CN115160413B

CN115160413B - Novel coronavirus vaccine

Info

Publication number: CN115160413B
Application number: CN202110369037.1A
Authority: CN
Inventors: 张林琦; 李明茜; 王若珂; 史宣玲; 张绮
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-04-06
Filing date: 2021-04-06
Publication date: 2023-11-14
Anticipated expiration: 2041-04-06
Also published as: CN115160413A

Abstract

The invention provides a recombinant adenovirus for preventing and/or treating SARS-CoV-2 south Africa strain 501Y.V2 infection and a vaccine composition containing the recombinant adenovirus. The recombinant adenovirus is obtained by transfecting adenovirus packaging cells having adenovirus E1 gene with a recombinant plasmid obtained by inserting SARS-CoV-2 south Africa strain 501Y.V2S protein encoding DNA molecule having mutation at S2 subunit and/or S1 and S2 cleavage site into delta E1 region of chimpanzee adenovirus vector AdC68 and culturing the cells. The vaccine for coronavirus SARS-CoV-2 south Africa strain 501Y.V2 developed by the invention provides possibility for radical treatment of coronavirus pneumonia.

Description

Novel coronavirus vaccine

Technical Field

The invention relates to a novel coronavirus vaccine, in particular to a novel coronavirus vaccine based on chimpanzee adenovirus 68 and SARS-CoV-2 south Africa strain 501Y.V2S protein.

Background

The novel coronavirus (SARS-CoV-2) is a coronavirus isolated from patients with unknown pneumonia. Most patients with SARS-CoV-2 infection develop severe respiratory diseases, the clinical symptoms of which are very similar to those of the disease caused by SARS-CoV, which has been outbreaked in 2003. A number of viral mutants have emerged during the transmission of SARS-CoV-2, of which mutant viruses 501Y.V2, which occur in south Africa, were first discovered in south Africa in the middle 10 th of 2020, and were the primary strains that were locally prevalent in the beginning of 11 th 2020. Since south Africa strains have important mutation sites in the receptor binding domain of membrane protein (S protein), the mutant strain is more infectious, and researches prove that the south Africa strain virus can escape the existing new coronal vaccine efficacy.

SARS-CoV-2 enters susceptible cells by using membrane protein S (also called S protein) on its surface. The S protein consists of three domains: an S1 domain at the N-terminus, an S2 domain at the proximal end, and a transmembrane domain. The susceptibility of SARS-CoV-2 to host cells is determined by the receptor binding domain RBD on the S1 domain.

Disclosure of Invention

In one aspect, the invention provides a variant of SARS-CoV-2 south Africa strain 501Y.V2S protein, which comprises one or more mutations in its S2 subunit relative to its wild-type S protein, which may further comprise one or more mutations at its S1 and S2 subunit cleavage sites.

In some embodiments, the mutation of the S2 subunit of the SARS-CoV-2 south africa strain 501y.v2S protein variant comprises K986P and/or V987P.

In some embodiments, the mutation site of the S1 and S2 subunit cleavage sites of the SARS-CoV-2 south Africa strain 501Y.V2S protein variant comprises one or more of R682, R683 and R685.

In some embodiments, the mutation at the R682 site of the SARS-CoV-2 south africa strain 501y.v2s protein variant is R682S or R682G, the mutation at the R683 site is R683S, and/or the mutation at the R685 site is R685G or R685S.

In some embodiments, the sequence of the SARS-CoV-2 south Africa strain 501Y.V2S protein variant is as shown in SEQ ID NO. 1 or is a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO. 1.

In some embodiments, the sequence of the SARS-CoV-2 south Africa strain 501Y.V2S protein variant is as shown in SEQ ID NO. 2 or is a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO. 2.

In another aspect, the invention also provides a DNA molecule encoding the SARS-CoV-2 south Africa strain 501Y.V2S protein variant as described above.

In some embodiments, the DNA molecule has a sequence as set forth in SEQ ID NO. 3, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO. 3.

In some embodiments, the DNA molecule has a sequence as set forth in SEQ ID NO. 4, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO. 4.

In a further aspect the invention provides a recombinant plasmid obtainable by inserting the aforementioned DNA molecule into the DeltaE 1 region of the chimpanzee adenovirus vector AdC 68.

In still another aspect, the present invention provides a recombinant adenovirus obtained by transfecting an adenovirus packaging cell having an adenovirus E1 gene using the aforementioned recombinant plasmid, and culturing the transfected cell.

In some embodiments, the adenovirus packaging cell is a HEK293A cell.

In another aspect, the invention provides a composition for preventing SARS-CoV-2 infection or for neutralizing SARS-CoV-2 or treating a disease associated with SARS-CoV-2 infection, wherein the composition comprises a recombinant adenovirus as described above.

In some embodiments, the composition is for preventing a SARS-CoV-2 south Africa strain 501Y.V2 infection or for neutralizing a SARS-CoV-2 south Africa strain 501Y.V2 or for treating a disease associated with a SARS-CoV-2 south Africa strain 501Y.V2 infection.

In another aspect, the invention provides a method of preventing SARS-CoV-2 infection or treating a disease associated with SARS-CoV-2 infection comprising administering to a subject in need thereof an effective amount of the aforementioned recombinant adenovirus or composition.

In some embodiments, the SARS-CoV-2 targeted in the prophylactic or therapeutic method is SARS-CoV-2 south Africa strain 501Y.V2.

In a further aspect, the invention provides the use of the aforementioned recombinant adenovirus or composition in the manufacture of a medicament for the prevention of SARS-CoV-2 infection, or for the neutralization of SARS-CoV-2, or for the treatment of a disease associated with SARS-CoV-2 infection.

In a further aspect, the invention provides a kit for preparing a recombinant adenovirus comprising the aforementioned recombinant plasmid and an adenovirus packaging cell having an adenovirus E1 gene.

In some embodiments, the adenovirus packaging cells used in the kit to package the recombinant adenovirus are HEK293A cells.

The recombinant adenovirus or the composition containing the recombinant adenovirus avoids the defect of pre-existing immunity of a human type 5 adenovirus vector, simultaneously maintains the advantages of high titer and easy production and storage of adenovirus, and provides an effective strategy for the effective treatment of novel coronavirus pneumonia caused by mutant viruses, thereby having wide application prospect.

Drawings

FIG. 1 shows Western Blot results of antigen expression following infection of cells with AdC68-2019S, adC-SV 2, adC68-SV2-GSAS and AdC68 in example one.

FIG. 2 shows the binding curves of serum to protein S after 2 weeks of mice immunized with AdC68-2019S and AdC68-SV2 in example two, step (four).

FIG. 3 shows the logarithmic base 10 ED50 values for binding of serum to S protein after 2 weeks of immunization of mice with AdC68-2019S, adC-SV 2 and AdC68 in example two, step (four).

FIG. 4 is a plot of neutralization of pseudoviruses by serum after 2 weeks of mice immunized with AdC68-2019S and AdC68-SV2 in example two, step (five).

FIG. 5 is a log 10-base neutralization ID50 value of serum against pseudovirus after 2 weeks of mice immunized with AdC68-2019S, adC-SV 2 and AdC68 in step (five) of example two.

Detailed Description

The invention aims to provide a recombinant adenovirus based on chimpanzee adenovirus 68 and a composition comprising the recombinant adenovirus, wherein the recombinant adenovirus comprises a sequence for encoding a heterologous protein, and the heterologous protein encoding sequence is a coding sequence for SARS-CoV-2 south Africa strain 501Y.V2 full-length S protein comprising mutation. The invention also includes administering the recombinant adenovirus composition of the invention to a subject in need thereof to induce effector and memory T cell and B cell immune responses in the subject to treat and/or prevent SARS-CoV-2, particularly SARS-CoV-2 south african strain 501y.v2 infection.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present test, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

As used herein, the terms "control" or "reference" may be used interchangeably and refer to a value used as a comparison standard.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. The polypeptide sequences of the invention may be produced by any suitable means, including recombinant production, chemical synthesis or other synthetic means. Suitable production techniques are well known to those skilled in the art. Alternatively, the peptide may be synthesized by a well-known solid phase peptide synthesis method.

As used herein, "fusion protein" refers to a protein in which the protein includes two or more proteins linked together by peptide bonds or other chemical bonds. Proteins may be linked together directly by peptide bonds or other chemical bonds, or have one or more amino acids between two or more proteins, which are referred to herein as spacers.

As used herein, a "mutation" is a change in a DNA sequence and its encoded protein that results in a change from its natural state. In the present invention, the S protein expressed by the DNA molecule inserted into the adenovirus is a variant of SARS-CoV-2 south African strain 501Y.V2S protein, which comprises one or more mutations in the region from about position 810 to position 990 of its S2 subunit relative to its wild-type S protein, e.g.the presence of F817P, A892 5292 893P, Q895 5298 899P, T912 2 921 54924 922P, A942 946P, S975P, N978 8238 986P, V985 987P mutation (numbering of amino acid positions in the present invention is based on the amino acid numbering of the S protein of the wild-type strain SARS-CoV-2), e.g.the variant K986P+V987P shown in SEQ ID NO:1 (meaning variants having only two mutations of K986P and V987P relative to the wild-type S protein, variants A892P being identical below), the variant A892P+A942P, the variant A892P+A899P, the variant F8172 P+A942P, the variant A99P 99% or the like, the variants 99.99% of which are at least as well as variants 99.99% or as variants of 99.99% of the amino acid sequence.

The SARS-CoV-2 south Africa strain 501Y.V2S protein variant of the present invention may further comprise one or more mutations at its S1 and S2 subunit cleavage sites, e.g. further one or more mutations at the R682, R683 and R685 sites, wherein the R682 site may be mutated to S or G or other amino acids of similar nature, the R683 site may be mutated to S or G or other amino acids of similar nature (see, e.g., stryer et al, biochemistry, 5 th edition, 2002, pages 44-49 for amino acid chemistry). For example, the SARS-CoV-2 south Africa strain 501Y.V2S protein variant can be a variant of SEQ ID NO. 2 or a variant sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to SEQ ID NO. 2.

As used herein, the term "nucleic acid" refers to a polynucleotide, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term should also be understood to include as equivalents analogs of RNA or DNA made from nucleotide analogs, and as applicable to the described embodiments, single-stranded polynucleotides (sense or antisense) and double-stranded polynucleotides.

As used herein, a "vector" is a composition of matter that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Herein, the term "vector" includes autonomously replicating viruses.

As used herein, "adenoviruses" are species-specific and have been isolated from multiple mammalian species of different serotypes (i.e., virus types that are not cross-neutralized by antibodies). For example, more than 50 serotypes have been isolated from humans, and numerous adenoviruses have also been isolated from primates such as chimpanzees, bonobos, rhesus and gorillas. The term "replication-defective" or "replication-defective" adenovirus refers to an adenovirus that is unable to replicate because it has been engineered to contain at least a loss of function (or "loss of function" mutation), i.e., a deletion or mutation that impairs the function of the gene without completely removing the gene, such as the introduction of an artificial stop codon, a deletion or mutation of an active site or interaction domain, a mutation or deletion of a regulatory sequence of the gene, etc., or the complete removal of a gene encoding a gene product necessary for viral replication. Particularly suitably E1 and optionally E3 and/or E4 are deleted. Preferably, the starting adenovirus strain used in the present invention is chimpanzee adenovirus type 68 or non-replicating chimpanzee adenovirus type 68.

As used herein, a "packaging cell" (sometimes also referred to in the industry as a "producer cell" or a "complement cell" or a "host cell") can be any packaging cell that can propagate the desired adenovirus. For example, propagation of recombinant adenovirus vectors is accomplished in packaging cells that compensate for adenovirus defects. These packaging cells preferably have at least the adenovirus E1 sequence in their genome and are thereby able to compensate for recombinant adenoviruses having a deletion of the E1 region. Any E1-compensating packaging cell may be used, for example, including but not limited to HEK293, A549, WEHI, 3T3 cells, preferably HEK293A cells.

The introduction of the vector into the host cell may be accomplished by any means known in the art, including transfection and infection. One or more adenovirus genes may be stably integrated into the genome of the host cell, stably expressed as episomes, or transiently expressed. The gene products may all be transiently expressed, episomally or stably integrated, or some may be stably expressed while others are transiently expressed. The introduction of the vector into the host cell may also be accomplished using techniques known to the skilled artisan. Suitably, standard transfection techniques are used, such as electroporation.

The assembly of selected DNA sequences of adenoviruses (as well as transgenes and other vector elements) into different intermediate plasmids and the use of said plasmids and vectors for the production of recombinant viral particles are accomplished using conventional techniques. Such techniques include conventional cloning techniques of cDNA, the use of overlapping oligonucleotide sequences of the adenovirus genome, the polymerase chain reaction, and any suitable method of providing the desired nucleotide sequence. Standard transfection and co-transfection techniques were used. Other conventional methods employed include homologous recombination of viral genomes, plaque of viruses in agar coats, methods of measuring signal generation, and the like.

It may be a single cell culture or a plurality of continuous cell cultures. When the cell culture is a plurality of continuous cell cultures, the first cell culture is to transfect adenovirus packaging cells with recombinant plasmids, then culture is carried out, and plaque formation can be observed on about 80% of cells cultured; starting from the second cell culture, the steps of each cell culture are: after the culture of the cells of the previous generation is completed, for example, when the cells are cultured to be in a floating state, the cells are collected, the cells are broken by a way such as repeated freezing and thawing, and then the supernatant is centrifugally collected and used for infecting new adenovirus packaging cells, and then the culture is carried out; repeating the steps for a plurality of times, collecting cells, crushing, and purifying the collected supernatant by adopting cesium chloride density gradient centrifugation to obtain virus liquid.

The recombinant adenoviruses of the invention can be formulated as pharmaceutical compositions. Such pharmaceutical compositions may be in a form suitable for administration to a subject, or the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or a combination of these.

Vaccine compositions comprising recombinant adenovirus particles prepared using the adenovirus vectors disclosed herein are useful for inducing immunity against encoded antigen proteins. Vaccines can be formulated using standard techniques and can include, in addition to replication-incompetent adenoviral vectors encoding the desired protein, pharmaceutically acceptable vehicles such as Phosphate Buffered Saline (PBS) or other buffer solutions, as well as other components such as antibacterial and antifungal agents, isotonic and absorption delaying agents, adjuvants, and the like. In some embodiments, the vaccine composition is administered in combination with one or more other vaccines.

As used herein, the term "prevent" means to provide a drug in advance, which may be before exposure to a pathogen (pre-exposure prevention) or before developing symptoms of the disease (post-exposure prevention). The term "treatment" means the administration of a drug during a disease.

Recombinant adenoviruses can be used for gene transfer to a subject in vitro, ex vivo, and in vivo. Recombinant adenoviruses may be administered in vaccine compositions. A vaccine composition as described herein is a composition comprising one or more recombinant adenoviruses capable of inducing an immune response, such as a humoral (e.g., antibodies) and/or cell-mediated (e.g., cytotoxic T-cell) response, upon delivery to a mammal (suitably a human), against a transgene product delivered by the vector. The recombinant adenovirus may comprise (suitably in any of its gene deletions) a gene encoding the desired immunogen and may therefore be used in a vaccine. Recombinant adenoviruses can be used as prophylactic or therapeutic vaccines against the corresponding pathogens.

As used herein, a "subject" or "patient" may be a human or non-human mammal. Non-human mammals include, for example, domestic animals and pets, such as sheep, cattle, swine, canine, feline, and murine mammals. Preferably, the subject is a human. The vaccine composition or pharmaceutical composition of the present invention may be administered by intramuscular injection, intravenous injection, intraperitoneal injection, subcutaneous injection, epidermal administration, intradermal administration, nasal administration, rectal administration or oral administration.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of virus-like particles produced from the vectors of the present invention that is required to prevent a particular condition, or to reduce the severity of a condition or at least one symptom thereof or a condition associated therewith and/or to improve a condition or at least one symptom thereof or a condition associated therewith.

The vectors, compositions and methods of the invention may have one or more of the following improved characteristics over the prior art, including but not limited to higher productivity, increased transgene expression, increased immunogenicity, improved antibody neutralization activity or unique serological cross-reactivity profiles. In particular increased immunogenicity, improved neutralizing activity of antibodies.

The following examples are presented to facilitate a better understanding of the invention and are not intended to limit the scope of the invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were all conventional reagents commercially available from biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Example one, preparation of recombinant Virus

Construction of recombinant plasmid

The pAdC68XY4 pAdC 68-delta E1 (GFP) -delta E3 (SwaI) -E4 (orf 3-6Hu 5) vector is a circular plasmid, and the sequence is shown as SEQ ID NO. 9. The pAdC68XY4 pAdC 68-. DELTA.E1 (GFP) -DELTA.E3 (SwaI) -E4 (orf 3-6Hu 5) vector was derived from Simian adenovirus 25 (NCBI Reference Sequence: AC_ 000011.1), and was modified as follows: both E1 and E3 partial regions are deleted, and the deletion of the E1 partial region prevents the generated recombinant virus from replicating in common cells so as to enhance the biological safety of the recombinant virus, and the deletion of the E3 partial region increases the insertion capacity of the exogenous gene. The pAdC68XY4 pAdC 68-. DELTA.E1 (GFP) -DELTA.E3 (SwaI) -E4 (orf 3-6Hu 5) vector has two SrfI cleavage recognition sequences with the EGFP gene in the small fragment between the two cleavage recognition sequences. When the pAdC68XY4 pAdC 68-. DELTA.E1 (GFP) -DELTA.E3 (SwaI) -E4 (orf 3-6Hu 5) vector was used to construct the recombinant plasmid, an insertion site between the two SrfI cleavage sites was selected as the foreign DNA molecule, and the insertion site was the DELTA.E1 region.

The DNA molecules shown in SEQ ID NO. 3 and 4 are inserted between two SrfI cleavage sites of pAdC68XY4 pAdC68- ΔE1 (GFP) - ΔE3 (SwaI) -E4 (orf 3-6Hu 5) vector by isothermal recombination method respectively, so as to obtain recombinant plasmids pAdC68-SV2 and pAdC68-SV2-GSAS.

The DNA molecule shown in SEQ ID NO. 3 encodes a protein shown in SEQ ID NO. 1, namely full-length SARS-CoV-2 south Africa strain 501Y.V2S protein containing K986P and V987P mutations; the DNA molecule shown in SEQ ID NO. 4 encodes the protein shown in SEQ ID NO. 2, namely the full-length SARS-CoV-2 south African strain 501Y.V2S protein containing K986P, V987P, R682G, R683S and R685S mutations.

According to the sequencing result, in the recombinant plasmids pAdC68-SV2 and pAdC68-SV2-GSAS, the GFP fragment between the two SrfI cleavage sites of the chimpanzee adenovirus vector AdC68 is replaced by the DNA molecules shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, and the insertion positions are all the delta E1 region in the chimpanzee adenovirus vector AdC 68.

(II) preparation of recombinant viruses

Cell culture conditions: 37 ℃ and 5% CO ₂ Is a constant temperature incubator.

1. HEK293A cells were cultured to a cell density of 80% using DMEM medium containing 10% fetal bovine serum.

2. Recombinant plasmids pAdC68-SV2 and pAdC68-SV2-GSAS are taken and digested with restriction enzymes PacI, respectively, to obtain linearized plasmids.

3. Plaque formation was observed by transfecting the cells cultured in step 1 with the linearized plasmid obtained in step 2 with X-tremgeNE HP DNA transfection reagent, culturing for 6 hours (serum-free DMEM medium), and then transferring the cells to DMEM medium containing 10% fetal bovine serum to about 80% cells (about 10-14 days).

4. After the completion of step 3, the cells were collected, resuspended in serum-free DMEM medium, and then repeatedly frozen and thawed 3 times, and then centrifuged at 4℃and 3000g for 10 minutes to collect the supernatant (P0 generation supernatant).

5. HEK293A cells were cultured to a cell density of 80% using DMEM medium containing 10% fetal bovine serum.

6. The cells cultured in step 5 were infected with the P0-generation supernatant, and the infected cells were cultured until the vast majority of the cells were in a floating state (about 24-48 hours).

7. After completion of step 6, the cells were collected, resuspended in serum-free DMEM medium, and then repeatedly frozen and thawed 3 times, and then centrifuged at 4℃and 3000g for 10 minutes to collect the supernatant (P1 generation supernatant).

8. HEK293A cells were cultured to a cell density of 80% using DMEM medium containing 10% fetal bovine serum.

9. The cells cultured in step 8 were infected with the P1-generation supernatant, and the infected cells were cultured until the vast majority of the cells were in a floating state (about 24-48 hours).

10. After completion of step 9, the cells were collected, resuspended in serum-free DMEM medium, and then repeatedly frozen and thawed 3 times, and then centrifuged at 4℃and 3000g for 10 minutes to collect the supernatant (P2-generation supernatant).

11. HEK293A cells were cultured to a cell density of 80% using DMEM medium containing 10% fetal bovine serum.

12. The cells cultured in step 11 were infected with the P2-generation supernatant, and the infected cells were cultured until the vast majority of the cells were in a floating state (about 24 to 48 hours).

13. After completion of step 12, the cells were collected, resuspended in serum-free DMEM medium, and then repeatedly frozen and thawed 3 times, and then centrifuged at 4℃and 3000g for 10 minutes to collect the supernatant (P3-generation supernatant).

14. Taking the P3 generation supernatant, and carrying out cesium chloride density gradient centrifugation and purification to obtain virus fluids, namely AdC68-SV2 and AdC68-SV2-GSAS virus fluids respectively.

15. Extracting genome DNA from AdC68-SV2 and AdC68-SV2-GSAS virus solutions. The genomic DNA is digested with restriction enzyme MfeI, and recombinant plasmids pAdC68-SV2 and pAdC68-SV2-GSAS recovered by the restriction enzyme PacI after the restriction enzyme MfeI is digested are used as positive controls of the genomic DNA. The positive control showed 10 bands after cleavage and the genomic DNA showed the same size of 10 bands after cleavage. The results showed that the correct recombinant virus was obtained.

(III) preparation of control virus

The recombinant plasmid was replaced with chimpanzee adenovirus vector pAdC68XY4 pAdC 68-. DELTA.E1 (GFP) -DELTA.E3 (SwaI) -E4 (orf 3-6Hu 5), as in step (two) of this example. The virus liquid obtained in the step 14 is AdC68 virus liquid.

The recombinant plasmid AdC68-2019S was prepared by the same method as in step (one) of this example, namely, the SARS-CoV-2 wild strain S protein sequence (SEQ ID NO: 10) was used to replace the GFP in the pAdC68XY4 pAdC 68-. DELTA.E1 (GFP) -DELTA.E3 (SwaI) -E4 (orf 3-6Hu 5) vector, to obtain the recombinant plasmid AdC68-2019S, and then the virus solution was obtained in step 14 by the method of step (two) of this example, namely, the AdC68-2019S virus solution.

Identification of viruses

1. Titer identification

Titers of AdC68-SV2, adC68-SV2-GSAS, adC68-2019S and AdC68 virus solutions were detected by absorbance methods.

Viral titer = OD260 x dilution x 1.1 x 10 ¹² 。

The unit of viral titer is the number of viral particles per milliliter (vp/mL)

The titer of the AdC68-SV2 virus liquid is 1.3X10 ¹³ vp/mL。

The titer of the AdC68-SV2-GSAS virus liquid is 1.9X10 ¹³ vp/mL。

The titer of the AdC68-2019S virus liquid is 1.1X10 ¹³ vp/mL。

The titer of the AdC68 virus liquid is 1.5X10 ¹³ vp/mL。

2. Identification of antigen expression

(1) HEK293A cells were grown at 5X 10 ⁵ Six well plates were seeded at a cell/well density and cultured to a cell density of about 90%.

(2) Infecting the cells cultured in step (1) with AdC68-SV2 and AdC68-SV2-GSAS virus at a dose of 10 per virus ¹⁰ vp/well, 3 replicates per dose were set. The cells were cultured for 24 hours and then collected.

(3) Infecting the cells cultured in step (1) with AdC68-2019S and AdC68 virus solution at a dose of 10 ¹⁰ vp/well, set 3 replicates. The cells were cultured for 24 hours and then collected.

(4) And (3) taking the cells collected in the step (2) and the step (3), performing cell lysis, collecting supernatant, performing polyacrylamide gel electrophoresis, and performing Western Blot (the primary antibody adopts a rabbit polyclonal antibody against SARS-CoV-2S). Beta-actin protein was used as an internal reference.

The information for the rabbit polyclonal antibody against SARS-CoV-2S is as follows: SARS-CoV-2 (2019-nCoV) Spike RBD Antibody, rabbit PAb: yiqiaoshenzhou 405892-T62.

The results show that: adC68-SV2 can express S protein with correct size, and meanwhile, shed S1 protein exists; adC68-SV2-GSAS also expressed S protein of the correct size, and did not produce shed S1 protein (see FIG. 1).

Example two use of recombinant adenoviruses

Animal immunization

Female BALB/C mice of 6 weeks of age were divided into four groups of 10 animals each, treated as follows:

first group (G1 group): a single immunization is carried out by intramuscular injection, and the immune substance is AdC68-SV2 virus liquid (virus amount is 2×10) ¹⁰ vp)；

Second group (G2 group): single immunization by intramuscular injection with AdC68-SV2-GSAS virus solution (viral load 2×10) ¹⁰ vp)；

Third group (G3 group): single immunization by intramuscular injection with AdC68-2019S virus solution (virus amount 2×10) ¹⁰ vp)；

Fourth group (G4 group): single immunization by intramuscular injection with AdC68 virus solution (virus amount of 2×10) ¹⁰ vp)；

In each of the above groups, the volume of the intramuscular immunization was 100. Mu.l, and the virus concentration was adjusted by using PBS buffer at pH7.2 as a solvent.

Blood collection was started at week 2 after the primary immunization, and blood was collected every two weeks (cheek blood collection).

Preparation of SARS-CoV-2 pseudovirus

The double-stranded DNA molecule shown in SEQ ID NO. 5 (encoding gene of SARS-CoV-2 south Africa strain 501Y.V2 full-length S protein) is inserted between BamHI and EcoRI cleavage sites of pcDNA3.1 (+) vector to obtain SARS-CoV-2 south Africa strain 501Y.V2S protein plasmid. The double-stranded DNA molecule shown in SEQ ID NO. 6 (encoding gene of SARS-CoV-2 wild strain full-length S protein) is inserted between BamHI and EcoRI cleavage sites of pcDNA3.1 (+) vector to obtain SARS-CoV-2 wild strain S protein plasmid.

The SARS-CoV-2 south Africa strain 501Y.V2S protein plasmid or SARS-CoV-2 wild strain S protein plasmid and skeleton plasmid pNL4-3R-E-luciferase (He J, choke S, walker R, di Marzio P, morgan DO, landau NR.J Virol 69:6705-6711,1995) are used for co-transfection of 293T cells, and after incubation, SARS-CoV-2 south Africa strain 501Y.V2 pseudotype virus and SARS-CoV-2 wild strain pseudotype virus with infectivity similar to that of live virus can be obtained. The method comprises the following steps: the preparation method comprises the steps of co-transfecting 293T cells with SARS-CoV-2 south Africa strain 501Y.V2S protein plasmid or SARS-CoV-2 wild strain S protein plasmid and skeleton plasmid pNL4-3R-E-luciferase, standing at 37 ℃ for incubation, and collecting cell culture supernatant after 48 hours of transfection, namely virus liquid containing SARS-CoV-2 south Africa strain 501Y.V2 pseudovirus (called SARS-CoV-2 south Africa strain 501Y.V2 virus liquid for short) and virus liquid containing SARS-CoV-2 wild strain pseudovirus (called SARS-CoV-2 wild strain virus liquid for short). ELISA kit for quantitative detection of SARS-CoV-2 south Africa strain 501Y.V2 virus liquid and SARS-CoV-2 wild strain virus liquid virus titer, OD of SARS-CoV-2 south Africa strain virus liquid by using P24 quantitative detection kit (HIV P24 antigen quantitative detection kit, KEY-BIO, 96T) _450nm The absorbance was 1 (1021 TCID 50/ml), and the OD of SARS-CoV-2 wild strain virus solution _450nm The absorbance was 1.7 (1735.7TCID50/ml), with a higher absorbance indicating a higher virus content.

Preparation of SARS-CoV-2 south Africa strain 501Y.V2S protein and SARS-CoV-2 wild strain S protein

1. The double-stranded DNA molecule shown in SEQ ID NO. 7 and the double-stranded DNA molecule shown in SEQ ID NO. 8 are respectively inserted into NotI and NheI cleavage sites of the pcDNA3.1 plasmid to obtain recombinant plasmids.

2. The recombinant plasmid obtained in step 1 was transfected into 293F cells grown to 90% density with PEI transfection reagent and incubated for 72 hours.

3. After the step 2 is completed, collecting supernatant, purifying protein by using a nickel column, and collecting protein solution.

4. Concentrating and system replacement are carried out on the protein solution obtained in the step 3, and the protein system is replaced by PBS buffer solution with pH of 7.2, so as to obtain SARS-CoV-2 south Africa strain 501Y.V2S protein solution or SARS-CoV-2 wild strain S protein solution.

(IV) detection of vaccine-induced Total antibodies

Taking the blood sample obtained in the step (one) of the embodiment, separating serum, and detecting total IgG by ELISA. In total IgG assay, the ELISA plate (100 ng/well) was coated with SARS-CoV-2 south Africa strain 501Y.V2S protein, serum was diluted to 200 volumes and then subjected to 3-fold gradient dilutions (200, 600, 1800, 5400, 16200, 48600, 145800 and 437400 for a total of 8 dilutions in PBS buffer pH 7.2) with Anti-mouse IgG HRP as the secondary antibody.

The ED50 values of the sera of each group of mice at week 2 are shown in Table 1 (N.D represents negative detection).

TABLE 1

Serum binding curves at week 2 for each group of mice are shown in figure 2, and log histograms with the ED50 values of each group at the bottom of 10 are shown in figure 3.

The results show that the serum immunized by the recombinant adenovirus of the invention can be very well combined with SARS-CoV-2 south Africa strain 501Y.V2S protein and SARS-CoV-2 wild strain S protein, and the combination of the serum and the SARS-CoV-2 south Africa strain 501Y.V2S protein is stronger than that of the serum immunized by AdC 68-2019S.

(V) detection of neutralizing Activity of antibodies in serum of animals after vaccine immunization

The solution to be measured is: taking the blood sample obtained in the step (one) of the embodiment, and separating the obtained serum.

1. The test solution was diluted 48-fold with DMEM medium containing 10% fbs, and then diluted 3-fold in a gradient, and dilutions with different serum concentrations were sequentially obtained (8 dilutions total of 48, 144, 432, 1296, 3888, 11664, 34992 and 104976).

2. 100 μl of the dilution obtained in step 1 was mixed with 50 μl of SARS-CoV-2 south Africa strain virus solution prepared in step (II) of this example or SARS-CoV-2 wild strain virus solution (virus content 100TCID 50), and incubated at 37deg.C for 1 hr. A blank was set up with 100 μl of DMEM medium containing 10% fbs instead of 100 μl of diluent.

3. After completion of step 2, 50. Mu.l of Huh7 cell broth (approximately 2X 10 in volume ⁴ Huh7 cells), and incubating at 37℃for 48 hours (in practical applications, 48-72 hours may be used).

4. After completion of step 3, 100. Mu.l of PBS buffer and 50. Mu.l of cell lysate (Bright-Glo ^TM Luciferase Assay System, promega, E2650), left for 2min, and then luciferase activity was detected with a chemiluminescent instrument.

3 duplicate wells were set for each treatment and the results averaged.

Neutralization activity= (fluorescence intensity of blank control-fluorescence intensity of experimental group added with diluent)/fluorescence intensity of blank control x 100%.

The corresponding serum dilution at 50% neutralization activity was ID50.

The ID50 values of the sera of each group of mice at week 2 are shown in Table 2 (N.D represents negative detection).

TABLE 2

Group of	ID50 against NanfAfrican pseudovirus	ID50 against wild strain pseudovirus
			G1 group	1690.3	119.9
G3 group	66.7	328.3
			G4 group	N.D.	N.D.

Serum neutralization plots for week 2 of each group of mice are shown in fig. 4, and a log histogram of each group of ID50 values, base 10, is shown in fig. 5.

The result shows that the recombinant adenovirus immune serum of the invention can well neutralize SARS-CoV-2 south Africa strain 501Y.V2 pseudovirus and SARS-CoV-2 wild strain virus liquid, and the neutralization of SARS-CoV-2 south Africa strain 501Y.V2 pseudovirus is stronger than that of AdC68-2019S immune serum.

Claims

1. A SARS-CoV-2 south Africa strain 501Y.V2S protein variant has the amino acid sequence shown in SEQ ID NO. 1 or has the amino acid sequence shown in SEQ ID NO. 2.

2. A DNA molecule encoding the SARS-CoV-2 south africa strain 501y.v2s protein variant of claim 1.

3. The DNA molecule according to claim 2, wherein the nucleotide sequence is shown in SEQ ID NO. 3.

4. The DNA molecule according to claim 2, wherein the nucleotide sequence is shown in SEQ ID NO. 4.

5. A recombinant plasmid obtained by inserting a DNA molecule according to any one of claims 2 to 4 into the Δe1 region of the chimpanzee adenovirus vector AdC 68.

6. A recombinant adenovirus obtained by transfecting an adenovirus packaging cell having an adenovirus E1 gene with the recombinant plasmid of claim 5 and culturing the transfected cell.

7. The recombinant adenovirus of claim 6, wherein the adenovirus packaging cell is a HEK293A cell.

8. A composition for preventing infection by SARS-CoV-2 or for neutralizing SARS-CoV-2 or for treating a disease associated with infection by SARS-CoV-2, the composition comprising the recombinant adenovirus of claim 6.

9. The composition of claim 8, wherein the SARS-CoV-2 is SARS-CoV-2 south african strain 501y.v2.

10. Use of the recombinant adenovirus of claim 6 or the composition of claim 8 in the manufacture of a medicament for preventing SARS-CoV-2 infection, or for neutralizing SARS-CoV-2, or for treating a disease associated with SARS-CoV-2 infection.

11. A kit for preparing a recombinant adenovirus comprising the recombinant plasmid of claim 5 and an adenovirus packaging cell having an adenovirus E1 gene.

12. The kit of claim 11, wherein the adenovirus packaging cell is a HEK293A cell.