A novel coronavirus polypeptide vaccine coupled to a TLR7 agonist and its application

Abstract

The present invention discloses a novel coronavirus polypeptide vaccine coupled to a TLR7 agonist and its application. Specifically, based on the RBD sequence and structural information analysis of the SARS-CoV-2 S protein, the present invention provides a vaccine polypeptide for novel coronavirus pneumonia, where the vaccine polypeptide has a structure such as Z-(JU)n, where Z, J, U, n, etc. are as defined in the instructions. The present invention also provides a vaccine composition containing the vaccine polypeptide and its application.

Description

[Technical Field]

[0001] The present invention relates to the field of polypeptide drugs and polypeptide vaccines, in particular to a novel coronavirus polypeptide vaccine coupled to a TLR7 small molecule agonist and its application. [Background technology]

[0002] Novel coronavirus pneumonia (Coronavirus disease 2019, COVID-19), caused by the coronavirus SARS-CoV-2, is highly contagious and has caused a severe epidemic worldwide, putting millions of people's lives at risk. However, there are currently no clear and effective preventive, therapeutic, or countermeasures for COVID-19, and clinical treatments are centered on supportive play therapy and symptomatic treatment.

[0003] Building herd immunity against SARS-CoV-2 through a vaccine is the ultimate way to control and interrupt the COVID-19 epidemic. Currently, various types of COVID-19 vaccines, including attenuated vaccines, inactivated virus vaccines, recombinant virus vector vaccines, recombinant protein vaccines, DNA vaccines, RNA vaccines, and polypeptide vaccines, are in preclinical and clinical trials.

[0004] However, the immune protection provided by currently developed vaccines has limitations, including low immunogenicity and viral immune escape.

[0005] Therefore, there is an urgent need in the art to develop new vaccines that can efficiently stimulate the human body to generate an immune response against SARS-CoV-2 and provide potent immune protection by generating blocking anti-SARS-CoV-2 antibodies in vaccinated individuals. Summary of the Invention [Problem to be solved by the invention]

[0006] The aim of the present invention is to provide a new vaccine that efficiently stimulates the human body to generate an immune response against SARS-CoV-2 and provides potent immune protection by generating blocking anti-SARS-CoV-2 antibodies in vaccinated individuals. [Means for solving the problem]

[0007] A first aspect of the present invention provides a vaccine polypeptide for a novel coronavirus, said vaccine polypeptide comprising an antigen polypeptide and optionally a TLR7 agonist coupled to said antigen polypeptide.

[0008] In another preferred embodiment, the vaccine polypeptide has the structure of Formula V or an oligomer comprising the structure of Formula V: Formula (V): Z-(U)n In the formula: Z is an antigenic polypeptide, the antigenic polypeptide having at least one T cell epitope and / or at least one B cell epitope of the novel coronavirus S protein, and the antigenic polypeptide having an amino acid sequence derived from the RBM region of the S protein; U are each independently a TLR7 agonist; n is 0 or a positive integer, "-" is a chemical bond or linker or connector.

[0009] In another preferred embodiment, the vaccine polypeptide has the structure of Formula I or an oligomer comprising the structure of Formula I: Formula (I): Z-(JU)n In the formula: Z is an antigenic polypeptide, the antigenic polypeptide having at least one T cell epitope and / or at least one B cell epitope of the novel coronavirus S protein, and the antigenic polypeptide having an amino acid sequence derived from the RBM region of the S protein; U are each independently a TLR7 agonist; n is 0 or a positive integer, J is a chemical bond or connector.

[0010] In another preferred embodiment, the vaccine polypeptide is capable of stimulating primates and rodents to generate neutralizing antibodies that block the binding of the RBD to ACE2.

[0011] In another preferred embodiment, the vaccine polypeptide is capable of stimulating a primate to develop cellular and humoral immunity.

[0012] In another preferred embodiment, the primates include human and non-human primates.

[0013] In another preferred example, the length of the antigen polypeptide is 8 to 100 amino acids, preferably 10 to 80 amino acids.

[0014] In another preferred embodiment, the antigenic polypeptide has an amino acid sequence derived from the RBD region of the novel coronavirus S protein.

[0015] In another preferred embodiment, the antigenic polypeptide has an amino acid sequence derived from the RBM region of the RBD region.

[0016] In another preferred example, the RBM region refers to amino acids 438 to 506 of the RBD protein of the novel coronavirus.

[0017] In another preferred example, the "amino acid sequence derived from the RBM region of an RBD protein" of the antigenic polypeptide means that the amino acid sequence of the antigenic polypeptide has homology (or identity) with the RBM region, and the homology is ≧80%, preferably ≧85%, more preferably ≧90%, and most preferably ≧95%.

[0018] In another preferred embodiment, the antigenic polypeptide competitively binds to the S protein of the novel coronavirus with the human ACE2 protein.

[0019] In another preferred example, the term "competitive binding" refers to the binding of the antigenic polypeptide to the same or substantially the same binding domain (or amino acid segment) of the human ACE2 protein as that of the S protein of the novel coronavirus.

[0020] In another preferred embodiment, the antigenic polypeptide binds to the same binding segment of the S protein of the novel coronavirus and the human ACE2 protein.

[0021] In another preferred embodiment, the competitive binding includes blocking or non-blocking competitive binding.

[0022] In another preferred embodiment, the antigen polypeptide is an artificially synthesized or recombinant antigen polypeptide.

[0023] In another preferred embodiment, the antigenic polypeptide is (a) a polypeptide having an amino acid sequence shown in any one of SEQ ID Nos. 1 to 12; (b) A polypeptide derived from the amino acid sequence of the polypeptide of (a) by adding one or more amino acids, substituting one or more amino acids, or deleting one to three amino acids, wherein the derived polypeptide has substantially the same function as the original polypeptide before derivation.

[0024] In another preferred embodiment, the term "substantially the same function" means that the derived polypeptide has substantially the same immunogenicity as the induced immune response, competitive binding activity to the S protein of the novel coronavirus and the human ACE2 protein, and / or has at least one T cell epitope and / or has at least one B cell epitope.

[0025] In another preferred embodiment, the structure of the antigenic polypeptide is as shown in Formula II: Formula (II): X1-X-X2 In the formula: (a) X is a core fragment, wherein the sequence of said core fragment is selected from one or more of SEQ ID NOs: 1-12 (see Table A); (b) X1 and X2 each independently represent none, 1, 2, or 3 amino acids, and the total number of amino acids in X1 and X2 is ≦4, preferably 3, 2, or 1, more preferably 0 or 1; (c) "-" indicates a peptide bond, a peptide linker, or other connector (i.e., X1 and X and / or X2 are connected by a peptide bond, a peptide linker (e.g., a flexible linker consisting of 1 to 15 amino acids), or other connector).

[0026] In another preferred embodiment, the antigenic polypeptide is selected from Table A.

[0027] [Table 1] In another preferred example, X1 and X2 are each independently none, K, C, G, L, or A.

[0028] In another preferred example, X1 is none, K, or C.

[0029] In another preferred example, X2 is none, K, or C.

[0030] In another preferred embodiment, the antigenic polypeptide has at least one T cell epitope and / or at least one B cell epitope in the RBD region of the novel coronavirus S protein.

[0031] In another preferred embodiment, the antigenic polypeptide has at least one T cell epitope and / or at least one B cell epitope in the RBM region of the novel coronavirus S protein.

[0032] In another preferred embodiment, the antigenic polypeptide has at least one T cell epitope, preferably 1, 2, 3 or 4 T cell epitopes, more preferably 1 or 2 T cell epitopes.

[0033] In another preferred embodiment, the antigenic polypeptide has at least one B cell epitope, preferably 1, 2, 3 or 4 B cell epitopes, more preferably 1 or 2 B cell epitopes.

[0034] In another preferred example, the antigen polypeptide has 1 to 2 T cell epitopes and 0 to 2 B cell epitopes, preferably 1 to 2 T cell epitopes and 0 to 1 B cell epitope.

[0035] In another preferred example, the number n of molecules of the TLR agonist is 1, 2, 3, 4, 5 or 6, preferably 1, 2, 3 or 4.

[0036] In another preferred embodiment, the TLR7 agonist is a small molecule agonist.

[0037] In another preferred embodiment, the TLR7 agonist comprises SZU-101.

[0038] [ka] In another preferred example, the TLR7 agonist (eg, SZU-101) is bound to the terminal amino group or a side chain amino group of the antigen polypeptide.

[0039] In another preferred example, the TLR7 agonist (eg, SZU-101) binds to a sulfhydryl group of the antigenic polypeptide.

[0040] In another preferred embodiment, the SZU-101 binds to an amino group of an antigen polypeptide to form a structure shown in S1, [ka] Formula (S1): or The SZU-101 binds to sulfhydryl groups of the antigenic polypeptide to form the structure shown in S2.

[0041] [ka] Formula (S2): In another preferred embodiment, the TLR7 agonist binds to the antigen polypeptide at a fixed point and / or randomly (ie, in Formula I, U binds to Z at a fixed point and / or randomly).

[0042] In another preferred embodiment, U is attached to Z at a fixed point.

[0043] In another preferred embodiment, U binds to an amino acid site selected from the group consisting of G, K, L, A, C, or a combination thereof of the antigenic polypeptide Z at a fixed point:

[0044] In another preferred embodiment, the vaccine polypeptide is selected from the following group of coupling peptides: The vaccine polypeptide is selected from the following group of coupling peptides: (S1)-GVEGFNCYFPLQSYGFQPTNGVGYQPYRK-(S1)(SEQ ID No:14) Here, SZU-101 binds to the N-terminal amino group of G and the side chain amino group of K in the amino acid sequence via the S1 structure, (S1)2-KGVEGFNCYFPLQSYGFQPTNGVGYQPYR(SEQ ID No:15) Here, SZU-101 binds to the N-terminus and side chain amino group of K in the amino acid sequence via the S1 structure, (S1)-LFRK(-S1)SNLK(-S1)PFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY(SEQ ID No:12) Here, SZU-101 binds to the N-terminal amino group of L and the side chain amino group of K in the amino acid sequence via the S1 structure, GVEGFNC(-S2)YFPLQSYGFQPTNGVGYQPYRK(SEQ ID No:14) Here, SZU-101 binds to the sulfhydryl on C in the amino acid sequence via the S2 structure, (S2)-CYRLFRKSNLKPFERDISTEIYQAGS(SEQ ID No:16) Here, SZU-101 binds to the sulfhydryl group on the C of the polypeptide via the S2 structure, (S2)-CYAWNRKRISN (SEQ ID No: 5) Here, SZU-101 binds to the sulfhydryl group on the C of the polypeptide via the S2 structure, (S2)-CVADYSVLYNSASFSTFK(SEQ ID No:6) Here, SZU-101 binds to the sulfhydryl group on the C of the polypeptide via the S2 structure, and the structures of S1 and S2 are as defined above.

[0045] In another preferred embodiment, the coupling peptide is selected from Table B.

[0046] [Table 2] In another preferred example, the vaccine polypeptide is selected from the group consisting of P-37 or a conjugate thereof with a TLR7 agonist, P-67-F1 or a conjugate thereof with a TLR7 agonist, P-71 or a conjugate thereof with a TLR7 agonist, LV54 or a conjugate thereof with a TLR7 agonist, or a combination thereof.

[0047] In another preferred embodiment, the TLR7 agonist is SUZ-101.

[0048] A second aspect of the present invention provides an isolated collection of peptides, said collection of peptides comprising vaccine polypeptides of at least two species of the novel coronavirus according to the first aspect of the present invention.

[0049] In another preferred example, the peptide collection includes at least 2 to 20, preferably 2 to 12 (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) types of vaccine polypeptides.

[0050] A third aspect of the present invention provides a pharmaceutical composition comprising a novel coronavirus vaccine polypeptide according to the first aspect of the invention or a peptide collection according to the second aspect of the invention and a pharmaceutically acceptable vector.

[0051] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0052] In another preferred embodiment, the vaccine composition is monovalent or multivalent.

[0053] In another preferred embodiment, the pharmaceutical composition further comprises an adjuvant, preferably an aluminum adjuvant, in which the molar or weight ratio of the active peptide to the adjuvant (e.g., aluminum) in the composition is between 1:100, preferably between 1:40 and 1:60.

[0054] In another preferred embodiment, the pharmaceutical composition comprises a single drug, a combination drug, or a synergistic drug.

[0055] In another preferred embodiment, the pharmaceutical composition is in the form of a liquid, solid, or gel.

[0056] In another preferred embodiment, the pharmaceutical composition is administered by a method selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oronasal spray and nebulizer inhalation.

[0057] A fourth aspect of the present invention provides the use of a novel coronavirus vaccine polypeptide according to the first aspect of the present invention or a peptide collection according to the second aspect or a pharmaceutical composition according to the third aspect, for the preparation of a medicament for preventing coronavirus SARS-CoV-2 infection or a disease associated therewith.

[0058] In another preferred embodiment, the coronavirus SARS-CoV-2 related disease is selected from the group consisting of respiratory tract infections, pneumonia and its complications, or a combination thereof.

[0059] In another preferred embodiment, the coronavirus SARS-CoV-2 related disease is novel coronavirus pneumonia (COVID-19).

[0060] A fifth aspect of the present invention provides a cell preparation, the cell preparation comprising: (a) immune cells activated by immunization with a novel coronavirus vaccine polypeptide according to the first aspect of the present invention or a peptide assembly according to the second aspect of the present invention; and (b) a pharmaceutically acceptable vector.

[0061] In another preferred embodiment, the immune cells are selected from the group consisting of dendritic cells, natural killer cells (NK), lymphocytes, monocytes / macrophages, granulocytes, or a combination thereof.

[0062] In another preferred embodiment, the activation is in vitro activation.

[0063] In another preferred example, the in vitro activation includes culturing the immune cells in the presence of the vaccine polypeptide for a certain period of time (for example, 6 to 48 hours) to obtain immunoactivated immune cells.

[0064] In another preferred embodiment, the cell preparation is a liquid preparation containing viable cells.

[0065] In another preferred embodiment, the cell preparation is reinfused by intravenous administration.

[0066] A sixth aspect of the present invention provides a method of generating an immune response against coronavirus SARS-CoV-2, comprising administering to a subject in need thereof a novel coronavirus vaccine polypeptide according to the first aspect of the present invention, a collection of peptides according to the second aspect or a pharmaceutical composition according to the third aspect.

[0067] In another preferred embodiment, the subject comprises a human or non-human mammal.

[0068] In another preferred embodiment, the non-human mammal comprises a non-human primate (eg, a monkey).

[0069] In another preferred embodiment, the method induces the production of neutralizing antibodies against coronavirus SARS-CoV-2 in the subject.

[0070] In another preferred embodiment, the neutralizing antibody blocks the binding of coronavirus SARS-CoV-2 to the human ACE2 protein.

[0071] A seventh aspect of the invention provides a fusion protein, said fusion protein comprising a carrier protein and a vaccine polypeptide according to the first aspect of the invention fused to said fusion protein.

[0072] In another preferred embodiment, the fusion protein is a non-naturally occurring protein.

[0073] In another preferred embodiment, the fusion protein has the structure of Formula IIIa or IIIb: Formula (IIIa): P1-P2 Formula (IIIb): P2-P1 In the formula, P1 is a vaccine polypeptide according to the first aspect of the invention and P2 is a carrier protein.

[0074] In another preferred example, P1 can be a single vaccine polypeptide, or multiple identical or different vaccine polypeptides (or antigen polypeptides) connected in series.

[0075] In another preferred embodiment, said P1 is coupled to one or more TLR7 agonists.

[0076] An eighth aspect of the present invention provides a pharmaceutical composition comprising (a) a fusion protein according to the seventh aspect of the invention or an immune cell activated by immunization therewith, and (b) a pharmaceutically acceptable vector.

[0077] A ninth aspect of the present invention provides the use of a fusion protein according to the seventh aspect of the invention or a pharmaceutical composition according to the eighth aspect of the invention, for the preparation of a medicament for the prevention of coronavirus SARS-CoV-2 infection or a disease associated therewith. [Effects of the Invention]

[0078] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief explanation of the drawings]

[0079] [Figure 1] Amino acid sequence analysis of the SARS-CoV-2 S protein and the key sites in the RBD that interact with ACE2. [Figure 2] A graph of the interaction structure and key sites of action between the RBD region of the S protein of SARS-CoV-2 and human ACE2 is shown. [Figure 3] 1 shows a comprehensive analysis of the binding potential of polypeptides P-37, P-67 and P-71 to molecules of 24 major population HLA class II molecule allele types. [Figure 4] 1 shows a graph of the linear B-cell epitopes contained in the S protein RBD predicted by the BepiPred software and their relative positions to polypeptides P-37, P-67, and P-71. [Figure 5] Figure 1 shows a structural diagram of the conformational B-cell epitope contained in the S protein RBD predicted by the Discope software. [Figure 6] 1 shows a diagram of the locations of polypeptides P-37, P-67, P-71, and LY54 on the S protein RBD structure. [Figure 7] 1 shows the chemical structure of the TLR7 small molecule agonist SZU-101. [Figure 8] 1 shows the results of cytokine release from mouse splenic lymphocytes in vitro induced by polypeptide P-71 and its coupled peptide bound to SZU-101. [Figure 9] 1 shows the results of in vitro proliferation of mouse splenic lymphocytes induced by polypeptide P-71 and its coupled peptide bound to SZU-101. [Figure 10] The production of anti-RBD antibodies after immunization of BALB / c mice with polypeptides P-37, P-71, and P-67-F2 and their coupled peptides conjugated to SZU-101 (see Table B), either alone or in combination, is shown. [Figure 11]The results of anti-RBD antibody production after immunization of crab-eating monkeys with LY54, LY54-S1, and mixed coupled peptides (see Table B) in which polypeptides P-37, P-71, and P-67-F2 were coupled to SZU-101 are shown. [Figure 12] We show that anti-RBD antibodies generated by immunizing crab-eating monkeys with LY54, LY54-S1, and mixed coupled peptides (see Table B) have neutralizing activity that blocks the binding of RBD to ACE2. [Figure 13] It shows that polypeptides P-37, P-71 and LY54 have better binding effect on human ACE2. DETAILED DESCRIPTION OF THE INVENTION

[0080] Through extensive and thorough research, the inventors analyzed the sequence and structure of the SARS-CoV-2 S protein RBD. They then analyzed the S protein's CD4+ T / CD8+ T cell epitopes, linear and conformational B cell epitopes, key interaction sites, surface features, glycosylation modification sites, and polypeptide physicochemical properties, among other characteristics, to screen for and identify vaccine polypeptides capable of effectively inducing an immune response against the coronavirus SARS-CoV-2 in mammals. Experiments have shown that the vaccine polypeptides of the present invention can effectively induce cellular and humoral immunity against SARS-CoV-2 in rodents (e.g., mice) and primates (e.g., crab-eating monkeys), thereby generating higher titers of neutralizing antibodies that block the binding of the RBD to ACE2. This makes the present invention potentially applicable to the prevention or treatment of novel coronavirus pneumonia. Based on this, the present invention was completed.

[0081] Specifically, the present invention determines the T / B cell epitopes of the S protein based on analysis of the RBD sequence and structural information of the SARS-CoV-2 S protein. By comprehensively considering the structural surface features of the S protein, the key sites for interaction with ACE2, and the physicochemical properties of the polypeptides, the present invention screens and determines the 12 antigen polypeptides listed in Table A. The antigen polypeptides (including P-37, P-67 (including P-67-F1 and P-67-F2), P-71, and LY54) are each conjugated to a TLR7 small molecule agonist to form the conjugated peptides listed in Table B. Experiments have shown that the conjugated polypeptide vaccine induces stronger cellular and humoral immune effects in mice and crab-eating monkeys, and generates higher titers of neutralizing antibodies that block the binding of RBD to ACE2.

[0082] term Coronavirus SARS-CoV-2 Coronaviruses (CoVs) are enveloped, positive-strand RNA viruses that belong to the family Coronaviridae in the order Nidovirales, and their subfamily includes four genera: α, β, δ, and γ.

[0083] Of the coronaviruses currently known to infect humans, HCoV-229E and HCoV-NL63 belong to the alpha coronavirus genus, while HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV, and SARS-CoV-2 all belong to the beta coronavirus genus.

[0084] The novel coronavirus (SARS-CoV-2) that emerged at the end of 2019 shares approximately 80% similarity with SARS-CoV and 40% similarity with MERS-CoV, and also belongs to the β-coronavirus genus.

[0085] The genome of this type of virus is a single-stranded positive-strand RNA, encoding replicase, spike protein, envelope protein, nucleocapsid protein, etc., making it one of the largest RNA viruses in terms of genome. In the early stage of viral replication, the genome is translated into two peptide chains consisting of several thousand amino acids, i.e., precursor polyproteins, which are then cleaved by proteases to generate nonstructural proteins (e.g., RNA polymerase and helicase), structural proteins (e.g., spike protein), and auxiliary proteins.

[0086] The S protein is the main structural protein of the coronavirus SARS-CoV-2, and its structure is shown in Figure 1. The RBD region is responsible for the structure of the human ACE2 receptor, while the RBM region contains a motif that binds to human ACE2. The amino acid sequence of a typical S protein is shown in SEQ ID No: 13.

[0087] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITG RLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVY DPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID No:13) The RBD region of the coronavirus SARS-CoV-2 is located at positions 333 to 527 of the S protein, and a representative amino acid sequence is as shown at positions 333 to 527 of SEQ ID No: 13 or as shown in Figure 4.

[0088] >RBD(333-527) TNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGP(SEQ ID No. 13 333~527) The RBM region of the coronavirus SARS-CoV-2 is located at positions 438-506 of the S protein, and a representative amino acid sequence is shown at positions 438-506 of SEQ ID No: 13 or in Figure 1.

[0089] >RBM(438-506) SNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ (SEQ ID No: 13, positions 438 to 506) In the present invention, it should be understood that the S protein, RBD region, and RBM region all include wild-type and mutant types.

[0090] Vaccine Polypeptides In the present invention, the terms "epitope peptide of the invention", "vaccine polypeptide of the invention", and "polypeptide of the invention" are used interchangeably and refer to a vaccine polypeptide according to the first aspect of the invention, in particular a polypeptide having the structure of Formula I. It should be understood that the terms include not only a single species of vaccine polypeptide of the invention, but also a peptide collection (or peptide combination) formed by multiple species of vaccine polypeptides of the invention.

[0091] In the present invention, the vaccine polypeptide further includes other forms such as pharmaceutically acceptable salts, conjugates, or fusion proteins. Preferably, the vaccine polypeptide of the present invention is a conjugate (coupled peptide) formed by binding to a TLR7 agonist, particularly a coupled peptide to which one or more TLR7 agonists are coupled, such as the conjugates shown in the tables.

[0092] Further, in the present invention, preferred vaccine polypeptides have the structure shown in Formula II: Formula (II): X1-X-X2 (a) X is a core fragment, wherein the sequence of said core fragment is selected from one or more of SEQ ID NOs: 1-12 (see Table A); (b) X1 and X2 each independently represent none, 1, 2, or 3 amino acids, and the total number of amino acids in X1 and X2 is ≦4, preferably 3, 2, or 1, more preferably 0 or 1; Here, the peptide bonds between X1 and X, and between X and X2 are connected by a peptide linker (for example, a flexible linker composed of 1 to 15 amino acids) or other connector.

[0093] In the present invention, the core fragment or vaccine polypeptide includes a derived polypeptide formed by adding one or more (e.g., 1 to 5, preferably 1 to 3) amino acids to any one of SEQ ID Nos: 1 to 12, substituting one or more (e.g., 1 to 5, preferably 1 to 3) amino acids, and / or deleting one to three amino acids, and the derived polypeptide has substantially the same function as the original polypeptide before derivatization.

[0094] Preferably, the core fragment or vaccine polypeptide comprises one to three amino acid additions (preferably at the N- or C-terminus) and / or one to two amino acid substitutions (preferably conservative amino acid substitutions) to any one of SEQ ID Nos: 1 to 12, and still has substantially the same function as the original polypeptide prior to derivation.

[0095] Preferably, the conservative amino acid substitutions are made according to Table C.

[0096] [Table 3] As used herein, the term "peptide collection" refers to a collection of peptides that is comprised of at least two species of vaccine polypeptides of the present invention or their derived polypeptides.

[0097] Preferably, the peptide collection according to the present invention comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 vaccine polypeptides or derived polypeptides thereof (including coupling peptides) selected from the first aspect of the present invention, more preferably said peptide collection comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 vaccine polypeptides or derived polypeptides thereof selected from SEQ ID NOs: 1 to 12. Furthermore, said peptide collection further comprises antigenic peptides or proteins of coronavirus SARS-CoV-2 other than SEQ ID NOs: 1 to 12.

[0098] As used herein, "isolated" refers to a substance that is separated from its environment (for a naturally occurring substance, the original environment is the natural environment). A polypeptide in its natural state within a living cell is not isolated and purified; the same polypeptide is isolated and purified when it is separated from other substances with which it is naturally present.

[0099] As used herein, "isolated peptide" refers to a polypeptide of the invention that is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. One skilled in the art can purify a polypeptide of the invention using standard protein purification techniques. A substantially purified polypeptide (fusion protein) will yield a single major band on a non-reducing polyacrylamide gel.

[0100] The polypeptides of the present invention can be recombinant or synthetic polypeptides, preferably synthetic polypeptides.

[0101] In the present invention, when the sequence of the vaccine polypeptide is relatively short (eg, ≦70 aa, more preferably ≦60 aa), it can be directly synthesized by chemical methods.

[0102] When the vaccine polypeptide sequence is relatively long or is provided in the form of a fusion protein, recombinant methods can also be used to obtain large amounts of the relevant peptide sequence, typically by cloning the coding sequence encoding the antigenic polypeptide or its fusion protein into a vector, transforming it into cells, and then isolating the relevant antigenic polypeptide or fusion protein from the grown host cells by conventional methods.

[0103] Pharmaceutical Compositions and Methods of Administration The present invention further provides pharmaceutical compositions. The pharmaceutical compositions of the present invention can be for therapeutic or prophylactic use (e.g., vaccines). The pharmaceutical compositions of the present invention comprise an effective amount of a vaccine polypeptide or peptide assembly of the present invention, or immune cells activated by the vaccine polypeptide (e.g., dendritic cells sensitized with the vaccine polypeptide of the present invention or T cells induced by dendritic cells), and at least one pharmaceutically acceptable vector, diluent, or excipient.

[0104] In another preferred embodiment, the associated disease caused by the novel coronavirus SARS-CoV-2 is selected from the group consisting of respiratory tract infections, pneumonia and its complications, or a combination thereof.

[0105] In the present invention, these (vaccine) compositions comprise an immunogenic antigen (including a vaccine polypeptide, peptide assembly, or derivative thereof of the present invention), and are typically combined with a "pharmaceutically acceptable vector," which includes any vector that does not itself induce the production of antibodies harmful to the individual receiving the composition. Examples of suitable vectors include, but are not limited to, proteins, lipid aggregates (e.g., oil droplets or liposomes), and the like. Additionally, these vectors can act as immunostimulatory agents ("adjuvants").

[0106] Furthermore, the (vaccine) composition of the present invention may further contain an additional adjuvant. Representative vaccine adjuvants include, but are not limited to, categories such as inorganic adjuvants such as aluminum hydroxide and alum, synthetic double-stranded polynucleotides (double-stranded polyadenylic acid, uridine acid), synthetic adjuvants such as levamisole and isoprinosine, Freund's adjuvant, peanut oil emulsion adjuvant, oils such as mineral oil and vegetable oil, etc.

[0107] Typically, vaccine or immunogenic compositions can be prepared as injectables, e.g., liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can be prepared. The formulations can also be emulsified or encapsulated in liposomes to enhance adjuvant effect.

[0108] The compositions can be prepared in unit dosage or multi-dosage form, each dosage containing a predetermined amount of active material calculated to produce a desired therapeutic effect, as well as suitable pharmaceutical excipients.

[0109] The prepared pharmaceutical compositions can be administered by conventional routes, including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, oral, or topical administration.

[0110] When using a (vaccine) composition, a safe and effective amount of the vaccine polypeptide or peptide assembly of the present invention is administered to a human, where the safe and effective amount is typically at least about 1 μg peptide / kg body weight, and in most cases, not more than about 8 mg peptide / kg body weight, and preferably the dose is about 1 μg to 1 mg peptide / kg body weight. Of course, the specific dose should take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0111] The main advantages of the present invention are:

[0112] (a) The vaccine polypeptides (including binding polypeptides) used in the present invention can generate neutralizing antibodies against the S protein RBD (receptor binding domain) in the body of a mammal such as a primate, and the neutralizing antibodies can block the binding of RBD to ACE2.

[0113] (b) Compared with non-optimized polypeptides, the coupled peptides formed by binding the optimized polypeptide sequences of the present invention to TLR7 small molecule agonists have an optimized structure and can efficiently generate cellular and humoral immunity against the SARS-CoV-2 virus.

[0114] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions are generally in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated as weight percentages and weight parts.

[0115] Example 1. Screening of antigenic polypeptides based on sequence and structural analysis of the S protein RBD In this example, we analyzed the RBD region of the SARS-CoV-2 S protein that interacts with human ACE2 and determined the key interaction sites in the RBD, as shown in Figures 1 and 2.

[0116] The inventors used software to predict and analyze CD8+ T cell epitopes in the RBD sequence, statistical analysis of the Allele Frequency Net Database database to obtain the major HLA class II molecule allele types of the world population, further analyzed and predicted binding peptides of HLA class II molecules in the RBD sequence, which were used as predicted CD4+ T cell epitopes, BepiPred and Discope software to predict linear and conformational B cell epitopes in the RBD sequence, Uniprot database to obtain glycosylation modification sites in the RBD region, and ProtParam software to predict the physicochemical properties of candidate polypeptides.

[0117] Based on the above analysis results and information, the present inventors finally screened 12 polypeptides, the amino acid sequences of which are as follows:

[0118] [Table 4] Furthermore, P-37, P-67 (including P-67-F1 and P-67-F2), P-71 and LY54 are determined according to their comprehensive characteristics.

[0119] The HLA class I molecule allele types corresponding to the CD8+ T cell epitopes contained in polypeptides P-37, P-67, and P-71 are shown in Table 1, suggesting that the three polypeptides have the potential to better induce antiviral responses from killer T cells.

[0120] Table 1. HLA class I molecule allele types corresponding to CTL epitopes contained in some polypeptides [Table 5] The binding affinities of the three polypeptides (P-37, P-67, and P-71) to the 24 predominant HLA class II molecules in the population are shown in Table 2 and Figure 3. This indicates that the presentation of the three polypeptides by the predominant HLA class II molecules in the population can induce a broad range of CD4+ T cell effects and promote the potential of B cells to produce antiviral antibodies.

[0121] Table 2. Prediction and analysis of binding affinities of polypeptides P-37, P-67, and P-71 to 24 major population HLA class I molecule allele types. [Table 6] Further analysis revealed that three polypeptides (P-37, P-67, and P-71) had good cross-overlap with the linear and conformational B cell epitopes of the RBD (Figures 4 and 5), suggesting that they could effectively activate B cell-mediated immune responses.

[0122] Furthermore, three polypeptides (P-37, P-67 and P-71) do not contain glycosylation modification sites and have suitable physicochemical properties.

[0123] LY54 is a long peptide containing both P-37 and P-71 epitopes, spanning the interaction surface between the RBD and ACE2, as shown in Figure 6.

[0124] Example 2. Preparation of Polypeptides In this example, polypeptides P-37, P-67, P-71 and LY54 are prepared using a polypeptide synthesizer.

[0125] [Table 7]

[0126] Example 3. Preparation of coupled peptides Artificially synthesized polypeptides such as P-37, P-67 (P-67-F1 and P-67-F2), P-71, and LY54 bind to TLR7 small molecule agonists to form corresponding coupling peptides.

[0127] The molecular structure of the TLR7 small molecule agonist used in the binding polypeptide is as follows:

[0128] [ka] SZU-101-NHS: [ka] SZU-101-Mal:

[0129] 3.1. Preparation of coupled peptide P-37-S2 Preparation of a conjugate of P-37 peptide (CYRLFRKSNLKPFERDISTEIYQAGS) with a TLR7 small molecule agonist. 18 mg of P-37 peptide was weighed and dissolved in a mixture of 270 μL of HO and 180 μL of DMF. 4.88 mg of SZU-101-Mal was added, and 30 mM NaHCO was added at the same time. The mixture was incubated at room temperature for 1 hour. The product was then separated using a C18 preparative column to obtain 15.5 mg of the coupling product P-37-S2 between SZU-101 and P-37 peptide. HRMS [M+5H]5+ 740.9976.

[0130] 3.2. Preparation of coupled peptide P-67-F1-S2 Preparation of the conjugate of P-67-F1 peptide (CYAWNRKRISN) with a TLR7 small molecule agonist. 16.5 mg of P-67-F1 peptide was weighed and dissolved in 330 μL of HO. 984 μL of SZU-101-Mal dissolved in DMF was added (20 mg of SZU-101-Mal was pre-dissolved in 2 mL of DMF to form a 10 mg / mL stock solution). At the same time, 30 mM NaHCO was added. After reacting at room temperature for 1 hour, the product was separated using a C18 preparative column to obtain 9.3 mg of the coupling product P-67-F1-S2 between SZU-101 and P-67-F1 peptide. HRMS [M+4H] 4+ 494.2456.

[0131] 3.3. Preparation of coupled peptide P-67-F2-S2 Preparation of the conjugate of P-67-F2 peptide (CVADYSVLYNSASFSTFK) with a TLR7 small molecule agonist. 20 mg of P-67-F2 peptide was weighed and dissolved in 2 mL of 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid (TFA). 675 μL of SZU-101-Mal dissolved in DMF was added (20 mg of SZU-101-Mal was pre-dissolved in 2 mL of DMF to form a 10 mg / mL stock solution). At the same time, 100 mM NaHCO3 was added. After incubation at room temperature for 1 hour, the product was separated using a C18 preparative column to obtain 13.5 mg of the coupling product P-67-F2-S2, with a HRMS [M+3H]3+ of 861.4077.

[0132] 3.4. Preparation of coupled peptide P-71-S2 Preparation of the conjugate of P-71-C peptide (GVEGFNCYFPLQSYGFQPTNGVGYQPYRK) with a TLR7 small molecule agonist. 20 mg of P-71-C peptide was weighed and dissolved in 400 μL of HO. 461 μL of SZU-101-Mal dissolved in DMF was added (20 mg of SZU-101-Mal was pre-dissolved in 2 mL of DMF to form a 10 mg / mL stock solution). At the same time, 30 mM NaHCO was added. After 1 hour of reaction at room temperature, the product was separated using a C18 preparative column to obtain a total of 20 mg of the coupling product P-71-S2 between SZU-101 and P-71-C peptide. HRMS [M+4H] 4+ 971.4554.

[0133] 3.4. Preparation of coupled peptide P-71-C-S1 Preparation of a TLR7 small molecule agonist conjugate with P-71-C peptide (GVEGFNCYFPLQSYGFQPTNGVGYQPYRK) (2). 18 mg of P-71-C peptide was weighed and dissolved in 360 μL of HO. 850 μL of SZU-101-NHS dissolved in DMF (pre-dissolved in DMF to form a 20 mg / mL stock solution) was added. NaHCO was added to a final concentration of 20 mM. After reacting at room temperature for 1 hour, three SZU-101 small molecule-conjugated products were formed. After adding DTT to a final concentration of 5 mM, the reaction was continued at 37 °C for 1 hour, forming two SZU-101-conjugated products. The products were separated using a C18 preparative column to obtain a total of 12 mg of the coupling product P-71-C peptide with SZU-101, P-71-C-S1. HRMS [M+4H] 4+ 1042.9789.

[0134] 3.5. Preparation of coupled peptide P-71-N-S1 Preparation of a conjugate of P-71-N peptide (KGVEGFNCYFPLQSYGFQPTNGVGYQPYR) with a TLR7 small molecule agonist. 22 mg of P-71-N peptide was weighed and dissolved in 440 μL of HO. 1000 μL of SZU-101-NHS dissolved in DMF (pre-dissolved in DMF to form a 20 mg / mL stock solution) was added. 20 mM NaHCO was added simultaneously. After 1 hour of reaction at room temperature, three SZU-101 small molecule-bound products were formed. After 1 hour of reaction at 37 °C, 5 mM DTT was added and two SZU-101 small molecule-bound products were formed. The product was separated using a C18 preparative column to obtain a total of 19 mg of the coupling product P-71-N-S1 between SZU-101 and P-71-N peptide. HRMS [M+4H] 4+ 1042.9789.

[0135] 3.6. Preparation of coupled peptide LY54-S1 Preparation of conjugates of LY54 peptide (LFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY) with TLR7 small molecule agonists. 10 mg of LY54 peptide was weighed and dissolved in 200 L of HO. 442 μL of SZU-101-NHS dissolved in DMF (pre-dissolved in DMF to form a 20 mg / mL stock solution) was added. 20 mM NaHCO3 was added at the same time. After 1 hour of reaction at room temperature, five SZU-101 small molecule-binding products were formed. 5 mM DTT was added and the reaction was continued at 37 °C for 1 hour to form three SZU-101-binding products. The products were separated using a C18 preparative column to obtain a total of 7.2 mg of the coupling product LY54-S1 between SZU-101 and LY54 peptide. HRMS [M+6H]6+ 1035.0771.

[0136] Example 4. In vitro mouse splenic lymphocyte proliferation and cytokine release experiments The polypeptides prepared in Example 3, such as P-37, P-67, and P-71, and the coupling peptides formed after binding to the TLR7 agonist SZU-101, were added to primary splenic lymphocyte cultures isolated and prepared from the spleens of BALB / c mice at four doses: 1 μM, 10 μM, 30 μM, and 60 μM. Cytokines IL-6, IL-12, and IFN-γ were detected after 24 hours of stimulation, and cell proliferation was detected after 72 hours of stimulation.

[0137] Taking P-71 and its corresponding coupled peptide as an example, the results are as follows, as shown in Figures 8 and 9.

[0138] (1) The polypeptide alone did not or only weakly induce an increase in cytokine release from splenic lymphocytes, whereas the coupled peptide after binding to SZU-101 could effectively induce an increase in cytokine release from splenic lymphocytes, suggesting that the immune response induced by the vaccine polypeptide can be significantly enhanced after binding to a TLR7 agonist.

[0139] (2) The Dandu polypeptide could not induce or could only weakly induce the proliferation of splenic lymphocytes, whereas the coupled peptide after binding to SZU-101 could effectively induce cytokine proliferation of splenic lymphocytes, suggesting that after coupling to a TLR7 agonist, the immune response induced by the vaccine polypeptide could be significantly improved.

[0140] Example 5. Mouse immunization experiment with conjugate polypeptide vaccine The polypeptides prepared in Example 3, such as P-37, P-67, and P-71, and the coupled peptides formed after binding to the TLR7 agonist SZU-101, were used alone or in combination. When combined, the mixing ratio was 1:1:1 (mass ratio). Aluminum hydroxide was used as an adjuvant, and then the mixture was intraperitoneally injected into BALB / c mice to immunize them.

[0141] Serum samples will be collected 29 and 57 days after the first immunization, and antibodies against the SARS-CoV-2 S protein RBD in the serum (1:100 dilution) will be detected using a bridging ELISA.

[0142] As shown in FIG. 10, the results are as follows:

[0143] (1) The mixed peptide + adjuvant group containing P-71-C-S1 or P-71-S2 can significantly generate antibodies against the RBD.

[0144] (2) The single conjugate P-37-S2 + adjuvant group can significantly generate antibodies against the RBD.

[0145] (3) Although the mixed peptides not coupled to SZU-101 could induce antibodies against the RBD, the number of antibodies produced was lower than that of the mixed peptides containing the coupled peptides, suggesting that the immune response induced by the vaccine polypeptides could be significantly enhanced after binding to a TLR7 agonist.

[0146] Example 6. Immunization experiment of coupled polypeptide vaccine in crab-eating monkeys In this example, the immune effect of the coupled polypeptide vaccine against SARS-CoV-2 is further verified in crab-eating monkeys.

[0147] (a) Mixed coupling peptides consisting of P-37, P-67-F2, and P-71 conjugated to SZU-101 (P-71-C-S1 / P-71-N-S1 / P-71-S2 / P-37-S2 / P-67-F2-S2), (b) LY54, and (c) LY54-S1 were used to prepare immunization preparations using TiterMax adjuvant. Crab-eating monkeys were immunized by multiple subcutaneous injections. 14 days after the second immunization, antibody titers were measured using a bridging ELISA to measure the ability of the antisera to block RBD binding to ACE2.

[0148] Neutralizing antibodies were detected by competitive ELISA. The specific measurement method was as follows: 10 μg / mL ACE2 was used to coat a microtiter plate overnight, followed by blocking. Anti-peptide serum was diluted to various degrees (1:128, 1:64, 1:32, 1:16, 1:8, and 1:4) in sample dilution buffer. The diluted antisera were then incubated with 12 μg / mL Bio-RBD at 37°C for 1 hour. 100 μL of the reaction mixture was then added to the wells of the blocked ACE2-coated microtiter plate. After incubation at 37°C for 1 hour, the plate was washed, and 1:10,000 diluted HRP-Streptavidin A was added. The mixture was then incubated at 37°C for 1 hour. After washing, TMB was added to develop the color. The plate was then read at 450 nm.

[0149] The results show that all animals produced antibodies to the RBD, and the antibody titers were all relatively high (Figure 11).

[0150] Furthermore, the antisera generated using the mixed coupling peptides, LY54, and LY54-S1, all had the ability to block the binding of RBD to ACE2 (Figure 12), thus blocking SARS-CoV-2 virus infection.

[0151] Example 7. In vitro binding experiment of polypeptide with ACE2 In this example, the polypeptides P-37, P-71, and LY54, whose spatial structures are located at the interaction surface between RBD and ACE2, were labeled with biotin, and the binding ability of these polypeptides to ACE2 was detected by ELISA.

[0152] The specific measurement method is as follows: 10 μg / mL ACE2 is used to coat a microtiter plate overnight, and after blocking, different concentrations (1, 0.5, and 0.25 μg / mL) of biotin-labeled polypeptide are added and incubated at 37°C for 1.5 hours. After that, 1:5000 diluted HRP-Streptavidin A is added and incubated at 37°C for 1 hour. After washing the plate, TMB is added to develop color, and the plate is read at a wavelength of 450 nm.

[0153] The results showed that the polypeptides P-37, P-71, and LY54 had strong binding ability to ACE2 (Figure 13), suggesting that the polypeptides P-37, P-71, and LY54 themselves have potential blocking effects.

[0154] Discussion Polypeptide vaccines are created by selecting one or more antigenic epitope fragments from highly immunogenic proteins for immunization. Because polypeptide vaccines have short amino acid chains, mature polypeptide synthesis technology makes them convenient for rapid in vitro synthesis and purification, ensuring the purity and reproducibility of each batch of product.

[0155] At the same time, computer-aided vaccine design can rapidly respond to sudden public health problems caused by novel viruses, etc., and predict and screen suitable candidate peptide segments for vaccine development. Furthermore, compared to other types of vaccines, polypeptide vaccines have distinct epitopes, excellent stability, high purity, and better safety. However, because the amino acid chain length of polypeptide vaccines is relatively short, usually about 10 to 30 amino acids, they also face the problem of low immunogenicity, and usually require modification or the addition of adjuvants to generate a better immune response.

[0156] Designing polypeptide vaccines requires the assistance of computer-aided vaccine design techniques, which typically require comprehensive analysis of the target protein's T / B cell epitopes, as well as its structural and modification information. CD8+ T cell epitopes primarily activate killer T cells to exert antiviral effects, while CD4+ T cell epitopes primarily activate helper T cells and deactivate B cells to generate antiviral antibodies. Linear and conformational B cell epitopes can directly activate B cells to generate antibodies.

[0157] SARS-CoV-2 enters host cells by binding its surface spike glycoprotein (S protein) to the angiotensin-converting enzyme 2 (ACE2) protein. Therefore, the S protein is a preferred target protein for COVID-19 vaccine design, with the hope of inducing the production of neutralizing antibodies that can block viral entry. However, currently known vaccines have difficulty effectively eliciting an immune response against the coronavirus SARS-CoV-2.

[0158] Therefore, activating highly efficient antigen-presenting cells against SARS-CoV-2 is key to developing a SARS-CoV-2 vaccine.

[0159] Through research, the inventors unexpectedly discovered that a vaccine polypeptide formed by combining an antigen polypeptide of the S protein of the coronavirus SARS-CoV-2 with a TLR7 small molecule agonist after screening and sequence optimization can effectively induce and generate antiviral antibodies with a blocking effect against the RBD region of the S protein in the body, including primates. Furthermore, the vaccine polypeptide combined with the TLR7 small molecule agonist SZU-101 can efficiently activate antigen-presenting cells, overcoming issues such as the low immunogenicity of the polypeptide and the occurrence of viral immune escape.

[0160] Therefore, the coupled peptides of the present invention can be used to develop novel coronavirus polypeptide vaccines to prevent and treat coronavirus SARS-CoV-2 infection and related diseases, including novel coronavirus pneumonia (Coronavirus disease 2019, COVID-19), by inducing cellular and humoral immunity in the human body.

[0161] Furthermore, the inventors have found that the strategy of coupling polypeptides formed by binding a TLR7 agonist to a specific antigen polypeptide (particularly binding polypeptides formed by coupling to the TLR7 small molecule agonist SZU-101, such as the S1 and / or S2 structure) can also be applied to the development of drugs or vaccines for preventing and treating other single-stranded RNA virus (ssRNA virus) infections or related diseases, particularly to the development of vaccines against various different coronaviruses, including known coronaviruses such as SARS and MERS, and unknown coronaviruses.

[0162] All documents mentioned in this application are incorporated by reference in this application as if each document were incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art will be able to make various changes or modifications to the present invention, and these equivalents will also fall within the scope defined by the appended claims of this application.

Claims

1. A vaccine polypeptide for the novel coronavirus SARS-CoV-2, comprising: The vaccine polypeptide has the structure of Formula I or an oligomer comprising the structure of Formula I, Formula (I): Z-(JU)n In the above formula, Z is an antigenic polypeptide, the antigenic polypeptide having at least one T cell epitope and / or at least one B cell epitope of the S protein of the novel coronavirus SARS-CoV-2, the antigenic polypeptide having an amino acid sequence derived from the RBM (receptor binding motif) region of the S protein, the length of the antigenic polypeptide is 8 to 100 amino acids, and the amino acid sequence of the antigenic polypeptide is set forth in SEQ ID NO: 2, 6, 7, or 12; each U is independently a TLR7 agonist, said TLR7 agonist being a small molecule agonist; n is a positive integer that is 1, 2, 3, 4, 5, or 6; J is a chemical bond or a linker; The vaccine polypeptide is capable of stimulating primates and rodents to generate neutralizing antibodies that block the binding of the RBD (receptor binding domain) region of the S protein of the novel coronavirus SARS-CoV-2 to ACE2. The vaccine polypeptide of the novel coronavirus SARS-CoV-2,

2. The vaccine polypeptide of claim 1, characterized in that the vaccine polypeptide can stimulate a primate to generate neutralizing antibodies that block the binding of the RBD region in the S protein of the novel coronavirus SARS-CoV-2 to ACE2.

3. The TLR7 agonist is SZU-101. 【Chemistry 1】 The vaccine polypeptide of claim 1, comprising:

4. The SZU-101 is bound to an amino group of an antigenic polypeptide and has the structure shown in S1. 【Chemistry 2】 Formula (S1): or The SZU-101 binds to sulfhydryl groups of antigenic polypeptides and has the structure shown in S2. 【Chemistry 3】 Formula (S2):

4. The vaccine polypeptide of claim 3, wherein the polypeptide forms:

5. said U is attached to Z at a fixed point; The vaccine polypeptide is selected from the following group of coupling peptides: (S1)-GVEGFNCYFPLQSYGFQPTNGVGYQPYRK-(S1) (SEQ ID NO: 14) Here, SZU-101 binds to the N-terminal amino group of G and the side chain amino group of K in the amino acid sequence via the S1 structure, (S1) ２ -KGVEGFNCYFPLQSYGFQPTNGVGYQPYR (SEQ ID NO: 15) Here, SZU-101 binds to the N-terminus and the side chain amino group of K in the amino acid sequence via the S1 structure, (S1)-LFRK(-S1)SNLK(-S1)PFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY (SEQ ID NO: 12) Here, SZU-101 binds to the N-terminal amino group of L and the side chain amino group of K in the amino acid sequence via the S1 structure, GVEGFNC (-S2) YFPLQSYGFQPTNGVGYQPYRK (SEQ ID NO: 14) Here, SZU-101 binds to the sulfhydryl on C in the amino acid sequence via the S2 structure, (S2) -CYRLFRKSNLKPFERDISTEIYQAGS (SEQ ID NO: 16) Here, SZU-101 binds via the S2 structure to the sulfhydryl group on the C of the polypeptide, (S2) -CYAWNRKRISN (SEQ ID NO:5) Here, SZU-101 binds via the S2 structure to the sulfhydryl group on the C of the polypeptide, (S2) -CVADYSVLYNSASFSTFK (SEQ ID NO: 6) 5. The vaccine polypeptide of claim 4, wherein SZU-101 is bound to a sulfhydryl group on C of the polypeptide via an S2 structure.

6. An isolated collection of peptides, characterized in that it comprises at least two species of vaccine polypeptides of the novel coronavirus SARS-CoV-2 according to claim 1.

7. A pharmaceutical composition comprising a vaccine polypeptide of novel coronavirus SARS-CoV-2 according to claim 1 or a collection of peptides according to claim 6, and a pharma- ceutical acceptable vector.

8. Use of a vaccine polypeptide according to claim 1 or a peptide collection according to claim 6 or a pharmaceutical composition according to claim 7, characterized in that it is used for the preparation of a medicament for preventing coronavirus SARS-CoV-2 infection or a disease associated therewith.

9. A cell preparation comprising: (a) immune cells activated by immunization with the vaccine polypeptide of claim 1 or the peptide assembly of claim 6; and (b) a pharma- ceutically acceptable vector.

10. The cell preparation according to claim 9, wherein the immune cells are selected from the group consisting of dendritic cells, natural killer cells (NK), lymphocytes, monocytes / macrophages, granulocytes, or a combination thereof.

11. A fusion protein comprising: A fusion protein comprising a carrier protein and a vaccine polypeptide according to claim 1 fused to said fusion protein.

12. A pharmaceutical composition comprising: (a) the fusion protein of claim 11 or an immune cell activated by the fusion protein or an immune cell activated by the fusion protein; and (b) a pharma- ceutically acceptable vector.

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