Adenovirus vector recombinant novel coronavirus vaccine as well as preparation method and application thereof

Abstract

The invention provides an adenovirus vector recombinant novel coronavirus vaccine as well as a preparation method and application thereof. XBB.1. 5 subtype S protein for coding novel coronavirus Omicro variant is selected as expression protein, the recombinant virus vector vaccine is prepared from an optimized nucleic acid sequence, an immunized body can generate a binding antibody, a neutralizing antibody and cellular immune response aiming at XBB.1. 5 variant virus, and the recombinant virus vector vaccine has good immunogenicity. After the novel coronavirus vaccine disclosed by the invention is used for immunizing an organism, neutralizing antibodies can be generated aiming at an original strain and other variants, and infection of the original strain and other variants can be effectively prevented.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and specifically to an adenovirus vector recombinant novel coronavirus SARS-CoV-2 vaccine, a preparation method and applications thereof, and more specifically to an adenovirus vector recombinant novel coronavirus Omicron variant XBB.1.5 subtype vaccine, a preparation method and applications thereof. Background Art

[0002] Omicron is less pathogenic than the original strain, but has a stronger ability to spread and a faster speed. This is mainly because its structure contains multiple mutation sites in the spike protein (Spike, S) compared to the original strain, and multiple mutation sites are located in the receptor binding region. The large number of mutations accumulated in the spike protein allows the epidemic strain to escape the antibodies stimulated by the new crown prototype vaccine to a certain extent, thereby causing the new crown prototype vaccine to fail or reduce its protective power. XBB is an Omicron subtype variant strain.

[0003] Internationally, XBB is a recombinant strain of BA.2 mutants BJ.1 (BA.2.10.1.1) and BA.2.75 (BA.2.75.3.1.1.1), rather than a single mutation. It was first discovered in India in mid-August 2022; according to the latest COVID-19 test data updated by the US Centers for Disease Control and Prevention (CDC) on December 30, 2022, the XBB.1.5 mutant has accounted for 40.5% of the newly detected strains in the United States, and is about twice as much as a week ago and four times as much as two weeks ago. XBB may cause continued infection in people who have been vaccinated or previously infected. Judging from the antibody efficacy obtained by vaccination, BQ.1.1.10, XBB, etc., which have been prevalent worldwide recently, have shown serious escape ability for the plasma of recipients of three-dose inactivated vaccines (CoronaVac); even for those who have recovered from three-dose vaccinations and then infected with BA.1, BA.2, BA.5 or even BF.7, their plasma has weak neutralizing titers for the latest XBB and other mutants.

[0004] During the evolution, based on BA.2, XBB had 14 new mutations in the S protein, including 5 sites in the N-terminal domain (NTD) and 9 mutations in the receptor binding domain (RBD). Seven of the key mutations in the RBD domain may cause XBB to have a stronger ability to spread than BQ.1.1. The large number of mutations on the RBD has given the XBB subtype strain a stronger ability to escape the immune system. XBB.1.16, XBB.1.5, and XBB.1 all belong to the XBB family. Among them, XBB.1 also has a g252v mutation; in addition to the high ability to escape the immune system, another "skill" of XBB.1.5 that may contribute to its spread is that the strain has a key mutation at site 486, which allows it to bind more tightly to ace2. XBB.1.16 has an additional mutation in the spike protein, and studies have shown that the transmission efficiency of XBB.1.16 is about 1.17 to 1.27 times higher than that of the XBB.1 and XBB.1.5 strains.

[0005] The new coronavirus is constantly mutating; even those who have been vaccinated in the early stages cannot resist the new mutant virus; at the same time, in the prevalence of new mutant strains, the original strains and previous mutant strains also pose a risk of infection. Therefore, it is very important to develop a new vaccine that can prevent new mutant viruses and further enhance the immune ability of previous vaccinations. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art. In one aspect of the present invention, the S protein encoding the XBB.1.5 subtype of the novel coronavirus Omicron variant is selected as the expression protein, and a recombinant viral vector vaccine is prepared with an optimized nucleic acid sequence. After immunization, the body can produce binding antibodies, neutralizing antibodies and cellular immune responses against the XBB.1.5 variant virus, and has good immunogenicity; at the same time, it can also produce neutralizing antibodies against the prototype strain, BA.4 / 5, BF.7, XBB.1.16, and BA.2.75 variants, and has more comprehensive protection.

[0007] The present invention provides an optimized design for the spike protein of the SARS-2-CoV coronavirus, which introduces at least one mutation into a specific site of the amino acid sequence of the spike protein, changes the conformation of the S protein, and improves the level of neutralizing antibodies produced by its immunity.

[0008] On the other hand, the use of the adenovirus vector recombinant novel coronavirus vaccine of the present invention will not affect the immunogenicity of each antigen component; the adenovirus vector recombinant novel coronavirus original strain antigen protein and the XBB.1.5 variant antigen protein have good chemical compatibility. There will be no mutual interference of immune response between them, and it can be prepared as an injectable or inhaled vaccine. There will be no antigen competition between the original strain antigen protein of the vaccine and the XBB.1.5 variant antigen protein. The types and ratios of excipients and antigens are appropriate. It can effectively protect the body from infection by the XBB.1.5 variant and the original strain.

[0009] On the other hand, the adenovirus vector recombinant new coronavirus vaccine of the present invention can also effectively protect the body from infection by BA.4 / 5, BF.7, XBB.1.16, and BA.2.75 mutant strains.

[0010] On the other hand, the adenovirus vector recombinant new coronavirus vaccine of the present invention can also effectively protect the body from infection by BA.4 / 5, BF.7, XBB.1.16, and BA.2.75 mutant strains.

[0011] On the other hand, the adenovirus vector recombinant new coronavirus vaccine of the present invention can trigger the body's secondary immune response and effectively protect the body from infection by XBB.1.5, BA.4 / 5, BF.7, XBB.1.16, and BA.2.75 mutant strains.

[0012] In order to achieve the above objectives, the present invention provides a recombinant adenovirus vector containing a polynucleotide, wherein the recombinant adenovirus expresses the spike protein of the new coronavirus XBB.1.5 variant encoded by the polynucleotide molecule.

[0013] Preferably, the adenovirus is a human adenovirus or a chimpanzee adenovirus; preferably, the human adenovirus is selected from: AdHu2, AdHu4, AdHu5, AdHu7, AdHu11, AdHu26, AdHu35, AdHu55; more preferably, it is AdHu5 type.

[0014] Preferably, the coding sequence is a mutation at position 666RRAR669 of the furin cleavage site between the S1 / S2 subunits of the sequence of the spike protein of the XBB.1.5 subtype of the novel coronavirus Omicron variant, preferably, comprising a mutation of 666RRAR669 to GSAG.

[0015] At the same time, in order to improve the expression level of antigen protein in the human body, preferably, the coding sequence is the sequence of the S protein of the XBB.1.5 subtype of the new coronavirus Omicron variant, and the signal peptide sequence of amino acids 1-12 is replaced by the signal peptide TPA.

[0016] Preferably, the signal peptide TPA sequence is MDAMKRGLCCVLLLCGAVFVSNS.

[0017] Specifically, the amino acid sequence encoding the S protein of the XBB.1.5 subtype of the novel coronavirus Omicron variant has at least 75% homology as shown in SEQ ID NO: 2, and preferably, has at least 85%, 90%, 95%, 97%, 98%, or 99% homology.

[0018] The present invention provides a recombinant adenovirus vector vaccine, specifically, the recombinant adenovirus vector and pharmaceutically acceptable excipients. Preferably, the vaccine is an injection, preferably, more preferably, an intramuscular injection.

[0019] Specifically, the vaccine further comprises pharmaceutically acceptable excipients; preferably, the pharmaceutically acceptable excipients can be selected from: one or more of: buffers, protectants, stabilizers, surfactants, osmotic pressure regulators, etc.; more preferably, the pharmaceutically acceptable excipients include sucrose, mannitol, sodium chloride, glycerol, HEPES, magnesium chloride, Tween 80, human albumin and gelatin; more preferably, the content of each component is: mannitol 1-100 mg / ml, sodium chloride 1-50 mg / ml, HEPES 0.1-5 mg / ml, polysorbate 80 0.05-5 mg / ml, glycerol 0.5-10 mg / ml, magnesium chloride 0.1-10 mg / ml, sucrose 5-50 mg / ml.

[0020] The present invention provides a recombinant adenovirus vector vaccine, comprising the recombinant adenovirus vector and a pharmaceutically acceptable excipient; preferably, the vaccine is a mucosal administration preparation, more preferably, the mucosal administration preparation is an inhalation administration preparation, more preferably, nasal inhalation or oral inhalation; more preferably, the inhalation administration preparation is a liquid inhalation preparation or a dry powder inhalation preparation. Preferably, the mucosa includes nasal mucosa, oral mucosa or lung mucosa.

[0021] Preferably, the pharmaceutically acceptable excipients include but are not limited to: one or more of a buffer, a protective agent, a stabilizer, a surfactant, and an osmotic pressure regulator.

[0022] Preferably, the mucosal administration preparation is selected from: liquid dosage form, solid dosage form, semisolid dosage form; preferably aerosol, spray, nasal drops, powder mist, powder, gel, microsphere, liposome, film, suspension; more preferably, the mucosal administration preparation is a spray or atomized administration preparation.

[0023] The present invention provides a method for preparing a vaccine, comprising preparing a recombinant adenovirus vector of a novel coronavirus Omicron variant XBB.1.5 subtype antigen protein, and optionally adding a pharmaceutically acceptable excipient.

[0024] Specifically, the preparation of the recombinant adenovirus vector includes the following steps:

[0025] (1) Construction of shuttle plasmid;

[0026] (2) co-transfecting the recombinant adenovirus shuttle plasmid obtained in step (1) and the backbone plasmid carrying most of the adenovirus genome into packaging cells to recombinantly package a replication-defective adenovirus;

[0027] The present invention provides a vaccine kit, comprising: a first immunogenic composition and a second immunogenic composition, which are packaged separately; preferably, the first immunogenic composition is a recombinant adenoviral vector containing the aforementioned adenovirus vector; and the second immunogenic composition comprises a recombinant adenoviral vector encoding a gene sequence of the S protein of the novel coronavirus prototype strain. More preferably, the immunogenic composition further comprises a pharmaceutically acceptable excipient.

[0028] Specifically, the amino acid sequence encoding the S protein of the novel coronavirus prototype strain as shown in SEQ ID NO:5 has at least 75% homology, preferably, at least 85%, 90%, 95%, 97%, 98%, 99% homology.

[0029] Specifically, the second immunogenic composition can be in liquid dosage form, solid dosage form, or semisolid dosage form; preferably, it is an oral preparation, an intramuscular injection preparation, an intravenous injection preparation, or an inhalation preparation.

[0030] Specifically, the content of the recombinant virus vector in the recombinant adenovirus vector vaccine or immunogenic composition is 1×10 8 ~1×10 12 VP / ml, preferably 5×10 10 VP / ml.

[0031] The present invention provides a method for preparing a vaccine kit, characterized in that it comprises the following steps:

[0032] S1: preparing a recombinant adenovirus vector encoding an antigen protein of a prototype strain of a novel coronavirus, and optionally adding a pharmaceutically acceptable excipient;

[0033] S2: Prepare a recombinant adenovirus vector of the novel coronavirus Omicron variant XBB.1.5 subtype antigen protein, and optionally add pharmaceutically acceptable excipients.

[0034] Specifically, the preparation of the recombinant adenovirus vector includes the following steps:

[0035] (1) construct plasmid;

[0036] (2) co-transfecting and packaging the recombinant adenovirus shuttle plasmid obtained in step (1) with a backbone plasmid carrying most of the adenovirus genome;

[0037] Preferably, (1) constructing the original strain plasmid and other novel coronavirus variant strain plasmids respectively;

[0038] (2) co-transfecting and packaging the recombinant adenovirus shuttle plasmid obtained in step (1) with a backbone plasmid carrying most of the adenovirus genome;

[0039] More preferably, the other novel coronavirus variants are variants encoding the novel coronavirus Omicron variant XBB.1.5 subtype variants.

[0040] Preferably, the packaging cell is a HEK293 cell or a cell line derived therefrom.

[0041] The present invention provides a use of a recombinant adenovirus vector vaccine or a vaccine kit in the preparation of a medicament for preventing and / or treating a disease; preferably, the disease is novel coronavirus infection (COVID-19).

[0042] The present invention provides an application of a recombinant adenovirus vector vaccine or a vaccine kit in the preparation of a medicament for preventing and / or treating a disease; the disease is an infection caused by a novel coronavirus prototype strain, XBB.1.5, BA.4 / 5, BF.7, XBB.1.16, or BA.2.75 mutant strains.

[0043] The present invention provides a use of a recombinant adenovirus vector vaccine or a vaccine kit in the preparation of a drug for preventing and / or treating a disease; the disease is infection caused by a novel coronavirus prototype strain, XBB.1.5, BA.4 / 5, BF.7, XBB.1.16, or BA.2.75 variant strain. Preferably, the recombinant adenovirus vector vaccine or vaccine kit is used to induce secondary immunity in the body.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The S protein encoding the XBB.1.5 subtype of the Omicron variant of the new coronavirus was selected as the expression protein, and the recombinant viral vector vaccine was prepared with the optimized nucleic acid sequence. After immunization, the body can produce binding antibodies, neutralizing antibodies and cellular immune responses against the XBB.1.5 variant virus, and has good immunogenicity.

[0046] The multivalent adenovirus vector recombinant novel coronavirus vaccine of the present invention is prepared by separately preparing a recombinant adenovirus vector vaccine encoding the antigen protein of the prototype strain of novel coronavirus and a recombinant adenovirus vector vaccine encoding the antigen protein of the subtype XBB.1.5 of the novel coronavirus Omicron variant, and then combining them into a multi-dose kit. The problem of immunosuppression of preparing adenovirus vector vaccines by co-expressing the plasmids of the S gene of the Omicron (XBB.1.5) variant and the RBD gene of the prototype strain is solved. The use of the multivalent vaccine of the present invention will not affect the immunogenicity of each antigen component; when applied at the same time, the adenovirus vector recombinant novel coronavirus original strain antigen protein and the XBB.1.5 variant antigen protein have good chemical compatibility. There will be no mutual interference of immune response between them; there will be no antigen competition between the original strain antigen protein and the XBB.1.5 variant antigen protein; the body can be effectively protected from infection by the XBB.1.5 variant and the original strain.

[0047] The multivalent adenovirus vector recombinant new coronavirus vaccine of the present invention can effectively protect the body from infection by the prototype strain, BA.4 / 5, BF.7, XBB.1.16, and BA.2.75 mutant strains.

[0048] The excipients used in the vaccine preparation of the present invention will not produce immunosuppression on the original strain vaccine stock solution, the XBB.1.5 variant vaccine stock solution and other variants, and have good compatibility.

[0049] The multi-dose kit for the novel coronavirus vaccine of the present invention can produce neutralizing antibodies against both the original strain and the variant strain XBB.1.5 after immunizing the body. Not only can the original strain of the novel coronavirus be prevented, but also the variant strain XBB.1.5 can be effectively prevented. In addition, neutralizing antibodies against the prototype strain, BA.4 / 5, BF.7, XBB.1.16, and BA.2.75 variant strains can be produced. The adenovirus vector recombinant novel coronavirus vaccine provided by the present invention has good stability, safety, and high efficiency, and can effectively fight against different differentiated strains of the novel coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 . Neutralizing antibody titers against XBB.1.5 variant pseudoviruses with different S protein sequences (injection)

[0051] Figure 2 . Serum S-IgG antibody titers against XBB.1.5 variants with different S protein sequences (injection)

[0052] Figure 3 .XBB.1.5S-specific CD8+ T cell response (spleen)

[0053] Figure 4.XBB.1.5S-specific CD4+ T cell response (spleen)

[0054] Figure 5 . Neutralizing antibody titers against XBB.1.5 variant pseudoviruses with different S protein sequences (inhalation)

[0055] Figure 6 . Anti-BA.4 / BA.5 variant S-IgG antibody titers of different S protein sequences (inhalation)

[0056] Figure 7 . Titers of neutralizing antibodies against pseudoviruses of various novel coronavirus strains induced by sequential booster immunization in mice

[0057] Figure 8 . Titers of neutralizing antibodies against pseudoviruses of various novel coronavirus strains induced by sequential booster immunization in mice (inhalation) DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0060] Example 1 Construction and preparation of mutant virus

[0061] The S protein gene sequence of the new coronavirus variant XBB is shown in Table 1.

[0062] Table 1. Mutation sites of the S protein gene of the novel coronavirus variant

[0063] Serial number Sequence Listing strain Mutation site 1 SEQ ID NO:1 XBB.1.5 none 2 SEQ ID NO:2 XBB.1.5 666GSAG669 3 SEQ ID NO:3 XBB.1 none 4 SEQ ID NO:4 XBB.1.16 none

[0064] The above-mentioned vectors that were correctly identified by sequencing were respectively recorded as: pDC316-nCoV-XBB.1.5-1, pDC316-nCoV-XBB.1.5-2, pDC316-nCoV-XBB.1, and pDC316-nCoV-XBB.1.16.

[0065] The shuttle plasmids pDC316-nCoV-XBB.1.5-1, pDC316-nCoV-XBB.1.5-2, pDC316-nCoV-XBB.1, pDC316-nCoV-XBB.1.16 and the backbone plasmid of the AdMax adenovirus system were packaged by co-transfection, and the packaged viruses were named Ad5-nCoV-XBB.1.5-1, Ad5-nCoV-XBB.1.5-2, Ad5-XBB.1-nCoV-3, and Ad5-XBB.1.16-nCoV-4, respectively.

[0066] The specific process is as follows: Before transfection, HEK293 cells were inoculated in a six-well plate. The backbone plasmid and shuttle plasmid were transfected with transfection reagent, and three replicate wells were transfected. After transfection, the overgrown cells were subcultured in a cell culture bottle and continued to be cultured with MEM medium. The signs of cell poisoning were observed every day. The poisoning phenomenon was that the cells became larger and rounder, grape-shaped, and obvious plaques began to appear. The poison was collected when most of the cells were diseased and fell off from the bottom.

[0067] Preparation of coronavirus injection vaccine (adenovirus type 5 vector): Take the coronavirus variant vaccine (adenovirus type 5 vector) stock solution (containing 5×10 virus particles) prepared above. 10 VP), add excipients 50 mg mannitol, 5 mg sodium chloride, 0.5 mg HEPES, 0.1 mg polysorbate 80, 1.5 mg glycerol, 0.5 mg magnesium chloride, and 25 mg sucrose, and mix to obtain 1 ml of recombinant novel coronavirus variant XBB vaccine (adenovirus type 5 vector) injection preparation. Mark.

[0068] Preparation of coronavirus inhalation vaccine (adenovirus type 5 vector): Take the coronavirus variant vaccine (adenovirus type 5 vector) prepared above (containing 1×10 virus particles) stock solution 10 VP), add 50 mg of mannitol, 5 mg of sodium chloride, 1 mg of HEPES, 0.2 mg of polysorbate 80, 4 mg of glycerol, 0.2 mg of magnesium chloride, and 30 mg of sucrose, and mix to obtain 1 ml of recombinant novel coronavirus variant XBB vaccine (type 5 adenovirus vector) inhalation preparation. Label.

[0069] Example 2 Evaluation test of coronavirus XBB.1.5 injection vaccine with different S protein sequences

[0070] The recombinant novel coronavirus variant XBB vaccine injection preparation prepared in Example 1 was used for single injection immunization for 14 days. The neutralizing antibody titer and S-IgG antibody titer against the XBB variant pseudovirus produced in the mouse serum were tested and the results are shown in Tables 2 and 3 below. The results are as follows Figure 1 and Figure 2 :

[0071] Table 2. Neutralizing antibody titers against XBB.1.5 variant pseudoviruses with different S protein sequences (injection)

[0072]

[0073]

[0074] Table 3. Serum S-IgG antibody titers against XBB.1.5 variants with different S protein sequences (injection)

[0075]

[0076] After a single injection of immunization for 28 days, the CD8+ and CD4+ T cell responses against the XBB.1.5 variant were produced in the spleen of mice. The results are shown in Tables 4 and 5 below. Figure 3 and Figure 4 :

[0077] Table 4. XBB.1.5S-specific CD8+ T cell responses (spleen)

[0078]

[0079]

[0080] Table 5. XBB.1.5S-specific CD4+ T cell responses (spleen)

[0081]

[0082] Experimental results showed that the Ad5-nCoV-XBB.1.5-2 injection preparation can simultaneously stimulate the body to produce high levels of anti-XBB.1.5 variant pseudovirus neutralizing antibodies and S-IgG antibodies; as well as S-specific CD8+T and CD4+T cell responses.

[0083] Example 3 Evaluation test of inhaled vaccine of coronavirus XBB.1.5 with different S protein sequences

[0084] The recombinant novel coronavirus variant XBB vaccine injection inhalation preparation prepared in Example 1 was used for single injection immunization for 14 days. The neutralizing antibody titer and S-IgG antibody titer against the XBB variant pseudovirus produced in the mouse serum were tested and the results are shown in Tables 6 and 7 below. The results are as follows Figure 5 and Figure 6 :

[0085] Table 6 Neutralizing antibody titers against XBB.1.5 variant pseudoviruses with different S protein sequences (inhalation)

[0086]

[0087]

[0088] Table 7. Anti-XBB.1.5 heterologous S-IgG antibody titers (inhalation) with different S protein sequences

[0089]

[0090] Experimental results show that the Ad5-nCoV-XBB.1.5-2 inhalation preparation can simultaneously stimulate the body to produce high levels of anti-XBB.1.5 variant pseudovirus neutralizing antibodies and S-IgG antibodies.

[0091] Example 4 Coronavirus injection vaccine sequential booster cross-protection evaluation test

[0092] Under the basic immunization background of two-dose bivalent (prototype strain and variant BA.5) new coronavirus vaccine (simulating population vaccination and infection), the immunogenicity study of sequential immunization of the recombinant new coronavirus XBB.1.5 variant vaccine was carried out; the titers of pseudovirus neutralizing antibodies against the new coronavirus strains XBB.1.5, XBB.1.16, BA.4 / 5, prototype strain, BA.2.75, and BF.7 were tested.

[0093] The preparation method of the bivalent (prototype strain and variant strain BA.4 / BA.5) novel coronavirus vaccine injection preparation (adenovirus type 5 vector) is as follows:

[0094] The S protein gene (SEQ ID NO:6) of the new coronavirus variant BA.4 / BA.5 was used to construct pDC316-nCoV-BA.4 / BA.5; the shuttle plasmid pDC316-nCoV-BA.4 / BA.5 and the backbone plasmid of the AdMax adenovirus system were co-transfected to package Ad5-nCoV-BA.4 / BA.5, and the packaged viruses were named Ad5-nCoV-BA.4 / BA.5. The specific process is as follows: Before transfection, HEK293 cells were inoculated in a six-well plate. Take the backbone plasmid and the shuttle plasmid, transfect them with a transfection reagent, and co-transfect 3 replicate wells. After transfection, the overgrown cells were passaged in a cell culture flask, and continued to be cultured with MEM medium, and the signs of cell poisoning were observed every day. The poisoning phenomenon is that the cells become larger and rounder, grape-shaped, and obvious plaques begin to appear. The virus is collected when most of the cells are diseased and fall off from the bottom.

[0095] The S protein gene sequence of the optimized novel coronavirus prototype strain is shown in SEQ ID NO:5, and the vector identified correctly by sequencing is recorded as pDC316-nCoV_Sopt. The shuttle plasmid pDC316-nCoV_Sopt and the backbone plasmid of the AdMax adenovirus system were packaged into Ad5-nCoV by co-transfection, and the packaged virus was named Ad5-nCoV. The specific process is as follows: Before transfection, HEK293 cells were inoculated in a six-well plate. Take the backbone plasmid and the shuttle plasmid, transfect them with a transfection reagent, and co-transfect 3 replicate wells. After transfection, the overgrown cells were passaged in a cell culture flask, and continued to be cultured with MEM medium, and the signs of cell poisoning were observed every day. The poisoning phenomenon is that the cells become larger and rounder, grape-shaped, and obvious plaques begin to appear. The virus is collected when most of the cells are diseased and fall off from the bottom.

[0096] Prototype strain: variant strain (Ad5-nCoV-BA.4 / BA.5) = 1:1; take the above-mentioned recombinant novel coronavirus prototype strain vaccine (adenovirus type 5 vector) stock solution (containing virus particles 2.5×10 10 VP); Recombinant novel coronavirus BA.4 / 5 variant vaccine (adenovirus vector type 5) stock solution (containing 2.5×10 10 VP) mix; add excipients 50 mg mannitol, 5 mg sodium chloride, 0.5 mg HEPES, 0.1 mg polysorbate 80, 1.5 mg glycerol, 0.5 mg magnesium chloride, and 25 mg sucrose to obtain 1 ml of bivalent (prototype strain and variant strain BA.4 / BA.5) new coronavirus vaccine injection preparation.

[0097] The preparation method of the bivalent (XBB.1.5 and variant BA.4 / BA.5) novel coronavirus vaccine (adenovirus type 5 vector) is as follows:

[0098] Variant (XBB.1.5): variant (Ad5-nCoV-BA.4 / BA.5-2) = 1:1; take the above-mentioned recombinant novel coronavirus prototype vaccine (adenovirus type 5 vector) stock solution (containing 2.5×10 10 VP); Recombinant novel coronavirus BA.4 / 5 variant vaccine (adenovirus vector type 5) stock solution (containing 2.5×10 10 VP) mix; add excipients 50 mg mannitol, 5 mg sodium chloride, 0.5 mg HEPES, 0.1 mg polysorbate 80, 1.5 mg glycerol, 0.5 mg magnesium chloride, and 25 mg sucrose to obtain 1 ml of bivalent (variant XBB.1.5 and variant BA.4 / BA.5) new coronavirus vaccine injection preparation.

[0099] The bivalent novel coronavirus vaccine injection preparation prepared above (prototype strain and variant strains BA.4 / BA.5 and XBB.1.5 and variant strains BA.4 / BA.5) was injected intramuscularly into BALB / c mice at a 1 / 10 human dose, for a total of one injection, and the immunization time point was day 0. The above was used as basic immunization.

[0100] On the 28th day, the second injection of sequential immunization was performed, and the above-prepared XBB.1.5 monovalent injection preparation, BA.4 / 5+XBB.1.5 bivalent injection preparation, and prototype strain+BA.4 / 5 bivalent injection preparation were respectively administered at 1 / 10 of the planned human dose, i.e., 5×10 9 BALB / c mice were immunized with VP doses by intramuscular injection.

[0101] Blood was collected on the 42nd day, and the geometric mean titer (GMT) of pseudovirus neutralizing antibodies produced in mouse serum against each novel coronavirus strain is shown in Table 8 and Figure 7 .

[0102] Table 8. GMT results of neutralizing antibody titers against pseudoviruses of various novel coronavirus strains induced by sequential booster immunization in mice

[0103]

[0104]

[0105]

[0106] Under the same immune background, sequential immunization with the XBB.1.5 monovalent injection preparation against the Omicron XBB.1.5 variant produced a high level of pseudovirus neutralizing antibodies against the Omicron XBB.1.5 variant, which was not significantly different from the BA.4 / 5+XBB.1.5 bivalent injection preparation, but was significantly higher than the prototype strain+BA.4 / 5 bivalent injection preparation;

[0107] For the Omicron XBB.1.16 variant, sequential immunization with a single injection of XBB.1.5 monovalent injection preparation produced a high level of neutralizing antibodies against the pseudovirus, which was not significantly different from the BA.4 / 5+XBB.1.5 bivalent injection preparation, but was significantly higher than the prototype strain+BA.4 / 5 bivalent injection preparation.

[0108] In addition, under the same immune background, sequential immunization with the XBB.1.5 monovalent injection preparation can also produce high levels of broadly neutralizing antibodies against previously prevalent strains, including: antigenic strains, anti-BA.4 / 5 variants, anti-BA.2.75 variants, and anti-BF.7 variants.

[0109] Example 5 Coronavirus inhaled vaccine sequential booster cross-protection evaluation test

[0110] The preparation method of the bivalent (prototype strain and variant strain BA.4 / BA.5) novel coronavirus vaccine inhalation preparation (adenovirus type 5 vector) is as follows:

[0111] The S protein gene (SEQ ID NO:6) of the new coronavirus variant BA.4 / BA.5 was used to construct pDC316-nCoV-BA.4 / BA.5; the shuttle plasmid pDC316-nCoV-BA.4 / BA.5 and the backbone plasmid of the AdMax adenovirus system were co-transfected to package Ad5-nCoV-BA.4 / BA.5, and the packaged viruses were named Ad5-nCoV-BA.4 / BA.5. The specific process is as follows: Before transfection, HEK293 cells were inoculated in a six-well plate. Take the backbone plasmid and the shuttle plasmid, transfect them with a transfection reagent, and co-transfect 3 replicate wells. After transfection, the overgrown cells were passaged in a cell culture flask, and continued to be cultured with MEM medium, and the signs of cell poisoning were observed every day. The poisoning phenomenon is that the cells become larger and rounder, grape-shaped, and obvious plaques begin to appear. The virus is collected when most of the cells are diseased and fall off from the bottom.

[0112] The S protein gene sequence of the optimized novel coronavirus prototype strain is shown in SEQ ID NO:5, and the vector identified correctly by sequencing is recorded as pDC316-nCoV_Sopt. The shuttle plasmid pDC316-nCoV_Sopt and the backbone plasmid of the AdMax adenovirus system were packaged into Ad5-nCoV by co-transfection, and the packaged virus was named Ad5-nCoV. The specific process is as follows: Before transfection, HEK293 cells were inoculated in a six-well plate. Take the backbone plasmid and the shuttle plasmid, transfect them with a transfection reagent, and co-transfect 3 replicate wells. After transfection, the overgrown cells were passaged in a cell culture flask, and continued to be cultured with MEM medium, and the signs of cell poisoning were observed every day. The poisoning phenomenon is that the cells become larger and rounder, grape-shaped, and obvious plaques begin to appear. The virus is collected when most of the cells are diseased and fall off from the bottom.

[0113] Prototype strain: variant strain (Ad5-nCoV-BA.4 / BA.5) = 1:1; take the above-mentioned recombinant novel coronavirus prototype strain vaccine (adenovirus type 5 vector) stock solution (containing virus particles 0.5×10 9 VP); Recombinant novel coronavirus BA.4 / 5 variant vaccine (adenovirus vector type 5) stock solution (containing virus particles 0.5×10 9VP) mix; add 50 mg of mannitol, 5 mg of sodium chloride, 1 mg of HEPES, 0.2 mg of polysorbate 80, 4 mg of glycerol, 0.2 mg of magnesium chloride, and 30 mg of sucrose to obtain 1 ml of bivalent (prototype strain and variant strain BA.4 / BA.5) new coronavirus vaccine inhalation preparation.

[0114] The preparation method of the bivalent (XBB.1.5 and variant BA.4 / BA.5) novel coronavirus vaccine (adenovirus type 5 vector) is as follows:

[0115] Variant (XBB.1.5): variant (Ad5-nCoV-BA.4 / BA.5-2) = 1:1; take the above-mentioned recombinant novel coronavirus prototype vaccine (adenovirus type 5 vector) stock solution (containing virus particles 0.5×10 9 VP); Recombinant novel coronavirus BA.4 / 5 variant vaccine (adenovirus vector type 5) stock solution (containing virus particles 0.5×10 9 VP) mix; add 50 mg of mannitol, 5 mg of sodium chloride, 1 mg of HEPES, 0.2 mg of polysorbate 80, 4 mg of glycerol, 0.2 mg of magnesium chloride, and 30 mg of sucrose to obtain 1 ml of bivalent (variant XBB.1.5 and variant BA.4 / BA.5) new coronavirus vaccine inhalation preparation.

[0116] The above-prepared bivalent novel coronavirus vaccine injection preparation (prototype strain and variant strains BA.4 / BA.5 and XBB.1.5 and variant strains BA.4 / BA.5) was injected into BALB / c mice at a 1 / 10 human dose, for a total of one injection, and the immunization time point was day 0. The above was used as basic immunization.

[0117] On the 28th day, the second injection of sequential immunization was performed, and the XBB.1.5 monovalent inhalation preparation and BA.4 / 5 + XBB.1.5 bivalent inhalation preparation prepared above were respectively administered at 1 / 10 of the human dose, i.e., 1×10 9 BALB / c mice were immunized with VP doses by intramuscular injection.

[0118] Blood was collected on the 42nd day, and the geometric mean titer (GMT) of pseudovirus neutralizing antibodies produced in mouse serum against each new coronavirus strain is shown in the table below.

[0119] Table 9. GMT results of neutralizing antibody titers against pseudoviruses of various novel coronavirus strains induced by sequential booster immunization in mice (inhalation)

[0120]

[0121]

[0122] Under the same immunization background, sequential immunization of the XBB.1.5 monovalent vaccine with one injection by nebulization inhalation produced a high level of pseudovirus neutralizing antibodies against the Omicron XBB.1.5 variant, which was not significantly different from the BA.4 / 5+XBB.1.5 bivalent vaccine; the level of pseudovirus neutralizing antibodies against the Omicron XBB.1.16 variant was significantly higher than that of the BA.4 / 5+XBB.1.5 bivalent vaccine.

[0123] In addition, under the same immune background, sequential immunization with the XBB.1.5 monovalent inhalation preparation can also produce high levels of broadly neutralizing antibodies against previously prevalent strains, including: antigenic strains, anti-BA.4 / 5 variants, anti-BA.2.75 variants, and anti-BF.7 variants.

[0124] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0125] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A recombinant adenovirus vector containing a polynucleotide, characterized in that: The recombinant adenovirus expresses the novel coronavirus XBB.1.5 variant spike protein encoded by the polynucleotide molecule; preferably, the adenovirus is a human adenovirus or a chimpanzee adenovirus; preferably, the human adenovirus is selected from: AdHu2, AdHu4, AdHu5, AdHu7, AdHu11, AdHu26, AdHu35, AdHu55; more preferably, it is AdHu5 type.

2. The recombinant adenoviral vector according to claim 1, characterized in that The coding sequence is a mutation at position 666RRAR669 of the furin cleavage site between the S1 / S2 subunits of the sequence of the XBB.1.5 subtype spike protein of the novel coronavirus Omicron variant, preferably comprising a mutation of 666RRAR669 to GSAG.

3. The recombinant adenovirus vector according to claim 1 or 2, characterized in that: The coding sequence is the signal peptide sequence of the S protein of the new coronavirus Omicron variant XBB.1.5 subtype replaced with the signal peptide TPA.

4. The recombinant adenovirus vector according to any one of claims 1 to 3, characterized in that The amino acid sequence encoding the XBB.1.5 subtype spike protein of the novel coronavirus Omicron variant has at least 75% homology as shown in SEQ ID NO: 2, preferably, at least 85%, 90%, 95%, 97%, 98%, 99% homology.

5. A recombinant adenovirus vector vaccine, characterized in that: Contains the recombinant adenovirus vector according to any one of claims 1 to 4 and pharmaceutically acceptable excipients; preferably, the vaccine is an injection, more preferably, an intramuscular injection.

6. The recombinant adenovirus vector vaccine according to claim 5, characterized in that The vaccine further comprises pharmaceutically acceptable excipients; preferably, the pharmaceutically acceptable excipients may be selected from: one or more of: buffers, protectants, stabilizers, surfactants, osmotic pressure regulators, etc.; more preferably, the pharmaceutically acceptable excipients include sucrose, mannitol, sodium chloride, glycerol, HEPES, magnesium chloride, Tween 80, human albumin and gelatin; more preferably, the content of each component is: mannitol 1-100 mg / ml, sodium chloride 1-50 mg / ml, HEPES 0.1-5 mg / ml, polysorbate 80 0.05-5 mg / ml, glycerol 0.5-10 mg / ml, magnesium chloride 0.1-10 mg / ml, sucrose 5-50 mg / ml.

7. A recombinant adenovirus vector vaccine, characterized in that: Contains the recombinant adenovirus vector according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient; preferably, the vaccine is a mucosal administration preparation, more preferably, the mucosal administration preparation is an inhalation administration preparation, more preferably, nasal inhalation or oral inhalation; more preferably, the inhalation administration preparation is a liquid inhalation preparation or a dry powder inhalation preparation.

8. The recombinant adenovirus vector vaccine according to claim 7, characterized in that: The mucosa includes nasal mucosa, oral mucosa or lung mucosa.

9. The recombinant adenovirus vector novel coronavirus vaccine according to any one of claims 7 or 8, characterized in that: The mucosal administration preparation is selected from: liquid dosage form, solid dosage form, semisolid dosage form; preferably aerosol, spray, nasal drops, powder mist, powder, gel, microsphere, liposome, film, suspension; more preferably, the mucosal administration preparation is a spray or atomized administration preparation.

10. The recombinant adenovirus vector novel coronavirus vaccine preparation according to any one of claims 7 to 9, characterized in that: The vaccine further comprises pharmaceutically acceptable excipients, which include but are not limited to: one or more of a buffer, a protective agent, a stabilizer, a surfactant, and an osmotic pressure regulator.

11. A method for preparing a vaccine according to any one of claims 5 to 10, characterized in that: It includes preparing a recombinant adenovirus vector for the novel coronavirus Omicron variant XBB.1.5 subtype antigen protein, and optionally adding a pharmaceutically acceptable excipient.

12. The method for preparing a vaccine according to claim 11, characterized in that: The preparation of recombinant adenovirus vector includes the following steps: (1) Construction of shuttle plasmid; (2) The recombinant adenovirus shuttle plasmid obtained in step (1) is co-transfected with a backbone plasmid carrying most of the adenovirus genome into packaging cells to recombinantly package a replication-defective adenovirus.

13. A vaccine kit, characterized in that: Including a first immunogenic composition and a second immunogenic composition; preferably, the first immunogenic composition is an adenovirus vector containing the adenovirus vector described in claims 1-4; the second immunogenic composition comprises a recombinant adenovirus vector encoding the gene sequence of the S protein of the novel coronavirus prototype strain; more preferably, the immunogenic composition also comprises a pharmaceutically acceptable excipient; more preferably, the compositions are packaged separately or the compositions are mixed.

14. The vaccine kit according to claim 13, characterized in that The amino acid sequence encoding the S protein of the novel coronavirus prototype strain has at least 75% homology as shown in SEQ ID NO:5, preferably, at least 85%, 90%, 95%, 97%, 98%, 99% homology.

15. The vaccine kit according to any one of claims 13-14, characterized in that The second immunogenic composition may be in liquid dosage form, solid dosage form, or semisolid dosage form; preferably, it may be an oral preparation, an intramuscular injection preparation, an intravenous injection preparation, or an inhalation preparation.

16. The recombinant adenovirus vector vaccine or vaccine kit according to any one of claims 5 to 10 or 13 to 15, characterized in that: The content of the recombinant virus vector in the recombinant adenovirus vector vaccine or immunogenic composition is 1×10 8 ~1×10 12 VP / ml, preferably 5×10 10 VP / ml.

17. A method for preparing the vaccine kit according to any one of claims 13 to 15, characterized in that: The following steps are involved: S1: preparing a recombinant adenovirus vector encoding an antigen protein of a prototype strain of a novel coronavirus, and optionally adding a pharmaceutically acceptable excipient; S2: Prepare a recombinant adenovirus vector of the novel coronavirus Omicron variant XBB.1.5 subtype antigen protein, and optionally add pharmaceutically acceptable excipients.

18. The method for preparing the vaccine kit according to claim 17, characterized in that: The preparation of recombinant adenovirus vector includes the following steps: (1) Construction of shuttle plasmid; (2) co-transfecting the recombinant adenovirus shuttle plasmid obtained in step (1) and the backbone plasmid carrying most of the adenovirus genome into packaging cells to recombinantly package a replication-defective adenovirus; Preferably, (1) constructing the original strain plasmid and other novel coronavirus variant strain plasmids respectively; (2) co-transfecting the recombinant adenovirus shuttle plasmid obtained in step (1) and the backbone plasmid carrying most of the adenovirus genome into packaging cells to recombinantly package a replication-defective adenovirus; More preferably, the other novel coronavirus variants are variants encoding the novel coronavirus Omicron variant XBB.1.5 subtype variants.

19. The method according to claim 18, characterized in that: The packaging cells in step (2) are HEK293 cells or cell lines derived therefrom.

20. Use of a recombinant adenovirus vector vaccine or a vaccine kit according to any one of claims 5-10 or 13-15 in the preparation of a medicament for preventing and / or treating a disease; preferably, the disease is novel coronavirus infection (COVID-19).

21. A use of a recombinant adenovirus vector vaccine or a vaccine kit according to any one of claims 5-10 or 13-15 in the preparation of a medicament for preventing and / or treating a disease; the disease is an infection caused by the novel coronavirus prototype strain, XBB.1.5, BA.4 / 5, BF.7, XBB.1.16, or BA.2.75 variant strains.

22. An application of a recombinant adenovirus vector vaccine or a vaccine kit according to any one of claims 5-10 or 13-15 in the preparation of a medicament for preventing and / or treating a disease; the disease is an infection caused by the novel coronavirus prototype strain, XBB.1.5, BA.4 / 5, BF.7, XBB.1.16, BA.2.75 mutant strains, and the recombinant adenovirus vector vaccine or vaccine kit is used to induce secondary immunity in the body.

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