CN113151184B - Method for cell membrane-based display of coronavirus immunogens to induce neutralizing antibodies - Google Patents

Abstract

The present application discloses a method for cell membrane-based display of coronavirus immunogens to induce neutralizing antibodies. Specifically, the present disclosure provides a cell displaying the spike protein S of the novel coronavirus SARS-CoV-2 on the surface of its cell membrane, a vaccine or vaccine combination against the novel coronavirus SARS-CoV-2 comprising the cell, the use of the cell in the preparation of a vaccine for preventing or treating the novel coronavirus SARS-CoV-2, and a preparation method thereof. The cell and the vaccine disclosed by the invention can efficiently activate B cells in vivo and induce neutralizing antibody response, and have wide application prospects in preventing and reducing new coronavirus infection.

Description

Method for cell membrane-based display of coronavirus immunogens to induce neutralizing antibodies

Technical Field

The present disclosure is in the field of biotechnology and vaccines. In particular, the present disclosure relates to a method for cell membrane-based display of coronavirus immunogens to induce neutralizing antibodies.

Background

To date, vaccines are the safest and most effective way for humans to actively prevent infectious diseases. The vaccine works on the principle that specific immune response including cellular immune response and antibody response aiming at pathogens is induced by actively immunizing inactivated pathogens or genetically engineered protein or nucleic acid components with higher immunogenicity, and meanwhile, immunological memory is formed, so that when infection risks exist again, the immune system of an organism can quickly react to generate sufficient specific immune response and prevent the pathogens from invading target cells.

The main forms of the existing vaccines are: inactivated virus vaccines, nucleic acid vaccines, protein subunit/virus-like particle vaccines, and bacterial/viral vector vaccines. The virus-like particle vaccine has high immunogenicity, and can generate good immune protection effect by adjuvant injection. However, for some enveloped viruses that do not self-assemble to form the native structure of the virus, the search for an immunogen display that expresses more closely to the native conformational form will help to increase the immunogenicity of the antigen and induce the production of neutralizing antibodies.

The antigen epitope expressed on the membrane surface of higher-order biological eukaryotic cells, particularly humanized cells, can be closer to the natural conformation form of the virus envelope protein, ensures that glycosyl contained on the surface of the higher-order biological eukaryotic cells is similar to that of an infected virus, truly reproduces the antigen characteristics required to be recognized by an organism, and can be used as a preferred cell carrier for displaying the envelope virus antigen. The K562 cell strain is derived from human erythroleukemia, is characterized by lacking endogenous expression HLA-A, B, C (MHC-I), HLA-DR (MHC-II) and blood group antigen (A, B, O) molecules, can avoid rejection reaction among organisms, is very sensitive to NK cell mediated killing, and has no tumorigenicity in organisms, so the application of the K562 cell strain asbase:Sub>A enveloped virus vaccine vector has better safety and effectiveness.

Coronaviruses are a positive strand single strand RNA virus with envelope, wherein SARS-CoV-2 belongs to a beta coronavirus subtype B coronavirus, has about 80% homology with severe acute respiratory syndrome coronavirus (SARS-CoV), and has extremely high transmission power and high pathogenicity in human population. Coronaviruses bind to host cell receptors primarily through the Spike protein Spike (S protein), mediate viral invasion and determine the host tropism of the virus. Wherein a receptor binding domain RBD positioned in an S1 subunit of the S protein can be combined with a host cell surface receptor angiotensin converting enzyme 2 (ACE 2), and then the S protein S2 subunit fuses virus and host cell membranes to promote the virus to enter susceptible cells. Therefore, the neutralizing antibody targeting the RBD, S1 and S protein epitopes can block the combination of the RBD of the virus, interfere the membrane fusion and invasion mediated by S2, inhibit the virus replication and can be used as a candidate target of the coronavirus vaccine immunogen.

However, how to prepare vaccines capable of inducing binding antibodies and neutralizing antibodies with high efficiency aiming at the immunogens still remains a challenge in the field. There is an urgent need in the art to develop novel vaccines that can efficiently produce coronavirus neutralizing antibodies.

Disclosure of Invention

It is within the present application to provide a cell and associated vaccine that can be effectively used to induce binding and neutralizing antibodies against novel coronaviruses.

In a first aspect of the present disclosure, there is provided a cell displaying on its cell membrane surface the spike protein S of the novel coronavirus SARS-CoV-2.

In some embodiments, the spike protein S is selected from the group consisting of: (a) a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 2; (b) A polypeptide homologous to the polypeptide of (a), e.g., having greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology to SEQ ID No. 2; (c) And (b) the protein or polypeptide which is derived from the (a) and has immunogenicity through substituting, deleting or adding one or more amino acids in the amino acid sequence defined by the (a).

In some embodiments, the spike protein S is comprised in a fusion peptide, e.g., the moiety fused thereto is selected from the group consisting of: proteins of viral or host origin, transferrin (Fn), the p24 protein of Human Immunodeficiency Virus (HIV), the stem of enveloped viruses, such as influenza virus HA2, gp41 of HIV, the Fc fragment of antibodies, GM-CSF, IL-21, CD40L or CD40 antibodies.

In some embodiments, the cell comprises a vector having a spike protein S coding sequence.

In some embodiments, the spike protein S encoding molecule is: (i) has the sequence shown in SEQ ID NO: 1; (ii) a molecule that hybridizes to (i) under stringent conditions; (iii) (iii) nucleotide molecules having a homology higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than or equal to 99% homology with the sequence in (i) or (ii); (iv) A nucleotide molecule which is formed by substituting, deleting or adding one or more nucleotides in the nucleotide sequence defined by (i) or (ii) and can express a functional RBD immunogenic peptide.

In some embodiments, the cell has been transformed with a vector comprising the spike protein S encoding molecule of any one of (i) to (iv).

In some embodiments, the vector is selected from viral vectors, such as poxviruses (e.g., tiantan strain, north American vaccine strain, whitberg derived strain, listeria strain, ankara derived strain, copenhagen strain, and New York strain poxviruses), adenoviruses (e.g., ad5, ad11, ad26, ad35, ad 68), lentiviral vectors, adeno-associated viruses, herpes simplex viruses, measles viruses, reoviruses, rhabdoviruses, forest encephalitis viruses, influenza viruses, respiratory syncytial viruses, poliovirus vectors.

In some embodiments, the cell is a mammalian cell or an insect cell, such as K562, A549, HEK293, heLa, CHO, NS0, SP2/0, PER. C6, vero, RD, BHK, HT 1080, A549, cos-7, ARPE-19, MRC-5 cells, high Five, sf9, se301, seIZD2109, seUCR1, sf9, sf900+, sf21, BTI-TN-5B1-4, MG-1, tn368, hzAm1, BM-N, ha2302, hz2E5, ao38.

In some embodiments, the cell is a K562 cell, an a549 cell, a HEK293 cell.

In some embodiments, the cell has an intact membrane structure displaying the spike protein S.

In some embodiments, the cell is an inactivated cell, e.g., using physical inactivation such as X-ray radiation, ultraviolet radiation; or chemical inactivation such as beta propiolactone, formaldehyde, paraformaldehyde fixation.

In one aspect of the disclosure, a vaccine or vaccine combination against a novel coronavirus SARS-CoV-2 is provided, comprising a cell of the disclosure.

In some embodiments, the vaccine or vaccine combination is in a form suitable for intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drops, aerosol inhalation, reproductive tract, rectal, oral administration, or any combination thereof, preferably intramuscular injection.

In some embodiments, the vaccine or vaccine combination comprises or is combined with an adjuvant including, but not limited to: aluminum adjuvant, cholera toxin and subunit thereof, oligodeoxynucleotide, manganese ion adjuvant, colloidal manganese adjuvant, freund's adjuvant, SAS adjuvant, MF59 adjuvant, AS03 adjuvant, QS-21 adjuvant, cpG adjuvant, poly I: C, E.coli adhesin and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, etc.

In some embodiments, the vaccine combination further comprises one or more additional vaccines against the novel coronavirus, e.g., the additional vaccines include vaccines against coronavirus S, S1 or RBD, e.g., the S, S1 or RBD is from a group including, but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV.

In some embodiments, the vaccine combination comprises a combination of a nucleic acid vaccine (DNA or RNA vaccine) and a recombinant human cell vector vaccine, and the components of the vaccine combination are sequentially vaccinated, preferably the DNA vaccine is vaccinated first.

In one aspect of the disclosure, there is provided the use of a cell of the disclosure in the preparation of a vaccine for the prevention or treatment of the novel coronavirus SARS-CoV-2.

In one aspect of the disclosure, there is provided a cell of the disclosure for use in the prevention or treatment of a novel coronavirus SARS-CoV-2.

In one aspect of the present disclosure, there is provided a method of preventing or treating a novel coronavirus SARS-CoV-2 infection or a condition associated therewith, the method comprising administering to a subject in need thereof a cell or vaccine composition of the present application.

In one aspect of the present disclosure, there is provided a method of preparing a vaccine or vaccine combination against a novel coronavirus SARS-CoV-2, the method comprising:

(a) Providing a cell as described herein;

(b) Combining the cells provided in (a) with an immunologically or pharmaceutically acceptable carrier.

In some embodiments of the disclosure, the cells of the disclosure display a coronavirus Spike protein immunogen on their cell membrane.

In some embodiments of the present disclosure, the cells of the present disclosure may be inactivated by means including physical inactivation such as X-ray radiation, ultraviolet radiation; or chemical inactivation such as beta propiolactone, formaldehyde paraformaldehyde fixation.

In some embodiments of the present disclosure, inactivation of the cells of the present disclosure and other treatments do not disrupt the integrity of their cell membranes.

In some embodiments of the disclosure, a vaccine comprising a cell of the disclosure is used as a prime and/or boost vaccine. In some embodiments, a vaccine comprising a cell of the present disclosure is used as a booster vaccine. In some embodiments, a vaccine comprising cells of the disclosure is used as a booster vaccine after priming of a DNA vaccine.

Any combination of the foregoing aspects and features may be made by those skilled in the art without departing from the spirit and scope of the present disclosure. Other aspects of the disclosure will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

The present disclosure is further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the disclosure only and not for the purpose of limiting the scope of the disclosure.

FIG. 1: construction of lentivirus expression vector plasmid and expression verification of cell membrane display Spike immunogen (S protein).

The expression profile of the S protein gene integrated lentiviral expression vector plasmid, pHAGE-S protein-puro (FIG. 1 a); after K562 cells are infected by lentivirus packaged by a lentivirus expression vector pHAGE-S protein-puro, the constructed K562-S protein cells successfully express S protein as shown by a western blotting result (figure 1 b); flow results showed successful expression of the S protein on K562 cell membranes and enrichment of K562-S protein cells by flow sorting after multiple staining (FIG. 1 c).

FIG. 2: identification of Human Leukocyte Antigen (HLA) and human blood group antigen (A, B) expressed on K562 cell membrane.

Expression and identification of human leukocyte antigen class I (HLA-A, B) and human leukocyte antigen class II (HLA-DR) molecules on K562 cell membranes, and western blotting result shows that two molecules are not expressed on K562 cell membranes, and membrane components thereof will not generate rejection reaction when entering into organism (FIGS. 2base:Sub>A and 2B); expressing and identifying human blood group antigen A and B molecules on K562 cell membrane, and displaying the result of western blotting that K562 cell does not express human blood group antigen basically, and membrane components can not stimulate different blood group organisms to generate hemolytic reaction (figure 2 c);

FIG. 3: comparing the difference between RBD specific binding antibody and neutralizing antibody generated by directly using S protein epitope related immunogen (RBD, S1) and K562 cell (K562-S protein) displaying S protein on membrane to immunize mice.

Immunizing a mouse by using an immunization strategy of table 1, and detecting titers of binding antibodies generated by RBD, S1 protein immunization and K562-S protein immunization respectively by using an ELISA method 2 weeks after immunization is finished, wherein the results show that RBD specific binding antibodies generated by RBD protein immunization induction are weak in a second needle, S1 protein and K562-S protein immunization can generate higher antibody response, and the titer of the K562 vaccine binding antibody displaying S protein on a membrane can reach 400000 (fig. 3 a); consistent with the trend of induced binding antibody, after 2 weeks of immunization, the titer of neutralizing antibody induced by K562-S protein and generated against SARS-CoV-2 pseudovirus was high, with an average value of about 1200, and one mouse could reach 4,500 (FIG. 3 b).

FIG. 4: different vaccination approaches are adopted to immunize K562-S protein carrier vaccine, and the difference of the RBD specific binding antibody and the neutralizing antibody is induced.

Mice were immunized according to the immunization strategy of table 2, and the serum binding antibody titers of mice at 1, 2 and 4 weeks after immunization were measured by ELISA. The immunization of K562 cells is divided into two modes of intraperitoneal injection and intramuscular injection. The results showed that the experimental group (K562-S protein) was able to increase the titer of RBD-specific binding antibodies by both intraperitoneal and intramuscular injection compared to the control group (K562), with a better intramuscular immunization effect (fig. 4 a); simultaneously detecting the titers of the neutralizing antibodies in the sera of the mice at 1 week, 2 weeks and 4 weeks after muscle immunization, wherein the results show that compared with a control group, the neutralizing antibodies in the experimental group at 1 week after immunization are improved weakly, the neutralizing antibodies in the experimental groups at 2 weeks and 4 weeks after immunization are improved gradually, and the titer of the neutralizing antibodies can reach more than 3500 (fig. 4 b); at 4 weeks after immunization, the neutralizing antibodies induced by intraperitoneal and intramuscular K562-S protein injections were compared, and the results showed that the mean value of the neutralizing antibodies induced in both groups was about 1200, and that one mouse was able to achieve a muscle immunization of 3700 or more (FIG. 4 c).

FIG. 5: the K562-S protein carrier vaccine is treated in different inactivation modes, and the induced RBD specific binding antibody and neutralizing antibody are different.

Mice were immunized with the immunization strategy of table 3 using x-ray radiation and paraformaldehyde fixation of K562-S protein cells, respectively, and the serum binding and neutralizing antibody titers in mice were measured at 1 week and 2 weeks after immunization. The results of the detection by the ELISA method show that the bound antibodies induced by the paraformaldehyde-treated group at weeks 1 and 2 after immunization are higher, and are basically equal to or even higher than those induced by the untreated group, while the bound antibodies induced by the x-ray-treated group are slightly higher than those induced by the untreated group at week 1 after immunization and are weaker than those induced by the untreated and paraformaldehyde-fixed groups at week 2 after immunization (FIG. 5 a); meanwhile, only the paraformaldehyde-treated group induces and generates a neutralizing antibody against SARS-CoV-2 pseudovirus in the 1 st week after immunization, and the neutralizing antibody generated by part of mice in the paraformaldehyde-treated group in the 2 nd week after immunization is remarkably improved to over 800 at most, while the neutralizing antibody is not effectively induced in the x-ray treated group, and the neutralizing antibody equivalent to the paraformaldehyde-treated group in the 1 st week after immunization can be induced in the untreated group (FIG. 5 b);

FIG. 6: the K562-S protein carrier vaccine is sequentially immunized and can induce to generate RBD specific binding antibody and neutralizing antibody.

Mice were randomized into 2 groups using paraformaldehyde fixation for K562-S protein cells and immunized with the immunization strategy of table 4, all immunogens were inoculated intramuscularly. The serum of mice was tested for binding and neutralizing antibody titers at week 1 and week 2 after 2 immunizations. The ELISA method is applied to detect results, the titer of the binding antibody induced by the K562-S protein immunization group at week 1 after 2 immunizations can reach 6400 on average, and the binding antibody at week 2 keeps higher level continuously (FIG. 6 a); meanwhile, no neutralizing antibody against SARS-CoV-2 pseudovirus was produced at week 1 after 2 immunizations, while mice in week 2 after immunization were induced to produce neutralizing antibody with a maximum titer of 324 (FIG. 6 b). K562-S protein cell membranes were extracted and mice were immunized with the immunization strategy of Table 5, and the immunogens were all inoculated intramuscularly. The serum of mice was tested for binding and neutralizing antibody titers at week 2 after 2 immunizations. The results of the ELISA procedure showed that only 2 mice produced a binding antibody with a titer of about 200 at week 2 after 2 immunizations (FIG. 6 c); meanwhile, only 1 mouse produced weak neutralizing antibody at 2 weeks after 2 immunizations, with a titer of about 20 (fig. 6 d).

FIG. 7: immunization with different vector vaccines based on S immunogen can induce the generation of RBD specific binding antibody and neutralizing antibody.

Mice were randomized into 4 groups and immunized according to the immunization strategy of table 6, all immunogens were inoculated intramuscularly. The K562-S protein cell is fixed by paraformaldehyde, and then the inactivated K562-S vaccine and the S trimer protein vaccine are used together with an aluminum hydroxide adjuvant (Alum). The serum of mice was tested for binding and neutralizing antibody titers at week 2 after 2 immunizations. The detection result of the ELISA method shows that the titer of the binding antibody induced by the DNA-S group after 2 times of immunization is weaker, the GMT of the binding antibody induced by the K562-S protein-Alum group is equivalent to that of the S trimer protein-Alum group, and the average value can reach 100000 (figure 7 a); meanwhile, the neutralizing antibody GMT of the anti-SARS-CoV-2 pseudovirus is basically consistent with the tendency of binding antibody, and the K562-S protein-Alum and S trimer protein-Alum group GMT are both higher than 1000 (FIG. 7 b). The S protein dose of every 1e 6K 562-S cells detected by the S protein ELISA quantitative kit is about 0.47 mug (figure 7 c), the antibody titer data of the S trimer protein group is converted into the same dose as the K562-S vaccine immunity in an equal proportion, the neutralizing antibody GMT of the protein vaccine group after conversion is over 100, the neutralizing antibody GMT of the K562-S protein vaccine group is over 1000, and the neutralizing antibody GMT of the cell vaccine can reach about 13 times of the protein vaccine (figure 7 d).

In the figure, n × represents a difference multiple, and × represents a significant difference.

FIG. 8: different adjuvants are compatible with K562-S protein carrier vaccine for use, and induced RBD specific binding antibody and neutralizing antibody difference

The mice were randomly divided into 7 groups, and the mice were immunized according to the immunization strategy of table 7, the vaccination mode and the vaccine treatment were the same as above, and the adjuvant is shown in table 7. 2 weeks after immunization is finished, the combined antibody titer and the neutralizing antibody titer generated by the immunization induction of the K562-S protein vaccine with different adjuvant combinations are respectively detected, and the result shows that the immunogenicity of the K562-S protein vaccine can be improved to different degrees by various adjuvants, in the compatibility form of various adjuvants, the AS03 emulsion adjuvant and two combined adjuvants of Alum + CpG and MnJ + CpG can induce relatively strongest immune response, the neutralizing antibody titer is highest, and the GMT is about 10000; mnJ adjuvant and emulsion adjuvant MF59 times alone; the improvement effect of the traditional adjuvant Alum is the weakest compared with other novel or combined adjuvants, but the improvement is obvious compared with a control without the adjuvant, and the GMT of a neutralizing antibody is about 1000; similarly, bound antibodies were also consistent with the trend for neutralizing antibodies (FIGS. 8a,8 b).

FIG. 9: the K562-S protein carrier vaccine is used by being matched with a dominant adjuvant, and can induce and generate a persistent RBD specific binding antibody and a neutralizing antibody.

ICR mice were randomized into 2 groups and mice were immunized with the immunization strategy of table 8 and the immunogens were all inoculated intramuscularly. And (3) respectively detecting the titer of the combined antibody and the titer of the neutralizing antibody generated by the immunization induction of the K562-S protein vaccine with different adjuvant combinations at different time points after the immunization is finished.

The results show that ICR mice bound antibody GMT to 102400 (Alum) and 557380 (MnJ + CpG) respectively 6 weeks after priming (2 weeks after boosting), neutralized antibody GMT to 9982 (Alum) and 33649 (MnJ + CpG) respectively, and antibody response gradually weakened with time, the strength and durability of antibody response induced by MnJ + CpG combined adjuvant are superior to those of traditional Alum adjuvant, antibody response is still maintained at high level 24 weeks after priming (5 months after immunization), bound antibody GMT reaches 13825 (Alum) and 32254 (MnJ + CpG) respectively, and neutralized antibody GMT reaches 535 (Alum) and 1045 (MnJ + CpG) (figure a, 9b), and the results show that MnJ + combined adjuvant is dominant adjuvant, and K562-S protein carrier vaccine can maintain durable immune CpG activity.

Detailed Description

The present disclosure relates to the field of vaccines. According to the application, the natural conformation of the enveloped virus protein is reduced to the maximum extent by utilizing the characteristics of a biological cell membrane and displaying the coronavirus S protein on the surface of a cell vector vaccine membrane; moreover, if the selected cells are, for example, K562 cells, the cells do not substantially express molecules which can cause rejection or hemolytic reactions such as Human Leukocyte Antigens (HLA) or blood group antigens (A, B, O) and the like except for the expressed immunogen, so that the cells have good safety as vaccine carriers for displaying the immunogen on the membranes; therefore, the carrier vaccine displaying the S protein immunogen on the cell membrane is expected to be used as an effective coronavirus vaccine to induce an organism to generate a specific neutralizing antibody and be used for preventing new corona or various coronavirus related diseases.

In tumor therapy, genetically engineered K562 cells have been engineered as a whole cell therapeutic vaccine that overexpresses GM-CSF cytokine, primarily by secreting cytokines to exert an adjuvant-like immune activation, stimulate antigen uptake and elicit a corresponding cellular immune response. However, there are no reports of using human cells as a prophylactic vaccine carrier, especially in infectious diseases distinct from tumors in terms of pathogenesis, disease state and development thereof, and prevention and treatment methods.

In the application, based on the principle that S protein is displayed as a natural conformation on a virus membrane in the natural infection process, a human cell membrane is used as a vaccine carrier matrix for S protein display, and a new coronavirus vaccine based on a human cell carrier is developed. The design enables the S protein to keep natural conformation, avoids rejection reaction of human body and reduces the carrier effect of the vaccine.

Experiments prove that the human cell vector vaccine K562-S has excellent immunogenicity and immunoreactivity for the first time; the antibody response can be effectively improved under the condition of lower dosage of the immunogenic substances; the immune effect can be further improved by using the adjuvant. The application proves that the S protein vaccine based on the human cells is a vaccine form for efficiently inducing neutralizing antibodies aiming at the new coronavirus, and the dominant effect of the S protein vaccine is unpredictable.

Animal experiment results prove that the vaccine disclosed by the invention is safe, can continuously generate high-titer neutralizing antibodies, and can be used for preventing and treating new coronavirus infection.

All numerical ranges provided herein are intended to expressly include all numbers between the end points of the ranges and numerical ranges there between. The features mentioned in the present disclosure or the features mentioned in the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

As used herein, "about" in the context of a value or range means ± 10% of the recited or claimed value or range.

It is to be understood that when ranges of parameters are provided, the invention likewise provides all integers and decimals thereof within the ranges. For example, "0.1-2.5 mg/day" includes 0.1 mg/day, 0.2 mg/day, 0.3 mg/day, etc. up to 2.5 mg/day.

As used herein, the term "a" or "an" refers to, "comprising," "having," or "including" includes "comprising," "consisting essentially of, 8230; \8230;," consisting essentially of, 8230; \8230; "consisting of, 8230;" and "consisting of, 8230;"; the Chinese medicine composition mainly comprises (8230) (\ 8230) ', (8230) ', essentially (8230) ', and (8230) ', the structure of (8230) ' belongs to the lower concepts of ' containing ', ' having ' or ' including '.

S protein and encoding molecule thereof

As used herein, the terms "S protein", "immunogenic peptide" and "immunogenic peptide of the present disclosure/application" and the like, used interchangeably, refer to a peptide that includes the Spike protein structure of SARS-CoV-2 virus and has the effect of eliciting binding and neutralizing antibodies.

In some embodiments of the disclosure, the S protein may be: (a) has the sequence of SEQ ID NO: 2; (b) A polypeptide homologous to the polypeptide of (a), e.g., having greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology to SEQ ID No. 2; (c) And (b) the protein or polypeptide which is derived from the (a) and has immunogenicity through substituting, deleting or adding one or more amino acids in the amino acid sequence defined by the (a).

In some cases, the S protein may include modifications and/or be linked to other moieties that facilitate enhancement of its immunogenicity or reactivity or stability, e.g., to enhance S protein stability, enhance neutralizing antibody responses, form multimers, increase cellular responses, etc. Moieties that may be attached to modified or unmodified S proteins include, but are not limited to: proteins derived from virus or host, transferrin, HIV p24, and envelope virus stem, such as influenza HA2, HIV gp41, antibody Fc fragment, GM-CSF, IL-21, CD40L or CD40 antibody.

S proteins may also include variants thereof, such as deletions, insertions and/or substitutions of one or more (typically 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, e.g. 1, 2, 3, 4,5, 6, 7, 8, 9 or 10) amino acids, and additions of one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein or polypeptide. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein or polypeptide.

The S proteins of the present application may be produced by recombinant expression under appropriate circumstances and conditions, e.g., produced by the encoding nucleotide molecules, vectors, host cells of the present disclosure; can also be obtained by chemical synthesis and the like. In the present application, it is preferred to display the S protein using a cell membrane (e.g., K562 cells) to maximize the reduction of the native conformation of the enveloped virus protein and to improve safety.

As used herein, the terms "S protein encoding molecule", "S protein coding sequence", and the like, are used interchangeably and all refer to a nucleotide molecule encoding an immunogenic S protein as described in the present disclosure. The nucleic acid molecule may be selected from, for example: (i) the sequence is as shown in SEQ ID NO: 1; (ii) a molecule that hybridizes to (i) under stringent conditions; (iii) (iii) a nucleotide molecule having greater than or equal to 90%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, greater than or equal to 99% homology with the sequence in (i) or (ii); (iv) (iii) a nucleotide molecule which is substituted, deleted or added with one or more nucleotides in the nucleotide sequence defined in (i) or (ii) and can express a functional immunogenic S protein.

As used herein, the term "stringent conditions" refers to: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42 ℃ etc. at the time of hybridization; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, more preferably 95% or more.

The full-length nucleotide sequence or a fragment thereof of the present disclosure can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in the present disclosure, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Vectors and host cells

The disclosure also relates to vectors comprising the S protein-encoding nucleotide molecules, and host cells genetically engineered with the vectors.

The coding sequences of the present disclosure can be used to express or produce recombinant immunogenic peptides by conventional recombinant DNA techniques (Science, 1984, 224. Generally, the following steps are performed:

(1) Transforming or transducing a suitable host cell with an encoding nucleotide molecule of the present disclosure, or with a recombinant expression vector containing the nucleotide molecule;

(2) A host cell cultured in a suitable medium;

(3) Isolating and purifying the protein or polypeptide from the culture medium or the cells.

In the present disclosure, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to a bacterial plasmid, phage, yeast plasmid, animal cell virus, mammalian cell virus or other vector well known in the art. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Expression vectors containing the S protein coding sequence and appropriate transcriptional/translational control signals can be constructed using methods conventional in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Expression systems such as pcDNA3.1 vector, pIRES2-EGFP vector, adMaxTM, and the like may be employed in the present disclosure.

In addition, the expression vector may contain one or more selectable marker genes to provide phenotypic traits useful for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform an appropriate host cell so that it can express the protein or polypeptide. Preferred in the present application are host cells which can display the S protein on the surface of the cell membrane, such as K562 cells, a549 cells, HEK293 cells, and the like.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples are: escherichia coli, streptomyces, agrobacterium; fungal cells such as yeast; animal cells, and the like. In the present disclosure, for example, a host cell selected from the group consisting of: HEK293, heLa, CHO, NS0, SP2/0, PER.C6, vero, RD, BHK, HT 1080, A549, cos-7, ARPE-19 and MRC-5 cells; high Five, sf9, se301, seIZD2109, seUCR1, sf9, sf900+, sf21, BTI-TN-5B1-4, MG-1, tn368, hzAm1, BM-N, ha2302, hz2E5, and Ao38.

The nucleotide molecules of the present disclosure will provide enhanced transcription when expressed in higher eukaryotic cells if enhancer sequences are inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase transcription of a gene. It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

The recombinant polypeptide in the above method may be expressed on a cell membrane. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

Vaccines and immunoconjugates

Also provided herein is a vaccine, or immunogenic composition, comprising the disclosed coronavirus Spike immunogen displayed on the cell membrane. The vaccine comprises a formulation of the S protein of the present disclosure in a form capable of being administered to a vertebrate, preferably a mammal, and which induces a protective immune response that enhances immunity to prevent and/or alleviate the novel coronavirus and/or at least one symptom thereof.

The term "protective immune response" or "protective response" refers to an immune response to an infectious agent or disease that is exhibited by a vertebrate (e.g., a human), which prevents or reduces infection or reduces at least one disease symptom thereof, mediated by an immunogen.

The term "vertebrate" or "subject" or "patient" refers to any member of the subphylum chordata, including, but not limited to: humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds include domesticated, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese. The terms "mammal" and "animal" are included in this definition and are intended to encompass adult, juvenile, and newborn individuals.

An effective amount of the immunogen herein is included in the vaccine compositions herein. The vaccine compositions of the present disclosure comprise an immunogen in an amount sufficient to achieve the desired biological effect. The term "effective amount" generally refers to an amount of immunogen that can induce a protective immune response sufficient to induce immunity to prevent and/or alleviate an infection or disease and/or to reduce at least one symptom of an infection or disease.

For example, the recombinant plasmid vaccine used in the present application has an immunizing dose in mice of 10-200. Mu.g/mouse; the recombinant human cell vector vaccine is 10 0000-500 0000 cells/mouse; the protein immunization dose is 1-20 mug/mouse. The human body immunizing dose of the recombinant plasmid vaccine can be 0.1-100 mg, such as 0.2-50mg, 0.5-10mg and 1-2 mg per person; the dosage of the recombinant human cell carrier vaccine for human use can be 1000000-10000 0000 cells/human; the human dosage for protein immunization can be 0.1-200 mu g, 0.5E

100 mug, 1-80 mug, 5-70 mug, 10-65 mug, 20-60 mug per person.

The cellular immunity of the application can obtain high immunity effect with extremely low S protein immunity dosage. For example, in some embodiments, every x 10 ⁶ The actual S protein immunizing dose provided by the K562-S cells of the present application is about 0.3-0.6. Mu.g, 0.4-0.5. Mu.g, e.g., 0.47. Mu.g. The membrane-displayed S protein vaccines of the present application compare to S trimer proteins, compared at equivalent S protein immunization dosesThe level of neutralizing antibodies induced by the white vaccine is increased by at least 10-fold, e.g., 10-50-fold, 10-30-fold, 10-15-fold. If supplemented with an appropriate adjuvant, the level may be increased by, for example, a further 2 to 9 fold.

Inactivated or inactivated cell vaccines can be used in the present application, for example, inactivation can be performed using a paraformaldehyde fixative, such as a paraformaldehyde fixative that can be at a concentration of 0.01% to 5% (g/ml; solvent is PBS).

In the course of vaccine preparation and treatment, X-ray irradiation is a common inactivation method in tumor-associated therapeutic cell vaccines, and does not affect the biological activity of the overexpressed GM-CSF cytokine, but the effect of X-ray irradiation on the immunogenicity of cell vaccine surface display proteins cannot be speculated on this basis. Irradiation inactivation may have some adverse effect on the immunological activity of the cell vaccine, making it less likely to produce neutralizing antibodies, and should be used with caution. Similarly, although formalin solution is also used for inactivating whole virus vaccines, the particle size, membrane protein density, membrane components and the like of human cell vaccines and whole virus vaccines are different, and the formalin solution is also adjusted in actual treatment, so that the formalin solution can maintain the immunogenicity to the maximum extent on the basis of ensuring complete inactivation.

Adjuvants may also be included in the vaccines herein. Adjuvants known to those of ordinary skill in the art may be employed, such as those described in Vogel et al, "A Complex of Vaccine Adjuvants and Excipients" (2 nd edition), which is incorporated herein by reference in its entirety. Examples of known adjuvants include, but are not limited to: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, lipopolysaccharide (LPS), RIBI adjuvant, MF-59, etc.

In some embodiments, the adjuvant useful in the vaccines herein may be one or more selected from the group consisting of: alum, AS03, MF59, mnJ, cpG, or any combination thereof, e.g., alum + CpG, mnJ + CpG, etc. Preferably, the adjuvanted vaccine is capable of inducing a significant increase in the level of neutralizing antibodies produced, e.g., 2-100 fold, 5-90 fold, 6-80 fold, 8-70 fold, or any range of fold therebetween, as compared to a corresponding vaccine not comprising an adjuvant. Preferably, the adjuvanted vaccine induces the production of neutralising antibodies for a duration of at least 5 months, e.g. half a year, 1 year, 1.5 years, 2 years, compared to a corresponding vaccine not comprising an adjuvant.

The vaccine compositions herein may further comprise pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, emulsifiers and like excipients. For example, pharmaceutically acceptable carriers are known and include, but are not limited to, water for injection, saline solution, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. Pharmaceutically acceptable carriers, diluents and other excipients can be found, for example, in Remington's Pharmaceutical Sciences.

The vaccine compositions herein may be in a form suitable for systemic or topical (especially in the respiratory tract) administration. Methods of administering vaccine compositions include, but are not limited to: intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drops, aerosol inhalation, reproductive tract, rectal, oral, or any combination thereof. In some embodiments, intramuscular injection, intraperitoneal injection, or a combination thereof is used.

In some embodiments, the vaccines herein prevent, eliminate or reduce a novel coronavirus infection or at least one symptom thereof in a subject, such as a respiratory symptom (e.g., nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, rales, wheezing, dyspnea, pneumonia due to infection, severe acute respiratory syndrome, renal failure, and the like.

Also contemplated herein is an immunoconjugate (also referred to as an immunoconjugate) comprising the immunogen herein and other materials coupled thereto. The additional substance may be a targeting substance (e.g., a moiety that specifically recognizes a particular target), a therapeutic substance (e.g., a drug, a toxin, a cytotoxic agent), a labeling substance (e.g., a fluorescent label, a radioisotope label).

Also provided in the present disclosure is a combination product comprising a host cell and/or vaccine of the present disclosure, and may further comprise one or more additional substances that help to better function or enhance the stability of the aforementioned substances in preventing and/or treating a novel coronavirus infection or a symptom thereof. For example, the other substances may include other vaccines against coronavirus S or S1, such as S or S1 vaccines from the group including, but not limited to, SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV; other active agents that benefit from T cell activation and/or memory immune response with T cells.

Immunization method

Also provided herein is a method for preventing and/or treating a novel coronavirus infection and/or symptoms thereof, comprising: administering at least once a prophylactically and/or therapeutically effective amount of one or more vaccines of the present disclosure. Inoculation regimes that may be used include, but are not limited to: systemic immunization modes, such as intramuscular injection, subcutaneous injection, intradermal injection and the like; and (3) immunization in respiratory tract, such as atomization, nasal drip and the like. In some embodiments, the primary immunization employs systemic or intrarespiratory vaccination, preferably systemic vaccination.

In some embodiments of the disclosure, the interval between each two vaccinations is at least 1 week, e.g., 2 weeks, 4 weeks, 2 months, 3 months, 6 months or longer intervals.

In some embodiments, a DNA vaccine is used for a primary immunization, and a cellular vaccine is used for one or more booster immunizations. The methods of immunization of the present disclosure may be by "prime-boost" or "prime-boost-re-boost", by a single systemic or local immunization of the respiratory tract, or by a combination of both immunization modalities.

In some preferred embodiments, a systemic priming with a recombinant DNA vaccine is used to establish a systemic immune response, followed by one or more boosts with a cellular vaccine.

The vaccine-specific immune response that can be effectively established in the local and systemic respiratory tract systems using the immunization methods herein helps to enhance the effectiveness of vaccine protection.

Providing the combination product herein in the form of a pharmaceutical pack or kit may, for example, be achieved by packaging one or more of the vaccine compositions herein or one or more of the ingredients thereof in one or more containers, for example in a sealed container such as an ampoule or sachet, indicating the amount of composition. The vaccine compositions may be provided as a liquid, sterile lyophilized powder, or anhydrous concentrate, etc., which may be diluted, reconstituted and/or formulated with an appropriate liquid (e.g., water, saline, etc.) immediately prior to use to obtain the appropriate concentration and form for administration to a subject.

Examples

The disclosure is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. Appropriate modifications, variations and changes may be made by those skilled in the art to the present disclosure, which modifications and changes are within the scope of the present disclosure.

The experimental procedures for the conditions not specified in the examples below can be carried out by methods conventional in the art, for example, by referring to the molecular cloning, A Laboratory Manual, new York, cold Spring Harbor Laboratory Press,1989 or according to the conditions recommended by the supplier. Methods for sequencing DNA are conventional in the art and tests are also available from commercial companies.

Unless otherwise indicated, percentages and parts are by weight. 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. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present disclosure. The preferred embodiments and materials described herein are intended to be exemplary only.

The experimental animals, immunization modes, immunogens, pseudoviruses and detection methods referred to in the examples are as follows:

I. experimental animals: 6-8 week old female C57/BL6 mice (examples 1-8); female ICR mice 6-8 weeks old (example 9).

Immunization protocol: the left, right and rear limbs of the mouse are respectively injected intramuscularly or the mouse is injected intraperitoneally.

Immunogen: the S protein sequence is from Genebank NC 045512.2, the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2

1. Recombinant plasmid vaccine (DNA): pcDNA3.1 (empty), pcDNA3.1-S protein;

2. protein subunit vaccine (protein): RBD protein (Nanjing Kinshire Biotech, inc., Z03483-1); s1 protein (beijing yi qiao shenzhou science ltd, Z03501); s trimer protein (near shore protein science and technology ltd, DRA 49);

3. recombinant human cell vector vaccine: k562, K562-S proteins;

4. human carrier cell membrane fragments: k562-cell membrane, K562-S protein-cell membrane;

immunogen preparation and immunization dose:

the immunization and inactivation doses of each immunogen in the combination were as follows:

1. recombinant plasmid vaccine (DNA): plasmid (dissolved in sterile PBS), 100. Mu.g/mouse, 100. Mu.L;

2. protein subunit vaccine (protein): immunization was performed after mixing the protein (dissolved in sterile PBS) with aluminum adjuvant (aluminuium, invivoGen, cat No. 5200) at a volume ratio of 1;

3. recombinant human cell vector vaccine (K562): 1000000 cells/mouse, 100 μ L, in sterile PBS;

k562 cell membrane fragment vaccine (K562-cell membrane): 100 μ L of 50 μ g/mouse dissolved in sterile physiological saline; (Thermo, mem-pertplus Membrane Protein Extraction Kit, 89842), about 50 μ g of cell Membrane was extracted from 1000000 cells, so the dose here was consistent with recombinant human cell carrier vaccine;

x-ray irradiation: 50Gy;

6. concentration of paraformaldehyde fixing solution: 4% in sterile PBS;

7.K562-S vaccine compatible adjuvant: after the K562-S vaccine is inactivated by paraformaldehyde, adjuvants AS03 and MF59 (InvivoGen) are mixed with the K562-S vaccine according to the volume ratio of the adjuvants to the vaccine of 1;

s protein content of K562-S cells: after a certain number of K562-S cells were lysed, the S protein content was determined using the S protein quantitative detection kit-ELISA method (Biodragon, BF 03087).

V. immunization interval:

specific immunization intervals are shown in the table below.

Immunogen-related vector construction:

1. the DNA sequence of the S protein (SEQ ID NO: 1) was synthesized by ligating the DNA sequence 5' to which the Not1 cleavage site and Xba1 cleavage site were ligated, and the ligated fragment was cleaved with the vector plasmid pcDNA3.1 (from Youbao/pHAGE-MCS-puro (from Shanghai Xinwan) using Not1 cleavage (Thermo Scientific Co., FD 0596) and Xba1 cleavage (Thermo Scientific Co., FD 0685), and recovered by gel electrophoresis followed by gel cutting, and the cleaved fragment was recovered using the Sanprep column DNA gel recovery kit (Promega, cat # A9282).

2. The gene recovery product was ligated to the digested linearized vector by T4 DNA ligase (Thermo Scientific, cat # 2011A): coli Stable was transformed with the ligation product and grown overnight on ampicillin-containing plates. On day 2, single colonies were randomly picked for sequencing, mutation site correction, and successful cloning of S protein gene expression plasmid (pcDNA3.1-S protein) and lentiviral vector plasmid (pHAGE-S protein-puro) was performed after all sequences were verified to be correct.

SARS-CoV-2 envelope pseudovirus packaging:

1. 293T cells were prepared the day before transfection and used for transfection and expression of packaging plasmids. Cells were diluted to 5X 10 with DMEM complete medium ⁶ cell/mL, 1mL diluted cells were plated in a 10cm dish at 37 ℃ and 5% CO ₂ Culturing overnight;

2. sucking 6 μ g SARS-CoV-2 membrane protein plasmid pcDNA3.1-S protein and 6 μ g pNL4-3 Δ env (NIH AIDS Reagent Program, 3418) skeleton plasmid, adding into 500 μ L DMEM without double antibody (serum-free, double antibody is mixed solution of streptomycin), and incubating at room temperature for 5min;

3. diluting 24 μ L TurboFect with double-no DMEM to a final volume of 500 μ L/sample, and incubating at room temperature for 5min;

4. and (3) uniformly mixing the liquid in the steps 2 and 3, incubating at room temperature for 20min at the final volume of 1000 mu L/sample, and adding 293T cells paved in a 10cm culture dish in advance after the incubation is finished. After 6h, replacing fresh 15mL of complete culture medium, and continuously culturing in a cell culture box for 48h;

5. after the culture, the cell culture supernatant was collected from a 10cm dish and placed in a 15mL centrifuge tube, 4000g, centrifuged at 4 ℃ for 10min, filtered through a 0.45 μm filter into a new 15mL centrifuge tube, frozen at-80 ℃ for storage, and titrated for future use.

Construction of 293T cells stably expressing hACE2 receptor:

1. an artificial synthetic human ACE2 (hACE 2) sequence (Genebank # NCBI _ NP _ 001358344.1) is shown as SEQ ID NO. 3, an amino acid sequence is shown as SEQ ID NO. 4, the 5 'end of the sequence is provided with an Age1 enzyme cutting site, and the 3' end is provided with an Xba1 enzyme cutting site. The synthesized fragment and the vector plasmid pHAGE-MCS-puro were digested with Age1 (Thermo Scientific, cat. No. FD 1464) and Xba1 (Thermo Scientific, cat. No. FD 0685), and recovered by gel electrophoresis followed by gel cutting, and the digested fragment was recovered using a Sanprep column DNA gel recovery kit (Promega, cat. No. A9282).

2. The gene recovery product was ligated to the digested linearized vector by T4 DNA ligase (Thermo Scientific, cat # 2011A): coli Stable was transformed with the ligation product and grown overnight on ampicillin-containing plates. On day 2, single colonies were randomly picked and sequenced, the mutation sites were corrected, and after confirming that the entire sequence was correct, a lentiviral expression plasmid (pHAGE-hACE 2-puro) for the hACE2 gene was successfully cloned.

3. 10cm dishes were taken and inoculated about 5X 10 in each dish ⁶ 293T cells, which ensures that the cell density reaches 90% when the transfection is carried out on the next day; three plasmids, namely pHAGE-hACE2-puro, a lentivirus packaging plasmid psPAX and VSVG are added according to the mass ratio of 1:2:1 ratio to transfect 293T cells.

Culturing at 4.37 deg.C in 5% incubator for about 48 hr, and collecting cell supernatant according to cell condition. The collected cell supernatant was filtered through a 0.45 μm filter and concentrated with PEG 8000 to obtain relatively purified hACE2 lentivirus.

5. Laying 5X 10 in advance one day ⁵ 293T cells were plated in one well of a 12-well plate, and 500. Mu.L of the virus concentrated in step 2, 1000g, was added to the plated cells the next day, followed by centrifugation for 2 hours.

6. After completion of the centrifugal infection, the cells were further cultured at 37 ℃ for about 12 hours in a 5% incubator, the medium was changed to a cell culture medium supplemented with 1. Mu.g/mL puromycin (puro), and the 293T cells which had the hACE2 gene integrated therein were finally survived, and 293T cells which stably expressed hACE2 (capable of binding to S protein) were selected by flow-sorting.

IV, a detection method:

blood collection:

mice: 4 weeks after the last immunization, the mice were taken off the neck and died, whole blood at the periphery of the mice was collected by an eyeball-picking method, collected in a 1.5mL EP tube, allowed to stand at room temperature for natural coagulation, and the coagulated serum of the mice was centrifuged at 7000g for 15min. Mouse sera were transferred to new 1.5mL EP tubes. Samples were inactivated at 56 ℃ for 30min prior to the experiment to destroy complement activity in the serum. And the tube is centrifuged for a short time before inactivation, so that the residual samples on the tube wall and the bottle cap are avoided. The bath level should be below the sample level but not above the cap.

Detection of bound antibodies by ELISA method

1. RBD protein was dissolved overnight in coating solution, coated in 96-well flat-bottom plates at a protein concentration of 1. Mu.g/ml, in a volume of 100. Mu.l/well, and placed in a 4-degree freezer overnight.

2. Washing the plate for 3 times: PBST was added at 300. Mu.l/well, the well was discarded after 1 minute residence time, and the final wash was performed on filter paper (same procedure for subsequent washing). 5% milk was blocked for 2 hours.

3. After washing the plate for 1 time, the sample was added, 100. Mu.l/well of diluted serum was added thereto, and the mixture was left at room temperature for 3 hours.

4. After washing the plate for 5 times, adding a detection antibody: diluted biotinylated anti-mouse antibody (purchased from Peking sequoia Jinqiao, cat # ZB-2305) was added at 100. Mu.l/well. The plate-sealing membrane was covered and incubated at room temperature for 1 hour.

5. Wash plate 5 times and add TMB: incubate 100. Mu.l/well at room temperature in the dark for 10-30 minutes.

6. And (3) terminating the reaction: the reaction was stopped by adding stop solution quickly at 100. Mu.l/well.

7. Reading a plate: readings at λ =450nm (microplate reader, from BioTek) were taken within 10min after addition of the stop solution.

Detection of neutralizing antibody in 293T-hACE2 cell:

1. a96-well transparent bottom blackboard is taken for carrying out a neutralization experiment, a Cell Control (CC) (150 mu L) is arranged in the first column, a Virus Control (VC) (100 mu L) is arranged in the second column, all the other wells are sample wells, and serum samples are diluted in a multiple proportion, so that the volume in the final wells is 100 mu L.

2. In addition to the cell control group, 50. Mu.L of SARS-CoV-2 pseudovirus diluent was added to each well, so that each well finally contained 200TCID pseudovirus ₅₀ 。

3. Gently shaking and mixing, placing the 96-well bottom blackboard in a cell incubator, incubating at 37 deg.C, 5% CO2 for 1h.

4. When the incubation time reached 20min, the 293T-hACE2 target cells were initially prepared and diluted to 10 with complete medium ⁵ Individual cells/mL.

5. When the incubation time is up to 1h, 100. Mu.L of target cells are added to each well of a 96-well transparent bottom blackboard, so that the cells in each well are 10 ⁴ And (4) respectively.

6. Gently shaking the 96-well transparent bottom blackboard all around to uniformly disperse the cells in the wells, placing the blackboard in a cell incubator at 37 deg.C and 5% CO ₂ Culturing for 48h.

7. After culturing for 48h, taking out a 96-well transparent bottom blackboard from a cell incubator, sucking off supernatant in the wells, adding 100 mu L of PBS into each well for washing once, sucking off the PBS, adding 50 mu L of 1 multiplied lysis buffer (purchased from Promega company Cat # E153A) into each well, and incubating on a horizontal shaker for 30min at room temperature to fully lyse the cells;

8. add 30. Mu.L of luciferase substrate (available from Promega, cat # E1501) to a 96-well blackboard and use the instrument

Luminescence-detection instrument for 96 microporous plateLuciferase activity was measured.

9. Reading values of fluorescein were derived, neutralization inhibition ratios were calculated, and ID50 was calculated by using Graphpad Prism 5.0 software in combination with the results of the neutralization inhibition ratios.

Example 1: construction of lentivirus expression vector pHAGE-S protein-puro and expression verification of coronavirus S protein displayed on K562 cell membrane

In order to study the function of S protein as a biological cell membrane carrier vaccine immunogen, we constructed a lentiviral expression vector for S protein, packaged lentiviruses to infect K562 cells, and screened positive clones expressing S protein on the membrane surface.

Firstly, an S protein gene is synthesized and is connected to a pHAGE-MCS-puro lentivirus expression vector through enzyme digestion to construct and form a pHAGE-S protein-puro plasmid (figure 1 a); infecting K562 cells after packaging pHAGE-S protein-puro into lentivirus, and identifying the expression of S protein in K562-S protein cells by Western blotting method (FIG. 1 b); and simultaneously performing flow cytometric staining identification, finding that the S protein is expressed on a cell membrane, and detecting that the related protein is expressed by the control infected cells, and further enriching the K562 cells expressing the S protein (figure 1 c).

The experimental procedure was as follows: preparation of 5X 10 ⁵ Individual K562 cells, resuspended in 500 μ L complete cell culture medium, placed in one well of a 12-well plate. Concentrated lentivirus was added to the plated cells at 1000g and centrifuged for 2 hours. After the completion of the centrifugal infection, the cells were further incubated at 37 ℃ in a 5% incubator for about 48 hours. After the infection, K562 cells were collected and cultured in RPMI (10% FBS) containing puromycin at a concentration of 4. Mu.g/ml because the expression vector plasmid was puromycin-resistant, and the cells which could survive were those in which the Spike gene had been integrated. Taking the infected cells, detecting S protein expression by Western blotting method, wherein the primary antibody is ACE2-C-AVI-6his (Shanghai near shore science and technology Co., ltd., model 0331753-4065), and the secondary antibody is HRThe goat anti-human antibody was labeled with P (China fir Jinqiao, cat # ZB 2304). The results show that the high expression of S protein can be detected by using a protein immunoblotting method for K562-S protein cells, and S protein is not expressed by uninfected K562 cells (FIG. 1 b).

The flow staining method was performed by indirect staining using ACE2-C-AVI-6his (Shanghai near technologies Co., ltd., model 0331753-4065)/PE-streptavidin (BD Pharminge, cat. 56259), and then finally by enrichment using flow cell sorting (BD Pharminge, model Arial). The results show that, after continuous enrichment, more than 80% of K562-S protein cells can highly express the target gene S protein (FIG. 1 c).

Example 2 identification of expression of Human Leukocyte Antigens (HLA) and human blood group antigens (A, B) on K562 cell membranes.

Human Leukocyte Antigen (HLA) is an expression product of human histocompatibility complex (MHC), and is an important antigen substance constituting transplant rejection. Classical HLA class I includes HLA-A, B, C; HLA class II includes mainly HLA-DP, DQ and DR; HLA class I is distributed on almost all cell surfaces of the body, and HLA class II is a glycoprotein that is mainly localized on the surface of macrophages and B lymphocytes. Therefore, the expression of HLA molecules on the surface of K562 cell membranes was detected by flow cytometry, and the safety of the membrane components as immunogen presenting vectors could be confirmed. Meanwhile, HLA-A, B and C staining takes 293T cells as positive control, the result shows that HLA class I molecules (HLA-A, B and C) are not expressed on K562 cell membranes (figure 2base:Sub>A), HLA-DR staining takes human B cells as positive control, and the result shows that the components of the K562 cell membranes also do not express HLA class II molecules (HLA-DR) (figure 2B), which indicates that the K562 cell membranes have certain safety, and the flow antibodies used are APC anti-human HLA-A, B and C antibodies (purchased from biolegend, article number 311409) and APC anti-human HLA-DR antibodies (purchased from biolegend, article number 327022). In addition, the expression of blood group antigens of K562 cells as human erythroid cells can also cause hemolytic reactions of organisms with different blood groups, and whether the blood group antigens are expressed in the K562 cells is detected by Western blotting.

The results show that K562 cells do not express Blood Group antigens A and B (FIG. 2 c), and can exclude the possibility of hemolytic reaction of different organisms caused by K562 cells, and the antibody used is Blood Group AB antigen (Z5H-2/Z2A) FITC (available from Santa, cat # sc-52370). Accordingly, it is believed that K562 cell membrane components as immunogen presentation carriers do not stimulate undesirable rejection reactions between different organisms.

Example 3 comparison of the differences between RBD-specific binding and neutralizing antibodies induced by immunization of mice with S protein epitope-associated immunogens (RBD, S1) and K562 cells displaying the S protein on their membranes (K562-S protein).

Mice were randomized into 4 groups and immunized according to the immunization strategy of table 1, all immunogens were inoculated intramuscularly. After 2 weeks of immunization, the titers of the binding antibodies generated by RBD, S1 protein immunization and K562-S protein immunization are respectively detected by using an ELISA method, and the results show that the RBD specific binding antibody generated by using RBD protein immunization induction is weaker in the second needle, and the S1 protein and K562-S protein immunization can generate higher antibody response, wherein the titer of the binding antibody of the K562 vaccine displaying the S protein on the membrane can reach more than 400000 at most (fig. 3 a).

Consistent with the trend of induced bound antibody, the titer of neutralizing antibody induced by K562-S protein and directed against SARS-CoV-2 pseudovirus was higher after 2 weeks of immunization, with an average value around 1200, with 4,500 being achieved in one mouse (FIG. 3 b). This experiment demonstrates that a K562 vector vaccine based on S protein displayed on cell membranes enhances immunity and can activate neutralizing antibodies at levels comparable to or even higher than those of common protein immunization.

TABLE 1 Experimental groups of mice boosted with protein and on-membrane displayed immunogens, respectively

Example 4 immunization of K562-S protein carrier vaccine with different routes of vaccination induced differences in RBD specific binding and neutralizing antibodies.

Mice were randomized into 4 groups, immunized according to the immunization strategy of table 2, and the titers of the bound antibodies in the sera of mice at 1 week, 2 weeks and 4 weeks after immunization were determined by ELISA. The plasmid DNA immunization of the first needle is intramuscular injection, and the immunization of the K562 cells of the second needle is divided into an abdominal cavity mode and an intramuscular injection mode. The results showed that both immunization by intraperitoneal and intramuscular injection increased the titer of RBD-specific binding antibodies in the experimental group (K562-S protein) compared to the control group (K562), with a better intramuscular immunization effect (fig. 4 a).

Meanwhile, the titers of neutralizing antibodies in mouse sera at 1 week, 2 weeks and 4 weeks after muscle immunization were measured. The results show that compared with the control group, the neutralizing antibody in the experimental group at 1 week after immunization is slightly improved, while the neutralizing antibody in the experimental groups at 2 weeks and 4 weeks after immunization is gradually improved, and the titer of the neutralizing antibody can reach more than 3500 at most (fig. 4 b); at 4 weeks after immunization, the neutralizing antibodies induced by intraperitoneal and intramuscular K562-S protein injections were compared, and the results showed that the mean value of the neutralizing antibodies induced in both groups was about 1200, and that one mouse was able to achieve a muscle immunization of 3700 or more (FIG. 4 c).

TABLE 2 Experimental groups of mice immunized with K562-S protein by different immunization regimens

Example 5. Treatment of K562-S protein carrier vaccine with different inactivation regimens induces differences in RBD-specific binding and neutralizing antibodies.

Mice were randomized into 4 groups using x-ray irradiation and paraformaldehyde fixation for K562-S protein cells, respectively, and immunized according to the immunization strategy of table 3, all immunogens were inoculated intramuscularly. The serum of mice was tested for binding and neutralizing antibody titers at week 1 and week 2 after immunization. The results of the test using the ELISA method showed that the bound antibodies induced by the paraformaldehyde-treated group at weeks 1 and 2 after immunization were higher, substantially equivalent to or even higher than those induced by the untreated group, whereas the bound antibodies induced by the x-ray-treated group were slightly higher at week 1 after immunization than those induced by the untreated group and were weaker at week 2 after immunization than those induced by the untreated and paraformaldehyde-fixed groups (FIG. 5 a).

Meanwhile, only the paraformaldehyde-treated group induced the generation of neutralizing antibody against SARS-CoV-2 pseudovirus at week 1 after immunization, and the neutralizing antibody generated by some mice in the paraformaldehyde-treated group at week 2 after immunization was significantly increased to over 800 at most, while the x-ray-treated group did not induce neutralizing antibody, and the untreated group could induce only neutralizing antibody equivalent to the paraformaldehyde-treated group at week 1 after immunization (FIG. 5 b).

TABLE 3 Experimental groups of mice immunized with K562-S protein by different inactivation modes

Example 6 sequential immunization with the K562-S protein carrier vaccine induced the production of RBD specific binding and neutralizing antibodies.

Mice were randomized into 2 groups using paraformaldehyde fixation for K562-S protein cells and immunized with the immunization strategy of table 4, all immunogens were inoculated intramuscularly. The serum of mice was tested for binding and neutralizing antibody titers at week 1 and week 2 after 2 immunizations. The ELISA method test result shows that after 2 immunizations, the titer of the binding antibody induced by the K562-S protein immunization group at week 1 can reach 6400 on average, and the binding antibody at week 2 keeps higher level continuously (FIG. 6 a).

Meanwhile, no neutralizing antibody against SARS-CoV-2 pseudovirus was produced in week 1 after 2 immunizations, while mice in week 2 after immunization were induced to produce neutralizing antibody with a maximum titer of 324 (FIG. 6 b); compared with the results, the 2 times of K562-S protein sequential immunization is found to have the level of the neutralizing antibody generated by DNA-pcDNA3.1-RBD priming and K562-S protein boosting induction to be equivalent, but the 2 times of K562-S protein sequential immunization does not have DNA-pcDNA3.1-Spike priming and K562-S protein boosting induction to have high level of the neutralizing antibody.

This result suggests that DNA-pcDNA3.1-Spike prime, K562-S protein boost are the preferred immunization strategy.

TABLE 4 sequential immunization of mice with protein K562-S vector vaccine experimental groups

In addition, immunization of mice with cell membranes extracted from K562-S protein cells was attempted with the protocol shown in Table 5. The results are shown in FIG. 6 c. The results show that immunization of mice with K562-extracted cell membranes as immunogens induces binding and neutralizing antibodies significantly less efficiently than can be achieved with intact K562 cells. The results indicate that the S protein is displayed on the K562 cell membrane with complete structure and has relatively better immunogenicity.

TABLE 5 sequential immunization of mouse with K562-S protein-cell Membrane Experimental groups

Example 7 comparison of the differences in induced RBD-specific binding and neutralizing antibodies in mice immunized with the polymorphic S protein immunogen vaccine and with K562 cells displaying the S protein on the membrane (K562-S protein).

C57/BL6 mice were randomized into 4 groups and mice were immunized with the immunization strategy of table 6, and the immunogens were all inoculated intramuscularly. 2 weeks after completion of the immunization, the binding antibody titer and the neutralizing antibody titer induced by the immunization with the different forms of the S protein vaccine DNA-S (i.e., the DNA-pcDNA3.1-spike described above), the S trimer protein-Alum and the K562-S protein-Alum were detected by ELISA, respectively, and the results are shown in FIG. 7.

The results show that: the titers of RBD-specific binding antibodies and neutralizing antibodies against pseudoviruses induced using DNA-S immunization were relatively low, whereas S-trimer protein-Alum and K562-S protein-Alum immunization were able to generate higher antibody responses with both Geometric Mean Titers (GMTs) of binding antibodies exceeding 100000 (fig. 7 a) and neutralizing antibodies GMTs exceeding 1000 (fig. 7 b).

Due to cellsThe immunization doses of the vaccine (K562-S) and the protein vaccine (S trimer protein) are different, the actual S protein immunization dose of the K562-S vaccine of each mouse is detected by an S protein ELISA quantitative kit, and the result shows that every 1 × 10 of the vaccines are used for immunization ⁶ The dose of S protein for each K562-S cell number was equal to about 0.47. Mu.g (FIG. 7 c).

The antibody titer data of the S trimer protein group are converted into the same dosage as the K562-S vaccine in an equal proportion, and the neutralizing antibody titer difference is compared, so that the neutralizing antibody GMT of the protein vaccine group is more than 100, the neutralizing antibody GMT of the K562-S protein vaccine group is more than 1000, and the neutralizing antibody GMT of the cell vaccine is about 13 times of that of the protein vaccine (figure 7 d).

The experiment proves that the K562 carrier vaccine based on the S protein displayed on the cell membrane can effectively activate high-level neutralizing antibodies, and compared with the protein vaccine with the same dose, the titer of the induced neutralizing antibodies is higher, which indicates that the immunogen displayed on the cell membrane has higher immunological activity.

TABLE 6 Experimental groups of mice immunized with DNA, protein and vaccine displaying S immunogen on membrane, respectively

Example 8 comparison of different types of adjuvants used with K562-S protein cell carrier vaccines induced differences in RBD-specific binding and neutralizing antibodies.

C57/BL6 mice were randomized into 7 groups and mice were immunized with the immunization strategy of table 7, and the immunogens were all inoculated intramuscularly. 2 weeks after the completion of the immunization, the titer of the combined antibody and the titer of the neutralizing antibody generated by the immunization of the K562-S protein vaccines with different adjuvant combinations are respectively detected, and the results are shown in FIG. 8.

The results show that: the immunogenicity of the K562-S protein vaccine can be improved to different degrees by various tested adjuvants, and in the compatibility of various adjuvants, the AS03 emulsion adjuvant and two combined adjuvants, namely Alum + CpG and MnJ + CpG, can induce relatively strongest immune response, the neutralizing antibody titer is highest, and the GMT is about 10000; mnJ adjuvant and emulsion adjuvant MF59 times alone; the boosting effect of the conventional adjuvant Alum is the weakest compared with other novel or combined adjuvants, the neutralizing antibody GMT is about 1000, but compared with a control group without the adjuvant, the conventional adjuvant Alum has obviously improved the amount of the neutralizing antibody. Similarly, bound antibodies are consistent with a trend to neutralize antibodies.

The experiment result proves that the immune response strength of the cell vaccine can be obviously improved by the compatibility of the K562-S and the dominant adjuvant.

In addition, the membrane display K562-S vaccine containing different adjuvants is compared with the immunization effect of the protein vaccine at the same dose in the conversion mode in example 7, and the membrane display K562-S vaccine can generate a remarkably improved and more excellent immunization effect compared with the protein vaccine after various adjuvants are adopted.

TABLE 7 Experimental groups of mice displaying S immunogen vaccines on membranes with different adjuvants

Example 9 K562-S protein Carrier vaccine used with the dominant adjuvant induced the production of persistent RBD specific binding and neutralizing antibodies.

This group of experiments tested the immune response of ICR strain mice to K562-S vector vaccine, selected the traditional adjuvant Alum or the novel combination adjuvant MnJ + CpG for compatibility, and observed the persistence of antibody response.

Mice were randomized into 2 groups and immunized with the immunization strategy of table 8, and the immunogens were all inoculated intramuscularly. At different time points after the completion of the immunization, the titer of the combined antibody and the titer of the neutralizing antibody generated by the immunization of the K562-S protein vaccine with different adjuvant combinations are respectively detected, and the results are shown in FIG. 9.

The results show that: ICR mice bound antibody GMT to 94810 (Alum) and 516064 (MnJ + CpG) respectively 2 weeks after priming, neutralized antibody GMT to 9791 (Alum) and 29716 (MnJ + CpG) respectively, and antibody response gradually weakened with time, the strength and durability of antibody response induced by MnJ + CpG combined adjuvant are superior to those of traditional Alum adjuvant, the antibody response is still maintained at higher level 24 weeks after priming (5 months after immunization), bound antibody GMT to 29863 (Alum) and 129016 (MnJ + CpG) respectively, and neutralized antibody GMT to 1849 (Alum) and 3249 (MnJ + CpG) (figure 9a, 9b).

The result shows that the K562-S protein carrier vaccine keeps better immunogenicity in different strains of mice, and the MnJ + CpG combined adjuvant is a superior adjuvant; the vaccine-induced antibody response was able to persist for at least 5 months, indicating the superiority and persistence of the adjuvanted K562-S vaccine.

TABLE 8 Experimental groups for on-Membrane display of S immunogen vaccine immunization of ICR mice

All documents referred to in this disclosure are incorporated by reference in this application as if each had been individually incorporated by reference. Furthermore, it should be understood that various changes or modifications to the disclosure may be made by those skilled in the art after reading the above teachings of the disclosure, and such equivalents may fall within the scope of the disclosure as defined by the appended claims.

Sequence listing

<110> Shanghai City public health clinic center

<120> method for inducing neutralizing antibody by displaying coronavirus immunogen based on cell membrane

<130> 214614 1CNCN

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 3846

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<213> Artificial sequence

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atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccaca 60

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aaggtgttcc ggagcagcgt gctgcactcc acacaggatc tgtttctgcc tttcttttct 180

aacgtgacct ggttccacgc catccacgtg agcggcacca atggcacaaa gcggttcgac 240

aatccagtgc tgccctttaa cgatggcgtg tacttcgcct ccaccgagaa gtctaacatc 300

atcagaggct ggatctttgg caccacactg gacagcaaga cacagtccct gctgatcgtg 360

aacaatgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa tgatccattc 420

ctgggcgtgt actatcacaa gaacaataag tcttggatgg agagcgagtt tcgcgtgtat 480

tcctctgcca acaattgcac atttgagtac gtgtcccagc ccttcctgat ggacctggag 540

ggcaagcagg gcaatttcaa gaacctgagg gagttcgtgt ttaagaatat cgatggctac 600

ttcaaaatct actccaagca caccccaatc aacctggtgc gcgacctgcc acagggcttc 660

tctgccctgg agccactggt ggatctgccc atcggcatca acatcacccg gtttcagaca 720

ctgctggccc tgcacagaag ctacctgaca ccaggcgaca gctcctctgg atggaccgca 780

ggagcagcag cctactatgt gggctatctg cagcccagga ccttcctgct gaagtacaac 840

gagaatggca ccatcacaga cgccgtggat tgcgccctgg atcccctgtc tgagaccaag 900

tgtacactga agagctttac cgtggagaag ggcatctatc agacaagcaa tttcagggtg 960

cagcctaccg agtccatcgt gcgctttccc aatatcacaa acctgtgccc ttttggcgag 1020

gtgttcaacg caacccgctt cgcaagcgtg tacgcctgga ataggaagcg catctccaac 1080

tgcgtggccg actattctgt gctgtacaac agcgcctcct tctctacctt taagtgctat 1140

ggcgtgagcc ccacaaagct gaatgacctg tgctttacca acgtgtacgc cgattccttc 1200

gtgatcaggg gcgacgaggt gcgccagatc gcaccaggac agacaggcaa gatcgcagac 1260

tacaattata agctgcctga cgatttcacc ggctgcgtga tcgcctggaa ctctaacaat 1320

ctggatagca aagtgggcgg caactacaat tatctgtacc ggctgtttag aaagtctaat 1380

ctgaagccat tcgagaggga catctccaca gaaatctacc aggccggctc taccccctgc 1440

aatggcgtgg agggctttaa ctgttatttc cctctgcaga gctacggctt ccagccaaca 1500

aacggcgtgg gctatcagcc ctaccgcgtg gtggtgctgt cttttgagct gctgcacgca 1560

cctgcaacag tgtgcggacc aaagaagagc accaatctgg tgaagaacaa gtgcgtgaac 1620

ttcaacttca acggactgac cggcacaggc gtgctgaccg agtccaacaa gaagttcctg 1680

ccttttcagc agttcggcag ggacatcgca gataccacag acgccgtgcg cgaccctcag 1740

accctggaga tcctggatat cacaccatgc tccttcggcg gcgtgtctgt gatcacacca 1800

ggcaccaata caagcaacca ggtggccgtg ctgtatcagg acgtgaattg taccgaggtg 1860

cccgtggcaa tccacgcaga tcagctgacc cctacatggc gggtgtactc taccggcagc 1920

aacgtgttcc agacaagagc aggatgcctg atcggagcag agcacgtgaa caatagctat 1980

gagtgcgaca tccctatcgg cgccggcatc tgtgcctcct accagaccca gacaaactcc 2040

ccaaggagag cacggtctgt ggcaagccag tccatcatcg cctataccat gagcctgggc 2100

gccgagaatt ccgtggccta ctccaacaat tctatcgcca tccctaccaa cttcacaatc 2160

tccgtgacca cagagatcct gccagtgagc atgaccaaga catccgtgga ctgcacaatg 2220

tatatctgtg gcgattccac cgagtgctct aacctgctgc tgcagtacgg ctctttttgt 2280

acccagctga atagagccct gacaggcatc gccgtggagc aggacaagaa cacacaggag 2340

gtgttcgccc aggtgaagca aatctacaag accccaccca tcaaggactt tggcggcttc 2400

aacttcagcc agatcctgcc cgatcctagc aagccatcca agcggtcttt tatcgaggac 2460

ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta tggcgattgc 2520

ctgggcgaca tcgccgccag agacctgatc tgtgcccaga agtttaatgg cctgaccgtg 2580

ctgcctccac tgctgacaga tgagatgatc gcccagtaca catctgccct gctggcaggc 2640

accatcacaa gcggatggac cttcggcgca ggagccgccc tgcagatccc ctttgccatg 2700

cagatggcct atcggttcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760

aagctgatcg ccaatcagtt taactccgcc atcggcaaga tccaggactc tctgagctcc 2820

acagcaagcg ccctgggcaa gctgcaggat gtggtgaatc agaacgccca ggccctgaat 2880

accctggtga agcagctgtc tagcaacttc ggcgccatct cctctgtgct gaatgatatc 2940

ctgagcaggc tggacaaggt ggaggcagag gtgcagatcg accggctgat cacaggcaga 3000

ctgcagtccc tgcagaccta cgtgacacag cagctgatca gggcagcaga gatcagggca 3060

tctgccaatc tggccgccac caagatgagc gagtgcgtgc tgggccagtc caagagagtg 3120

gacttttgtg gcaagggcta tcacctgatg agcttcccac agtccgcccc tcacggagtg 3180

gtgtttctgc acgtgaccta cgtgccagcc caggagaaga acttcaccac agcaccagca 3240

atctgccacg atggcaaggc acactttcct agggagggcg tgttcgtgag caacggcacc 3300

cactggtttg tgacacagcg caatttctac gagccacaga tcatcaccac agacaataca 3360

ttcgtgtccg gcaactgtga cgtggtcatc ggcatcgtga acaataccgt gtatgatcct 3420

ctgcagccag agctggactc ttttaaggag gagctggata agtacttcaa gaatcacacc 3480

agccccgacg tggatctggg cgacatctct ggcatcaatg ccagcgtggt gaacatccag 3540

aaggagatcg acaggctgaa cgaggtggcc aagaatctga acgagtccct gatcgatctg 3600

caggagctgg gcaagtatga gcagtacatc aagtggccct ggtatatctg gctgggcttc 3660

atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacaagctgc 3720

tgttcctgcc tgaagggctg ctgttcttgt ggcagctgct gtaagtttga tgaggacgat 3780

agcgagcctg tgctgaaggg cgtgaagctg cactacacca ccggtctgca gctagctcga 3840

gtctag 3846

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<212> PRT

<213> Artificial sequence

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Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1025 1030 1035

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1055 1060 1065

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1100 1105 1110

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1115 1120 1125

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1160 1165 1170

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1175 1180 1185

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu

1205 1210 1215

Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Val Leu Lys Gly Val Lys Leu His Tyr Thr Thr Gly Leu Gln Leu

1265 1270 1275

Ala Arg Val

1280

<210> 3

<211> 2418

<212> DNA

<213> Artificial sequence

<400> 3

atgtcaagct cttcctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60

attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120

tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180

aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240

caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300

cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360

aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420

tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480

aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540

gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600

gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660

cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720

catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780

tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840

ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900

gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960

cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020

aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080

tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140

tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200

catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260

attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320

aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380

tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440

aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500

gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560

taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620

aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680

ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740

gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800

aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860

aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920

tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980

cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040

tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100

gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160

agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220

atatggctga ttgtttttgg agttgtgatg ggagtgatag tggttggcat tgtcatcctg 2280

atcttcactg ggatcagaga tcggaagaag aaaaataaag caagaagtgg agaaaatcct 2340

tatgcctcca tcgatattag caaaggagaa aataatccag gattccaaaa cactgatgat 2400

gttcagacct ccttttag 2418

<210> 4

<211> 805

<212> PRT

<213> Artificial sequence

<400> 4

Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala

1 5 10 15

Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe

20 25 30

Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp

35 40 45

Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn

50 55 60

Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala

65 70 75 80

Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln

85 90 95

Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys

100 105 110

Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser

115 120 125

Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu

130 135 140

Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu

145 150 155 160

Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu

165 170 175

Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg

180 185 190

Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu

195 200 205

Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu

210 215 220

Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu

225 230 235 240

His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile

245 250 255

Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly

260 265 270

Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys

275 280 285

Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala

290 295 300

Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu

305 310 315 320

Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro

325 330 335

Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly

340 345 350

Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp

355 360 365

Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala

370 375 380

Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe

385 390 395 400

His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys

405 410 415

His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn

420 425 430

Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly

435 440 445

Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe

450 455 460

Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met

465 470 475 480

Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr

485 490 495

Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe

500 505 510

Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala

515 520 525

Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile

530 535 540

Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu

545 550 555 560

Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala

565 570 575

Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe

580 585 590

Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr

595 600 605

Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu

610 615 620

Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met

625 630 635 640

Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu

645 650 655

Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val

660 665 670

Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro

675 680 685

Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile

690 695 700

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn

705 710 715 720

Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln

725 730 735

Pro Pro Val Ser Ile Trp Leu Ile Val Phe Gly Val Val Met Gly Val

740 745 750

Ile Val Val Gly Ile Val Ile Leu Ile Phe Thr Gly Ile Arg Asp Arg

755 760 765

Lys Lys Lys Asn Lys Ala Arg Ser Gly Glu Asn Pro Tyr Ala Ser Ile

770 775 780

Asp Ile Ser Lys Gly Glu Asn Asn Pro Gly Phe Gln Asn Thr Asp Asp

785 790 795 800

Val Gln Thr Ser Phe

805

Claims (23)

1. A cell displaying a novel coronavirus SARS-CoV-2 spike protein S on the surface of the cell membrane, wherein the cell is a K562 cell, and the amino acid sequence of the spike protein S is shown as SEQ ID NO. 2.

2. The cell of claim 1, wherein the cell comprises a vector having a spike protein S coding sequence.

3. The cell according to claim 1, wherein the cell is transformed with a vector having a nucleotide molecule represented by SEQ ID NO. 1.

4. The cell of claim 1, wherein each 1 x 10 ⁶ Each of the cells provides 0.3-0.6 micrograms of spike protein S.

5. The cell of claim 1, wherein each 1 x 10 ⁶ Each of the cells provided 0.47 micrograms of spike protein S.

6. The cell of claim 1, wherein the cell is inactivated by paraformaldehyde fixation.

7. A vaccine or vaccine combination against the novel coronavirus SARS-CoV-2, comprising the cell of any one of claims 1-6.

8. The vaccine or vaccine combination according to claim 7, wherein the form of the vaccine or vaccine combination is suitable for intramuscular vaccination, intradermal vaccination, subcutaneous vaccination, reproductive tract, rectal or any combination thereof.

9. The vaccine or vaccine combination according to claim 7, wherein the vaccine or vaccine combination is in a form suitable for intramuscular injection.

10. The vaccine or vaccine combination according to claim 7, wherein the vaccine combination further comprises one or more additional vaccines against novel coronaviruses.

11. The vaccine or vaccine combination of claim 10, wherein the further vaccine comprises a vaccine against coronavirus S, S1 or RBD.

12. The vaccine or vaccine combination according to claim 11, wherein the S, S1 or RBD is from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 or bat-CoV.

13. The vaccine or vaccine combination according to claim 7, wherein the vaccine combination comprises a nucleic acid vaccine in combination with a recombinant human cell vector vaccine, and the components of the vaccine combination are administered sequentially.

14. The vaccine or vaccine combination according to claim 13, wherein the nucleic acid vaccine is a DNA or RNA vaccine.

15. The vaccine or vaccine combination according to claim 14, wherein the sequential vaccination is preceded by a DNA vaccine.

16. The vaccine or vaccine combination according to any one of claims 7-15, wherein the vaccine or vaccine combination further comprises an adjuvant.

17. The vaccine or vaccine combination according to claim 16, wherein the adjuvant is selected from the group consisting of: AS03, MF59, mnJ, cpG, aluminum hydroxide adjuvant, complete freund's adjuvant, incomplete freund's adjuvant, lipopolysaccharide, RIBI adjuvant, or any combination thereof.

18. The vaccine or vaccine combination according to claim 16, wherein the adjuvant is selected from the group consisting of: alum, a combination of Alum and CpG, and a combination of MnJ and CpG.

19. Use of a cell according to any one of claims 1-6 for the preparation of a vaccine for the prevention or treatment of the novel coronavirus SARS-CoV-2.

20. A method of making a vaccine or vaccine combination against a novel coronavirus SARS-CoV-2, the method comprising:

(a) Providing a cell according to any one of claims 1-6;

(b) Combining the cells provided in (a) with an immunologically or pharmaceutically acceptable carrier or adjuvant.

21. The method of claim 20, wherein the vaccine or vaccine combination comprises an adjuvant.

22. The method of claim 20, wherein the adjuvant is selected from the group consisting of: AS03, MF59, mnJ, cpG, aluminum hydroxide adjuvant, complete freund's adjuvant, incomplete freund's adjuvant, lipopolysaccharide, RIBI adjuvant, or any combination thereof.

23. The method of claim 20, wherein the adjuvant is selected from the group consisting of: alum, a combination of Alum and CpG, and a combination of MnJ and CpG.

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