventors have conducted extensive research to solve the above problems and have completed the present invention.

[0012] The present invention relates to the following (1) to (25): (1) (a) a receptor binding domain (RBD) consisting of the amino acid sequence set forth in SEQ ID NO: 1, (b) an RBD consisting of an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids in the amino acid sequence set forth in SEQ ID NO: 1 have been substituted, deleted, inserted, or added, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1, or (c) A coronavirus spike protein variant comprising an RBD consisting of an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1.(1-1)(b) an RBD consisting of an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added at amino acid residues other than positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 of the amino acid sequence set forth in SEQ ID NO: 1, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1; or (c) The coronavirus spike protein variant according to item 1, comprising an RBD having a substitution, deletion, insertion or addition at an amino acid residue other than positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 of the amino acid sequence set forth in SEQ ID NO: 1, and having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1. (1-2) (a) an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32; (b) an RBD consisting of an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids in the amino acid sequence set forth in SEQ ID NO: 32 have been substituted, deleted, inserted, or added, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32; or (c) A coronavirus spike protein variant comprising an RBD consisting of an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 32, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32.(1-3)(b) an RBD consisting of an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added at amino acid residues other than positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173, and 175 of the amino acid sequence set forth in SEQ ID NO: 32, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32; or (c) The coronavirus spike protein variant according to Aspect 1-2, comprising an RBD having substitutions, deletions, insertions or additions at amino acid residues other than those at positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173 and 175 in the amino acid sequence set forth in SEQ ID NO: 32, and having 95% (preferably 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 32, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32. (1-4) A coronavirus spike protein variant comprising an RBD consisting of the amino acid sequence of positions 325 to 527 of the amino acid sequence set forth in any one of SEQ ID NOs: 65, 68, 71, 74, 76, 82, 84, 87, 89, 92, 93, 94, 96, 97, 100, 102 to 104, and 106 to 110. (2) The spike protein variant according to item (1) or (1-1), wherein the amino acid sequence set forth in SEQ ID NO: 1 is an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 5. (3) The spike protein variant according to any one of items (1), (2), and (1-1) to (1-4), comprising an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence of positions 14 to 1211 of the amino acid sequence set forth in SEQ ID NO: 6.(4) The spike protein variant according to any one of items (1) to (3) and (1-1) to (1-4), comprising an N-terminal domain (NTD) consisting of: (a) the amino acid sequence of SEQ ID NO: 7; (b) an amino acid sequence in which one to several (preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids in the amino acid sequence of SEQ ID NO: 7 have been substituted, deleted, inserted, or added; or (c) an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 7. (5) The spike protein mutant according to item (4), wherein the amino acid sequence set forth in SEQ ID NO: 7 is the amino acid sequence set forth in either SEQ ID NO: 8 or 9. (5-1) (a) an amino acid sequence of a region corresponding to the spike protein NTD derived from a beta coronavirus (preferably derived from a sarbecovirus); (b) an amino acid sequence in which one to several (preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added in the amino acid sequence of a region corresponding to the spike protein NTD derived from a beta coronavirus (preferably derived from a sarbecovirus); or (c) an amino acid sequence having a sequence identity of 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) with the amino acid sequence of a region corresponding to the spike protein NTD derived from a beta coronavirus (preferably derived from a sarbecovirus). (5-2) The spike protein variant according to any one of items (1) to (3) and (1-1) to (1-4), comprising an NTD consisting of: (5-3) The spike protein variant according to item (5-1), wherein the betacoronavirus is a sarbecovirus selected from the viruses listed in the table below. (5-3) The spike protein variant according to item (5-1), wherein the beta coronavirus is a Middle East respiratory syndrome coronavirus (MERS-CoV). (5-4) The spike protein variant according to any one of items (1) to (5) and (1-1) to (1-4), comprising an NTD consisting of the amino acid sequence of positions 14 to 301 of the amino acid sequence set forth in any one of SEQ ID NOs: 66, 67, 69, 70, 72, 73, 75, 77 to 81, 83, 85, 86, 88, 90, 91, 95, 98, 99, 101, 105, and 111 to 122. (6) (a) the amino acid sequence of SEQ ID NO: 10; (b) an amino acid sequence in which one to several (preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids are substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 10; or (c) an amino acid sequence having a sequence identity of 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of SEQ ID NO: 10. (7) The spike protein variant according to any one of Items (1) to (5), (1-1) to (1-4), and (5-1) to (5-4), comprising a region consisting of SD1, SD2, and S2 subunits, consisting of the amino acid sequence of positions 528 to 1209 of the amino acid sequence of any one of SEQ ID NOs: 66, 67, 69, 70, 72, 73, 75, 77 to 81, 83, 85, 86, 88, 90, 91, 95, 98, 99, 101, 105, and 111 to 122.(8) (a) the amino acid sequence of SEQ ID NO: 16; (b) an amino acid sequence in which one to several (preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids in the amino acid sequence of SEQ ID NO: 16 have been substituted, deleted, inserted, or added; or (c) an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence of SEQ ID NO: 16. (9) The spike protein variant of any of items (8) to (9), having amino acid substitutions corresponding to the Q434C substitution and the S472C substitution in the amino acid sequence of SEQ ID NO: 16. (10) The spike protein variant of item (8) or (9), having amino acid substitutions corresponding to the K455P substitution and / or the V456P substitution in the amino acid sequence of SEQ ID NO: 16. (11) The spike protein variant of any of items (8) to (10), having amino acid substitutions corresponding to one or more amino acid substitutions selected from the group consisting of F286P substitution, A361P substitution, A368P substitution, and A411P substitution in the amino acid sequence of SEQ ID NO: 16. (12) The spike protein variant of any of Items (8) to (11), which has an amino acid substitution corresponding to the T201P substitution and / or the K264P substitution in the amino acid sequence of SEQ ID NO: 16. (13) The spike protein variant of any of Items (8) to (12), which has an amino acid mutation in one or more amino acid residues corresponding to RRAR at positions 151 to 154 of the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 17), and which is not cleaved by furin protease. (14) The spike protein variant of Item (13), in which the amino acid residues corresponding to RRAR at positions 151 to 154 of the amino acid sequence of SEQ ID NO: 16 are substituted with GSAS (SEQ ID NO: 18).(15) The spike protein variant according to any one of items (8) to (14), having one or more amino acid substitutions corresponding to one or more amino acid substitutions selected from the group consisting of N543Q substitution, N567Q substitution, and N642L substitution in the amino acid sequence of SEQ ID NO: 16. (16) The spike protein variant according to any one of items (1) to (15), (1-1) to (1-4), (5-1) to (5-4), and (7-1), wherein the transmembrane domain and the cytoplasmic tail are deleted. (17) The spike protein variant according to item (16), wherein a trimerization domain is added to the C-terminus. (17-1) The spike protein variant according to item (17), wherein the trimerization domain is a Foldon sequence. (18) The spike protein variant according to any one of items (1) to (17), (1-1) to (1-4), (5-1) to (5-4), (7-1), and (17-1), wherein a His tag is added to the C-terminus. (19) The spike protein variant according to any one of items (1) to (18), (1-1), (5-1), and (17-1), comprising an amino acid sequence from position 14 to the C-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27. (19-1) The spike protein variant according to any one of items (1) to (19) and (17-1), comprising an amino acid sequence from position 14 to 1236 of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27. (19-2) (a) an amino acid sequence in which one to several (preferably 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids are substituted, deleted, inserted, or added in the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27; or (b) an amino acid sequence having a sequence identity of 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) with the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27. The spike protein variant according to any one of items (1) to (19), (1-1), (5-1) to (5-4), (7-1), and (17-1), comprising:(19-3) The spike protein variant according to item (19-2), which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to a spike protein variant comprising an amino acid sequence from positions 14 to 1236 in the amino acid sequence set forth in any of SEQ ID NOs: 21 to 27. (19-4) The spike protein variant according to item (19-2) or (19-3), which comprises an amino acid sequence from positions 14 to 1236 in the amino acid sequence set forth in any of SEQ ID NOs: 65 to 122. (19-5) The spike protein variant according to any of items (1) to (19), (1-1), (1-4), (5-1), (5-4), (7-1), or (17-1), which comprises an amino acid sequence from positions 14 to 1209 in the amino acid sequence set forth in any of SEQ ID NOs: 21 to 27. (19-6) (a) an amino acid sequence in which one to several (preferably 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids are substituted, deleted, inserted, or added in the amino acid sequence of positions 14 to 1209 of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27; or (b) an amino acid sequence having a sequence identity of 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) with the amino acid sequence of positions 14 to 1209 of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27. (19-7) The spike protein variant according to any one of items (19-6), (19-7) or (19-8), which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of a spike protein variant comprising an amino acid sequence from positions 14 to 1209 of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27. (19-8) The spike protein variant according to item (19-6) or (19-8), which comprises an amino acid sequence from positions 14 to 1209 of the amino acid sequence set forth in any one of SEQ ID NOs: 65 to 122.(20) A coronavirus spike protein variant having 99% or more (preferably 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.9% or more, or 99.9% or more) sequence identity with the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in SEQ ID NO: 26, and comprising the amino acid sequences of positions 14 to 324 and 528 to 1236 of the amino acid sequence set forth in SEQ ID NO: 26. (20-1) A coronavirus spike protein variant having an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted, or added at positions 14 to 1236 of the amino acid sequence set forth in SEQ ID NO: 26, and comprising the amino acid sequences of positions 14 to 324 and 528 to 1236 of the amino acid sequence set forth in SEQ ID NO: 26. (20-2) The spike protein variant according to item (20) or (20-1), which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to a spike protein variant comprising the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in SEQ ID NO: 26. (21) The spike protein variant according to item (20) or (20-1), which comprises the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in any one of SEQ ID NOs: 65, 68, 71, 74, 76, 82, 84, 87, 89, 92, 93, 94, 96, 97, 100, 102 to 104, and 106 to 110. (22) A coronavirus spike protein variant having 99% or more (preferably 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.9% or more, or 99.9% or more) sequence identity with the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in SEQ ID NO: 26, and comprising the amino acid sequence of positions 325 to 527 of the amino acid sequence set forth in SEQ ID NO: 26. (22-1) A coronavirus spike protein variant having an amino acid sequence in which one to several amino acids are substituted, deleted, inserted, or added at positions 14 to 1236 of the amino acid sequence set forth in SEQ ID NO: 26, and comprising the amino acid sequence of positions 325 to 527 of the amino acid sequence set forth in SEQ ID NO: 26.(22-2) The spike protein variant according to item (22) or (22-1), which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to a spike protein variant comprising the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in SEQ ID NO: 26. (23) The spike protein variant according to item (22) or (22-1), which comprises the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in any one of SEQ ID NOs: 66, 67, 69, 70, 72, 73, 75, 77 to 81, 83, 85, 86, 88, 90, 91, 95, 98, 99, 101, 105, and 111 to 122. (23-1) An RBD consisting of the amino acid sequence of (a) or (b) below: (a) the amino acid sequence of SEQ ID NOs: 1 to 5; (b) the amino acid sequence of positions 325 to 527 of the amino acid sequence of any one of SEQ ID NOs: 65, 68, 71, 74, 76, 82, 84, 87, 89, 92, 93, 94, 96, 97, 100, 102 to 104, and 106 to 110; An NTD consisting of the amino acid sequence of (c) or (d) below; and (c) the amino acid sequence of SEQ ID NOs: 7 to 9; (d) the amino acid sequence of positions 14 to 301 of the amino acid sequence of any one of SEQ ID NOs: 66, 67, 69, 70, 72, 73, 75, 77 to 81, 83, 85, 86, 88, 90, 91, 95, 98, 99, 101, 105, and 111 to 122; (24) A coronavirus spike protein variant comprising: a region consisting of SD1, SD2, and S2 subunits, which consists of the amino acid sequence of (e) or (f) below: (e) an amino acid sequence set forth in SEQ ID NOs: 7 to 9, or (f) an amino acid sequence from positions 528 to 1209 of the amino acid sequence set forth in any of SEQ ID NOs: 66, 67, 69, 70, 72, 73, 75, 77 to 81, 83, 85, 86, 88, 90, 91, 95, 98, 99, 101, 105, and 111 to 122. (25) An immunogenic composition comprising the spike protein variant set forth in any of items (1) to (23), (1-1) to (1-4), (5-1) to (5-4), (7-1), (17-1), (19-1) to (19-8), (20-1), (20-2), (22-1), (22-2), or (23-1).(25) The immunogenic composition according to item (24), comprising only one type of spike protein variant according to any one of items (1) to (23), (1-1) to (1-4), (5-1) to (5-4), (7-1), (17-1), (19-1) to (19-8), (20-1), (20-2), (22-1), (22-2), and (23-1) as an antigen derived from coronavirus spike protein, and not containing any other antigen derived from coronavirus spike protein. (26) The immunogenic composition according to item (24) or (25), comprising an adjuvant. (27) The immunogenic composition according to item (26), wherein the adjuvant is an adjuvant containing squalene, tocopherol, and polysorbate 80. (28) A nucleic acid encoding the spike protein variant according to any one of items (1) to (23), (1-1) to (1-4), (5-1) to (5-4), (7-1), (17-1), (19-1) to (19-8), (20-1), (20-2), (22-1), (22-2), and (23-1). (29) An expression vector comprising the nucleic acid according to item (28). (30) A method for preventing infection with SARS-CoV-2 and / or other coronaviruses (preferably betacoronaviruses, more preferably sarbecoviruses), comprising administering to an individual an effective amount of the immunogenic composition according to any one of items (24) to (27), the nucleic acid according to item (28), or the expression vector according to item (29). (31) The immunogenic composition according to any one of items (24) to (27), the nucleic acid according to item (28), or the expression vector according to item (29), for preventing infection with SARS-CoV-2 and / or other coronaviruses (preferably, betacoronaviruses, more preferably sarbecoviruses). (32) Use of the immunogenic composition according to any one of items (24) to (27), the nucleic acid according to item (28), or the expression vector according to item (29), for the manufacture of a medicament for preventing infection with SARS-CoV-2 and / or other coronaviruses (preferably, betacoronaviruses, more preferably sarbecoviruses). (33) A spike protein variant comprising the amino acid sequence of positions 14 to 1209 of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27 and 65 to 122.(34) The spike protein variant according to item (33), comprising the amino acid sequence from position 14 to position 1236 of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27 and 65 to 122. (35) The spike protein variant according to item (34), comprising the amino acid sequence from position 14 to the C-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27 and 65 to 122. (36) An immunogenic composition comprising the spike protein variant according to any one of items (33) to (35). (37) The immunogenic composition according to item (36), comprising only one type of spike protein variant set forth in any one of items (33) to (35) as an antigen derived from coronavirus spike protein, and not comprising any other antigen derived from coronavirus spike protein. (38) The immunogenic composition according to item (36) or (37), comprising an adjuvant. (39) The immunogenic composition according to item (37), wherein the adjuvant contains squalene, tocopherol, and polysorbate 80. (40) A nucleic acid encoding the spike protein variant according to any one of items (33) to (35). (41) An expression vector comprising the nucleic acid according to item (40). (42) The immunogenic composition according to any one of items (36) to (39), the nucleic acid according to item (40), or the expression vector according to item (41), for preventing infection with SARS-CoV-2 and / or other coronaviruses (preferably betacoronaviruses, more preferably sarbecoviruses).

[0013] The coronavirus spike protein mutant of the present invention has the excellent effect of being a monovalent antigen that exhibits broad-spectrum immunogenicity.

[0014] FIG. 1 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen conducted in Example 5. The "Pseudovirus" label in the figure indicates the name (strain) of the pseudovirus, and the "Immunization" label indicates the type of antigen used for immunization. The vertical axis in the figure represents the neutralizing antibody titer (NT50), the vertical bars indicate the geometric mean value for each group of combinations of each antigen and each pseudovirus, the error bars indicate the 95% confidence interval, and the small circles indicate the neutralizing antibody titer for each individual in each group (the same applies to FIGS. 2 to 24). FIG. 2 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen conducted in Example 6. FIG. 3 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen conducted in Example 7. FIG. 4 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen conducted in Example 8. FIG. 5 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen, performed in Example 9. FIG. 6 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen, performed in Example 10. FIG. 7 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen, performed in Example 11. FIG. 8 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen, performed in Example 12. FIG. 9 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen, performed in Example 13. FIG. 10 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen, performed in Example 14. FIG. 11 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen, performed in Example 15. FIG. 12 shows the results of a neutralizing activity test against each pseudovirus of sera from mice immunized with each antigen, performed in Example 16. Figure 13 shows the results of a test of the neutralizing activity of sera from mice immunized with each antigen against each pseudovirus, performed in Example 17. Figure 14 shows the results of a test of the neutralizing activity of sera from mice immunized with each antigen against each pseudovirus, performed in Example 18.FIG. 15 shows the results of a neutralizing activity test performed in Example 19 on the sera of mice immunized with each antigen against each pseudovirus. FIG. 16 shows the results of a neutralizing activity test performed in Example 19 on the sera of mice immunized with each antigen against each pseudovirus. FIG. 17 shows the results of a neutralizing activity test performed in Example 19 on the sera of mice immunized with each antigen against each pseudovirus. FIG. 18 shows the results of a neutralizing activity test performed in Example 19 on the sera of mice immunized with each antigen against each pseudovirus. FIG. 19 shows the results of a neutralizing activity test performed in Example 19 on the sera of mice immunized with each antigen against each pseudovirus. FIG. 20 shows the results of a neutralizing activity test performed in Example 19 on the sera of mice immunized with each antigen against each pseudovirus. FIG. 21 shows the results of a neutralizing activity test performed in Example 19 on the sera of mice immunized with each antigen against each pseudovirus. FIG. 22 shows the results of a neutralizing activity test performed in Example 19 on the sera of mice immunized with each antigen against each pseudovirus. Figure 23 shows the results of a test of the neutralizing activity of sera from mice immunized with each antigen against each pseudovirus, performed in Example 19. Figure 24 shows the results of a test of the neutralizing activity of sera from mice immunized with each antigen against each pseudovirus, performed in Example 19.

[0015] The term "consisting of" means having only the elements. The term "including" means being open-ended and not excluding unrecited elements.

[0016] The present invention will be described below with reference to embodiments. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the above-mentioned field unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0017] "Sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with those in the reference polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining sequence identity can be accomplished in a variety of ways within the skill of one in the art, for example, using publicly available computer software such as BLAST.

[0018] As used herein, "conventional strain spike protein" refers to the spike protein of SARS-CoV-2 as set forth in NCBI Reference Number: YP_009724390.1.

[0019] In the present invention, "immunogenicity" refers to the property of stimulating the immune system of a target individual to induce an immune response, such as the production of antibodies against a target pathogen. Furthermore, an "immunogenic composition" refers to a composition that, when administered to a target individual, induces an immune response in the individual.

[0020] As used herein, the term "neutralizing antibody-inducing activity" refers to the ability of an antigen protein to induce neutralizing antibodies, and can be evaluated by the neutralizing antibody titer in immune serum obtained by inoculating a test animal with the antigen protein. The strength of the neutralizing activity of the serum can be evaluated by the dilution factor of the serum required to reduce the number of infections with the test virus by 50%, i.e., the neutralizing antibody titer (NT50).

[0021] As used herein, the phrase "having equivalent neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1" means that the spike protein variant has a neutralizing antibody titer of 12.5% ​​or more (preferably 25%, 50%, 70%, 80%, 90%, 95%, or 100% or more) of the neutralizing antibody titer of the spike protein being compared against the D614G strain of SARS-CoV-2 (GISAID ID: EPI_ISL_529135, hereinafter also referred to as SARS-CoV-2(D614G)), and a neutralizing antibody titer of 12.5% ​​or more (preferably 25%, 50%, 70%, 80%, 90%, 95%, or 100% or more) of the neutralizing antibody titer of the spike protein being compared against SARS-CoV-1 (Genbank "The neutralizing antibody-inducing activity of a spike protein variant can be confirmed, for example, by producing the spike protein variant to be measured according to the method described in Example 1, and immunizing mice using the method described in Example 4 to measure the neutralizing antibody titer of the resulting serum.

[0022] The present invention relates to coronavirus spike protein mutants. The term "coronavirus spike protein mutant" refers to a coronavirus having some amino acid mutation in the amino acid sequence of the spike protein of a natural coronavirus, and may also be referred to simply as "spike protein mutant." As used herein, "amino acid mutation" refers to the substitution, deletion, insertion, or addition of one or more amino acids, or a combination thereof.

[0023] The spike protein variant of the present invention comprises: (a) a receptor binding domain (RBD) consisting of the amino acid sequence set forth in SEQ ID NO: 1; (b) an RBD consisting of an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids in the amino acid sequence set forth in SEQ ID NO: 1 have been substituted, deleted, inserted, or added, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1; or (c) An RBD consisting of an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0024] The term "receptor binding domain (RBD)" refers to a region of a coronavirus spike protein that contributes to binding to a cellular receptor. However, in the present invention, "receptor binding domain (RBD)" is a term used to identify a region in the spike protein, and does not necessarily have binding activity to a cellular receptor. In the case of a conventional strain spike protein, the amino acid sequence from positions 329 to 531 corresponds to the RBD.

[0025] The amino acid sequence set forth in SEQ ID NO:1 includes the amino acid sequence set forth in any of SEQ ID NOs:2 to 5.

[0026] In order to design an RDB sequence that has neutralizing antibody-inducing activity against both SARS-CoV-2 and SARS-CoV-1, the present inventors created various hybrid RBDs based on the RDB sequences of SARS-CoV-2 and SARS-CoV-1, and discovered the RBD sequence of SEQ ID NO: 3. As its variations, they also obtained the RBD sequences of SEQ ID NOs: 2, 4, and 5 below.

[0027] SEQ ID NO: 1 is an amino acid sequence (RBD consensus sequence) that generalizes the amino acid sequences of the RBDs contained in the spike virus mutants of the present invention described below (SEQ ID NOs: 2 to 5), and the amino acid sequences are as follows:

[0028]

[0029] In the amino acid sequence of SEQ ID NO: 1, X at position 3 is Q or N, X at position 132 is N or K, X at position 149 is S or Y, and X at position 173 is N or Y.

[0030] Examples of the amino acid sequence set forth in SEQ ID NO:1 above include the amino acid sequences set forth in any of SEQ ID NOs:2 to 5.

[0031] (b) RBDs comprising an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 1, and which have neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to the RBD consisting of the amino acid sequence of SEQ ID NO: 1 include RBDs consisting of the amino acid sequence of positions 325 to 527 in the amino acid sequence of any of SEQ ID NOs: 65, 68, 71, 74, 76, 82, 84, 87, 89, 92, 93, 94, 96, 97, 100, 102 to 104, and 106 to 110.

[0032] In the present invention, "RBD having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1" means an RBD that exhibits a neutralizing antibody titer of 12.5% ​​or more (preferably 25%, 50%, 70%, 80%, 90%, 95%, or 100% or more) of the neutralizing antibody titer of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1 against the D614G strain of SARS-CoV-2 (GISA ID: EPI_ISL_529135, hereinafter also referred to as SARS-CoV-2(D614G)), and exhibits a neutralizing antibody titer of 12.5% ​​or more (preferably 25%, 50%, 70%, 80%, 90%, 95%, or 100% or more) of the neutralizing antibody titer of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1 against SARS-CoV-1 (Genbank The term "RBD" refers to an RBD that exhibits a neutralizing antibody titer of 12.5% ​​or more (preferably, 25%, 50%, 70%, 80%, 90%, 95%, or 100% or more) of the neutralizing antibody titer against SARS-CoV-2 and SARS-CoV-1 (SEQ ID NO: AY278741.1). The neutralizing antibody-inducing activity of an RBD can be confirmed, for example, by preparing the RBD to be measured, forming it into nanoparticles, immunizing mice with the nanoparticles, and measuring the neutralizing antibody titer of the serum obtained by immunizing mice according to the method described in Example 6. The definition of "RBDs having equivalent neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1" for sequences other than SEQ ID NO: 1 is the same as above.

[0033] SEQ ID NO: 3 is a sequence contained in H-00149 of Example 5, H-00428 of Example 6, H-00557 of Example 8, and H-00657 of Example 14, and has been shown to have neutralizing antibody-inducing activity against both SARS-CoV-2 (D614G) and SARS-CoV-1.

[0034] SEQ ID NO: 2 is a sequence contained in the amino acid sequence (SEQ ID NO: 62) encoded by H-00646, H-00669, H-00672, and H-00675 in Example 14 and mRNA-001 in Example 16, and has been shown to have neutralizing antibody-inducing activity against both SARS-CoV-2 (D614G) and SARS-CoV-1.

[0035] SEQ ID NO: 4 is a sequence contained in H-00739 of Example 15, and SEQ ID NO: 5 is a sequence contained in H-00740 of Example 15, and it has been shown that they each have neutralizing antibody-inducing activity against both SARS-CoV-2 (D614G) and SARS-CoV-1.

[0036] In another embodiment, the spike protein variant of the present invention is: (b) an RBD consisting of an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added at amino acid residues other than positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 of the amino acid sequence of SEQ ID NO: 1, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence of SEQ ID NO: 1; (c) an RBD having substitutions, deletions, insertions or additions at amino acid residues other than those at positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 of the amino acid sequence set forth in SEQ ID NO: 1, and having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0037] In the above, "an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added at amino acid residues other than positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 of the amino acid sequence set forth in SEQ ID NO: 1" means an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added at positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 of the amino acid sequence set forth in SEQ ID NO: 1 This refers to a mutant sequence of the amino acid sequence set forth in SEQ ID NO: 1, in which the amino acid residues at positions 5, 74, 75, and 78 are not substituted or the like (i.e., the amino acid residues at positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 are maintained), and one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids are substituted, deleted, inserted, or added at the amino acid residues at other positions.

[0038] In the above, "an amino acid sequence having substitutions, deletions, insertions or additions at amino acid residues other than those at positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75 and 78 of the amino acid sequence set forth in SEQ ID NO: 1 and having a sequence identity of 90% or more (preferably, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence set forth in SEQ ID NO: 1" means an amino acid sequence having substitutions, deletions, insertions or additions at positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75 and 78 of the amino acid sequence set forth in SEQ ID NO: 1. "A" refers to a mutant sequence of the amino acid sequence of SEQ ID NO: 1, which has not been substituted at positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 (i.e., the amino acid residues at positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 are maintained), but has been substituted, deleted, inserted, or added at amino acid residues at other positions, resulting in a sequence identity of 90% or more (preferably, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of SEQ ID NO: 1. The same applies to SEQ ID NOs: 2 to 5.

[0039] Based on the experimental results of Examples 5 and 6, the amino acid residues at positions 18, 20, 26, 29, 44, 45, 56, 65, 74, 75, and 78 in the amino acid sequence set forth in SEQ ID NO: 1 are considered to be important amino acid residues for having neutralizing activity against both SARS-CoV-2 and SARS-CoV-1.

[0040] In another embodiment, the spike protein variant of the present invention comprises: (a) an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32; (b) an RBD consisting of an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids in the amino acid sequence set forth in SEQ ID NO: 32 have been substituted, deleted, inserted, or added, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32; or (c) an RBD consisting of an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 32, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32.

[0041] In another embodiment, the spike protein variant of the present invention is: (b) an RBD consisting of an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added at amino acid residues other than positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173, and 175 of the amino acid sequence set forth in SEQ ID NO: 32, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32; (c) an RBD having substitutions, deletions, insertions or additions at amino acid residues other than those at positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173 and 175 of the amino acid sequence set forth in SEQ ID NO: 32, and consisting of an amino acid sequence having 95% (preferably 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 32, and having neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of an RBD consisting of the amino acid sequence set forth in SEQ ID NO: 32.

[0042] In the above, "an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added at amino acid residues other than positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173, and 175 of the amino acid sequence set forth in SEQ ID NO: 32" means an amino acid sequence in which one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added at positions other than positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173, and 175 of the amino acid sequence set forth in SEQ ID NO: 32 This refers to a mutant sequence of the amino acid sequence of SEQ ID NO: 32 in which the amino acid residues at positions 116, 117, 118, 170, 171, 173, and 175 are not substituted or the like (i.e., the amino acid residues at positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173, and 175 are maintained), and one to several (preferably 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids are substituted, deleted, inserted, or added at the amino acid residues at other positions.

[0043] In the above, "an amino acid sequence having substitutions, deletions, insertions or additions at amino acid residues other than those at positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173 and 175 of the amino acid sequence set forth in SEQ ID NO: 32 and having 95% (preferably 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence set forth in SEQ ID NO: 32" means an amino acid sequence having substitutions, deletions, insertions or additions at amino acid residues other than those at positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173 and 175 of the amino acid sequence set forth in SEQ ID NO: 32. The amino acid sequence of SEQ ID NO: 32 refers to a mutant sequence of the amino acid sequence of SEQ ID NO: 32 that has not been substituted at positions 0, 171, 173, and 175 (i.e., the amino acid residues at positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173, and 175 are maintained), and has been substituted, deleted, inserted, or added at amino acid residues at other positions, resulting in a sequence identity of 90% or more (preferably, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of SEQ ID NO: 32.

[0044] Based on the experimental results of Examples 5 and 6, the amino acid residues at positions 44, 45, 56, 65, 74, 75, 78, 110, 111, 113, 115, 116, 117, 118, 170, 171, 173, and 175 in the amino acid sequence set forth in SEQ ID NO: 32 are considered to be important amino acid residues for having neutralizing activity against both SARS-CoV-2 and SARS-CoV-1.

[0045] SEQ ID NO: 32 is a sequence contained in H-000148 of Example 5, H-00081 of Example 6, and H-00556 of Example 8, and has been shown to have neutralizing antibody-inducing activity against both SARS-CoV-2 (D614G) and SARS-CoV-1.

[0046] In a further aspect, the spike protein variant of the present invention may comprise an amino acid sequence that has 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence consisting of positions 14 to 1211 of the amino acid sequence shown in SEQ ID NO: 6 (full-length spike of the Delta strain).

[0047] Sequence number 6 is the amino acid sequence of the full-length spike protein of the Delta strain, and the sequence is as follows:

[0048]

[0049] The antigens described in the Examples of the present application (e.g., H-00646, H-00657, H-00669, H-00672, H-00675, H-00739, and H-00740) contain amino acid sequences that share 90% or more sequence identity with the amino acid sequence of positions 14 to 1211 of SEQ ID NO: 6. For example, H-00657 (SEQ ID NO: 22) contains an amino acid sequence that shares 96% or more identity with the amino acid sequence of positions 14 to 1211 of SEQ ID NO: 6.

[0050] The spike protein variants of the present invention may comprise an N-terminal domain (NTD) consisting of: (a) the amino acid sequence of SEQ ID NO: 7; (b) an amino acid sequence in which one to several (preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 7; or (c) an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0051] Examples of the amino acid sequence of SEQ ID NO:7 include the amino acid sequence of either SEQ ID NO:8 or SEQ ID NO:9.

[0052] (b) NTDs consisting of an amino acid sequence in which one to several (preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added in the amino acid sequence set forth in SEQ ID NO: 7 include NTDs consisting of the amino acid sequence of positions 14 to 301 in the amino acid sequence set forth in any of SEQ ID NOs: 66, 67, 69, 70, 72, 73, 75, 77 to 81, 83, 85, 86, 88, 90, 91, 95, 98, 99, 101, 105, and 111 to 122.

[0053] The N-terminal domain (NTD) refers to the N-terminal region of the coronavirus spike protein. In the case of conventional spike proteins, the region of the amino acid sequence from positions 14 to 305 corresponds to the NTD.

[0054] SEQ ID NOs: 8 and 9 are included in the NTD sequences contained in the spike virus mutants of the present invention, and SEQ ID NO: 7 is a generalized amino acid sequence (NTD consensus sequence) of SEQ ID NOs: 8 and 9, and the respective amino acid sequences are as follows:

[0055]

[0056] X at position 150 in the amino acid sequence of SEQ ID NO: 7 is T or L.

[0057] The above NTD sequence 1 is contained in H-00646, H-00657, H-00669, H-00672, H-00739, and H-00740, and NTD sequence 2 is contained in H-00675.

[0058] In another embodiment, the spike protein variant of the present invention is: (a) an amino acid sequence of a region corresponding to the NTD of a spike protein derived from a betacoronavirus (preferably derived from a sarbecovirus); (b) an amino acid sequence in which one to several (preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added in the amino acid sequence of a region corresponding to the NTD of a spike protein derived from a betacoronavirus (preferably derived from a sarbecovirus); (c) An NTD consisting of an amino acid sequence having 90% or more (preferably, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the amino acid sequence of a corresponding region of a spike protein NTD derived from a betacoronavirus (preferably derived from a sarbecovirus).

[0059] The betacoronavirus is preferably a sarbecovirus. Preferred sarbecoviruses include:

[0060]

[0061] Examples of preferred non-sarbecovirus betaviruses include Middle East respiratory syndrome coronavirus (MERS-CoV).

[0062] The spike protein variant of the present invention may comprise a region consisting of SD1, SD2 and S2 subunits, which consist of: (a) the amino acid sequence of SEQ ID NO: 10; (b) an amino acid sequence in which one to several (preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 10; or (c) an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0063] Examples of the amino acid sequence of SEQ ID NO: 10 include the amino acid sequences of any of SEQ ID NOs: 11 to 15.

[0064] The region consisting of SD1, SD2, and S2 subunits refers to the region of the coronavirus spike protein from subdomain 1 (SD1), subdomain 2 (SD2), and up to the S2 subunit. In the case of conventional strain spike proteins, the region of the amino acid sequence from positions 532 to 1211 corresponds to the region consisting of the SD1, SD2, and S2 subunits. Note that, in this specification, the region consisting of the SD1, SD2, and S2 subunits does not include the transmembrane domain and the cytoplasmic tail. The region consisting of the SD1, SD2, and S2 subunits is sometimes referred to as "SD1 / 2-S2."

[0065] SEQ ID NO: 10 is a generalized sequence of SEQ ID NOs: 11 to 15, which have been identified as suitable regions consisting of SD1, SD2 and S2 subunits contained in the spike virus mutant of the present invention, and the amino acid sequences thereof are as follows:

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] In the amino acid sequence of SEQ ID NO: 10, X at position 264 is K or P, X at position 434 is Q or C, X at position 455 is P or K, X at position 472 is S or C, X at position 543 is N or Q, X at position 567 is N or T, and X at position 642 is N or L.

[0073] Region 1 (SEQ ID NO: 11) consisting of the SD1, SD2, and S2 subunits is the amino acid sequence contained in H-00646 in Example 14, region 2 (SEQ ID NO: 12) consisting of the SD1, SD2, and S2 subunits is the amino acid sequence contained in H-00657 in Example 14, region 3 (SEQ ID NO: 13) consisting of the SD1, SD2, and S2 subunits is the amino acid sequence contained in H-00669 in Example 14, region 4 (SEQ ID NO: 14) consisting of the SD1, SD2, and S2 subunits is the amino acid sequence contained in H-00672, H-00739, and H-00740 in Examples 14 or 15, and region 5 (SEQ ID NO: 15) consisting of the SD1, SD2, and S2 subunits is the sequence contained in H-00675 in Example 14. These antigens exhibit broad immunogenicity as shown in Examples 14 and 15.

[0074] (b) an amino acid sequence in which one to several (preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added in the amino acid sequence of SEQ ID NO: 10; The region consisting of the SD1, SD2 and S2 subunits includes a region consisting of the SD1, SD2 and S2 subunits consisting of the amino acid sequence of positions 528 to 1209 of the amino acid sequence set forth in any one of SEQ ID NOs: 66, 67, 69, 70, 72, 73, 75, 77 to 81, 83, 85, 86, 88, 90, 91, 95, 98, 99, 101, 105, and 111 to 122.

[0075] In another aspect, the spike protein variant of the present invention comprises a region consisting of SD1, SD2 and S2 subunits, which comprises: (a) the amino acid sequence of SEQ ID NO: 16; (b) an amino acid sequence in which one to several (preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 16; or (c) an amino acid sequence having 90% or more (preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 16; may also include:

[0076] SEQ ID NO: 16 corresponds to the amino acid sequence consisting of positions 529 to 1211 of the amino acid sequence of the full-length spike protein of the Delta strain (SEQ ID NO: 6), and is the following amino acid sequence.

[0077]

[0078] In the above-mentioned "amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 16 in which one to several (preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) amino acids have been substituted, deleted, inserted, or added," examples of the amino acid substitution, deletion, insertion, or addition include the following.

[0079] The spike protein mutants of the present invention may contain amino acid substitutions corresponding to the Q434C substitution and the S472C substitution in the amino acid sequence set forth in SEQ ID NO: 16. "Amino acid substitutions corresponding to the Q434C substitution and the S472C substitution in the amino acid sequence set forth in SEQ ID NO: 16" refers to the following amino acid substitutions. That is, when the amino acid sequence of the spike protein mutants of the present invention and the amino acid sequence set forth in SEQ ID NO: 16 are aligned, with gaps introduced as necessary to increase sequence identity, the amino acid residues on the spike protein mutants of the present invention corresponding to Q at position 434 and S at position 472 in SEQ ID NO: 16 are both substituted with C. Examples of spike protein mutants having such amino acid substitutions include H-00346 in Example 12.

[0080] The spike protein variants of the present invention may comprise an amino acid substitution corresponding to the K455P substitution and / or the V456P substitution in the amino acid sequence set forth in SEQ ID NO: 16. "An amino acid substitution corresponding to the K455P substitution and / or the V456P substitution in the amino acid sequence set forth in SEQ ID NO: 16" means that when the amino acid sequences of the spike protein variants of the present invention and the amino acid sequence set forth in SEQ ID NO: 16 are aligned, with gaps introduced as necessary to increase sequence identity, one or both of the amino acid residues in the spike protein variants of the present invention corresponding to K at position 455 and V at position 456 in SEQ ID NO: 16 are substituted with P. Examples of spike protein variants having such amino acid substitutions include H-00646, H-00657, H-00669, H-00672, H-00675, H-00739, and H-00740 in Example 14 or Example 15.

[0081] The spike protein variants of the present invention may comprise amino acid substitutions corresponding to one or more amino acid substitutions selected from the group consisting of F286P, A361P, A368P, and A411P in the amino acid sequence of SEQ ID NO: 16. "Amino acid substitutions corresponding to one or more amino acid substitutions selected from the group consisting of F286P, A361P, A368P, and A411P in the amino acid sequence of SEQ ID NO: 16" means that, when the amino acid sequence of the spike protein variants of the present invention and the amino acid sequence of SEQ ID NO: 16 are aligned, with gaps introduced as necessary to increase sequence identity, one or more of the amino acid residues in the spike protein variants of the present invention corresponding to F at position 286, A at position 361, A at position 368, and A at position 411 in SEQ ID NO: 16 are substituted with P. Spike protein variants in which all of the corresponding amino acid residues are substituted with P are preferred. Examples of spike protein variants having such amino acid substitutions include H-00646, H-00657, H-00669, H-00672, H-00675, H-00739 and H-00740 in Example 14 or Example 15.

[0082] The spike protein variants of the present invention may contain amino acid substitutions corresponding to the T201P substitution and / or the K264P substitution in the amino acid sequence set forth in SEQ ID NO: 16. The phrase "an amino acid substitution corresponding to the T201P substitution and / or the K264P substitution in the amino acid sequence set forth in SEQ ID NO: 16" means that, when the amino acid sequence of the spike protein variants of the present invention and the amino acid sequence set forth in SEQ ID NO: 16 are aligned, with gaps introduced as necessary to increase sequence identity, one or both of the amino acid residues in the spike protein variants of the present invention corresponding to T at position 201 and K at position 264 in SEQ ID NO: 16 are substituted with P. An example of the T201P substitution is H-00391 in Example 13, and an example of the K264P substitution is H-00399 in Example 12.

[0083] The spike protein variants of the present invention may be spike protein variants that have an amino acid mutation in one or more amino acid residues corresponding to RRAR at positions 151 to 154 of the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 17), and that are not cleaved by furin protease. The phrase "having an amino acid mutation in one or more amino acid residues corresponding to RRAR at positions 151 to 154 of the amino acid sequence of SEQ ID NO: 16 (SEQ ID NO: 17)" means that, when the amino acid sequence of the spike protein variants of the present invention and the amino acid sequence of SEQ ID NO: 16 are aligned, with gaps introduced as necessary to increase sequence identity, amino acid mutations (substitution, deletion, insertion, or addition) have been introduced into all or one or more of the amino acid residues in the spike protein variants of the present invention that correspond to RRAR at positions 151 to 154 of SEQ ID NO: 16 (SEQ ID NO: 17). The phrase "not cleaved by furin protease" means that the introduction of the amino acid mutations prevents the site from being cleaved by furin protease.

[0084] As the amino acid mutation, for example, amino acid residues corresponding to RRAR (SEQ ID NO: 17) at positions 151 to 154 of the amino acid sequence of SEQ ID NO: 16 may be substituted with GSAS (SEQ ID NO: 18), QQAQ (SEQ ID NO: 39), etc. Substitution with GSAS (SEQ ID NO: 18) is preferred.

[0085] Examples of substitutions in the above GSAS (SEQ ID NO: 18) include H-00646, H-00657, H-00669, H-00672, H-00675, H-00739 and H-00740 in Example 14 or Example 15.

[0086] RRAR at positions 151 to 154 of the amino acid sequence of SEQ ID NO: 16 is the furin protease cleavage site RRAR located at positions 682 to 685 in the spike protein of the conventional strain. By adding a mutation to this site, it is possible to prevent the protein from being cleaved by furin protease, which is expected to improve stability, protein yield, etc. when expressed in cultured cells, etc.

[0087] The spike protein variants of the present invention may comprise amino acid substitutions corresponding to one or more amino acid substitutions selected from the group consisting of N543Q, N567Q, and N642L substitutions in the amino acid sequence of SEQ ID NO: 16. "Amino acid substitutions corresponding to one or more amino acid substitutions selected from the group consisting of N543Q, N567Q, and N643L substitutions in the amino acid sequence of SEQ ID NO: 16" means that when the amino acid sequence of the spike protein variants of the present invention and the amino acid sequence of SEQ ID NO: 16 are aligned, with gaps introduced as necessary to increase sequence identity, one or more of the amino acid residues in the spike protein variants of the present invention corresponding to N at positions 543, N at positions 567, and N at positions 642 in SEQ ID NO: 16 are substituted with Q, Q, or L, respectively. An example of a spike protein variant having the above-mentioned N543Q and N567Q substitutions is H-00511 in Example 11, and an example of a spike protein variant having an N642L substitution is H-00180 in Example 10.

[0088] The spike protein mutants of the present invention may lack the transmembrane domain and the cytoplasmic tail.

[0089] The "transmembrane domain and cytoplasmic end" in the above context refers to the lipid transmembrane domain located at the C-terminus of the spike protein (positions 1211 to 1273 in the conventional spike protein) and the sequence corresponding to the cytoplasmic end of that C-terminus. Deleting this region in the spike protein allows for efficient extracellular secretion when expressed in animal or insect cells, and allows for efficient recovery of the expressed protein, making it suitable as an antigen for use in a vaccine.

[0090] For example, H-00646, H-00657, H-00669, H-00672, H-00675, H-00739 and H-00740 in Example 14 or Example 15 lack the transmembrane domain and the cytoplasmic tail.

[0091] The spike protein variants of the present invention may have a trimerization domain, i.e., an amino acid sequence that contributes to trimer formation of the polypeptide, added to them. Preferably, the trimerization domain is added to the C-terminus of the S2 subunit.

[0092] The spike protein mutant of the present invention may have a Foldon sequence added as a trimerization domain. Preferably, the Foldon sequence is added to the C-terminus of the S2 subunit.

[0093] The Foldon sequence is an amino acid sequence of approximately 27 amino acids called the Foldon domain derived from bacteriophage T4 fibritin. It contributes to trimer formation, and by expressing it as a fusion protein with the Foldon sequence, it is possible to stabilize the protein.

[0094] A preferred Foldon sequence includes, but is not limited to, GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 19). The Foldon sequences described in WO2008 / 025516 and WO2018 / 081318 can also be used.

[0095] For example, H-00646, H-00657, H-00669, H-00672, H-00675, H-00739 and H-00740 in Example 14 or Example 15 contain the foldon sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 19).

[0096] The spike protein mutant of the present invention may have a His tag added to the C-terminus. An example of the His tag is the amino acid sequence of SEQ ID NO: 20 (HHHHHH).

[0097] By adding a His tag, it can be purified using a carrier containing nickel (Ni).

[0098] For example, H-00646, H-00657, H-00669, H-00672, H-00675, H-00739 and H-00740 in Example 14 or Example 15 contain the amino acid sequence of SEQ ID NO: 20 (HHHHHH) at the C-terminus.

[0099] Examples of spike protein mutants of the present invention include spike protein mutants comprising the amino acid sequence from position 14 to the C-terminus of the amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27. The amino acid sequence (HHHHHH) contained at the C-terminus is used for protein purification and is not an essential element for use as an antigen.

[0100] Therefore, examples of spike protein variants of the present invention also include spike protein variants comprising the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in any of SEQ ID NOs: 21 to 27. Furthermore, since spike protein variants of the present invention may not comprise a trimerization domain, examples of spike protein variants of the present invention also include spike protein variants comprising the amino acid sequence of positions 14 to 1209 of the amino acid sequence set forth in any of SEQ ID NOs: 21 to 27.

[0101] The amino acid sequence set forth in any one of SEQ ID NOs: 21 to 27 is shown below.

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] The amino acid sequences from positions 1 to 13 on the N-terminus of any of SEQ ID NOs: 21 to 27 are a secretory signal sequence, which is necessary for extracellular secretion when produced in mammalian-derived cultured cells, insect-derived cultured cells, etc., but is cleaved during secretion.

[0110] Examples of spike protein mutants of the present invention include spike protein mutants comprising the amino acid sequence from position 14 to the C-terminus of the amino acid sequence set forth in any of SEQ ID NOs: 65 to 122. The amino acid sequence (HHHHHH) contained at the C-terminus is used for protein purification and is not an essential element for use as an antigen.

[0111] Therefore, examples of spike protein variants of the present invention also include spike protein variants comprising the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in any of SEQ ID NOs: 65 to 122. Furthermore, since spike protein variants of the present invention may not comprise a trimerization domain, examples of spike protein variants of the present invention also include spike protein variants comprising the amino acid sequence of positions 14 to 1209 of the amino acid sequence set forth in any of SEQ ID NOs: 65 to 122.

[0112] The amino acid sequences set forth in any of SEQ ID NOs: 65 to 122 are shown below.

[0113]

[0114] The immunogenic compositions of the invention contain the spike protein variants of the invention.

[0115] Preferably, the immunogenic composition of the present invention contains only one type of spike protein variant of the present invention as an antigen derived from a coronavirus spike protein, and does not contain any other coronavirus spike protein-derived antigens. That is, the spike protein variant of the present invention has broad immunogenicity as a monovalent antigen and is effective as a broad-spectrum vaccine.

[0116] The immunogenic composition of the present invention can be used to prevent infection with SARS-CoV-2 and other coronaviruses (preferably betacoronaviruses, more preferably sarbecoviruses).

[0117] The immunogenic compositions of the invention may include an adjuvant.

[0118] Preferred adjuvants include adjuvants containing squalene, tocopherol, and polysorbate 80. A specific example is A-910823, a squalene-based adjuvant. Details of A-910823 are described in, for example, Front Immunol. 2023 Feb 20;14:1116238 (Reference 1).

[0119] Preferred adjuvants include an adjuvant in which Alum, an aluminum salt, is mixed with ODN2006, an oligodeoxynucleotide, or an adjuvant in which Alum, an aluminum salt, is mixed with CpG1018, an oligodeoxynucleotide.

[0120] The present invention includes nucleic acids encoding the spike protein mutants of the present invention. Specifically, the coronavirus spike protein mutants of the present invention may be encoded in a DNA plasmid expression vector, mRNA, or the like and expressed in vivo. Note that the spike protein mutants of the present invention encoded by the nucleic acids encoding the spike protein mutants of the present invention may not contain a C-terminal trimerization domain and / or a His tag, and may contain a transmembrane domain and a cytoplasmic tail.

[0121] The present invention includes an expression vector comprising a nucleic acid encoding the spike protein mutant of the present invention. Expression vectors include viral vectors (e.g., adenoviral vectors).

[0122] The coronavirus spike protein mutants of the present invention can be produced by known genetic engineering techniques. For example, they may be expressed using hosts such as cultured mammalian cells or cultured insect cells, or by methods well known to those skilled in the art. Alternatively, the coronavirus spike protein mutants of the present invention may be encoded in a plasmid expression vector, a viral vector (e.g., an adenoviral vector), or mRNA, and then expressed in vivo.

[0123] The formulation of the immunogenic composition can be selected appropriately, and may be a liquid formulation, a powder formulation, or a lyophilized formulation.

[0124] The route of administration of the immunogenic composition of the present invention can be selected appropriately, and it can be administered orally or parenterally. Examples of parenteral administration include, but are not limited to, nasal administration, inhalation, intranasal administration, intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, intradermal administration, sublingual administration, intravenous administration, enteral administration, and transmucosal administration.

[0125] The immunogenic composition of the present invention may contain, as appropriate, buffering agents, isotonicity agents, soothing agents, preservatives, antibacterial agents, antioxidants, pH adjusters, dispersants, fragrances, colorants, antifoaming agents, etc., depending on the purpose, use, etc.

[0126] The dosage of the immunogenic composition of the present invention is appropriately selected depending on the type of active ingredient, the route of administration, the recipient, the age, weight, sex, symptoms, and other conditions of the patient.

[0127] The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

[0128] Method for producing coronavirus spike protein mutants using Expi293F cells Expi293F cells were used as a production system for coronavirus spike protein mutants. An expression vector encoding the spike protein mutant (a mammalian expression plasmid consisting of a cytomegalovirus immediate early enhancer-chicken β-actin hybrid (CAG) promoter, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence, and a rabbit β-globin polyadenylation signal sequence) was transfected into Expi293F cells using ExpiFectamine. ＴＭ Transfection was performed using the 293 Transfection Kit (Thermo Fisher Scientific). The experiment was basically performed according to the protocol attached to the kit, but kifunenesine (final concentration 10 μM) was added simultaneously with transfection. One day after transfection, enhancers 1 and 2 included in the kit were mixed and added immediately before transfection. Culture supernatants were collected 4 to 5 days after transfection, and each spike protein mutant was obtained in the culture supernatant.

[0129] The resulting culture supernatant containing each spike protein mutant was purified using Talon® Metal Affinity Resin (Takara Bio) or NGL COVID-19 Spike Protein Affinity Resin (REPLIGEN) to obtain purified spike protein mutants. The purified antigen protein was then concentrated and substituted with PBS buffer (GIBCO) using an Amicon® Ultra-2 Centrifugal Filter Unit 100 kDa NWMCO (Merck Millipore). The purity of the target spike protein was confirmed for the resulting purified spike protein mutants by gel electrophoresis and CBB staining. The concentration of the purified spike protein was also measured by protein quantification using bicinchoninic acid (BCA) reagent.

[0130] Method for nanoparticle production of RBD-only antigens: Each 6xHis-Avi-tagged RBD protein was transfected using the same method as in Example 1. After transfection, the cells were cultured in the presence of 100 μM biotin. Biotinylated RBD protein was purified from the culture supernatant containing each spike protein mutant using Talon Metal Affinity Resin (Takara Bio). Biotinylated RBD was mixed with streptavidin-coated nanoparticles (Bangs Laboratories) to prepare biotinylated RBD-bound nanoparticles. Excess biotinylated RBD and non-biotinylated RBD that did not bind to the nanoparticles were removed using a Nanosep centrifugal filter device 300 kDa NWMCO (Nihon Pall) and replaced with PBS buffer (GIBCO).

[0131] Method for Producing Coronavirus Spike Protein Mutants Using a Baculovirus Expression System (BEVS) Insect cells, such as Spodoptera frugiperda (Sf) cells (e.g., Sf9 cells) or Trichoplusia ni (Tn) cells (e.g., High Five cells), were used as hosts for producing coronavirus spike protein mutants. Baculovirus vectors (e.g., pVL1392) encoding spike protein mutants were co-transfected with the baculovirus genome (e.g., Oxford Expression Technologies flashBAC) and allowed to undergo homologous recombination in the insect cells to produce baculovirus. Cells were cultured in a bioreactor, spinner flask, or other device, and infected with baculovirus to express each spike protein mutant. Cells or culture supernatants were collected 3 days after infection. Full-length spike proteins, including the transmembrane domain, were recovered from the host cells by extraction with a detergent. The extracellular domain, the ectodomain, was recovered from the culture supernatant and concentrated and buffer-exchanged using tangential flow filtration (TFF). The first stage of purification for the full-length spike protein and ectodomain was performed using His-tag affinity resin or spike protein receptor-binding domain (RBD) affinity resin (e.g., Repligen NGL COVID-19 Spike Protein Affinity Resin). The second stage of purification was performed using a hydrophobic interaction resin or ion exchange resin. After removing nucleic acids using an anion exchange membrane, the sample was concentrated and buffer-exchanged using a TFF system or a combination of ultrafiltration and dialysis, and then passed through a 0.22 μm filter to obtain the antigen drug substance. The protein content of the resulting antigen drug substance was measured using the bicichiconic acid (BCA) method, and the purity was measured by staining the proteins separated by SDS-PAGE.

[0132] Neutralizing Activity Test of Mutant Spike Proteins (Drug Efficacy Test Method) (1) Immunization of Mice Spike proteins derived from Expi293F cells prepared by the method of Example 1 or 2 or from BEVS (Baculovirus Expression Vector System) prepared by the method of Example 3 were mixed 1:1 with squalene-based emulsion adjuvant A-910823 (Reference 1) to prepare a 20 μg / ml antigen solution, and 50 μl of this solution was intramuscularly inoculated into female BALB / c mice (5 mice per group) to give a concentration of 1 μg / mouse. Two weeks after the first inoculation, the immunogen was inoculated in the same manner as described above.

[0133] The mRNA antigen described in Example 16 was administered intramuscularly to female BALB / c mice (5 mice per group) in 50 μl at a dose of 0.3 μg / mouse or 3 μg / mouse without adjuvant. Three weeks after the first vaccination, the same immunogen was administered in the same manner as above.

[0134] (2) Serum Collection Two weeks after the final inoculation, blood was collected from the inferior vena cava of the mice under isoflurane anesthesia, and serum was separated in a separation tube containing a coagulation promoter. The serum was inactivated by heating at 56°C for 30 minutes.

[0135] (3) Neutralizing Activity Test: A pseudovirus neutralization test was performed using the inactivated serum obtained in (2) above. Specifically, a mixture of serially diluted serum and pseudovirus was added to 293T-AT cells seeded in a 96- or 384-well plate and cultured at 37°C in the presence of 5% carbon dioxide for 2 days. ONE-Glo Reagent was then added to the cells, and luminescence was measured using a plate reader. The neutralizing antibody titer (NT50) is the serum dilution ratio that inhibits 50% infection in the pseudovirus assay. The neutralizing antibody titer (NT50) was calculated using XLfit. Note that if the percent inhibition at the lowest dilution ratio was below 50%, the NT50 value was set to half the value of the lowest dilution ratio. If the percent inhibition at the highest dilution ratio was above 50%, the NT50 value was set to the value of the highest dilution ratio. The graphs in Figures 1 to 24 show the neutralizing antibody titers for each individual and the geometric mean and 95% confidence interval for each group. The method for producing pseudoviruses is described in Vaccine. 2022 Jul 29; 40(31): 4231-4241 (Reference 2). The accession numbers of the spike proteins of each virus strain used for the pseudoviruses are as shown in the table below.

[0136]

[0137] To design an RBD sequence that can confer neutralizing activity against both SARS-CoV-1 and SARS-CoV-2, hybrid sequences of SARS-CoV-1 and SARS-CoV-2 (Delta strain) were designed. Each of the designed hybrid sequences (Designs 1 to 5) was inserted into the RBD of the SARS-CoV-2 Delta strain spike protein (ectodomain, i.e., a deletion of the transmembrane domain and cytoplasmic tail) to create spike protein mutants, and neutralizing activity tests against SARS-CoV-1 and SARS-CoV-2 were performed.

[0138] The spike proteins listed in the table below were prepared using Expi293F cells by the method described in Example 1. Note that positions 1 to 13 of each full-length amino acid sequence are signal sequences and are not included in the prepared antigens.

[0139]

[0140] Mice were immunized with the prepared spike protein using the method described in Example 4, and serum neutralizing antibody titers were measured. The administration groups were designated Groups 1 to 6 below. Groups 5 and 6 were coronavirus spike protein mutants of the present invention, Groups 2 to 4 were reference examples, and Group 1 was a comparative example. (Group 1) H-00120, (Group 2) H-00145, (Group 3) H-00146, (Group 4) H-00147, (Group 5) H-00148, (Group 6) H-00149.

[0141] As a result, H-00148 (Group 5) and H-00149 (Group 6) induced neutralizing antibodies against SARS-CoV-1 in addition to wild-type D614G (Figure 1). In particular, antigen H-00149 in Group 6 was found to have high neutralizing antibody-inducing activity against each pseudovirus strain tested.

[0142] Neutralizing antibody induction test for RBD chimeras To confirm whether the RBDs of Design 5 and Design 4 can induce neutralizing antibodies against both SARS-CoV-1 and SARS-CoV-2 using the RBD alone, a neutralizing activity test was conducted using an RBD-only antigen. RBD sequences of RBD designs 1, 2, 4, and 5 were prepared, along with RBD sequences of the Delta strain and SARS-CoV-1 as controls. Specifically, each RBD in the table below was prepared using the method of Example 1, and nanoparticles were produced using the method of Example 2.

[0143]

[0144] Mice were immunized with the prepared spike protein using the method described in Example 4, and serum neutralizing antibody titers were measured. The administration groups were designated Groups 1 to 8 below. Groups 6 and 7 represent the coronavirus spike protein mutants of the present invention, Groups 4 and 5 are reference examples, and Groups 1 to 3 and 8 are comparative examples. (Group 1) SARS-CoV-2 Delta strain RBD antigen (H-00033), (Group 2) SARS-CoV-1 RBD antigen (H-00082, SARS-1 is shown in the figure), (Group 3) SARS-CoV-2 Delta strain RBD antigen (H-00033) and SARS-CoV-1 (H-00082) mixed in equal amounts, (Group 4) H-00078, (Group 5) H-00079, (Group 6) H-00081, (Group 7) H-00428, (Group 8) Original strain S-full antigen (SEQ ID NO: 64, prepared by the method described in Example 3)

[0145] As a result, among the monovalent vaccines, H-00079 (Design 2), H-00081 (Design 4), and H-00428 (Design 5) induced neutralizing antibodies against SARS-CoV-1 in addition to wild-type D614G, and H-00428 (Design 5) in particular induced neutralizing antibodies against both SARS-CoV-1 and SARS-CoV-2 in a balanced manner (Figure 2). These results demonstrate that the RBDs of Designs 5 and 4 are excellent antigen sequences that have the activity of inducing neutralizing antibodies against both SARS-CoV-1 and SARS-CoV-2, whether administered as the full-length spike protein or as the RBD alone.

[0146] Effect of NTD substitution on neutralizing antibody-inducing activity 1 To examine the effect of NTD substitution on the neutralizing activity induction of spike protein NTD, antigens shown in the table below in which the NTD of the spike protein (ectodomain) of the Delta strain was replaced were prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0147]

[0148] Mice were immunized with the spike protein prepared above using the method described in Example 4, and serum neutralizing antibody titers were measured. The administration groups were designated Groups 1 to 3 below. Groups 2 and 3 are reference examples used to examine the effect of NTD substitution on neutralizing antibody-inducing activity, while Group 1 is a comparative example. (Group 1) SARS-CoV-2 Delta strain spike ectodomain (H-00120); (Group 2) SARS-CoV-2 Delta strain spike protein ectodomain in which the NTD was replaced with the BA.5 strain NTD sequence (H-00545, SEQ ID NO: 46); (Group 3) SARS-CoV-2 Delta strain spike protein ectodomain in which the NTD sequence was replaced with the SARS-CoV-1 NTD sequence (H-00632, SEQ ID NO: 47).

[0149] As a result, compared to H-00120, H-00545 had elevated neutralizing antibody titers against BA.5, while H-00632 had elevated neutralizing antibody titers against SARS-CoV-1 (Figure 3). This result indicates that the NTD of the spike protein contributes to neutralizing antibody induction activity.

[0150] Effect of NTD substitution on neutralizing antibody-inducing activity 2 To examine the effect of replacing the NTD of the spike protein on the neutralizing activity directionality of the RBD, the antigens in the table below were prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0151]

[0152] Mice were immunized with the spike protein prepared above using the method described in Example 4, and serum neutralizing antibody titers were measured. The administration groups were designated Groups 1 to 5 below. Groups 3 and 4 were coronavirus spike protein mutants of the present invention, Group 2 was a reference example, and Groups 1 and 5 were comparative examples. (Group 1) H-00120, (Group 2) SARS-CoV-2 Delta strain spike protein ectodomain (H-00445) in which the NTD and S2 sequences were replaced with those of the BA.5 strain, (Group 3) H-00149 NTD and SD1 / 2-S2 sequences were replaced with those of the BA.5 strain, (Group 4) H-00148 NTD and SD1 / 2-S2 sequences were replaced with those of the BA.5 strain. Ectodomain replaced with the sequence of 5 strains (H-00556), (Group 5) Original strain S-full antigen prepared with BEVS.

[0153] As a result, H-00557, which has Design 5 in the RBD, had neutralizing antibody-inducing activity not only against D614G but also against both BA.4 / 5 and SARS-CoV-1, and induced neutralizing activity in a balanced manner against both virus strains (Figure 4). These results demonstrate that the sequence introduced into the NTD not only induces neutralizing antibodies against the strain, but also simultaneously induces neutralizing antibodies against SARS-CoV-1 and SARS-CoV-2 when combined with the RBD chimeric sequence discovered in Examples 5 and 6.

[0154] Effect of amino acid mutations on neutralizing antibody-inducing activity 1 To examine the effect of deleting amino acid residues in the glycan-binding site of the spike protein on neutralizing activity directionality, the antigens in the table below were prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0155]

[0156] In the above table, positions 167 and 331 refer to the amino acid residues corresponding to positions 167 and 331 in the amino acid sequence of the spike protein of the conventional strain. Mice were immunized with the prepared spike protein using the method described in Example 4, and the neutralizing antibody titers in the serum were measured. The administration groups are designated Groups 1 to 3 below. Groups 2 and 3 are reference examples for investigating the effects of amino acid mutations at positions 167 and 331, while Group 1 is a comparative example. (Group 1) SARS-CoV-2 Delta strain spike ectodomain (H-00120); (Group 2) SARS-CoV-2 Delta strain spike ectodomain (H-00161) with the T167L mutation introduced; or (Group 3) SARS-CoV-2 Delta strain spike ectodomain with the N331Q mutation introduced.

[0157] As a result, it was found that H-00161 and H-00168 had higher neutralizing antibody-inducing activity against BA.4 / 5 than H-00120 (FIG. 5).

[0158] Effect of amino acid mutations on neutralizing antibody-inducing activity 2 To examine the effect of deleting amino acid residues in the glycan-binding site of the spike protein on neutralizing activity directionality, the antigens in the table below were prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0159]

[0160] Note that position 1173 in the above table refers to the amino acid residue corresponding to position 1173 in the amino acid sequence of the spike protein of the conventional strain. Mice were immunized with the prepared spike protein using the method described in Example 4, and the neutralizing antibody titers in the serum were measured. The administration groups are shown below as Groups 1 and 2. Group 2 is a reference example for investigating the effect of the N1173L mutation, and Group 1 is a comparative example. (Group 1) SARS-CoV-2 Delta strain spike ectodomain (H-00120), (Group 2) SARS-CoV-2 Delta strain spike ectodomain into which the N1173L mutation was introduced (H-00189).

[0161] As a result, H-00189 showed higher neutralizing antibody-inducing activity against BA.4 / 5 than H-00120 (FIG. 6).

[0162] Effect of amino acid mutations on neutralizing antibody induction 3 To examine the effect of deleting amino acid residues in the glycan-binding site of the spike protein on neutralizing activity directionality, the antigens in the table below were prepared according to the method in Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0163]

[0164] In the above table, positions 1074 and 1098 refer to the amino acid residues corresponding to positions 1074 and 1098 in the amino acid sequence of the spike protein of the conventional strain. Mice were immunized with the prepared spike protein using the method described in Example 4, and the neutralizing antibody titers in the serum were measured. The administration groups are designated Groups 1 and 2 below. Group 2 is a reference example for investigating the effects of the N1074Q and N1074T mutations, and Group 1 is a comparative example. (Group 1) SARS-CoV-2 Delta strain spike ectodomain (H-00120). (Group 2) SARS-CoV-2 Delta strain spike ectodomain into which the N1074Q and N1074T mutations were introduced (H-00511).

[0165] As a result, H-00511 showed higher neutralizing antibody induction activity against BA.4 / 5 than H-00120 (Figure 7). The above results indicate that the neutralizing antibody induction activity can be improved by amino acid substitutions that eliminate the carbohydrate binding site.

[0166] Effect of structure-stabilizing mutations on neutralizing antibody-inducing activity 1 To investigate the effect on neutralizing activity directionality of the following amino acid residue substitutions, which may contribute to the stabilization of the spike protein structure, the antigens in the table below were prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0167]

[0168] In the above table, positions 795, 965, and 1003 refer to the amino acid residues corresponding to positions 795, 965, and 1003 in the amino acid sequence of the spike protein of the conventional strain. Mice were immunized with the prepared spike protein using the method described in Example 4, and the neutralizing antibody titers in the serum were measured. The administration groups are as follows: Groups 1 to 3. Groups 2 and 3 are reference examples for examining the effects of structure-stabilizing mutations, and Group 1 is a comparative example. (Group 1) SARS-CoV-2 Delta strain spike ectodomain (H-00120), (Group 2) SARS-CoV-2 Delta strain spike ectodomain with Q965C and S1003C mutations introduced (H-00346), (Group 3) SARS-CoV-2 Delta strain spike ectodomain with K795P mutation introduced (H-00399).

[0169] As a result, compared to H-00120, H-00346 and H-00399 showed higher neutralizing antibody-inducing activity against BA.4 / 5 (FIG. 8).

[0170] Effect of structure-stabilizing mutations on neutralizing antibody-inducing activity 2 To investigate the effect on neutralizing activity directionality of the following amino acid residue substitutions, which may contribute to the stabilization of the spike protein structure, the antigens in the table below were prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0171]

[0172] In the above table, position 732 refers to the amino acid residue corresponding to position 732 in the amino acid sequence of the spike protein of the conventional strain. Mice were immunized with the prepared spike protein using the method described in Example 4, and the neutralizing antibody titers in the serum were measured. The administration groups are designated Groups 1 to 3 below. Group 2 is a reference example for investigating the effects of structure-stabilizing mutations, and Group 1 is a comparative example. (Group 1) SARS-CoV-2 Delta strain spike ectodomain (H-00120), (Group 2) SARS-CoV-2 Delta strain spike ectodomain with the T732P mutation introduced (H-00391).

[0173] As a result, H-00391 showed higher neutralizing antibody-inducing activity against BA.4 / 5 than H-00120 (Figure 9). These results demonstrate that substitution of each of the above amino acid residues in the spike protein can improve neutralizing antibody-inducing activity.

[0174] Neutralizing antibody induction test 1 using antigens designed by combining the above results Based on the findings obtained in Examples 5 to 13, the spike protein mutants listed in the table below were designed, and antigen proteins were prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0175]

[0176] Mice were immunized with the spike protein prepared above or each antigen prepared by the method described in Example 3 using the method described in Example 4, and serum neutralizing antibody titers were measured. The administration groups were designated Groups 1 to 7 below. Groups 3 to 7 were coronavirus spike protein mutants of the present invention, while Groups 1 and 2 were comparative examples. (Group 1) A bivalent vaccine comprising an equal mixture of the S-full antigen of the origin strain (SEQ ID NO: 64, prepared by the method described in Example 3), (Group 2) an S-full antigen of the origin strain (same as Group 1) and the S-full antigen of the BA.5 strain (SEQ ID NO: 63, prepared by the method described in Example 3), (Group 3) H-00646, (Group 4) H-00657, (Group 5) H-00669, (Group 6) H-00672, and (Group 7) H-00675.

[0177] The results showed that the original strain vaccine in Group 1 induced neutralizing antibodies against a limited number of strains, and the bivalent vaccine in Group 2 did not sufficiently induce neutralizing activity against each strain of SARS-CoV-2 or other sarbecoviruses (SARS-CoV-1, WIV-1). On the other hand, H-00646, H-00657, H-00669, H-00672, and H-00675 showed high neutralizing antibody induction activity against all sarbecoviruses (Figure 10).

[0178] Neutralizing antibody induction test 2 using antigens designed by combining the above results Based on the findings obtained in Examples 5 to 13, the spike protein mutants listed in the table below were designed, and antigen proteins were prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0179]

[0180] Mice were immunized with the spike protein prepared above or each antigen prepared by the method described in Example 3 using the method described in Example 4, and the neutralizing antibody titers in the serum were measured. The administration groups were designated Groups 1 to 3 below. Groups 2 and 3 were coronavirus spike protein mutants of the present invention, and Group 1 was a comparative example. (Group 1) Original strain S-full antigen (SEQ ID NO: 64, prepared by the method described in Example 3), (Group 2) H-00739, (Group 3) H-00740.

[0181] As a result, the original strain vaccine induced neutralizing antibodies against a limited number of strains, while H-00739 and H-00740 showed high neutralizing antibody-inducing activity against all sarbecoviruses (FIG. 11).

[0182] Test for Induction of Neutralizing Antibodies by Antigens Administered in Vivo as mRNA The following experiment was carried out to examine the neutralizing antibody-inducing activity of the above-designed antigens when administered in vivo as mRNA.

[0183] (1) Preparation of template DNA for in vitro transcription (IVT) To prepare template DNA for in vitro transcription (IVT), plasmid DNA was amplified by PCR and purified. A DNA fragment containing a sequence in which the T7 promoter sequence, 5'-UTR sequence, KOZAK sequence, ORF, and 3'-UTR sequence were linked in order was introduced into the plasmid. 1.8 ng of the plasmid was dissolved in nuclease-free water (207 μL), to which 2× Prime STAR MAX DNA polymerase Premix (120 μL, Takara catalog #R045), 10 μM sense primer (9 μL, SEQ ID NO: 60), and 10 μM antisense primer (9 μL, SEQ ID NO: 61) were added. After incubation at 98°C for 2 minutes, 35 cycles of 98°C for 10 seconds, 55°C for 5 seconds, and 72°C for 10 seconds were carried out to amplify DNA encoding S full of H-00672 (SEQ ID NO: 62, a sequence in which the C-terminal Foldon sequence and His tag sequence of H-00672 have been deleted and a transmembrane domain and cytoplasmic tail have been added). After the reaction, the template DNA was purified using NucleoSpin (registered trademark) Gel and PCR Clean-up (Takara catalog #740609.250).

[0184] (2) Preparation of SARS-CoV-2 S-full mRNA-001 (SEQ ID NO: 59) by in vitro transcription 358 μg / mL template DNA obtained above (8.4 μL), 75 mM ATP (30 μL Thermo Fisher catalog # AMB13345), 75 mM GTP (30 μL Thermo Fisher catalog # AMB13345), 75 mM CTP (30 μL Thermo Fisher catalog # AMB13345), 100 mM N1-methylpseudouridine Triphosphate (22.5 μL), nuclease-free water (119 μL), 10X Reaction Buffer (30 μL Thermo Fisher catalog # AMB13345), and Enzyme mix T7 RNA Polymerase (30 μL Thermo Fisher catalog # AMB13345) were mixed and incubated at 37°C for 2 hours. TURBO® DNase (15 μL Thermo Fisher catalog # AM2239) was added and incubated at 37°C for 15 minutes. This was mixed with 7.5 M LiCl solution (150 μL Thermo Fisher catalog #AM9480) and allowed to stand overnight at −20° C. After centrifugation (4° C., 15,000×g, 15 minutes), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4° C., 15,000×g, 10 minutes). The supernatant was discarded, and the mixture was air-dried.The resulting residue was dissolved in nuclease-free water, and a portion of the resulting solution (285 μL, 450 μg in UV equivalent) was mixed with nuclease-free water (254 μL), 10 mM GTP (90 μL New England Biolab catalog #M2080), 20 mM SAM (90 μL New England Biolab catalog #M2080), 10X Capping Buffer (90 μL New England Biolab catalog #M2080), Vaccinia Capping Enzyme (10 U / μL, 45 μL New England Biolab catalog #M2080), and 10 mM GTP (90 μL New England Biolab catalog #M2080). The mixture was mixed with mRNA Cap2'-O-methyltransferase (50 U / μL, 45 μL, New England Biolab catalog #M2080) and incubated at 37°C for 2 hours. 7.5 M LiCl solution (450 μL, Thermo Fisher catalog #AM9480) was added and the mixture was left standing overnight at -20°C. After centrifugation (4°C, 15,000 × g, 15 minutes), the supernatant was discarded. 70% ethanol was added, followed by centrifugation (4°C, 15,000 × g, 10 minutes). The supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water, and the resulting solution (450 μL, 421 μg in UV equivalent) was mixed with 10× Antarctic Phosphatase Reaction Buffer (51 μL New England Biolab catalog #B0289) and Antarctic Phosphatase (10 μL New England Biolab catalog #M0289). The mixture was incubated at 37°C for 30 minutes and then purified using Monarch® RNA Cleanup Kit (New England Biolab catalog #M0289). The target mRNA-001 was obtained by purification using a PCR product (PCR kit #T2050) according to the accompanying manual. The obtained mRNA was analyzed using TapeStation (Agilent) and confirmed to be of the desired length.

[0185] (3) Preparation of mRNA-encapsulated nucleic acid lipid particles using SARS-CoV-2 S full mRNA SM-102 (Medchemexpress, HY-134541) and a cationic lipid described in WO2022168884 (hereinafter referred to as Lipid), distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine: hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (cholesterol: hereinafter referred to as Chol, NIPPON FINE CHEMICAL CO., LTD.), and 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol having a molecular weight of about 2000. Glycol (1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol, hereinafter referred to as PEG-DMG, NOF CORPORATION) was dissolved in ethanol at a molar ratio of DSPC:Chol:Lipid:PEG-DMG = 7.9:30.2:60.4:1.5 to a total lipid concentration of 13 mM.

[0186] On the other hand, the mRNA obtained in the preparation of mRNA-001 above was adjusted to 35 μg / mL in acetate buffer (pH 4.0).

[0187] The lipid solution and mRNA solution were mixed in a volume ratio of 1:3 using NanoAssembler® IGNITE. ＴＭ The nucleic acid-lipid particle dispersion was then mixed in a microchannel using a Precision Nanosystems Inc. to obtain a crude dispersion of nucleic acid-lipid particles. The nucleic acid-lipid particle dispersion was dialyzed (Slide-A-Lyzer) for 12-18 hours against approximately 300 times the volume of 4 mM phosphate buffered saline (pH 7.4). ＴＭ The ethanol was removed by filtering the particles in a Dialysis Cassette G2, MWCO: 10 kD, Spectra / Por) to obtain a dispersion of purified mRNA-encapsulating nucleic acid-lipid particles, which was then subjected to the following neutralizing antibody induction experiment.

[0188] (4) mRNA Neutralizing Antibody Induction Test Mice were immunized with the mRNA prepared by the above method, the H-00672 antigen prepared by the method described in Example 1, or the original strain antigen prepared by the method described in Example 3, using the method described in Example 4, and the neutralizing antibody titer in the serum was measured. The administration groups were as follows: (Group 1) original strain S-full antigen (SEQ ID NO: 64, prepared by the method described in Example 3), (Group 2) H-00672 protein antigen (SEQ ID NO: 24, prepared by the method described in Example 1; positions 1 to 13 of the amino acid sequence are a signal sequence and are not contained in the prepared antigen), (Group 3) 0.3 μg of mRNA antigen (nucleic acid sequence: SEQ ID NO: 59, amino acid sequence: SEQ ID NO: 62, dispersion prepared as described above), and (Group 4) 3 μg of mRNA antigen (nucleic acid sequence: SEQ ID NO: 59, amino acid sequence: SEQ ID NO: 62, dispersion prepared as described above). Group 2 is the coronavirus spike protein mutant of the present invention, Groups 3 and 4 are nucleic acids encoding the coronavirus spike protein mutant of the present invention, and Group 1 is a comparative example.

[0189] In this test, (Group 3) and (Group 4) were given a booster immunization 3 weeks after the primary immunization, and sera were collected 2 weeks after that to measure the neutralizing antibody titer.

[0190] As a result, both the protein antigen and the mRNA antigen induced high neutralizing antibody-inducing activity against all the sarbecoviruses evaluated (FIG. 12).

[0191] Comparison of neutralizing antibody-inducing activity between antigen expressed in Expi293F cells and antigen prepared in BEVS For H-00672, the neutralizing antibody-inducing activity of the antigen expressed in Expi293F cells described in Example 1 was compared with that of the antigen prepared in BEVS described in Example 3. Mice were immunized using the method described in Example 4, and the neutralizing antibody titer in the serum was measured. The administration groups are shown as Groups 1 to 3 below. Groups 2 and 3 represent the coronavirus spike protein mutants of the present invention, and Group 1 is a comparative example. (Group 1) Original strain S-full antigen (SEQ ID NO: 64, prepared by the method described in Example 3), (Group 2) H-00672 (SEQ ID NO: 24, prepared by the method described in Example 1, positions 1 to 13 of the amino acid sequence are a signal sequence that is not contained in the prepared antigen), (Group 3) H-00672 (SEQ ID NO: 24, prepared by the method described in Example 3, positions 1 to 13 of the amino acid sequence are a signal sequence that is not contained in the prepared antigen).

[0192] As a result, all antigens produced by either method showed high neutralizing antibody-inducing activity against the pseudoviruses evaluated (Figure 13).

[0193] Adjuvant Study: To study adjuvants, the H-00672 antigen was used to compare A-910823 (Group 1, below), which was described in Example 4 and used in Examples 5 to 17 above for non-mRNA, with the following two alternative adjuvants (Groups 2 and 3, below). Mice were immunized using the same method as in Example 4, except for the type of adjuvant, and serum neutralizing antibody titers were measured. The administration groups were as follows: (Group 1) squalene-based emulsion adjuvant A-910823; (Group 2) adjuvant consisting of a mixture of aluminum salt Alum and oligodeoxynucleotide ODN2006; and (Group 3) adjuvant consisting of a mixture of aluminum salt Alum and oligodeoxynucleotide CpG1018. Note that Groups 1 to 3 all contain immunogenic compositions containing the coronavirus spike protein mutants of the present invention.

[0194] As a result, all adjuvants had similar activity in inducing neutralizing antibodies against the pseudovirus (Figure 14).

[0195] Based on the findings obtained in Examples 5 to 15, a total of 58 coronavirus spike protein mutants of the present invention were designed as shown in the table below, and each spike protein was prepared according to Example 1. Note that positions 1 to 13 of each amino acid sequence are signal sequences and are not included in the prepared antigens.

[0196]

[0197] Mice were immunized with each of the prepared spike proteins and serum neutralizing antibody titers were measured using the method described in Example 4. The spike proteins of each virus strain used in the pseudovirus neutralization test were three types: D614G, XBB.1, and SARS-1, as shown in Table 18.

[0198] As a result, all of the spike proteins in the table above exhibited high neutralizing antibody-inducing activity against the pseudoviruses of the three types of sarbecoviruses tested (FIGS. 15 to 24).

[0199] The spike protein mutant of the present invention or an immunogenic composition containing the spike protein mutant has the excellent effect of exhibiting broad-spectrum neutralizing antibody-inducing activity with a monovalent antigen.

Claims

1. A coronavirus spike protein mutant comprising: (a) a receptor binding domain (RBD) consisting of the amino acid sequence set forth in SEQ ID NO:1; (b) an RBD consisting of an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in the amino acid sequence set forth in SEQ ID NO:1, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of the RBD consisting of the amino acid sequence set forth in SEQ ID NO:1; or (c) an RBD consisting of an amino acid sequence which has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:1, and which has neutralizing antibody-inducing activity against SARS-CoV-2 and SARS-CoV-1 equivalent to that of the RBD consisting of the amino acid sequence set forth in SEQ ID NO:

1.

2. The spike protein mutant of claim 1, wherein the amino acid sequence set forth in SEQ ID NO: 1 is an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 5.

3. The spike protein mutant of claim 1 or 2, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence from positions 14 to 1211 of the amino acid sequence set forth in SEQ ID NO:

6.

4. A spike protein variant according to any one of claims 1 to 3, comprising an N-terminal domain (NTD) consisting of: (a) the amino acid sequence set forth in SEQ ID NO: 7; (b) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 7; or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

7.

5. The spike protein mutant of claim 4, wherein the amino acid sequence set forth in SEQ ID NO:7 is the amino acid sequence set forth in either SEQ ID NO:8 or 9.

6. A spike protein variant according to any one of claims 1 to 5, comprising a region consisting of SD1, SD2 and S2 subunits, which consist of: (a) the amino acid sequence set forth in SEQ ID NO: 10; (b) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 10; or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

10.

7. The spike protein mutant of claim 6, wherein the amino acid sequence set forth in SEQ ID NO: 10 is an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 15.

8. A spike protein mutant according to any one of claims 1 to 5, comprising a region consisting of SD1, SD2 and S2 subunits, which consist of: (a) the amino acid sequence set forth in SEQ ID NO: 16; (b) an amino acid sequence in which one to several amino acids have been substituted, deleted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 16; or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:

16.

9. The spike protein variant of claim 8, having amino acid substitutions corresponding to the Q434C and S472C substitutions in the amino acid sequence set forth in SEQ ID NO:

16.

10. The spike protein variant of claim 8 or 9, having an amino acid substitution corresponding to a K455P substitution and / or a V456P substitution in the amino acid sequence set forth in SEQ ID NO:

16.

11. A spike protein variant according to any one of claims 8 to 10, having amino acid substitutions corresponding to one or more amino acid substitutions selected from the group consisting of F286P substitution, A361P substitution, A368P substitution and A411P substitution in the amino acid sequence set forth in SEQ ID NO:

16.

12. A spike protein mutant according to any one of claims 8 to 11, having an amino acid substitution corresponding to the T201P substitution and / or the K264P substitution in the amino acid sequence set forth in SEQ ID NO:

16.

13. A spike protein mutant described in any one of claims 8 to 12, which has an amino acid mutation in one or more amino acid residues corresponding to RRAR (sequence number 17) at positions 151 to 154 of the amino acid sequence described in sequence number 16, and is not cleaved by furin protease.

14. The spike protein mutant of claim 13, in which the amino acid residues corresponding to RRAR at positions 151 to 154 of the amino acid sequence set forth in SEQ ID NO:16 are replaced with GSAS (SEQ ID NO:18).

15. A spike protein variant according to any one of claims 8 to 14, having amino acid substitutions corresponding to one or more amino acid substitutions selected from the group consisting of an N543Q substitution, an N567Q substitution, and an N642L substitution in the amino acid sequence set forth in SEQ ID NO:

16.

16. A spike protein mutant according to any one of claims 1 to 15, wherein the transmembrane domain and the cytoplasmic tail are deleted.

17. The spike protein mutant of claim 16, having a trimerization domain added to the C-terminus.

18. A spike protein mutant described in any one of claims 1 to 17, having a His tag added to the C-terminus.

19. A spike protein mutant described in any one of claims 1 to 18, comprising the amino acid sequence of positions 14 to 1236 of the amino acid sequence described in any one of SEQ ID NOs: 21 to 27.

20. A coronavirus spike protein variant having 99% or more sequence identity with the amino acid sequence of positions 14 to 1236 of the amino acid sequence set forth in SEQ ID NO:26, and comprising the amino acid sequences of positions 14 to 324 and 528 to 1236 of the amino acid sequence set forth in SEQ ID NO:

26.

21. The spike protein variant of claim 20, comprising an amino acid sequence from positions 14 to 1236 of an amino acid sequence set forth in any one of SEQ ID NOs: 65, 68, 71, 74, 76, 82, 84, 87, 89, 92, 93, 94, 96, 97, 100, 102-104, and 106-110.

22. A coronavirus spike protein mutant having 99% or more sequence identity with the amino acid sequence of positions 14 to 1,236 of the amino acid sequence set forth in SEQ ID NO:26 and comprising the amino acid sequence of positions 325 to 527 of the amino acid sequence set forth in SEQ ID NO:

26.

23. The spike protein variant of claim 22, comprising an amino acid sequence from positions 14 to 1236 of an amino acid sequence set forth in any one of SEQ ID NOs: 66, 67, 69, 70, 72, 73, 75, 77-81, 83, 85, 86, 88, 90, 91, 95, 98, 99, 101, 105, and 111-122.

24. An immunogenic composition comprising a spike protein variant described in any one of claims 1 to 23.

25. The immunogenic composition described in claim 24, which contains only one type of spike protein mutant described in any one of claims 1 to 23 as an antigen derived from a coronavirus spike protein, and does not contain any other antigens derived from coronavirus spike proteins.

26. The immunogenic composition according to claim 24 or 25, which contains an adjuvant.

27. The immunogenic composition of claim 26, wherein the adjuvant is an adjuvant containing squalene, tocopherol and polysorbate 80.

28. A nucleic acid encoding a spike protein mutant according to any one of claims 1 to 23.

29. An expression vector comprising the nucleic acid of claim 28.