CN115160413B - Novel coronavirus vaccine - Google Patents

Abstract

The present invention provides a recombinant adenovirus for preventing and/or treating SARS-CoV-2 South African strain 501Y.V2 infection and a vaccine composition containing the recombinant adenovirus. The recombinant adenovirus is obtained by transfecting adenovirus packaging cells with the adenovirus E1 gene using a recombinant plasmid that contains mutations in its S2 subunit and/or at the S1 and S2 cleavage sites and culturing the cells. The DNA molecule encoding the 501Y.V2S protein of the SARS-CoV-2 South African strain was inserted into the △E1 region of the chimpanzee adenovirus vector AdC68. The vaccine developed by this invention against the new coronavirus SARS-CoV-2 South African strain 501Y.V2 provides the possibility of a radical cure for new coronavirus pneumonia.

Description

A novel coronavirus vaccine

Technical field

The present invention relates to a novel coronavirus vaccine, specifically, to a novel coronavirus vaccine based on chimpanzee adenovirus type 68 and SARS-CoV-2 South African strain 501Y.V2 S protein.

Background technique

The novel coronavirus (SARS-CoV-2) is a coronavirus isolated from patients with pneumonia of unknown origin. Most patients infected with SARS-CoV-2 will develop severe respiratory illness, and their clinical symptoms are very similar to the disease caused by SARS-CoV in the 2003 outbreak. Many viral mutant strains have emerged during the spread of SARS-CoV-2. Among them, the mutated virus 501Y.V2 that appeared in South Africa was first discovered in South Africa in mid-October 2020, and became the main strain circulating locally from early November 2020. Because the South African strain has an important mutation site in the receptor-binding domain of the membrane protein (S protein), the mutant strain is more infectious. Studies have proven that the South African strain of the virus can evade the efficacy of existing COVID-19 vaccines.

SARS-CoV-2 uses the membrane protein S (also known as S protein) on its surface to enter susceptible cells. The S protein consists of the following three domains: the S1 domain located at the N-terminus, the S2 domain located at the proximal membrane end, and the transmembrane domain. The susceptibility of SARS-CoV-2 to host cells is determined by the receptor binding domain RBD on the S1 domain.

Contents of the invention

One aspect of the present invention provides a SARS-CoV-2 South African strain 501Y.V2 S protein variant. Compared with its wild-type S protein, the S protein variant contains one or more mutations in its S2 subunit. The S protein The variant may further comprise one or more mutations in its S1 and S2 subunit cleavage sites.

In some embodiments, the mutations in the S2 subunit of the SARS-CoV-2 South African strain 501Y.V2 S protein variant include K986P and/or V987P.

In some embodiments, the mutation sites of the S1 and S2 subunit cleavage sites of the SARS-CoV-2 South African strain 501Y.V2 S protein variant include one or more of R682, R683 and R685.

In some embodiments, the mutation at the R682 site of the SARS-CoV-2 South African strain 501Y.V2 S 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 African strain 501Y.V2 S protein variant is as shown in SEQ ID NO: 1, or is at least 90%, 95%, 96% identical to SEQ ID NO: 1 , 97%, 98%, 99% or 99.9% identity.

In some embodiments, the sequence of the SARS-CoV-2 South African strain 501Y.V2 S protein variant is as shown in SEQ ID NO: 2, or is at least 90%, 95%, 96% identical to SEQ ID NO: 2 , 97%, 98%, 99% or 99.9% identity.

Another aspect of the present invention also provides a DNA molecule encoding the aforementioned SARS-CoV-2 South African strain 501Y.V2S protein variant.

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

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

Yet another aspect of the present invention provides a recombinant plasmid, which is obtained by inserting the aforementioned DNA molecule into the ΔE1 region of the chimpanzee adenovirus vector AdC68.

Another aspect of the present invention provides a recombinant adenovirus, which is obtained by transfecting adenovirus packaging cells with the adenovirus E1 gene using the aforementioned recombinant plasmid, and cultivating the transfected cells.

In some embodiments, the adenovirus packaging cells are HEK293A cells.

Another aspect of the present invention provides a composition for preventing SARS-CoV-2 infection or for neutralizing SARS-CoV-2 or treating SARS-CoV-2 infection-related diseases, wherein the composition contains the aforementioned Recombinant adenovirus.

In some embodiments, the composition is used to prevent SARS-CoV-2 South African strain 501Y.V2 infection or to neutralize SARS-CoV-2 South African strain 501Y.V2 or to treat SARS-CoV-2 South African strain 501Y. Diseases related to V2 infection.

Another aspect of the present invention provides a method for preventing SARS-CoV-2 infection or treating SARS-CoV-2 infection-related diseases, including administering an effective amount of the aforementioned recombinant adenovirus or composition to a subject in need.

In some embodiments, the SARS-CoV-2 targeted in the preventive or therapeutic method is SARS-CoV-2 South African strain 501Y.V2.

Another aspect of the present invention provides the aforementioned recombinant adenovirus or composition for use in preventing SARS-CoV-2 infection, or for neutralizing SARS-CoV-2, or for treating diseases related to SARS-CoV-2 infection. Uses in medicines.

Yet another aspect of the present invention provides a kit for preparing recombinant adenovirus, which includes the aforementioned recombinant plasmid and adenovirus packaging cells containing the adenovirus E1 gene.

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

The recombinant adenovirus of the present invention or the composition containing it avoids the defects of pre-existing immunity of human type 5 adenovirus vector, while retaining the advantages of high titer and easy production and storage of adenovirus, and is effective against new coronavirus pneumonia caused by mutant viruses. Effective treatment strategies have been proposed, which have broad application prospects.

Description of the drawings

Figure 1 shows the Western Blot results of antigen expression after AdC68-2019S, AdC68-SV2, AdC68-SV2-GSAS and AdC68 infected cells in Example 1.

Figure 2 is the binding curve of serum to S protein 2 weeks after AdC68-2019S and AdC68-SV2 immunized mice in step (4) of Example 2.

Figure 3 is the base 10 logarithmic result of the ED50 value of serum binding to S protein in mice immunized with AdC68-2019S, AdC68-SV2 and AdC68 in step (4) of Example 2 2 weeks later.

Figure 4 is the neutralization curve of serum against pseudovirus 2 weeks after immunizing mice with AdC68-2019S and AdC68-SV2 in step (5) of Example 2.

Figure 5 is the base 10 logarithmic result of the ID50 value of the pseudovirus neutralized by the serum of mice immunized with AdC68-2019S, AdC68-SV2 and AdC68 for 2 weeks in step (5) of Example 2.

Detailed ways

The object of the present invention is to provide a recombinant adenovirus based on chimpanzee adenovirus type 68 and a composition containing the recombinant adenovirus. The recombinant adenovirus contains a sequence encoding a heterologous protein. The heterologous protein encoding sequence is SARS containing mutations. -Coding sequence of the full-length S protein of CoV-2 South African strain 501Y.V2. The invention also includes administering to a subject in need thereof a recombinant adenoviral composition of the invention to induce effector and memory T cell and B cell immune responses in the subject to treat and/or prevent SARS-CoV-2, in particular Infection with SARS-CoV-2 South African strain 501Y.V2.

Unless otherwise defined, 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 testing of the present invention, 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" are used interchangeably and refer to a value used as a standard for comparison.

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 containing 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, peptides can be synthesized by well-known solid-phase peptide synthesis methods.

"Fusion protein" as used herein refers to one 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 they may have one or more amino acids between two or more proteins, referred to herein as a spacer.

"Mutation" as used herein is a change in a DNA sequence and the protein it encodes that results in an alteration from its native state. In the present invention, the S protein expressed by the DNA molecule inserted into the adenovirus is the SARS-CoV-2 South African strain 501Y.V2 S protein variant. Compared with its wild-type S protein, the S protein variant has a higher concentration in its S2 subunit. The segment from approximately position 810 to position 990 contains one or more mutations, for example, one of the mutations F817P, A892P, A893P, Q895P, A899P, T912P, K921P, L922P, A942P, G946P, S975P, N978P, K986P, V987P or more (the numbering of the amino acid sites in the present invention is based on the amino acid numbering of the S protein of the wild-type SARS-CoV-2 strain), for example, the variant K986P+V987P shown in SEQ ID NO: 1 (referring to the wild-type S protein The protein only has two mutations of K986P and V987P, the same below), variant A892P+A942P, variant A892P+A899P+A942P, variant F817P+A892P+A942P, variant F817P+A892P+A899P+A942P, etc., Or a variant sequence that is at least 90%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to these variants.

The SARS-CoV-2 South African strain 501Y.V2 S protein variant of the present invention may further include one or more mutations at its S1 and S2 subunit cleavage sites, for example, there may be one or more mutations at the R682, R683 and R685 sites. Multiple mutations, in which the R682 position can be mutated to S or G or other amino acids with similar properties, the R683 position can be mutated to S or other amino acids with similar properties, and the R685 position can be mutated to S or G or other amino acids with similar properties ( Regarding the chemical properties of amino acids see, for example, Stryer et al., Biochemistry, 5th edition, 2002, pp. 44-49). For example, the SARS-CoV-2 South African strain 501Y.V2 S protein variant may be a variant of SEQ ID NO:2, or may be at least 90%, 95%, 96%, or 97% identical to SEQ ID NO:2. , 98%, 99% or 99.9% identical variant sequences.

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

As used herein, a "vector" is a composition that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into 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. As used herein, the term "vector" includes autonomously replicating viruses.

"Adenovirus" as used herein is species specific, and different serotypes (i.e., virus types that are not cross-neutralized by antibodies) have been isolated from multiple mammalian species. For example, more than 50 serotypes have been isolated from humans, and numerous adenoviruses have been isolated from primates such as chimpanzees, bonobos, rhesus monkeys, and gorillas. The term "replication-deficient" or "replication-deficient" 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., without completely removing the gene Deletion or mutation that impairs the function of the gene, such as the introduction of artificial stop codons, deletion or mutation of the active site or interaction domain, mutation or deletion of the regulatory sequence of the gene, etc., or encoding the virus necessary for replication Complete removal of the gene product of the gene. 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 "complementing cell" or "host cell") can be any packaging cell that can propagate the desired adenovirus. For example, propagation of recombinant adenoviral vectors is accomplished in packaging cells that compensate for adenovirus deficiencies. These packaging cells preferably have at least the adenovirus E1 sequence in their genome and are thereby able to compensate for recombinant adenoviruses with deletions of the E1 region. Any El compensation packaging cell can be used, for example, including but not limited to HEK293, A549, WEHI, 3T3 cells, preferably HEK293A cells.

Introduction of the vector into the host cell can be accomplished by any means known in the art, including transfection and infection. One or more adenoviral genes can be stably integrated into the genome of the host cell, stably expressed as an episome, or transiently expressed. The gene products may all be transiently expressed, episomal or stably integrated, or some gene products may be stably expressed while other gene products are transiently expressed. Introduction of the vector into the host cell can also be accomplished using techniques known to the skilled person. Suitably, standard transfection techniques such as electroporation are used.

The assembly of selected DNA sequences of adenovirus (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, use of overlapping oligonucleotide sequences from adenoviral genomes, polymerase chain reaction, and any suitable method that provides the desired nucleotide sequence. Standard transfection and co-transfection techniques were used. Other conventional methods employed include homologous recombination of viral genomes, plaques of viruses in agar overlays, methods of measuring signal production, etc.

Single cell culture or multiple continuous cell cultures can be used. When the cell culture is multiple continuous cell cultures, the first cell culture is to transfect adenovirus packaging cells with recombinant plasmid, and then culture until about 80% of the cells can be observed to form plaques; from the second time, When cell culture begins, the steps for each cell culture are: after the previous generation of cells has been cultured, for example, when most of the cells are cultured in a floating state, the cells are collected, broken by repeated freezing and thawing, and then centrifuged to collect the supernatant. The supernatant is used to infect new adenovirus packaging cells, and then cultured; after repeated many times, the cells are collected and broken, and the collected supernatant is purified using cesium chloride density gradient centrifugation to obtain virus liquid.

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

Vaccine compositions comprising recombinant adenoviral particles prepared using the adenoviral vectors disclosed herein can be used to induce immunity against the encoded antigenic protein. Vaccines may be formulated using standard techniques and may include, in addition to a replication-incompetent adenoviral vector encoding the desired protein, a pharmaceutically acceptable vehicle, such as phosphate buffered saline (PBS) or other buffered solutions, and other components, Such as antibacterial and antifungal agents, isotonic and absorption delaying agents, adjuvants, etc. In some embodiments, the vaccine composition is administered in combination with one or more other vaccines.

As used herein, the term "prophylaxis" means providing the drug in advance, either before exposure to the pathogen (pre-exposure prophylaxis) or before the development of disease symptoms (post-exposure prophylaxis). The term "treatment" means the administration of a drug during a disease.

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

A "subject" or "patient" as used herein may be a human or non-human mammal. Non-human mammals include, for example, domestic animals and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human. The vaccine composition or pharmaceutical composition of the present invention can 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 invention that is necessary to prevent a particular condition, or to reduce the condition or at least one symptom thereof or the severity of a condition associated therewith and/or an amelioration of a condition or at least one symptom thereof or a condition associated therewith.

The vectors, compositions and methods of the present 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, improved immunogenicity, improved antibody and active or unique serological cross-reactivity profiles. In particular, improved immunogenicity, improved antibody neutralizing activity.

The following examples facilitate a better understanding of the invention but are not meant to limit the scope of the invention in any way.

The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, are all conventional reagents available from biochemical reagent stores. The quantitative experiments in the following examples were repeated three times, and the results were averaged.

Example 1. Preparation of recombinant virus

(1) Construction of recombinant plasmid

pAdC68XY4 pAdC68-ΔE1(GFP)-ΔE3(Swal)-E4(orf3-6Hu5) vector is a circular plasmid, and the sequence is shown in SEQ ID NO: 9. pAdC68XY4 pAdC68-ΔE1(GFP)-ΔE3(SwaI)-E4(orf3-6Hu5) vector is derived from Simian adenovirus 25 (NCBI Reference Sequence:AC_000011.1) and has been modified as follows: E1 and E3 parts have been deleted, and E1 Partial deletion of the region prevents the resulting recombinant virus from replicating in ordinary cells to enhance its biological safety. Partial deletion of the E3 region increases the insertion capacity of foreign genes. pAdC68XY4 pAdC68-ΔE1(GFP)-ΔE3(Swal)-E4(orf3-6Hu5) vector has two SrfI restriction recognition sequences, and the small fragment between the two restriction recognition sequences contains the EGFP gene. When constructing the recombinant plasmid using pAdC68XY4 pAdC68-ΔE1(GFP)-ΔE3(SwaI)-E4(orf3-6Hu5) vector, select the space between the two SrfI restriction sites as the insertion site for foreign DNA molecules. The insertion position is △E1 area.

The DNA molecules shown in SEQ ID NO: 3 and 4 were inserted between the two SrfI restriction sites of the pAdC68XY4 pAdC68-ΔE1(GFP)-ΔE3(Swal)-E4(orf3-6Hu5) vector through the isothermal recombination method. Recombinant plasmids pAdC68-SV2 and pAdC68-SV2-GSAS were obtained.

The DNA molecule shown in SEQ ID NO:3 encodes the protein shown in SEQ ID NO:1, that is, the full-length SARS-CoV-2 South African strain 501Y.V2 S protein containing K986P and V987P mutations; shown in SEQ ID NO:4 The DNA molecule encodes the protein shown in SEQ IDNO:2, which is the full-length SARS-CoV-2 South African strain 501Y.V2 S protein containing K986P, V987P, R682G, R683S and R685S mutations.

According to the sequencing results, in the recombinant plasmids pAdC68-SV2 and pAdC68-SV2-GSAS, the two SrfI enzyme cutting sites of the chimpanzee adenovirus vector AdC68 were replaced with the DNA molecules shown in SEQ ID NO:3 and SEQ ID NO:4 respectively. The insertion position of the GFP fragments between the dots is the ΔE1 region in the chimpanzee adenovirus vector AdC68.

(2) Preparation of recombinant viruses

Cell culture conditions: 37°C, 5% _CO2 constant temperature incubator.

1. Use DMEM medium containing 10% fetal bovine serum to culture HEK293A cells until the cell density reaches 80%.

2. Take the recombinant plasmids pAdC68-SV2 and pAdC68-SV2-GSAS and digest them with restriction endonuclease PacI respectively to obtain linearized plasmids.

3. Use X-tremeGENE HP DNA transfection reagent to transfect the cells cultured in step 1 with the linearized plasmid obtained in step 2, and culture for 6 hours (serum-free DMEM medium), then transfer the cells to a medium containing 10% Culture in DMEM medium with fetal calf serum until about 80% of the cells can observe plaque formation (about 10-14 days).

4. After step 3 is completed, collect the cells, resuspend them in serum-free DMEM medium, freeze and thaw repeatedly three times, and then centrifuge at 4°C and 3000g for 10 minutes to collect the supernatant (P0 generation supernatant).

5. Use DMEM medium containing 10% fetal bovine serum to culture HEK293A cells until the cell density reaches 80%.

6. Use the P0 generation supernatant to infect the cells cultured in step 5, and culture the infected cells until most of the cells are in a floating state (about 24-48 hours).

7. After step 6 is completed, collect the cells, resuspend them in serum-free DMEM medium, freeze and thaw repeatedly three times, and then centrifuge at 4°C and 3000g for 10 minutes to collect the supernatant (P1 generation supernatant).

8. Use DMEM medium containing 10% fetal bovine serum to culture HEK293A cells until the cell density reaches 80%.

9. Use the P1 generation supernatant to infect the cells cultured in step 8, and culture the infected cells until most of the cells are in a floating state (about 24-48 hours).

10. After step 9 is completed, collect the cells, resuspend them in serum-free DMEM medium, freeze and thaw repeatedly three times, and then centrifuge at 4°C and 3000g for 10 minutes to collect the supernatant (P2 generation supernatant).

11. Use DMEM medium containing 10% fetal calf serum to culture HEK293A cells until the cell density reaches 80%.

12. Use the P2 generation supernatant to infect the cells cultured in step 11, and culture the infected cells until most of the cells are in a floating state (about 24-48 hours).

13. After step 12 is completed, collect the cells, resuspend them in serum-free DMEM medium, freeze and thaw repeatedly three times, and then centrifuge at 4°C and 3000g for 10 minutes to collect the supernatant (P3 generation supernatant).

14. Take the P3 generation supernatant and perform cesium chloride density gradient centrifugation purification to obtain virus liquids, namely AdC68-SV2 and AdC68-SV2-GSAS virus liquids respectively.

15. Take the AdC68-SV2 and AdC68-SV2-GSAS virus liquids respectively and extract the genomic DNA. Genomic DNA was digested with restriction endonuclease MfeI, and the recombinant plasmids pAdC68-SV2 and pAdC68-SV2-GSAS that had been digested with restriction endonuclease PacI and recovered from the gel were used as genomic DNA. Positive control. The positive control showed 10 bands after digestion, and the genomic DNA showed 10 bands of the same size after digestion. The results showed that the correct recombinant virus was obtained.

(3) Preparation of control virus

Chimpanzee adenovirus vector pAdC68XY4 pAdC68-ΔE1(GFP)-ΔE3(Swal)-E4(orf3-6Hu5) was used instead of the recombinant plasmid, and the other steps were the same as step (2) of this example. Obtain the virus liquid in step 14, which is the AdC68 virus liquid.

The AdC68-2019S recombinant plasmid was prepared using the same method as step (1) of this example, that is, using the SARS-CoV-2 wild strain S protein sequence (SEQ ID NO: 10) to replace pAdC68XY4 pAdC68-ΔE1(GFP)-ΔE3( SwaI)-E4(orf3-6Hu5) vector to obtain the recombinant plasmid AdC68-2019S, and then use the method of step (2) of this embodiment to obtain the virus liquid in step 14, which is the AdC68-2019S virus liquid.

(4) Identification of viruses

1. Titer identification

The titers of AdC68-SV2, AdC68-SV2-GSAS, AdC68-2019S and AdC68 virus liquids were detected using the absorbance photometric method.

Virus titer=OD260×dilution factor×1.1×10 ¹² .

The unit of virus titer is virus particles per milliliter (vp/mL)

The titer of AdC68-SV2 virus liquid is 1.3×10 ¹³ vp/mL.

The titer of the AdC68-SV2-GSAS virus liquid was 1.9×10 ¹³ vp/mL.

The titer of AdC68-2019S virus liquid is 1.1×10 ¹³ vp/mL.

The titer of AdC68 virus liquid is 1.5×10 ¹³ vp/mL.

2. Identification of antigen expression

Cell culture conditions: 37°C, 5% _CO2 constant temperature incubator.

① Inoculate HEK293A cells into a six-well plate at a density of 5×10 ⁵ cells/well and culture until the cell density is about 90%.

② Infect the cells cultured in step ① with AdC68-SV2 and AdC68-SV2-GSAS virus liquids. The infection dose of each virus liquid is 10 ¹⁰ vp/well, and 3 replicate treatments are set for each dose. Incubate for 24 hours, then collect cells.

③ Use AdC68-2019S and AdC68 virus liquid to infect the cells cultured in step ①. The infection dose is 10 ¹⁰ vp/well, and 3 repeated treatments are set. Incubate for 24 hours, then collect cells.

④ Take the cells collected in steps ② and ③, perform cell lysis, and then collect the supernatant for polyacrylamide gel electrophoresis, and then perform Western Blot (the primary antibody uses anti-SARS-CoV-2S rabbit polyclonal antibody). β-actin protein served as internal reference.

The information of rabbit polyclonal antibodies against SARS-CoV-2S is as follows: SARS-CoV-2 (2019-nCoV) Spike RBD Antibody, Rabbit PAb: Yiqiao Shenzhou, 405892-T62.

The results show that: AdC68-SV2 can express the S protein of the correct size without producing the shed S1 protein; AdC68-SV2-GSAS can also express the S protein of the correct size without producing the shed S1 protein (see Figure 1).

Example 2. Application of recombinant adenovirus

(1) Animal immunity

Six-week-old female BALB/C mice were divided into four groups, with 10 mice in each group, and were treated as follows:

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

The second group (G2 group): A single immunization was carried out by intramuscular injection, and the immune substance was AdC68-SV2-GSAS virus liquid (the virus amount was 2×10 ¹⁰ vp);

The third group (G3 group): A single immunization was carried out by intramuscular injection, and the immune substance was AdC68-2019S virus liquid (the virus amount was 2×10 ¹⁰ vp);

The fourth group (G4 group): A single immunization was carried out by intramuscular injection, and the immune substance was AdC68 virus liquid (the virus amount was 2×10 ¹⁰ vp);

In each of the above groups, the immune volume for intramuscular injection was 100 μl, and PBS buffer with pH 7.2 was used as the solvent to adjust the virus concentration.

Blood collection will begin 2 weeks after the initial immunization, and blood collection will be done every two weeks (blood collection from the cheek).

(2) Preparation of SARS-CoV-2 pseudovirus

Insert the double-stranded DNA molecule shown in SEQ ID NO:5 (the gene encoding the full-length S protein of SARS-CoV-2 South African strain 501Y.V2) between the BamHII and EcoRI restriction sites of the pcDNA3.1(+) vector , the SARS-CoV-2 South African strain 501Y.V2 S protein particle was obtained. Insert the double-stranded DNA molecule shown in SEQ ID NO: 6 (the gene encoding the full-length S protein of the SARS-CoV-2 wild strain) between the BamHII and EcoRI restriction sites of the pcDNA3.1(+) vector to obtain SARS-CoV-2 wild strain S protein particle.

The above SARS-CoV-2 South African strain 501Y.V2 S protein plasmid or SARS-CoV-2 wild strain S protein plasmid and backbone plasmid pNL4-3R-E-luciferase (He J, Choe S, Walker R, Di Marzio P , Morgan DO, Landau NR.J Virol 69:6705–6711, 1995) were co-transfected into 293T cells. After incubation, the SARS-CoV-2 South African strain 501Y.V2 pseudotype virus and SARS could be obtained that are infectious but have no replication ability. - CoV-2 wild strain pseudotyped virus, whose infectivity is similar to that of live virus. Specifically: co-transfect 293T cells with SARS-CoV-2 South African strain 501Y.V2 S protein plasmid or SARS-CoV-2 wild strain S protein plasmid and backbone plasmid pNL4-3R-E-luciferase, and incubate at 37°C. , collect the cell culture supernatant 48 hours after transfection, which is the virus liquid containing the SARS-CoV-2 South African strain 501Y.V2 pseudovirus (referred to as the SARS-CoV-2 South African strain 501Y.V2 virus liquid) and the virus liquid containing the SARS-CoV-2 South African strain 501Y.V2. -2 Viral fluid of wild strain pseudovirus (referred to as SARS-CoV-2 wild strain viral fluid). Use the ELISA kit for quantitative p24 detection (HIV P24 antigen quantitative detection kit, KEY-BIO, 96T) to detect virus droplets in SARS-CoV-2 South African strain 501Y.V2 virus fluid and SARS-CoV-2 wild strain virus fluid. The OD _450nm absorbance value of the SARS-CoV-2 South African strain virus liquid is 1 (1021TCID50/ml), and the OD _450nm absorbance value of the SARS-CoV-2 wild strain virus liquid is 1.7 (1735.7TCID50/ml). The larger the number, the higher the virus content.

(3) Preparation of SARS-CoV-2 South African strain 501Y.V2 S protein and SARS-CoV-2 wild strain S protein

1. Insert the double-stranded DNA molecule shown in SEQ ID NO:7 and the double-stranded DNA molecule shown in SEQ ID NO:8 into the NotI and NheI restriction sites of the pcDNA3.1 plasmid respectively to obtain the recombinant plasmid.

2. With the help of PEI transfection reagent, transfect the recombinant plasmid obtained in step 1 into 293F cells grown to a density of 90% and culture for 72 hours.

3. After step 2 is completed, collect the supernatant, use a nickel column for protein purification, and collect the protein solution.

4. Take the protein solution obtained in step 3, concentrate and replace the system, replace the protein system with PBS buffer at pH 7.2, and obtain the SARS-CoV-2 South African strain 501Y.V2 S protein solution or SARS-CoV-2 wild strain Strain S protein solution.

(4) Detection of total antibodies induced by vaccines

Take the blood sample obtained in step (1) of this example, separate the serum, and use ELISA to detect total IgG. In total IgG detection, the SARS-CoV-2 South African strain 501Y.V2 S protein was used to coat the enzyme plate (100ng/well). The serum was first diluted to 200 times the volume, and then subjected to 3 times gradient dilution (200, 600, 1800, 5400, 16200, 48600, 145800 and 437400, a total of 8 dilutions, the dilution solvent is PBS buffer with pH 7.2), and the secondary antibody is Anti-mouse IgG HRP.

In the second week, the ED50 values of mouse serum in each group are shown in Table 1 (N.D represents negative test result).

Table 1

The serum binding curve of each group of mice at week 2 is shown in Figure 2, and the logarithmic histogram of the ED50 value of each group with base 10 is shown in Figure 3.

The results show that the serum immunized with the recombinant adenovirus of the present invention can bind to the SARS-CoV-2 South African strain 501Y.V2 S protein and the SARS-CoV-2 wild strain S protein very well, which is very effective against SARS-CoV-2 South Africa strain. The binding of strain 501Y.V2 S protein is stronger than that of AdC68-2019S immune serum.

(5) Detection of antibody neutralizing activity in animal serum after vaccine immunization

The solution to be tested is: take the blood sample obtained in step (1) of this embodiment and separate the obtained serum.

1. Use DMEM medium containing 10% FBS to dilute the solution to be tested to 48 times, and then dilute it 3 times in a gradient to obtain dilutions with different serum concentrations (48, 144, 432, 1296, 3888, 11664, 34992 and 104976 8 dilutions in total).

2. Mix 100 μl of the dilution obtained in step 1 with 50 μl of the SARS-CoV-2 South African strain virus liquid or SARS-CoV-2 wild strain virus liquid (virus content is 100 TCID50) prepared in step (2) of this example, 37 Incubate at ℃ for 1 hour. Set up a blank control with 100 μl of DMEM medium containing 10% FBS instead of 100 μl of diluent.

3. After step 2 is completed, add 50 μl of Huh7 cell solution (containing approximately 2×10 ⁴ Huh7 cells) and incubate at 37°C for 48 hours (in practical applications, 48-72 hours can be used).

4. After step 3 is completed, add 100 μl PBS buffer and 50 μl cell lysis buffer (Bright-Glo ^TM Luciferase Assay System, Promega, E2650), let stand for 2 minutes, and then use a chemiluminescence instrument to detect luciferase activity.

Three replicate wells were set for each treatment, and the results were averaged.

Neutralizing activity = (fluorescence intensity of the blank control group - fluorescence intensity of the experimental group with diluent added)/fluorescence intensity of the blank control group × 100%.

The corresponding serum dilution factor when the neutralizing activity is 50% is ID50.

In the second week, the ID50 values of mouse serum in each group are shown in Table 2 (N.D represents negative test result).

Table 2

Group ID50 for the South African strain of pseudovirus ID50 of wild strain pseudovirus G1 group 1690.3 119.9 G3 group 66.7 328.3 G4 group N.D. N.D.

The serum neutralization curve of each group of mice at week 2 is shown in Figure 4, and the logarithmic histogram of the ID50 value of each group with base 10 is shown in Figure 5.

The results show that the recombinant adenovirus immune serum of the present invention can very well neutralize the SARS-CoV-2 South African strain 501Y.V2 pseudovirus and the SARS-CoV-2 wild strain virus liquid, which is very effective against SARS-CoV-2 South Africa strain. The neutralization of strain 501Y.V2 pseudovirus was stronger than that of AdC68-2019S immune serum.

Claims (12)

1. A SARS-CoV-2 South African strain 501Y.V2 S protein variant, the amino acid sequence of which is shown in SEQ ID NO: 1 or the amino acid sequence of which is shown in SEQ ID NO: 2. 2. A DNA molecule encoding the SARS-CoV-2 South African strain 501Y.V2 S protein variant according to claim 1. 3. The DNA molecule according to claim 2, whose nucleotide sequence is shown in SEQ ID NO:3. 4. The DNA molecule according to claim 2, whose nucleotide sequence is shown in SEQ ID NO:4. 5. A recombinant plasmid obtained by inserting the DNA molecule of any one of claims 2 to 4 into the ΔE1 region of the chimpanzee adenovirus vector AdC68. 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 cells. 7. The recombinant adenovirus according to claim 6, wherein the adenovirus packaging cells are HEK293A cells. 8. A composition for preventing SARS-CoV-2 infection or for neutralizing SARS-CoV-2 or treating diseases related to SARS-CoV-2 infection, 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. The recombinant adenovirus of claim 6 or the composition of claim 8 is used in preparation for preventing SARS-CoV-2 infection, or for neutralizing SARS-CoV-2, or for treating SARS-CoV-2 infection. Use in medicines for diseases. 11. A kit for preparing recombinant adenovirus, which contains the recombinant plasmid of claim 5 and an adenovirus packaging cell having an adenovirus E1 gene. 12. The kit according to claim 11, wherein the adenovirus packaging cells are HEK293A cells.