Pharmaceutical composition for resisting infection by SARS-CoV-2 or its variants and its combination drugs

Abstract

The present invention relates to a pharmaceutical composition and a combined agent thereof for resisting infection by SARS-CoV-2 or its variants, and particularly to a broad-spectrum nasal spray of a compound preparation for resisting infection by SARS-CoV-2 or its variants belonging to the medical field and a combined agent thereof. To solve the problem that there is no effective preventive and therapeutic drug for infection by SARS-CoV-2 or its mutant virus, the present invention provides a recombinant protein vaccine and / or an adenovirus vaccine for preventing and / or treating infection by SARS-CoV-2 or its variants, particularly a nasal spray of a compound preparation containing the active ingredients of two vaccines, namely, a recombinant protein vaccine and an adenovirus vaccine, and a combination of two vaccines for nasal spray administration, which can induce strong antibody and cellular immune responses in vivo and block the binding of the protein S of SARS-CoV-2 to the ACE2 receptor of host cells, thus enabling the host to resist coronavirus infection. In particular, the present invention has good preventive and therapeutic effects against various mutant viruses.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition and a combined agent thereof for resisting the infection of SARS-CoV-2 or its variants, and in particular, to a broad-spectrum nasal spray of a compound preparation for resisting the infection of SARS-CoV-2 or its variants belonging to the medical field and a combined agent thereof.

Background Art

[0002] SARS-CoV-2 is a novel beta coronavirus named by the World Health Organization (WHO). The virus has an envelope, and the particles are circular or elliptical, and are mostly polymorphic with a diameter of 60 nm to 140 nm. Its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV, and SARS-CoV-2 is a variant of a novel coronavirus that has not been found in humans so far. Currently, there are five main types of the newly mutated novel coronavirus, namely alpha, beta, gamma, delta, and omicron. The omicron variant is further divided into several subtypes such as BA.1, BA.2, BA.2.12.1, BA.4, and BA.5. So far, the novel coronavirus SARS-CoV-2 has infected 545 million people worldwide, including more than 6.3 million deaths.

[0003] The main structural proteins of SARS-CoV-2 include spike (S), envelope (E), membrane (M), and nucleocapsid (N). Among these, the S protein plays an important role in virus infection and pathogenicity. It is often used as an antigen in vaccines. Since SARS-CoV-2 variants contain multiple mutation sites and the virus's S protein also contains multiple mutation sites, the variants can escape to some extent from the antibodies stimulated by the SARS-COV-2 prototype strain (i.e., the "wild strain of the novel coronavirus") vaccine, resulting in the invalidation or reduced protection of the SARS-COV-2 vaccine, which poses great pressure on the prevention and control of the SARS-COV-2 pandemic. Therefore, the development of vaccines against various variants of the SARS-CoV-2 virus, especially broad-spectrum vaccines against various variants of the SARS-CoV-2 virus, is very important for the prevention and treatment of novel coronavirus pneumonia.

[0004] Currently, hundreds of companies and departments around the world are mainly involved in the development of SARS-COV-2 vaccines through five technical routes: inactivated vaccines, recombinant protein vaccines, adenovirus vector vaccines, attenuated influenza virus vector vaccines, and nucleic acid vaccines (including mRNA vaccines and DNA vaccines). On the other hand, various vaccines have been marketed. The vaccination routes mainly include subcutaneous injection, intradermal injection, intramuscular injection, oral administration, and inhalation (including nasal spray). Many viruses such as influenza virus and novel coronavirus mainly spread through the respiratory tract, and nasal spray and inhaled vaccines are developed by simulating the natural infection process of the human body. Therefore, it is important to develop vaccines that can induce strong mucosal immunity to effectively prevent the invasion and infection of these viruses from the respiratory tract.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention aims to solve one of the technical problems existing in the prior art. For this purpose, the present invention provides a nasal spray of a compound preparation containing a recombinant protein vaccine and an adenovirus vaccine for resisting the infection of SARS-CoV-2 or its variants, and a combined nasal spray.

Means for Solving the Problems

[0006] The present invention provides a pharmaceutical composition for preventing and / or treating the infection of SARS-CoV-2 or its variants, which is a compound preparation containing an active ingredient of a recombinant protein vaccine and / or an adenovirus vaccine for resisting the infection by SARS-CoV-2 or its variants. Further, the compound preparation also contains other pharmaceutically acceptable excipients or complementary components.

[0007] The present invention further provides a combined agent for preventing and / or treating the infection of SARS-CoV-2 or its variants, which contains a recombinant protein vaccine for resisting the infection of SARS-CoV-2 or its variants and an adenovirus vaccine, and the recombinant protein vaccine and the adenovirus vaccine are administered separately or simultaneously.

[0008] Furthermore, the pharmaceutical composition or the combined agent is an intramuscular injection, a nasal drop, a spray, a nasal spray or an inhalant, and preferably, the pharmaceutical composition or the combined agent is a nasal spray.

[0009] Furthermore, the recombinant protein vaccine and / or the adenovirus vaccine contains a protein and / or a protein precursor for resisting the infection of SARS-CoV-2 or its variants.

[0010] Furthermore, the protein and / or the protein precursor contains a full-length S protein, or a protein formed by at least one RBD sequence and / or at least one HR sequence in the S protein of SARS-CoV-2 or its variants.

[0011] Preferably, the RBD sequence is shown in SEQ ID NO: 1, or the RBD sequence has homology with SEQ ID NO: 1 and the same or similar biological activity, and is a variant obtained by substitution and / or deletion and / or insertion of at least one amino acid in the sequence of SEQ ID NO: 1. Preferably, the RBD sequence is a variant obtained by substitution and / or deletion and / or insertion of 1 to 400 amino acids in the sequence of SEQ ID NO: 1. Preferably, the RBD sequence is a variant obtained by substitution and / or deletion and / or insertion of 5 to 30 amino acids in the sequence of SEQ ID NO: 1.

[0012] In addition, the protein formed by the RBD sequence and the HR sequence in the S protein can spontaneously form a trimer.

[0013] Furthermore, the homologous amino acid sequence is selected from at least one of the RBD sequences of alpha, beta, gamma, delta and omicron.

[0014] Furthermore, the protein precursor has a signal peptide and / or a protein tag bound to the protein to resist infection by SARS-CoV-2 or its variants. Preferably, the signal peptide includes the signal peptide of the S protein or its variant and / or the human tPA signal peptide further provided outside the front of the self-provided signal peptide. Preferably, the protein tag is selected from at least one of a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like modified protein tag, a maltose binding protein tag, a c-Myc protein tag and an Avi tag protein tag. More preferably, the protein tag is a Trx tag and / or a 6His tag.

[0015] Furthermore, the protein for resisting the infection of SARS-CoV-2 or its variants is further bound to a protease recognition region for removing the protein tag, and preferably, the protease is selected from at least one of enterokinase, TEV protease, thrombin, factor Xa of coagulation, carboxypeptidase A and rhinovirus 3c protease.

[0016] Furthermore, the amino acid sequence of the protein and / or the protein precursor is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70 and SEQ ID NO: 73.

[0017] Furthermore, the nucleotide sequence for encoding the amino acid sequence is shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74 or SEQ ID NO: 75.

[0018] Furthermore, the recombinant protein vaccine and / or the adenovirus vaccine contains a nucleic acid for resisting the infection of SARS-CoV-2 or its variants.

[0019] Furthermore, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO: 76.

[0020] Furthermore, the nucleotide sequence is obtained by codon or cell optimization based on the encoded amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73 or SEQ ID NO: 77, and furthermore, the cell is a mammalian cell CHO or an insect cell.

[0021] The present invention further provides a recombinant vector or an adenovirus vector, which contains a polynucleotide sequence in a recombinant protein vaccine or an adenovirus vaccine in a pharmaceutical composition or a combined agent, and the polynucleotide sequence is selected from at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76.

[0022] Furthermore, the recombinant vector is selected from at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an E. coli expression vector and a yeast expression vector. Preferably, the insect baculovirus expression vector is pFastBac1. Preferably, the mammalian cell expression vector is a CHO cell expression vector. More preferably, the CHO cell expression vector is pTT5 or FTP-002. Preferably, the E. coli expression vector is pET32a. Preferably, the yeast expression vector is pPICZaA.

[0023] Furthermore, the adenovirus vector is a human replication-deficient recombinant adenovirus vector. Preferably, the adenovirus vector is selected from human type 5, 35 or 26 replication-deficient adenoviruses and / or chimpanzee type AdC68 or AdC7 replication-deficient adenoviruses. More preferably, it is selected from human type 5 replication-deficient adenoviruses having a combined deletion of E1 and E3.

[0024] The present invention also provides a host cell containing the above recombinant vector or adenovirus vector.

[0025] Furthermore, the host cell is selected from at least one of insect cells, mammalian cells, Escherichia coli, and yeast. Preferably, the insect cell is selected from at least one of sf9 cells, sf21 cells, and Hi5 cells. Preferably, the mammalian cell is a CHO cell or a HEK293 cell.

[0026] Furthermore, the recombinant protein vaccine and / or adenovirus vaccine further contains a pharmaceutically acceptable excipient or complementary component.

[0027] Furthermore, the complementary component is an immunoadjuvant. Preferably, the immunoadjuvant is selected from at least one of squalene water-in-oil emulsion, aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoryl lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid, and cationic liposome material.

[0028] Furthermore, the immune adjuvant satisfies that at least one squalene water-in-oil emulsion is MF59, the aluminum salt is selected from at least one of aluminum hydroxide and alum, the calcium salt is tricalcium phosphate, the plant saponin is QS-21 or ISCOM, the plant polysaccharide is astragalus polysaccharide, the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin, the lipid is selected from at least one of phosphatidylethanolamine, phosphatidylcholine, cholesterol and dioleoylphosphatidylethanolamine, or the cationic liposome material is (2,3-dioleyloxypropyl) trimethylammonium chloride, N-[1-(2,3-dioleoyl chloride) propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-arginine formylamino) ethylammonium trimethyldodecylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, dimethyldioctadecylammonium bromide and CpG ODN.

[0029] Furthermore, the vaccine preparations of the recombinant protein vaccine and the adenovirus vaccine are intramuscular injections, injections, nasal drops, sprays, nasal sprays or inhalants, and preferably, the vaccine is a nasal spray.

[0030] The present invention further provides the use of a pharmaceutical composition or a combined agent in the preparation of a medicament for preventing and / or treating SARS-CoV-2 or its variant infection.

[0031] The present invention further provides the use of a pharmaceutical composition or a combined agent in the preparation of a medicament for treating and / or preventing the infection or onset of SARS-COV-2 variant strains.

[0032] Furthermore, the SARS-COV-2 variant includes at least one of alpha, beta, gamma, delta, and omicron.

[0033] The present invention provides the use of a pharmaceutical composition or a combination drug in the preparation of a drug for treating and / or preventing the infection or onset of influenza virus or other respiratory viruses.

[0034] The present invention further provides a method for preparing a protein, the method including the steps of culturing a host cell to express the required protein or precursor, and then collecting the required protein.

[0035] The present invention further provides a method for preparing an adenovirus, the method including the steps of constructing a plasmid vector containing a polynucleotide, transfecting a host cell and culturing the transfected host cell, obtaining a recombinant adenovirus, extending the culture of the recombinant adenovirus, and purifying the recombinant adenovirus.

[0036] The polynucleotide sequence is selected from at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76.

[0037] The present invention further provides a method for preparing a recombinant protein, the method including the steps of constructing a recombinant vector containing a polynucleotide, and immunizing a human body to generate the protein.

[0038] Furthermore, the vector is selected from at least one of mRNA, DNA vaccine, adenovirus, vaccinia Ankara virus, and adeno-associated virus.

[0039] The present invention further provides a second pharmaceutical composition containing a recombinant protein vaccine and an inactivated virus vaccine as active ingredients. Furthermore, the inactivated virus vaccine is an inactivated influenza virus vaccine.

[0040] The present invention further provides a third pharmaceutical composition containing an adenovirus vaccine and an influenza virus recombinant protein vaccine as active ingredients.

[0041] The present invention further provides the use of the second or third pharmaceutical composition in the preparation of a medicament for treating and / or preventing the infection or onset of respiratory viruses.

[0042] In the three pharmaceutical compositions of the present invention, the active ingredients are combined in any combination ratio.

[0043] Furthermore, in the pharmaceutical composition, the volume ratio of the recombinant protein vaccine to the adenovirus vaccine is 1 to 5 to 1 to 5.

[0044] The present invention further provides a second combination drug in which the recombinant protein vaccine and the inactivated virus vaccine are administered separately or simultaneously. Furthermore, the inactivated virus vaccine is an inactivated influenza virus vaccine.

[0045] The present invention further provides a combination drug in which the adenovirus vaccine and the influenza virus recombinant protein vaccine are administered separately or simultaneously.

[0046] Preferably, the influenza virus is selected from influenza A virus and / or influenza B virus.

[0047] More preferably, the influenza A virus is an influenza A (H1N1) virus.

[0048] SEQ ID NO: 1 original strain (Wuhan strain) VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNG

[0049] SEQ ID NO: 1 shares the same amino acid sequence as positions 320 - 545 in the S protein of SARS-CoV-2.

[0050] SEQ ID NO: 2 RBD-HR-beta sequence MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0051] The above sequence consists of a signal peptide - Trx tag - 6His tag - EK restriction enzyme cleavage site - RBD sequence - HR1 sequence - HR2 sequence. For the RBD sequence, the amino acid at position 98 of the sequence of SEQ ID NO: 1 is substituted from K to N, the amino acid at position 133 is substituted from L to R, the amino acid at position 165 is substituted from E to K, the amino acid at position 182 is substituted from N to Y, and the amino acid at position 219 is substituted from C to S.

[0052] SEQ ID NO: 3 RBD-HR - Delta sequence MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0053] The above sequence consists of a signal peptide - Trx tag - 6His tag - EK restriction enzyme cleavage site - RBD sequence - HR1 sequence - HR2 sequence. For the RBD sequence, the amino acid at position 133 of the sequence of SEQ ID NO: 1 is substituted from L to R, the amino acid at position 159 is substituted from T to K, and the amino acid at position 219 is substituted from C to S.

[0054] SEQ ID NO: 4 RBD-HR - Omicron BA.1 MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0055] The above sequence consists of a signal peptide - Trx tag - 6His tag - EK restriction enzyme cleavage site - RBD sequence - HR1 sequence - HR2 sequence. The RBD sequence is obtained by mutating 5 or more amino acid sequences in the sequence of SEQ ID NO: 1.

[0056] SEQ ID NO: 5 RBD-HR - Omicron BA.2 MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0057] The above sequence consists of a signal peptide - Trx tag - 6His tag - EK restriction enzyme cleavage site - RBD sequence - HR1 sequence - HR2 sequence. The RBD sequence is obtained by mutating 5 or more amino acid sequences in the sequence of SEQ ID NO: 1.

[0058] SEQ ID NO: 6 RBD-HR - Omicron BA.4 / .5 MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0059] The above sequence consists of a signal peptide - Trx tag - 6His tag - EK restriction enzyme cleavage site - RBD sequence - HR1 sequence - HR2 sequence. The RBD sequence is obtained by mutating 5 or more amino acid sequences in the sequence of SEQ ID NO: 1.

[0060] SEQ ID NO: 7 RBD-HR - Omicron BA2.12.1 MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYQYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0061] The above sequence consists of a signal peptide - Trx tag - 6His tag - EK restriction enzyme cleavage site - RBD sequence - HR1 sequence - HR2 sequence. The RBD sequence is obtained by mutating 5 or more amino acid sequences in the sequence of SEQ ID NO: 1.

[0062] SEQ ID NO: 8 RBD-HR-BF.7 MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATTFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0063] The above sequence consists of a signal peptide - Trx tag - 6His tag - EK restriction enzyme cleavage site - RBD sequence - HR1 sequence - HR2 sequence. The RBD sequence is obtained by mutating 5 or more amino acid sequences in the sequence of SEQ ID NO: 1.

[0064] SEQ ID NO: 9 RBD-HR-BQ.1 MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGDDDDKVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSTVGGNYNYRYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL

[0065] The above sequence consists of a signal peptide - Trx tag - 6His tag - EK restriction enzyme cleavage site - RBD sequence - HR1 sequence - HR2 sequence. The RBD sequence is obtained by mutating 5 or more amino acid sequences in the sequence of SEQ ID NO: 1.

[0066] SEQ ID NO: 10 (nucleotide sequence encoding SEQ ID NO: 2)

[0067] Signal peptide coding sequence - Trx tag coding sequence - 6His tag coding sequence - EK restriction enzyme cleavage site coding sequence - RBD coding sequence - HR1 coding sequence - HR2 coding sequence

[0068] SEQ ID NO: 11 (nucleotide sequence encoding SEQ ID NO: 3)

[0069] Signal peptide coding sequence - Trx tag coding sequence - 6His tag coding sequence - EK restriction enzyme cleavage site coding sequence - RBD coding sequence - HR1 coding sequence - HR2 coding sequence

[0070] SEQ ID NO: 12 (nucleotide sequence encoding SEQ ID NO: 4)

[0071] Signal peptide coding sequence - Trx tag coding sequence - 6His tag coding sequence - EK restriction enzyme cleavage site coding sequence - RBD coding sequence - HR1 coding sequence - HR2 coding sequence

[0072] SEQ ID NO: 13 (nucleotide sequence encoding SEQ ID NO: 5)

[0073] Signal peptide coding sequence - Trx tag coding sequence - 6His tag coding sequence - EK restriction enzyme cleavage site coding sequence - RBD coding sequence - HR1 coding sequence - HR2 coding sequence

[0074] SEQ ID NO: 14 (nucleotide sequence encoding SEQ ID NO: 6)

[0075] Signal peptide coding sequence - Trx tag coding sequence - 6His tag coding sequence - EK restriction enzyme cleavage site coding sequence - RBD coding sequence - HR1 coding sequence - HR2 coding sequence

[0076] SEQ ID NO: 15 (nucleotide sequence encoding SEQ ID NO: 7)

[0077] Signal peptide coding sequence - Trx tag coding sequence - 6His tag coding sequence - EK restriction enzyme cleavage site coding sequence - RBD coding sequence - HR1 coding sequence - HR2 coding sequence

[0078] SEQ ID NO: 16 (nucleotide sequence encoding SEQ ID NO: 8)

[0079] Signal peptide coding sequence - Trx tag coding sequence - 6His tag coding sequence - EK restriction enzyme cleavage site coding sequence - RBD coding sequence - HR1 coding sequence - HR2 coding sequence

[0080] SEQ ID NO: 17 (nucleotide sequence encoding SEQ ID NO: 9)

[0081] Signal peptide coding sequence - Trx tag coding sequence - 6His tag coding sequence - EK restriction enzyme cleavage site coding sequence - RBD coding sequence - HR1 coding sequence - HR2 coding sequence

[0082] Construction mode of M65, RBD(BA.1)-HR1-RBD(delta)-HR2-RBD(beta)

[0083] The amino acid sequence constructed for M65 is as follows.

[0084] SEQ ID NO: 18 RBD(BA.1)-HR1-RBD(delta)-HR2-RBD(beta) MDAMKRGLCCVLLLCGAVFVSPVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNG

[0085] tPA signal peptide - Omicron BA.1 RBD sequence - HR1 sequence - Delta RBD sequence - HR2 sequence - Beta RBD sequence

[0086] SEQ ID NO: 19 (nucleotide sequence encoding SEQ ID NO: 18)

[0087] Construction of M65-BA.4 / 5, replacement of BA.1 RBD in M65 with the RBD sequence of BA.4 / 5, and its construction pattern, RBD(BA.4 / 5)-HR1-RBD(delta)-HR2-RBD(WT)

[0088] The amino acid sequence constructed for M65-BA.4 / 5 is as follows.

[0089] SEQ ID NO: 20 RBD(BA.4 / 5)-HR1-RBD(delta)-HR2-RBD(WT) MDAMKRGLCCVLLLCGAVFVSPVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNG

[0090] tPA signal peptide - RBD sequence of Omicron BA.4 / 5 - HR1 sequence - RBD sequence of Delta - HR2 sequence - RBD sequence of WT.

[0091] SEQ ID NO: 21 (nucleotide sequence encoding SEQ ID NO: 20)

[0092] Construction of M65-HR-opt, further optimization of the HR1 and HR2 sequences in M65, and its construction pattern, RBD(BA.4 / 5)-HR1(opt)-RBD(delta)-HR2(opt)-RBD(WT)

[0093] The amino acid sequence constructed for M65-HR-opt is as follows.

[0094] SEQ ID NO: 22 RBD(BA.4 / 5)-HR1(opt)-RBD(delta)-HR2(opt)-RBD(WT) MDAMKRGLCCVLLLCGAVFVSPVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNGVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYGKYEQYIKWPWYIWLGFIAGVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSVNFNFNG

[0095] tPA signal peptide - Omicron BA.4 / 5 RBD sequence - HR1 sequence - Delta RBD sequence - HR2 sequence - WT RBD sequence.

[0096] SEQ ID NO: 23 (nucleotide sequence encoding SEQ ID NO: 22)

[0097] SEQ ID NO: 24 Nucleotide sequence of the S antigen of the wild-type Ad-S vaccine

[0098] SEQ ID NO: 25 Amino acid sequence of the S antigen of the wild-type Ad-S vaccine

[0099] Nucleotide sequence of the S antigen of Array No. 26 Ad-S-beta vaccine

[0100] Amino acid sequence of the S antigen of Array No. 27 Ad-S-beta vaccine

[0101] Nucleotide sequence of the S antigen of Array No. 28 Ad-S-Delta vaccine

[0102] Amino acid sequence of the S antigen of Array No. 29 Ad-S-delta vaccine

[0103] Nucleotide sequence of the S antigen of the array number 30 Ad-S-beta-delta vaccine

[0104] Amino acid sequence of the S antigen of Array No. 31 Ad-S-beta-delta vaccine

[0105] Nucleotide sequence of the S antigen of the array number 32 Ad-S-Omicron-BA.1 vaccine

[0106] Amino acid sequence of the S antigen of the array number 33 Ad-S-Omicron-BA.1 vaccine

[0107] Nucleotide sequence of the S antigen of the array number 34 Ad-S-Omicron-BA.2 vaccine

[0108] Amino acid sequence of the S antigen of the array number 35 Ad-S-Omicron-BA.2 vaccine

[0109] Nucleotide sequence of the S antigen of the array number 36 Ad-S-Omicron-BA.2.12.1 vaccine

[0110] Amino acid sequence of the S antigen of the array number 37 Ad-S-Omicron-BA.2.12.1 vaccine

[0111] Nucleotide sequence of the S antigen of the array number 38 Ad-S-Omicron-BA.4 / 5 vaccine

[0112] Amino acid sequence of the S antigen of the array number 39 Ad-S-Omicron-BA.4 / 5 vaccine

[0113] Sequence number 40: Full-length amino acid sequence of wild-type S antigen

[0114] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0115] SEQ ID NO: 41 Full-length nucleic acid sequence of wild-type S antigen (optimized for CHO cells)

[0116] Note: The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0117] SEQ ID NO: 42 Full-length nucleic acid sequence of wild-type S antigen (optimized for insect cells)

[0118] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0119] SEQ ID NO: 43 Extracellular amino acid sequence of wild-type S antigen

[0120] Note The above array consists of a signal peptide - extracellular domain.

[0121] SEQ ID NO: 44 Extracellular nucleic acid sequence of wild - type S antigen (optimized for CHO cells)

[0122] Note The above array consists of a signal peptide - extracellular domain.

[0123] SEQ ID NO: 45 Extracellular nucleic acid sequence of wild - type S antigen (optimized for insect cells)

[0124] Note: The above array consists of a signal peptide - extracellular domain.

[0125] SEQ ID NO: 46 Full-length amino acid sequence of beta S antigen

[0126] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0127] SEQ ID NO: 47 Full-length nucleic acid sequence of beta S antigen (optimized for CHO cells)

[0128] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0129] SEQ ID NO: 48 Full-length nucleic acid sequence of beta S antigen (optimized for insect cells)

[0130] Note The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0131] SEQ ID NO: 49 Extracellular amino acid sequence of the beta S antigen

[0132] Note The above array consists of a signal peptide - extracellular domain.

[0133] SEQ ID NO: 50 Extracellular nucleic acid sequence of beta S antigen (optimized for CHO cells)

[0134] Note The above-mentioned array consists of a signal peptide - extracellular domain.

[0135] SEQ ID NO: 51 Extracellular nucleic acid sequence of beta S antigen (optimized for insect cells)

[0136] Note The above array consists of a signal peptide - extracellular domain.

[0137] SEQ ID NO: 52 Full - length amino acid sequence of the delta S antigen

[0138] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0139] SEQ ID NO: 53 Full-length nucleic acid sequence of the delta S antigen (optimized for CHO cells)

[0140] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0141] SEQ ID NO: 54 Full-length nucleic acid sequence of the delta S antigen (optimized for insect cells)

[0142] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0143] SEQ ID NO: 55 Extracellular amino acid sequence of the delta S antigen

[0144] Note The above array consists of a signal peptide - extracellular domain.

[0145] SEQ ID NO: 56 Extracellular nucleic acid sequence of the delta S antigen (optimized for CHO cells)

[0146] Note The above array consists of a signal peptide - extracellular domain.

[0147] SEQ ID NO: 57 Extracellular nucleic acid sequence of delta S antigen (optimized for insect cells)

[0148] Note The above-mentioned array consists of a signal peptide and an extracellular domain.

[0149] SEQ ID NO: 58 The full-length amino acid sequence of the Omicron-BA.1 S antigen

[0150] Note The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0151] SEQ ID NO: 59 Full-length nucleic acid sequence of Omicron-BA.1 S antigen (optimized for CHO cells)

[0152] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0153] SEQ ID NO: 60 Full-length nucleic acid sequence of Omicron-BA.1 S antigen (optimized for insect cells)

[0154] Note The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0155] SEQ ID NO: 61 Extracellular amino acid sequence of Omicron-BA.1 S antigen

[0156] Note The above array consists of a signal peptide - extracellular domain.

[0157] SEQ ID NO: 62 Extracellular nucleic acid sequence of Omicron - BA.1 S antigen (optimized for CHO cells)

[0158] Note The above array consists of a signal peptide - extracellular domain.

[0159] SEQ ID NO: 63 Extracellular nucleic acid sequence of Omicron - BA.1 S antigen (optimized for insect cells)

[0160] Note The above-mentioned array consists of a signal peptide - extracellular domain.

[0161] SEQ ID NO: 64 Full-length amino acid sequence of Omicron-BA.2S antigen

[0162] Note The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0163] SEQ ID NO: 65 Full-length nucleic acid sequence of Omicron-BA.2S antigen (optimized for CHO cells)

[0164] Note The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0165] SEQ ID NO: 66 Full-length nucleic acid sequence of Omicron-BA.2S antigen (optimized for insect cells)

[0166] Note The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0167] SEQ ID NO: 67 Extracellular amino acid sequence of Omicron-BA.2S antigen

[0168] Note The above array consists of a signal peptide - extracellular domain.

[0169] SEQ ID NO: 68 Extracellular nucleic acid sequence of Omicron - BA.2S antigen (optimized for CHO cells)

[0170] Note The above array consists of a signal peptide - extracellular domain.

[0171] SEQ ID NO: 69 Extracellular nucleic acid sequence of Omicron-BA.2S antigen (optimized for insect cells)

[0172] Note The above array consists of a signal peptide - extracellular domain.

[0173] SEQ ID NO: 70 Full-length amino acid sequence of Omicron-BA.4 / 5S antigen

[0174] Note The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0175] SEQ ID NO: 71 Full-length nucleic acid sequence of Omicron-BA.4 / 5S antigen (optimized for CHO cells)

[0176] Note The above array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0177] SEQ ID NO: 72 Full-length nucleic acid sequence of Omicron-BA.4 / 5S antigen (optimized for insect cells)

[0178] Note The above-mentioned array consists of a signal peptide - extracellular domain - transmembrane domain - intracellular domain.

[0179] Sequence number 73 Extracellular amino acid sequence of Omicron-BA.4 5S antigen

[0180] Note The above array consists of a signal peptide - extracellular domain.

[0181] SEQ ID NO: 74 Extracellular nucleic acid sequence of Omicron - BA.4 / 5S antigen (optimized for CHO cells)

[0182] Note The above array consists of a signal peptide - extracellular domain.

[0183] SEQ ID NO: 75 Extracellular nucleic acid sequence of Omicron - BA.4 / 5S antigen (optimized for insect cells)

[0184] Note: The above array consists of a signal peptide - extracellular domain.

[0185] Nucleotide sequence of the S protein of the BQ.1.1 variant with accession number 76

[0186] Amino acid sequence of the S protein of the BQ.1.1 variant with accession number 77 [Effect of the Invention]

[0187] The present invention provides a broad-spectrum protein and a vaccine thereof for resisting infection by SARS-CoV-2 or its variants. Specifically, the present invention provides a recombinant protein vaccine for resisting infection by SARS-CoV-2 or its variants, and an adenovirus vector vaccine using a human type 5 replication-deficient adenovirus as a vector. The nucleotide sequences encoding these two vaccines are selected from at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76. On the other hand, in the present invention, these two vaccines are combined. Here, the recombinant protein vaccine is mainly prepared based on the optimized sequence consisting of the RBD sequence of SARS-CoV-2, the RBD and HR of SARS-CoV-2 variants, and the full-length S protein sequence of optimized expression in CHO cells. The adenovirus vaccine is constructed by optimizing the full-length sequence of the S antigen of SARS-CoV-2 or its variants in order to prepare a pharmaceutical composition or a combined drug for preventing and / or treating infection by SARS-CoV-2 or its variants, that is, a bivalent vaccine. The present invention can induce antibodies and other immune responses in the body by intramuscular injection, nasal spray administration or combined administration of the two vaccines, and can block the binding between the S protein of SARS-CoV-2 and the ACE2 receptor of host cells, thereby helping the host to resist coronavirus infection, and having a better preventive and therapeutic effect especially against mutant viruses.

Brief Description of Drawings

[0188]

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Figure 27

Modes for Carrying Out the Invention

[0189] Abbreviations of Terms

[0190] Monophosphoryl lipid A (MPL), squalene water - in - oil emulsion (MF59), recombinant cholera toxin (rCTB), astragalus polysaccharide (APS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), (2,3 - dioleyloxypropyl) trimethylammonium chloride (DOTAP), N - [1 - (2,3 - dioleyloxypropyl)] - N,N,N - trimethylammonium chloride (DOTMA), cationic cholesterol (DC - Chol), dimethyl - 2,3 - dioleyloxypropyl - 2 - (2 - arginine formylamino) ethylammonium trimethyldodecylammonium bromide (DOSPA), dodecyltrimethylammonium bromide (DTAB), tetradecyltrimethylammonium bromide (TTAB), hexadecyltrimethylammonium bromide (CTAB), dimethyldioctadecylammonium bromide (DDAB), or CpG ODN (nucleotide sequence containing unmethylated cytosine - phosphate - guanine as a core sequence, synthetic CpG).

[0191] In the present invention, the recombinant protein vaccine and the adenovirus vaccine for resisting SARS-CoV-2 infection are mainly designed based on the amino acid sequences of heptapeptide repeat region 1 (HR1) and heptapeptide repeat region 2 (HR2) at positions 320-545 of the S protein of SARS-CoV-2 or its variants. On the other hand, a new sequence is constructed based on the RBD sequences, HR1 and HR2 sequences of different SARS-CoV-2 variants, and finally, a drug resistant to SARS-CoV-2 infection is constructed based on the full-length nucleotides of the S protein of SARS-CoV-2 or its mutant virus, extracellular domain, transmembrane domain or intracellular domain. In order to improve the stability and expression level of the S protein, the amino acid sequence of the S protein of SARS-CoV-2 or its variant is further artificially mutated when designing a drug for resisting SARS-CoV-2 infection.

[0192] The present invention further provides a recombinant protein vaccine and an adenovirus vaccine for resisting SARS-CoV-2 or its variant infection, or a pharmaceutical composition or a combined drug of these two vaccines. This mainly targets the S protein of the SARS-CoV-2 virus or its variant. In particular, by blocking the ACE2 receptor binding domain of the S protein to induce an immune response such as antibodies in the body, the binding of the S protein of SARS-CoV-2 or its variant to the ACE2 receptor of host cells is blocked, helping the host resist coronavirus infection, and having a better preventive and therapeutic effect especially against mutant viruses such as alpha, beta, gamma, delta, omicron and omicron variants.

[0193] Furthermore, the present invention demonstrates that a pharmaceutical composition containing a recombinant protein vaccine and an inactivated influenza virus vaccine as active ingredients for resisting infection by SARS-CoV-2 or its variants, or a pharmaceutical composition containing an adenovirus vaccine and an influenza recombinant protein vaccine as active ingredients for resisting infection by SARS-CoV-2 or its variants has a better preventive and therapeutic effect against respiratory viral infections or diseases derived therefrom.

[0194] Hereinafter, the solution means of the present invention will be described with reference to embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. When a specific technique or condition is not specified in the embodiment, it shall be implemented according to the techniques or conditions described in the literature of the technical field or according to the product specifications. The reagents or devices used are conventional products that can be purchased on the market when the manufacturer is not indicated.

[0195] Part I. Immunoprotection Induced by Individual Adenoviruses

[0196] Embodiment 1.1 Construction and Preparation of Recombinant Adenovirus Vaccine

[0197] 1. Optimization and Synthesis of S Protein Gene (Wild-Type and Mutant Strains) Sequences Using the Wuhan virus strain (GenBank accession number YP_009724390.1) as a template, the gene sequence of wild-type spike (S) is obtained, and the signal peptide of the S protein is retained. Based on this, in order to improve the expression level of the protein, the codons are optimized and then the optimized sequence is synthesized.

[0198] Based on the spike (S) sequence of the Wuhan virus strain (GenBank accession number YP_009724390.1), according to the mutation sites of SARS-CoV-2 beta, delta, omicron and other mutant strains provided on the website of https: / / covariant.org / , the nucleotide sequences of the S proteins of each mutant strain are obtained, and the signal peptides of each S protein mutant are retained. The gene sequence of the beta-delta chimeric S protein is based on the mutation sites of the S protein in the beta mutant strain and the mutation sites of the S protein RBD in the delta mutant strain. Based on this, in order to ensure the stability of the protein and improve its expression level, the protein structure is mutated. First, to improve the stability and expression level of the S protein, lysine (L) at site 986 is replaced with proline (P), and valine (V) at site 987 is replaced with proline (P). Next, the codons are optimized. Finally, according to the mutated and optimized sequences, the S protein genes of each mutant strain are synthesized.

[0199] The S protein genomes of SARS-CoV-2 and its mutant strains synthesized according to the above method include WT (wild type), beta, delta, beta / delta chimeric type, omicron BA.1 and omicron BA.4 / 5, and their nucleotide sequences are SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 38.

[0200] 2. Packaging of Recombinant Adenovirus SARS-CoV-2 Vaccine In the gene synthesis process, the S gene is cloned into the pDC316 vector by a recombinant cloning strategy to obtain a shuttle plasmid (pDC316-S). The above pDC316-S containing the S genes of the wild strain and the mutant strain are co-transfected into HEK293 cells with the backbone plasmid pBHGlox_E1,3Cre of the AdMax adenovirus system, and the recombinant adenovirus is packaged by the following steps.

[0201] 1) HEK293A cells were seeded at 8×10 5Inoculate cells / wells into a 6-well plate and culture overnight at 37°C in a cell incubator with 5% CO2 using high-glucose DMEM + 10% FBS medium.

[0202] 2) The next day, replace the above solution with high-glucose DMEM + 2% FBS and co-transfect HEK293A cells with the backbone plasmid (pBHGlox_E1,3Cre) and the shuttle plasmid using Lipofectamine 3000. Specifically, collect 4 μg of the backbone plasmid and 2 μg of the shuttle plasmid into each transfection well, dilute with 125 μL of Opti-MEM medium, add 12 μL of diluted P3000 reagent to the diluted plasmid, collect another 1.5 ml EP tube, dilute 7.5 μL of Lipofectamine 3000 with 125 μL of Opti-MEM medium, mix the diluted plasmid and the diluted Lipofectamine 3000 in a 1:1 ratio, incubate at room temperature for 10 - 15 minutes, then add to the cells and continue culturing the cells. After overgrowth of the cells, transfer them to a 25 cm 2 cell culture bottle, observe the signs of virus generation in the cells daily, and after overgrowth of the cells at the bottom of the bottle, transfer them to a 75 cm 2 cell culture bottle until the cells show obvious plaques, and collect the virus when most of the cells are affected and detached from the bottom.

[0203] 3) Collect the cell culture containing the virus, centrifuge at 1200 rpm for 3 minutes, aspirate the supernatant containing the virus, resuspend the cell pellet with 1 / 10 of the culture volume together with the supernatant containing the virus, repeat freeze-thawing 3 times in a -80°C refrigerator and a 37°C water bath, centrifuge at 3000 rpm for 20 minutes, collect the supernatant containing the virus, and mix with the supernatant containing the above virus. The resulting product is the virus strain of the adenovirus vaccine.

[0204] 4) Collect 50 μL of the vaccine candidate virus strain solution, mix it with 2 μL of proteinase K, dissociate it at 50 °C for 30 minutes to release the viral genome, and use this as the PCR-amplified S gene sequence. After collecting the PCR product electrophoresis gel, perform sequencing and identification. The conditions for PCR amplification are as follows.

[0205] Denaturation at 95 °C for 10 minutes, denaturation at 95 °C for 10 seconds, annealing at 64 °C for 30 seconds, extension at 72 °C for 2 minutes, extension at 72 °C for 5 minutes, number of cycles 40. The primers for PCR amplification are as follows. pDC516-F1 ACACGTCAATGGGAAGTGAAA (SEQ ID NO: 78) pDC516-R1 GCTAGACGATCCAGACATGAT (SEQ ID NO: 79)

[0206] 3. Amplification of recombinant adenovirus SARS-COV-2 vaccine The accurately identified recombinant adenovirus vaccine strain is amplified step by step in 293 cells by the following specific steps. Add 80% - 90% of 293 overgrown cells according to MOI = 10. After most of the cell viruses become round, collect the virus culture, and prepare the master virus seed stock and working virus seed stock by the above repeated freeze-thaw method. Amplify the recombinant adenovirus vaccine in a cell factory or bioreactor, and collect the virus culture after most of the cells are affected. Amplify the cells and virus in the bioreactor by the following steps. Add 3 - 5 g / L of Cytodex1 microvector to the bioreactor and sterilize it. Add the cell culture medium to the bioreactor. When the operating conditions are maintained at 37 °C, pH 7.0, DO 50% and 50 rpm, dissociate and collect the HEK293 cells amplified in the cell factory, inoculate them into the bioreactor, where the inoculated cells are 1.0 - 5.0×10 5Having a density of cells / ml, replenishing the cell culture medium to 5 L, when the culture conditions of the cells in the bioreactor are 37°C, 30 - 50 rpm, pH 7.15 - 7.25, and DO 30% - 50%, samples are taken daily to test the glucose concentration, cell density, and cell morphology on the microvector. When the cell density in the bioreactor reaches 1.0 - 5.0×10 6 cells / ml, the recombinant adenovirus vaccine strain is inoculated into the bioreactor at an MOI of 5 - 30. After inoculation, samples are taken daily to test the virus titer in the glucose concentration, culture supernatant, and cell pellet, observe the morphology of the cells on the microvector, and terminate the culture when most of the cells have detached from the microvector. Then, the virus lysate is added to the bioreactor at a final concentration of 0.05% - 1% Tween 20, lysed at 37°C for 2 - 4 hours, and then the virus solution is collected.

[0207] 4. Purification of Recombinant Adenovirus SARS-COV-2 Vaccine The collected virus is purified by cesium chloride ultracentrifugation or ion exchange column chromatography according to the following specific process steps.

[0208] (1) Purification of Adenovirus Vaccine by Cesium Chloride Ultracentrifugation The collected virus culture was centrifuged at 1200 g for 10 minutes, the culture supernatant containing the virus was aspirated, the cell pellet was resuspended at 1 / 10 of the culture volume together with the culture supernatant containing the virus, and freeze-thaw cycles were repeated three times in a -80°C refrigerator and a 37°C water bath. After centrifugation at 3000 rpm for 10 - 20 minutes, the supernatant was aspirated. The culture supernatant containing the virus was concentrated 10 times with a 100K - 300K ultrafiltration membrane. A 1.4 g / ml cesium chloride solution (53 g of cesium chloride + 87 ml of 10 mM Tris-HCl, pH 7.9) and a 1.2 g / ml cesium chloride solution (26.8 g of cesium chloride + 92 ml of 10 mM Tris-HCl, pH 7.9) were prepared. 8 ml of the 1.4 g / ml cesium chloride solution and 6 ml of the 1.2 g / ml cesium chloride solution were slowly added sequentially to the centrifuge tube. Finally, 20 ml of the supernatant containing the virus was added to the top of the discontinuous gradient. After equilibration, centrifugation was performed at 100,000×g for 90 minutes at 4°C. After centrifugation, the blue virus band was aspirated with a syringe, cesium chloride was removed by dialysis, and it was stored at -80°C.

[0209] (2) Purification of adenovirus by ion exchange chromatography Collect the virus culture, dissolve it at 37°C for 2 - 4 hours using 0.05% - 1% Tween 20, filter the dissolved culture, clarify it with 1.2μm and 0.45μm bag filters, concentrate the sample 10 times with a tangential flow membrane with a molecular weight of 100kD - 300kD, then diafiltrate with a buffer (10 times the volume of the concentrated sample) (50mM Tris - HCl, 2mM MgCl2, 0mM - 500mM NaCl, pH 8.0), collect the filtered sample, mix it with nuclease, the mixture has a final concentration of 10U / ml - 50U / ml, dissociate at 37°C for 1 - 3 hours, subject the sample to anion - exchange chromatography using Q Sepharose XL, Source 30Q or Source 15Q and other fillers by the following specific steps: the step of equilibrating the buffer at a flow rate of 20ml / min to equilibrate 5 column volumes, after equilibration, the step of loading the sample at a flow rate of 10ml / min, after loading the sample, the step of equilibrating the buffer to the conductivity level, under the elution conditions of 100% low - salt buffer to 100% high - salt buffer, a linear gradient, an elution volume of 10V of the column and a flow rate of 10ml / min, elute the sample through a linear gradient, collect each elution peak, after elution, regenerate 5 - 10 column volumes of the column at a flow rate of 20ml / min using 2M NaCl buffer, collect the virus peak, then perform buffer replacement of the eluted virus sample through dialysis or tangential flow filtration (buffer: 10mM Tris, 10mM Na - PO4, 150mM NaCl, 2mM MgCl2, 2% sucrose, 0.15% glycerol and 0.02% Tween 80, pH 7.6). Fill the purified adenovirus directly and store it in the dark at - 20°C.

[0210] The adenovirus or vaccine prepared according to Embodiment 1 is Ad5 respectively WT (SEQ ID NO: 24), Ad5 ベータ (SEQ ID NO: 26), Ad5 デルタ (SEQ ID NO: 28), Ad5 BA.1 (SEQ ID NO: 32), Ad5 BA.4 / 5 (SEQ ID NO: 38), Ad5 ベータ / デルタ (SEQ ID NO: 30) recombinant adenovirus.

[0211] The efficacy of the recombinant adenovirus SARS-CoV-2 vaccine is demonstrated by the following experimental examples.

[0212] Experiment 1.1 Identification of Recombinant Adenovirus Vaccine

[0213] The inventors expressed the full-length spike glycoproteins of the SARS-CoV-2 wild-type strain, beta strain, delta strain, omicron BA.1, and omicron BA.4 / 5, respectively, using a human replication-deficient Ad5 adenovirus vector. On the other hand, based on the full-length spike glycoprotein of the beta strain, the inventors designed and constructed the adenovirus Ad5-beta / delta (Figure 1) with two delta strain variants L452R and T478K added to the RBD domain. The expression level of spike in 293T cells infected with the recombinant Ad5 ベータ / デルタ virus was detected by Western blot at 48 hours post-infection. As shown in Figure 1, 293T cells infected with the adenovirus Ad5 ベータ / デルタ were able to induce high-level spike expression, while such cells infected with the empty adenovirus control Ad5 empty were unable to do so, indicating that the inventors' recombinant adenovirus protein vaccine was successfully prepared.

[0214] Experiment 1.2 Animal Preparation, Mouse Immunization, and Sample Collection

[0215] Female BALB / c mice aged 6 - 8 weeks were purchased from Charles River and housed in a pathogen-free environment in the State Key Laboratory of Biotherapy at Sichuan University. The mice were divided into the following 7 groups: (1) PBS group, (2) Ad5 空 , (3) Ad5 WT , (4) Ad5 ベータ , (5) Ad5 デルタ , (6) Ad5 BA.1 , and (7) Ad5 ベータ / デルタ groups. The BALB / c mice were immunized at weeks 0, 4, and 8 with 5×10 9Intranasal immunization was performed with the dose of viral particle VP / fragment. Blood samples were collected through the orbital vein at the 3rd and 7th weeks, and the mice were sacrificed at the 11th week. Blood, bronchoalveolar lavage fluid, lungs, and mediastinal lymph node tissues were collected. The tissues were prepared into single-cell suspensions and subjected to flow cytometry. Blood was centrifuged at 4°C and 6000 rpm for 10 minutes. Serum samples were stored at -20°C before use for the subsequent detection of binding and neutralizing antibodies in the serum.

[0216] Experiment 1.3 Detection of anti-RBD specific antibodies

[0217] RBD-specific antibodies in the serum were detected by ELISA. The inventors coated a 96-well NUNC-MaxiSorp plate (Thermo Fisher Scientific, USA) with recombinant RBD protein (0.1 μg / well) at 4°C for 12 hours. Such plates were washed three times with 1×PBST (1×PBS + 0.1% Tween-20) and blocked with 1% bovine serum albumin (BSA) at 37°C for 1 hour. The culture plates and serum samples with a 2-fold dilution gradient were incubated at room temperature for 1 hour and then washed three times with 1×PBST. A 1:10000 diluted horseradish peroxidase (HRP)-goat anti-mouse IgG antibody was added to the culture plates, incubated at room temperature for 1 hour, and washed five times. 3,3’,5,5’-Tetramethylbenzidine (TMB) was added to the culture plates and incubated in the dark for 10 minutes. The reaction was stopped with 1 M H2SO4 (100 μl / well), and the absorbance was measured at 450 nm.

[0218] As shown in Figure 2, recombinant adenovirus vaccines Ad5 WT , Ad5 ベータ , Ad5 デルタ , Ad5 BA.1 and Ad5 ベータ / デルタ all induced high levels of anti-RBD specific binding antibodies in the blood at 3, 7, and 11 weeks after vaccination. Therefore, the levels of neutralizing antibodies in the serum were further detected to evaluate the broad-spectrum neutralizing immune responses induced by various recombinant adenovirus vaccines.

[0219] Experiment 1.4 Pseudovirus Neutralization Assay

[0220] In addition to the BA.3 and BA.4 / 5 pseudoviruses purchased from Vazem Biotech (China), the inventors purchased other SARS-CoV-2 variant pseudoviruses of SARS-CoV-2 pseudovirus (GFP-luciferase) from Genomeditech (Shanghai). Serum samples collected after sacrificing mice at the 11th week were inactivated at 60 °C for 30 minutes and then diluted 3-fold with DMEM double culture medium containing serum and antibiotics. The diluted serum was incubated with luciferase pseudoviruses (wild type, B.1.1.7, B.1.351, P.1, B.1.617, C.37, BA.1, BA.2, BA.2.12.1, BA.3, and BA.4 / 5) at 37 °C for 1 hour. 1.2×10 4 Individual 293T / ACE2 cells were added to each well to express the reporter gene. After 48 hours, the supernatant of the infected cells was removed, and 100 μl of lysis reagent containing luciferase substrate was added to each well. Finally, a multimode microplate reader (PerkinElmer, USA) was used for detection.

[0221] As shown in Figure 3, Ad5 WT , Ad5 ベータ and Ad5 デルタ induced high levels of neutralizing antibodies against their own viruses, but such vaccines induced low levels of neutralizing antibodies against Omicron subtypes due to the strong immune evasion ability of the Omicron strain. The Ad5-Omicron vaccine was able to induce high levels of neutralizing antibodies against Omicron subtypes, but was unable to induce high levels of neutralizing antibodies against WT, Alpha, Beta, Delta, etc. Compared with other adenovirus vaccines, Ad5 ベータ / デルタ was able to induce high levels of broad-spectrum neutralizing antibodies against a wide range of viruses including WT, Alpha, Beta, Delta, and Omicron BA.1, BA.2, BA.2.12.1, BA.3, and BA.4 / 5.

[0222] Experiment 1.5 Detection of anti-RBD specific IgG and IgA antibodies in bronchoalveolar lavage fluid

[0223] RBD-specific antibodies IgG and IgA in bronchoalveolar lavage fluid were detected by ELISA. The inventors coated a 96-well NUNC-MaxiSorp plate (Thermo Fisher Scientific, USA) with recombinant RBD protein (0.1 μg / well) at 4 °C for 12 hours. Such plates were washed three times with 1× PBST (1× PBS + 0.1% Tween-20), and blocked with 1% bovine serum albumin (BSA) at 37 °C for 1 hour. The culture plates and two-fold dilution gradient bronchoalveolar lavage fluid samples were incubated at room temperature for 1 hour and then washed three times with 1× PBST. Horseradish peroxidase (HRP)-goat anti-mouse IgG antibody or IgA (diluted 1:5000) antibody diluted 1:10000 was added to the culture plates, incubated at room temperature for 1 hour, and washed five times. 3,3’,5,5’-Tetramethylbenzidine (TMB) was added to the culture plates and incubated in the dark for 10 minutes. The reaction was stopped with 1 M H2SO4 (100 μl / well), and the absorbance was measured at 450 nm.

[0224] As shown in Figure 4, the recombinant adenovirus vaccine Ad5 ベータ / デルタ induced high levels of anti-RBD specific conjugate antibodies IgG and IgA in bronchoalveolar lavage fluid at 11 weeks after vaccination, indicating that the inventors' recombinant adenovirus vaccine can induce strong antibody protection in the mucosal part of the airway.

[0225] Experiment 1.6 Detection of tissue-resident T cells in bronchoalveolar lavage fluid

[0226] Tissue-resident T cells (T RM) numbers were detected using flow cytometry. 1 ml of collected bronchoalveolar lavage fluid was centrifuged (400×g, 5 minutes), the supernatant was removed to obtain a cell pellet. After resuspending the cells in 100 μl of PBS buffer, PerCP / Cyanine5.5 conjugated anti-mouse CD3 (BioLegend, 100718), Brilliant Violet421 conjugated anti-mouse CD4 (BioLegend, 100412), Brilliant Violet510 conjugated anti-mouse CD8, PE conjugated anti-mouse CD44, FITC conjugated anti-mouse CD69, and APC conjugated anti-mouse CD103 were added and incubated at 4°C for 30 minutes. The cells were washed once with PBS and then reselected and detected by flow cytometry.

[0227] As shown in Figure 5, Ad5 ベータ / デルタ When such mice immunized intranasally with it were compared with PBS-treated mice, the adenovirus vaccine could mobilize a large number of tissue-resident CD4 + and CD8 + T cells, resulting in cell-mediated immunity protection.

[0228] Experiment 1.7 Detection of antigen-specific T cells in lung tissue

[0229] Antigen-specific T cells in lung tissue were detected by flow cytometry. The collected lung tissue was cut into 1 mm 3After cutting to the size of [[ID=]], the prepared collagenase digestion solution was added and incubated at 37 °C for 1 hour. The tissue digestion solution was filtered through a 70 μM sieve to obtain a single-cell suspension. The single cells of the lung tissue were stimulated and cultured in a double 1640 medium containing a spike peptide library for 12 hours and then subjected to intracellular cytokine staining (ICS). To the 1640 medium, 10% fetal bovine serum, 100 μg / ml streptomycin, 100 U / ml penicillin, 1 mM pyruvic acid (all purchased from Gibco), 50 μM β-mercaptoethanol, and 20 U / ml IL-2 (all purchased from Sigma-Aldrich) were added. To block the secretion of intracellular cytokines, brefeldin A (BFA, BD Biosciences) was incubated for 6 hours before staining. The cells were washed with 1× PBS cooling solution and stained with PerCP / Cyanine5.5 conjugate anti-mouse CD3 (BioLegend, 100718), APC conjugate anti-mouse CD4 (BioLegend, 100412), FITC conjugate anti-mouse CD8, and PE conjugate anti-mouse CD44 antibodies at 4 °C for 30 minutes. Then, the cells were fixed and permeabilized and left at room temperature for 2 hours, after which they could be stained with PE-Cy7 conjugate anti-mouse IFN-γ and Brilliant Violet510 conjugate anti-mouse TNF-α (all flow antibodies manufactured by BioLegend). After the cells were washed with 1× PBS, they were detected by flow cytometry.

[0230] T cell responses are major determinants of clinical outcomes and play an important role in the prevention of SARS-CoV-2. As shown in Figure 6, the cells immunized intranasally with the Ad5 ベータ / デルタ vaccine induce strong antigen-specific cellular immunity including T cells that secrete IFN-γ and TNF-α.

[0231] Experiment 1.8 Detection of germinal center reactions

[0232] After preparing a single-cell suspension from longitudinal lymphoid cells, the inventors used PerCP / Cyanine5.5-conjugated anti-mouse CD3, PE-Cy7-conjugated anti-mouse CD45R / B220, Brilliant Violet 421-conjugated anti-mouse GL-7, APC-conjugated anti-mouse CD95, Brilliant Violet 421-conjugated anti-mouse CD19, APC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CXCR5, and Brilliant Violet 510-conjugated PD-1 antibodies to stain Tfh cells and GC B cells (flow cytometry antibodies were from BioLegend). The cells were stained in the dark at 4°C. After 30 minutes, the cells were washed with 1× PBS and then detected by flow cytometry.

[0233] Persistent prior responses provide rapid and effective protective immunity in the case of reinfection. The increase in Tfh and GCB cells indicates that the vaccine can provide protective immunity that persists in the body for a long time. As shown in Figure 7, Ad5 ベータ / デルタ Cells immunized intranasally with the vaccine can induce a significant increase in Tfh and GCB cells in the mediastinal lymph nodes, demonstrating that our recombinant adenovirus vaccine can improve long-lasting immune protection.

[0234] Part II Immune protection induced by individual proteins

[0235] Embodiment 2.1 Preparation of recombinant protein using an insect baculovirus expression system (taking S-RBD-HR BA.4 / 5 as an example)

[0236] 1. Structural design of S-RBD (Omicron_BA.4 / .5)-HR

[0237] The S protein of SARS-CoV-2 is a membrane-localized protein. To ensure the simulation of its secretion process, when constructing the SARS-CoV-2 S-RBD (Omicron_BA.4 / .5)-HR protein expression, the inventors added the signal peptide sequence of GP67 to the N-terminus of the protein to assist in the secretory expression of the protein. This signal peptide is spontaneously removed by insect cells during the protein secretion process. Furthermore, to assist in the folding of S-RBD (Omicron_BA.4 / .5)-HR, the inventors added the thioredoxin (Trx) tag of Spodoptera frugiperda, S. frugiperda, behind the GP67 signal peptide, added a 6xhis tag to assist in subsequent purification, and added an EK restriction enzyme cleavage site to remove the Trx and 6xhis tags. Protein expression can remove all non-S-RBD (Omicron_BA,4 / 5)-HR redundant amino acids by the EK restriction enzyme. The designed protein amino acid sequence is shown in SEQ ID NO: 6. The expression construct design pattern is shown in Figure 8.

[0238] 2. Identification of recombinant plasmid construction

[0239] The designed coding fragment is cloned into the pFastBac1 vector plasmid and identified by bacterial PCR. The identification results by bacterial PCR show that the GP67-Trx-His-EK-S-RBD (Omicron_BA.4 / .5)-HR fragment is successfully amplified in all five selected clones as shown in Figure 9.

[0240] 3. Identification of recombinant bacmid

[0241] Select the correct pFastBac1-GP67-Trx-His-EK-S-RBD (Omicron_BA.4 / .5)-HR recombinant clone, extract the recombinant plasmid, transform DH10b competent cells, and identify them by colony PCR. Detect the colony PCR products by 1% agarose gel electrophoresis, and the identification results are shown in Figure 10. The white colonies are recombinant bacmid clones, and the blue colonies are non-recombinant bacmid clones.

[0242] 4. Packaging of Recombinant Baculovirus

[0243] Transfect the recombinant bacmid into sf9 insect cells. After 5 - 6 days, collect the recombinant baculovirus P0 and inoculate it into fresh sf9 insect cells at a ratio of 1:100. After 4 days, collect the recombinant baculovirus P1 and inoculate it into fresh sf9 insect cells at a ratio of 1:100. After 4 days, collect the recombinant baculovirus P2. The flowchart of the packaging and amplification of the recombinant baculovirus is shown in Figure 11.

[0244] 5. Verification of Target Protein Expression

[0245] Baculovirus amplification is accompanied by the expression of the target protein. Before removing the tag, since the target protein contains a His tag, we verify the expression of the recombinant protein by anti-His WB experiment. The verification results show that obvious bands are observed between the 40KD marker band and the 55KD marker band, and their sizes are consistent with the size of the Trx-His-EK-S-RBD (Omicron_BA.4 / .5)-HR protein, indicating that the baculovirus amplification and the expression of the target protein are successful. The detection results are shown in Figure 12.

[0246] Recombinant Protein Vaccines RBD-WT, RBD-HR デルタ 、RBD-HR BA.4 / .5 、and RBD-HR BA.1 / デルタ / ベータThey are constructed by the method for constructing recombinant proteins based on the amino acid sequence of the S protein RBD of SARS-CoV-2 or the amino acid sequence of the S protein RBD-HR of SARS-CoV-2 variants, namely, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 18, respectively, and all of them are used for animal immunization and subsequent research.

[0247] Embodiment 2.2 Preparation of recombinant proteins using the CHO cell expression system (taking S-BA.4 / 5 as an example)

[0248] The recombinant protein vaccine produced by CHO cells mainly targets the full-length S protein. These fragments are genetically synthesized according to codon selection. Polyhistidine is used as a purification tag (6His), and the complete nucleotide sequence is shown in SEQ ID NO: 71. Subsequently, the high-expression vector pTT5 is constructed to express the precursor protein with the amino acid sequence shown in SEQ ID NO: 70. After vector construction, CHO cells are transfected, and as shown in Figure 13, the collected solution is taken for SDS-PAGE and Western blot tests. This proves that the target protein was successfully expressed. The recombinant proteins and vaccines S-WT, S-Beta, S-Delta, S-BA.1, S-BA.2, and S-BA.4 / 5 are constructed by the method for constructing recombinant proteins based on the full-length amino acid sequence of the S protein of SARS-CoV-2, namely, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 58, SEQ ID NO: 64, and SEQ ID NO: 70, respectively, and all of them are used for animal immunization and subsequent research.

[0249] The effect of the recombinant protein vaccine is proven by the following experimental examples.

[0250] Experiment 2.1 Preparation of animals, mouse immunization, and sample collection

[0251] Female BALB / c mice aged 6 - 8 weeks were purchased from Charles River and housed in a pathogen-free environment in the State Key Laboratory of Biotherapy at Sichuan University.

[0252] BALB / c mice were immunized intranasally on days 0, 14, and 28. Blood samples were collected through the orbital vein on day 42, and the mice were sacrificed at 11 weeks. Blood, bronchoalveolar lavage fluid, and lung and mediastinal lymph node tissues were collected. The tissues were prepared into single-cell suspensions and subjected to flow cytometry. Blood was centrifuged at 4°C and 6000 rpm for 10 minutes. Serum samples were stored at -20°C before use for the subsequent detection of bound and neutralizing antibodies in the serum.

[0253] Experiment 2.2 Pseudovirus neutralization assay after immunization with single-protein vaccine

[0254] The inventors used pseudovirus to detect neutralizing antibodies in the sera of immunized mice. Here, BA.3 and BA.4 / 5 pseudoviruses were purchased from Vazem (China), and wild-type B.1.1.7, B.1.351, P.1, B.1.617, C.37, BA.1, BA.2, BA.2.12.1, and other pseudoviruses were purchased from Genomeditech (Shanghai). Serum samples collected after sacrificing the mice at 11 weeks were inactivated at 60°C for 30 minutes and then diluted 3-fold in DMEM double-culture medium containing serum and antibiotics. The diluted sera were incubated with luciferase pseudovirus (wild-type, B.1.1.7, B.1.351, P.1, B.1.617, C.37, BA.1, BA.2, BA.2.12.1, BA.3, and BA.4 / 5) at 37°C for 1 hour. 1.2×10 4 individual 293T / ACE2 cells were added to each well to express the reporter gene. After 48 hours, the supernatant of the infected cells was removed, and 100 μl of lysis reagent containing luciferase substrate was added to each well. Finally, a multimode microplate reader (PerkinElmer, USA) was used for detection.

[0255] As shown in Figure 14, wild-type RBD-WT, RBD-HR デルタ and RBD-HR BA.1 / デルタ / ベータThe protein induces higher levels of neutralizing antibodies against WT, Alpha, Beta, Delta, and Omicron viruses, and individual RBD-HR デルタ proteins as well as chimeric RBD-HR containing Beta and Delta variants BA.1 / デルタ / ベータ The protein shows strong immune protection against WT, Alpha, Beta, Delta, Omicron, and other viruses. Individual RBD-WT proteins show strong immune protection only against WT and Delta variants, weak immune protection against Omicron, and individual RBD-HR BA.4 / .5 proteins did not show strong immune protection against WT, Delta, and Omicron. RBD-HR BA.1 / デルタ / ベータ proteins showed strong immune protection against various types of SARS-COV-2.

[0256] Experiment 2.3 Neutralizing antibodies induced by bivalent RBD proteins

[0257] In the above experiment, the inventors found that individual RBD-HRBA.4 / .5 proteins have no obvious immune protection against WT, Delta, Omicron, and other viruses. After immunizing mice with a bivalent mixed vaccine (5 micrograms of RBD-HR デルタ + 5 micrograms of RBD-HR BA.4 / .5 protein, SEQ ID NO: 3 + SEQ ID NO: 6), high-titer neutralizing antibodies were induced, which had significantly stronger immune protection than individual RBD-HRBA.4 / .5 proteins, and as shown in Figure 15, the effectiveness of the bivalent vaccine was demonstrated.

[0258] Experiment 2.4 Pseudovirus neutralization assay against different S proteins

[0259] To verify the immune protection induced by the S proteins (full-length trimers) of different virus strains, the inventors immunized mice with the prepared S proteins on days 0, 14, and 28, where the S proteins of BA.2.12.1 and BA.3 were commercially available S proteins. S-WT, S-Beta, S-Delta, S-BA.1, S-BA.2, and S-BA.4 / 5 were all recombinant S proteins prepared with reference to Embodiment 2.2. In the immunization protocol, 10 micrograms of protein + MF59 adjuvant was provided. Pseudovirus neutralizing antibodies were determined by removing the antiserum on day 42 as shown in Figures 16 and 17.

[0260] As shown in Figure 16, the S proteins of the WT strain, Beta strain, and Delta strain have strong immune protection against the WT strain, Beta strain, and Delta strain, but weak immune protection against the Omicron strain. The S proteins of the Omicron strain (S-BA.1 and S-Ba.2) have significantly stronger immune protection against the Omicron strain than other strains.

[0261] As can be seen from Figure 17, the full-length S proteins of B.1, BA.2, BA.2.12.1, BA.3, and BA.4 / 5 have significantly stronger immune protection against the Omicron strain than the WT, Alpha, Beta, and Delta strains and show a certain specificity.

[0262] Part III Stronger immune protection induced by adenovirus combined with trimeric recombinant protein

[0263] Experiment 3.1 Preparation of a mixed preparation of an adenovirus vaccine and a recombinant protein vaccine

[0264] The inventors expressed the full-length spike glycoprotein of the SARS-CoV-2 BA.4 / 5 strain using a human replication-deficient Ad5 adenovirus vector. Recombinant Ad5 BA.4 / 5The expression level of spike in 293T cells infected with the virus for 48 hours was detected by Western blot. The detailed procedure is shown in Embodiment 1. As shown in FIG. 18, the inventors mixed different doses of adenovirus Ad5 BA.4 / 5 with the RBD-HR delta generated in Embodiment 2.1 to prepare a preparation for intranasal immunization. Adenovirus Ad5 BA.4 / 5 was prepared based on SEQ ID NO: 38. The recombinant protein RBD-HR delta was prepared based on SEQ ID NO: 3.

[0265] Similarly, adenovirus Ad5 WT was prepared based on SEQ ID NO: 24, the S-BA.4 / 5 protein was prepared based on SEQ ID NO: 71, and the RBD-WT protein was prepared based on SEQ ID NO: 1. The inventors mixed adenovirus Ad5 WT with the RBD-WT protein generated in Embodiment 2 to prepare preparations respectively. Adenovirus Ad5 BA.4 / 5 was mixed with the S-BA.4 / 5 protein generated in Embodiment 2.2 to prepare a preparation for intranasal immunization.

[0266] Experiment 3.2 Preparation of animals, immunization of mice and collection of samples

[0267] Female BALB / c mice aged 6 - 8 weeks were purchased from Charles River and housed in a pathogen-free environment in the State Key Laboratory of Biotherapy at Sichuan University. The mice were divided into the following six groups: (1) RBD-HR デルタ (10 μg / section) control group, (2) Ad5 空 (5×10 9 VP / section) + RBD-HR デルタ (10 μg / section), (3) Ad5 BA.4 / 5 (2.5×10 9 VP / section) low-dose group, (4) Ad5 BA.4 / 5 (2.5×10 9 VP / section) low-dose + RBD-HR デルタ (10 μg) group, (5) Ad5 BA.4 / 5 (5×10 9 VP / section) high-dose group, (6) Ad5 BA.4 / 5 (5×109 High dose of VP / tablet + RBD-HR デルタ (10 μg) group.

[0268] Also, Ad5 WT The experimental grouping of the combined agent of + RBD-WT was as follows: (a) PBS (5 tablets), (b) RBD-WT vaccine group (10 μg / tablet, 5 tablets), (c) adenovirus Ad5 WT (5×10 9 VP / tablet), (d) Ad5 WT (5×10 9 VP / tablet) + RBD-WT group (10 μg / tablet) (5 tablets).

[0269] Ad5 BA.4 / 5 The experimental grouping of the combined agent of + S-BA.4 / 5 was as follows: (a) PBS (5 tablets), (b) S-BA.4 / 5 vaccine group (10 μg / tablet, 5 tablets), (c) Ad5 BA.4 / 5 group (5×10 9 VP / tablet, 5 tablets), (d) Ad5 BA.4 / 5 (5×10 9 High dose of VP / tablet) + S-BA.4 / 5 (10 μg / tablet) group (5 tablets).

[0270] BALB / c mice were immunized intranasally at 0, 4, and 8 weeks. Blood samples were collected through the orbital vein at 3 and 7 weeks, and the mice were sacrificed at 11 weeks. Blood, bronchoalveolar lavage fluid, lungs, and mediastinal lymph node tissues were collected. The tissues were prepared into single-cell suspensions and subjected to flow cytometry. Blood was centrifuged at 4°C and 6000 rpm for 10 minutes. Serum samples were stored at -20°C before being used for subsequent detection of binding and neutralizing antibodies in the serum. The immunization protocol is shown in Figure 19.

[0271] Experiment 3.3 Detection of anti-RBD specific antibodies in serum and bronchoalveolar lavage fluid

[0272] RBD-specific antibodies in the serum were detected by ELISA. The inventors coated a 96-well NUNC-MaxiSorp plate (Thermo Fisher Scientific, USA) with recombinant RBD protein (0.1 μg / well) at 4 °C for 12 hours. Such plates were washed three times with 1× PBST (1× PBS + 0.1% Tween-20) and blocked with 1% bovine serum albumin (BSA) at 37 °C for 1 hour. The culture plates and two-fold dilution gradients of serum samples or bronchoalveolar lavage fluid samples were incubated at room temperature for 1 hour and then washed three times with 1× PBST. A 1:10,000 dilution of horseradish peroxidase (HRP)-goat anti-mouse IgG antibody was added to the culture plates, incubated at room temperature for 1 hour, and washed five times. 3,3’,5,5’-Tetramethylbenzidine (TMB) was added to the culture plates and incubated in the dark for 10 minutes. The reaction was stopped with 1 M H2SO4 (100 μl / well), and the absorbance was measured at 450 nm.

[0273] As shown in Figure 20, after immunization, the preparation of recombinant RBD-HR デルタ combined with adenovirus Ad5 BA.4 / 5 induced stronger anti-RBD specific binding antibodies in the serum and bronchoalveolar lavage fluid than the preparation of Ad5 BA.4 / 5 , indicating that the preparation of adenovirus combined with recombinant subunits can provide stronger humoral immune protection. Furthermore, intranasal immunization with RBD-HR デルタ cannot induce strong blood conjugate antibodies, but RBD-HR 空 conjugated with Ad5 デルタ can significantly increase its immunogenicity. This demonstrates that adenovirus can be used as an adjuvant for recombinant subunits to enhance the immunogenicity of protein antigens.

[0274] Experiment 3.4 Pseudovirus neutralization assay

[0275] In addition to the BA.3 and BA.4 / 5 pseudoviruses purchased from Vazem Biotech (China), the inventors purchased other SARS-CoV-2 variant pseudoviruses (GFP-luciferase) of SARS-CoV-2 from Genomeditech (Shanghai). Serum samples collected after sacrificing the mice at the 11th week were inactivated at 60 °C for 30 minutes and then diluted in a three-fold gradient in DMEM dual culture medium containing serum and antibiotics. The diluted serum was incubated with luciferase pseudoviruses (wild type, B.1.617, BA.1, BA.2, BA.2.12.1, BA.3, and BA.4 / 5) at 37 °C for 1 hour. 1.2×10 4 individual 293T / ACE2 cells were added to each well to express the reporter gene. After 48 hours, the supernatant of the infected cells was removed, and 100 μl of lysis reagent containing luciferase substrate was added to each well. Finally, a multimode microplate reader (PerkinElmer, USA) was used for detection.

[0276] As shown in Figure 21, the preparation of adenovirus Ad5 デルタ combined with recombinant RBD-HR BA.4 / 5 induced stronger neutralizing antibodies in serum and bronchoalveolar lavage fluid than the preparation of adenovirus Ad5 BA.4 / 5 , indicating that the preparation of adenovirus combined with recombinant subunits can provide stronger blood and local mucosal neutralizing protection for preventing virus infection. As shown in Figure 22, Ad5 WT +RDB-WT generated serum neutralizing antibodies against viruses such as WT, BA.1, and BA.4 / 5. As shown in Figure 23, Ad5 BA.4 / 5 +S-BA.4 / 5 generated higher levels of serum neutralizing antibodies against WT, BA.1, BA.4 / 5, and other viruses, and the amount of antibodies was significantly higher than that of the serum neutralizing antibodies of Ad5 WT +RBD-WT. Therefore, it was suggested that the adenovirus vaccine of the Omicron S protein combined with the S protein could provide stronger immune protection against the Omicron variant.

[0277] Experiment 3.5 Challenge Test of SARS-CoV-2 Virus-Infected Mice

[0278] Female BALB / c mice aged 6 - 8 weeks were purchased from Charles River and tested in an animal biosafety level 4 (ABSL-4) facility at the High-Level Primate Research Center in Kunming, Yunnan Province, China. Twenty mice were divided into the following groups for the live SARS-CoV-2 antigen administration test. (a) PBS (5 tablets), (b) Adenovirus Ad5 BA.4 / 5 group (5×10 9 VP / tablet, 5 tablets), (c) S-BA.4 / 5 protein vaccine group (10 μg / tablet, 5 tablets), and (d) AD5 BA.4 / 5 (5×10 9 VP / tablet) high-dose + S-BA.4 / 5 (10 μg) group (5 tablets).

[0279] The mice were immunized intranasally at 0, 4, and 8 weeks, and challenged intranasally with SARS-CoV-2 (0.5 ml, 10 6 pfu / ml) 21 days after the final immunization. Lung tissues of the mice were collected 4 days after the virus challenge. As shown in Figure 24, after treatment with the immune serum induced by the Ad5 BA.4 / 5 (5×10 9 VP / tablet) high-dose + S-BA.4 / 5 (10 μg) combined vaccine, the virus amounts in the turbinate, trachea, and lung tissues were not detected, the lung tissue structure was normal, the alveolar structure was intact, and no obvious inflammation was present. In the PBS control group, mild pathological changes including multiple sclerosis regions, mild thickening of the alveolar septum, and alveolar congestion were observed. Also, small inflammatory patches composed of macrophages, neutrophils, and lymphocytes were sometimes seen near small blood vessels. The effects of the adenovirus or protein in the nasal drops were significantly weaker than those of the combined agent of adenovirus and protein.

[0280] Experiment 3.6 Detection of Tissue-Resident T Cells in Bronchoalveolar Lavage Fluid

[0281] Tissue-resident T cells (T RMThe number of () was detected using flow cytometry. 1 ml of the collected bronchoalveolar lavage fluid was centrifuged (400×g, 5 minutes), and the supernatant was removed to obtain a cell pellet. After resuspending the cells in 100 μl of PBS buffer, PerCP / Cyanine5.5-conjugated anti-mouse CD3 (BioLegend, 100718), Brilliant Violet421-conjugated anti-mouse CD4 (BioLegend, 100412), Brilliant Violet510-conjugated anti-mouse CD8, PE-conjugated anti-mouse CD44, FITC-conjugated anti-mouse CD69, and APC-conjugated anti-mouse CD103 were incubated at 4°C for 30 minutes. The cells were washed once with PBS and then reselected and detected by flow cytometry.

[0282] As shown in Figure 25, the recombinant RBD-HR デルタ in combination with high-dose adenovirus Ad5 BA.4 / 5 preparations mobilized more lung-resident T BA.4 / 5 cells than preparations of adenovirus Ad5 RM Furthermore, intranasal immunization with RBD-HR デルタ was unable to induce sufficient lung tissue T RM cells. However, intranasal immunization with RBD-HR 空 in combination with Ad5 デルタ significantly improved its immunogenicity and was able to induce more lung tissue T RM cells, indicating that preparations of adenovirus combined with recombinant subunits can generate a stronger local mucosal cellular immune response and prevent viral infection.

[0283] Experiment 3.7 Detection of antigen-specific T cells in lung tissue

[0284] Antigen-specific T cells in lung tissue were detected by flow cytometry. The collected lung tissue was cut into 1 mm 3After cutting to the size, the prepared collagenase digestive solution was added and incubated at 37 °C for 1 hour. The tissue digestive solution was filtered through a 70 μM sieve to obtain a single-cell suspension. The single cells of lung tissue were stimulated and cultured in a double 1640 medium containing a spike peptide library for 12 hours and subjected to intracellular cytokine staining (ICS). To the 1640 medium, 10% fetal bovine serum, 100 μg / ml streptomycin, 100 U / ml penicillin, 1 mM pyruvic acid (all purchased from Gibco), 50 μM β-mercaptoethanol and 20 U / ml IL-2 (all purchased from Sigma-Aldrich) were added. To block the secretion of intracellular cytokines, brefeldin A (BFA, BD Biosciences) was incubated for 6 hours before staining. The cells were washed with 1× PBS cooling solution and stained with PerCP / Cyanine5.5 conjugated anti-mouse CD3 (BioLegend, 100718), APC conjugated anti-mouse CD4 (BioLegend, 100412), FITC conjugated anti-mouse CD8, and PE conjugated anti-mouse CD44 antibodies at 4 °C for 30 minutes. Then, the cells were fixed and permeabilized and left at room temperature for 2 hours, and then could be stained with PE-Cy7 conjugated anti-mouse IFN-γ and Brilliant Violet510 conjugated anti-mouse TNF-α (all flow antibodies manufactured by BioLegend). After the cells were washed with 1× PBS, they were detected by flow cytometry.

[0285] As shown in Figure 26, the recombinant RBD-HR デルタ in combination with the high-dose adenovirus Ad5 BA.4 / 5 preparation mobilized more antigen-specific T cells than the adenovirus Ad5 BA.4 / 5 preparation. Furthermore, intranasal immunization with RBD-HR デルタ was unable to induce sufficient antigen-specific T cells. However, RBD-HR 空 in combination with Ad5 デルタIntranasal immunization by [specific agent] significantly improves its immunogenicity, can induce more antigen-specific T cells, and indicates that a preparation of adenovirus combined with a recombinant subunit can produce a stronger local mucosal cell immune response and prevent viral infection.

[0286] Experiment 3.8 Detection of germinal center reaction

[0287] After preparing a single-cell suspension from mediastinal lymphocytes, the inventors stained Tfh cells and GC B cells using PerCP / Cyanine5.5-conjugated anti-mouse CD3, PE-Cy7-conjugated anti-mouse CD45R / B220, Brilliant Violet 421-conjugated anti-mouse GL-7, APC-conjugated anti-mouse CD95, Brilliant Violet 421-conjugated anti-mouse CD19, APC-conjugated anti-mouse CD4, PE-conjugated anti-mouse CXCR5, and Brilliant Violet 510-conjugated PD-1 antibodies (flow cytometry antibodies are from BioLegend). The cells were stained in the dark at 4°C. After 30 minutes, the cells were washed with 1× PBS and then detected by flow cytometry.

[0288] Persistent prior responses provide rapid and effective protective immunity in the event of reinfection. The increase in Tfh and GCB cells indicates that the vaccine can provide protective immunity that persists in the body for a long time. As shown in Figure 27, a high-dose adenovirus Ad5 BA.4 / 5 preparation combined with recombinant RBD-HR delta can significantly increase Tfh cells in the mediastinal lymph nodes, demonstrating that the adenovirus vaccine-binding protein vaccine can improve long-term persistent immune protection.

Claims

1. A pharmaceutical composition for preventing and / or treating SARS-CoV-2 or a variant thereof, which is a compound preparation containing an active ingredient of a recombinant protein vaccine and an adenovirus vaccine for resisting infection by SARS-CoV-2 or a variant thereof. The pharmaceutical composition is characterized by this.

2. A combined agent for preventing and / or treating SARS-CoV-2 or a variant thereof, which contains a recombinant protein vaccine and an adenovirus vaccine for resisting infection by SARS-CoV-2 or a variant thereof, and the recombinant protein vaccine and the adenovirus vaccine are administered separately or simultaneously. The combined agent is characterized by this.

3. The pharmaceutical composition or the combined agent is an intramuscular injection, a nasal drop, a spray, a nasal spray or an inhalant. Preferably, the pharmaceutical composition or the combined agent is a nasal spray. The pharmaceutical composition according to Claim 1 or the combined agent according to Claim 2 is characterized by this.

4. The recombinant protein vaccine and / or the adenovirus vaccine contains a protein and / or a protein precursor for resisting infection by SARS-CoV-2 or a variant thereof. The pharmaceutical composition according to Claim 1 or the combined agent according to Claim 2 is characterized by this.

5. The protein and / or the protein precursor contains a full-length S protein, or a protein formed by at least one RBD sequence and / or at least one HR sequence in the S protein of SARS-CoV-2 or a variant thereof. Preferably, the RBD sequence is shown in SEQ ID NO: 1, or the RBD sequence has homology with SEQ ID NO: 1 and the same or similar biological activity, and is a variant obtained by substitution and / or deletion and / or insertion of at least one amino acid in the sequence of SEQ ID NO:

1. Preferably, the RBD sequence is a variant obtained by substitution and / or deletion and / or insertion of 1 to 400 amino acids in the sequence of SEQ ID NO:

1. Preferably, the RBD sequence is a variant obtained by substitution and / or deletion and / or insertion of 5 to 30 amino acids in the sequence of SEQ ID NO:

1. The pharmaceutical composition or combined agent according to Claim 4 is characterized by this.

6. The pharmaceutical composition or combination drug according to claim 5, characterized in that the protein formed by the RBD sequence and the HR sequence in the S protein can spontaneously form a trimer.

7. The pharmaceutical composition or combination drug according to claim 5, characterized in that the homologous amino acid sequence is selected from at least one of the RBD sequences of alpha, beta, gamma, delta and omicron.

8. The protein precursor is one in which a signal peptide and / or a protein tag is bound to the protein to resist the infection of SARS-CoV-2 or its variant. Preferably, the signal peptide contains the signal peptide of the S protein or its variant and / or the human tPA signal peptide further provided outside the front part of the self-provided signal peptide. Preferably, the protein tag is selected from at least one of a histidine tag, a thioredoxin tag, a glutathione transferase tag, a ubiquitin-like modified protein tag, a maltose binding protein tag, a c-Myc protein tag and an Avi tag protein tag. More preferably, the protein tag is a Trx tag and / or a 6His tag. The pharmaceutical composition or combination drug according to claim 5.

9. The protein for resisting the infection of SARS-CoV-2 or its variant is further bound to a protease recognition region for removing the protein tag. Preferably, the protease is selected from at least one of enterokinase, TEV protease, thrombin, factor Xa of coagulation, carboxypeptidase A and rhino virus 3c protease. The pharmaceutical composition or combination drug according to claim 8.

10. The amino acid sequence of the protein and / or the protein precursor is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70 and SEQ ID NO:

73. The pharmaceutical composition or combination drug according to claim 5.

11. The nucleotide sequence for encoding the amino acid sequence is shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74 or SEQ ID NO:

75. The pharmaceutical composition or combined agent according to claim 10 is characterized by this.

12. The recombinant protein vaccine and / or the adenovirus vaccine according to claim 1 or the combined agent according to claim 2 is characterized by containing a nucleic acid for resisting the infection of SARS-CoV-2 or its variant.

13. The nucleotide sequence of the nucleic acid is shown in SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75 or SEQ ID NO:

76. The pharmaceutical composition or combined agent according to claim 12 is characterized by this.

14. The nucleotide sequence is obtained by codon or cell optimization based on the encoded amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73 or SEQ ID NO:

77. Further, the cell is mammalian cell CHO or insect cell. The pharmaceutical composition or combined agent according to claim 13 is characterized by this.

15. A recombinant vector or adenovirus vector, characterized by containing a polynucleotide sequence in a recombinant protein vaccine or an adenovirus vaccine in the pharmaceutical composition or combined agent according to any one of claims 1 to 14, wherein the polynucleotide sequence is selected from at least one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO:

76.

16. The recombinant vector is selected from at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector and a yeast expression vector. Preferably, the insect baculovirus expression vector is pFastBac1. Preferably, the mammalian cell expression vector is a CHO cell expression vector. More preferably, the CHO cell expression vector is pTT5 or FTP-002. Preferably, the Escherichia coli expression vector is pET32a. Preferably, the yeast expression vector is pPICZaA. The recombinant vector or adenovirus vector according to claim 15, characterized in that.

17. The adenovirus vector is a human replication-deficient recombinant adenovirus vector. Preferably, the adenovirus vector is selected from human type 5, type 35 or type 26 replication-deficient adenoviruses, and / or chimpanzee type AdC68 or AdC7 replication-deficient adenoviruses. More preferably, it is selected from human type 5 replication-deficient adenoviruses having a combined deletion of E1 and E3. The recombinant vector or adenovirus vector according to claim 15, characterized in that.

18. A host cell comprising the recombinant vector or adenovirus vector according to any one of claims 15 to 17.

19. The host cell is selected from at least one of insect cells, mammalian cells, Escherichia coli, and yeast, preferably, the insect cell is selected from at least one of sf9 cells, sf21 cells, and Hi5 cells, preferably, the mammalian cell is a CHO cell or a HEK293 cell. The host cell according to claim 18.

20. The recombinant protein vaccine and / or the adenovirus vaccine further comprises a pharmaceutically acceptable excipient or complementary component. The pharmaceutical composition according to claim 1 or the combined agent according to claim 2.

21. The complementary component is an immune adjuvant. Preferably, the immune adjuvant is selected from at least one of squalene water-in-oil emulsion, aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoryl lipid A, muramyl dipeptide, muramyl tripeptide, bacterial toxin, GM-CSF cytokine, lipid and cationic liposome material. Further, the immune adjuvant satisfies that at least one of the squalene water-in-oil emulsions is MF59, the aluminum salt is selected from at least one of aluminum hydroxide and alum, the calcium salt is tricalcium phosphate, the plant saponin is QS-21 or ISCOM, the plant polysaccharide is astragalus polysaccharide, the bacterial toxin is selected from at least one of recombinant cholera toxin and diphtheria toxin, the lipid is selected from at least one of phosphatidylethanolamine, phosphatidylcholine, cholesterol and dioleoylphosphatidylethanolamine, or the cationic liposome material is (2,3-dioleyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloride)propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-arginineformylamino)ethylammonium trimethyldodecylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, dimethyldioctadecylammonium bromide and CpG ODN. The pharmaceutical composition or combined agent according to claim 20 is characterized by this.

22. The vaccine preparations of the recombinant protein vaccine and the adenovirus vaccine are intramuscular injection agents, injection agents, nasal drops, sprays, nasal sprays or inhalants. Preferably, the recombinant protein vaccine or the adenovirus vaccine is a nasal spray. The pharmaceutical composition or combined agent according to claim 20 is characterized by this.

23. Use of the pharmaceutical composition or combination drug according to any one of claims 1 to 15 and 20 to 22 in the preparation of a medicament for preventing and / or treating infection with SARS-CoV-2 or a variant thereof.

24. Use of the pharmaceutical composition or combination drug according to any one of claims 1 to 15 and 20 to 22 in the preparation of a medicament for treating and / or preventing infection or onset of a SARS-CoV-2 variant strain, wherein the SARS-CoV-2 variant strain includes at least one of alpha, beta, gamma, delta, and omicron.

25. Use of the pharmaceutical composition or combination drug according to any one of claims 1 to 15 and 20 to 22 in the preparation of a medicament for treating and / or preventing infection or onset of a respiratory virus.

26. A pharmaceutical composition characterized by containing a recombinant protein vaccine and an inactivated virus vaccine as active ingredients in the pharmaceutical composition or combination drug according to claims 1 to 15 and 20 to 22, wherein the inactivated virus vaccine is an inactivated influenza virus vaccine.

27. A pharmaceutical composition characterized by containing an adenovirus vaccine and an influenza virus recombinant protein vaccine as active ingredients in the pharmaceutical composition or combination drug according to claims 1 to 15 and 20 to 22.

28. The pharmaceutical composition according to any one of claims 26 or 27 in the preparation of a medicament for treating and / or preventing infection or onset of a respiratory virus.

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