CN114213548A

CN114213548A - Method for simultaneously inducing immune response against multiple viruses

Info

Publication number: CN114213548A
Application number: CN202111597361.5A
Authority: CN
Inventors: 徐建青; 张晓燕; 周东明; 曹康丽; 王祥; 胡杨洋; 丁龙飞
Original assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Current assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Priority date: 2020-12-24
Filing date: 2021-12-24
Publication date: 2022-03-22
Also published as: WO2022135563A1

Abstract

The present application relates to methods of simultaneously inducing immune responses against multiple viruses (e.g., influenza virus as well as new corona virus). In particular, the present application provides an immunogenic peptide comprising (a) an immunogenic stem: it comprises the HA2 region of influenza virus hemagglutinin HA; (b) immunogen crown: comprising a viral membrane protein or an immunogenic fragment thereof, wherein the viral membrane protein is derived from a virus other than the virus from which the HA2 region of (a) was derived; and (c) optionally, a further moiety fused to the aforementioned moiety. The vaccine of the application is safe, can continuously generate high-titer neutralizing antibodies and T cell responses, and can be used for preventing and treating influenza and new coronavirus.

Description

Method for simultaneously inducing immune response against multiple viruses

Technical Field

The present invention relates to the field of vaccines, and in particular to a method for simultaneously inducing an immune response against a plurality of viruses including influenza, such as coronaviruses, and in particular neocoronaviruses.

Background

Influenza, called influenza for short, is an acute respiratory infectious disease caused by influenza virus, and is mainly divided into seasonal and pandemic influenza. It is estimated that the adult accounts for 5-10% and the child accounts for 20-30% of the influenza infection patients all over the world. Influenza not only causes a severe disease burden, but also causes a huge direct and indirect economic burden.

The influenza virus genome is divided into 8 segments, and 10 proteins such as Hemagglutinin (HA), Neuraminidase (NA), matrix proteins 1 and 2(M1 and M2), nonstructural proteins 1 and 2(NS1 and NS2), Nucleocapsid Protein (NP), and three polymerase complexes (PB1, PB2, and PA) are encoded respectively. Influenza viruses can be classified into influenza A, influenza B and influenza C types, for example, Avian Influenza Virus (AIV) is influenza A virus (influenza A virus), based on the antigenic difference of nucleoprotein, M1 and M2. Influenza a can be classified into 18H subtypes and 9N subtypes according to antigenicity differences of HA and NA. These 18H subtypes can be classified into group I (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, H18) and group II (H3, H4, H7, H10, H14, H15). Among the subtypes known to directly infect humans are subtypes H1, H2, H3, H5, H7, H9, and H10, and viruses causing highly pathogenic avian influenza are all subtypes H5 and H7.

Most protective antibodies raised against influenza virus target the HA protein, a trimeric surface glycoprotein consisting of HA1 and HA2 domains. Most of the HA1 chains formed a spherical crown comprising the receptor binding site, while HA2 formed a stem, supporting HA1 and formed a trimer. Although the HA crown (HA1) HAs significant immune advantages in inducing the production of neutralizing antibodies, it is highly susceptible to mutation by itself, leading to antigenic drift and antigenic variation of the virus, forming a new influenza virus subtype. The HA2 region is more conserved than HA1, and 3GBN of currently isolated neutralizing antibodies against H7N9 HA2 HAs broad protection against influenza a, 3SDY HAs broad protection against influenza a group II, and 4FQV HAs broad protection against influenza a and b. Therefore, HA2 of H7N9 is crucial as an influenza neutralizing antibody that induces a broad spectrum.

At present, although some vaccines have entered clinical trials, marketing, production and use of vaccines remain an urgent challenge.

Coronaviruses comprise four structural proteins, including the spike protein (S protein), the envelope protein, the membrane protein, and the nucleocapsid protein. Among them, the S protein plays the most important role in the attachment, fusion and entry processes of viruses, and is also a main target of antibodies, entry inhibitors and vaccines. The S protein mediates entry of the virus into the host cell by first binding to the host receptor via the Receptor Binding Domain (RBD) of the S1 subunit, and then fusing the virus and host cell membranes via the S2 subunit. According to WHO report: animals immunized with coronavirus vaccines may develop more severe symptoms when exposed to live virus again. Non-neutralizing antibodies or lower antibody levels produced by vaccine immunization may cause antibody-dependent enhancement (ADE) effects that enhance viral pathogenicity. Therefore, to reduce the side effects of ADE, the RBD region in the S protein of the novel coronavirus would be the most effective target for vaccine development.

Autumn and winter are high-incidence seasons of respiratory diseases, including influenza virus infection, and how to prevent the risk of overlapping epidemic of influenza and respiratory infectious diseases such as new crown becomes important. In the prevention of infectious diseases, vaccines have been the most effective and economical measure since the advent, and most of the currently used preventive vaccines are aimed at activating neutralizing antibodies. Therefore, designing an immunogen that can simultaneously activate neutralizing antibodies against multiple influenza viruses or against influenza viruses and other viruses (such as new coronaviruses) at the same time is a direction we need to explore and develop urgently.

Disclosure of Invention

Immunogenic peptides capable of simultaneously inducing immune responses against a variety of viruses, including influenza viruses, their encoding nucleotide molecules, vectors, host cells, vaccines and uses thereof are provided in the present disclosure.

In one aspect of the present disclosure, an immunogenic peptide is provided comprising the following moieties:

(a) immunogen stem: it comprises the HA2 region of influenza virus hemagglutinin HA;

(b) immunogen crown: comprising a viral membrane protein or an immunogenic fragment thereof, wherein the viral membrane protein is derived from a virus other than the virus from which the HA2 region of (a) was derived;

(c) optionally, other moieties attached to the foregoing moieties.

In some embodiments, the HA2 region is derived from a source selected from the group consisting of: any of H1 to H18, in particular from the widely prevalent human influenza H1, H2, H3 and the multiple-appearing human infections with avian influenza H5 and H7, for example from 2009 prevalent H1(H1N1) and 2013 prevalent H7(H7N 9).

In some embodiments, the amino acid sequence of the HA2 region is set forth in SEQ ID NO. 1, or is encoded by a nucleotide molecule having the sequence set forth in SEQ ID NO. 2.

In some embodiments, the viral membrane protein is an immunogenic membrane protein.

In some embodiments, the source of the immunogen crown is selected from the group consisting of: an influenza virus of a different origin from the HA2 region in (a); other viruses than influenza virus.

In some embodiments, the source of the immunogen crown is selected from the group consisting of: coronavirus (such as neocoronavirus), hiv, influenza virus (such as the HA1 region of influenza virus hemagglutinin HA), rabies virus, hog cholera virus, porcine reproductive and respiratory syndrome virus, measles virus, ebola virus, herpes virus, arbovirus (such as zika virus, epidemic encephalitis b virus, forest encephalitis virus, dengue virus, hantavirus, hemorrhagic fever virus).

In some embodiments, the source of the immunogen crown is selected from the group consisting of: coronavirus SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, e.g., an S1 protein comprising the receptor binding domain of a coronavirus, or a Receptor Binding Domain (RBD), or an engineered receptor binding domain (e.g., the RBD region modified with a terminal cysteine to form an sRBD region), or an immunogenic fragment thereof.

In some embodiments, the immunogenic crown is selected from S1 of coronavirus SARS-CoV-2, particularly the RBD region from S1 (e.g., the sequence shown in SEQ ID NO:3 or encoded by the nucleotide molecule of SEQ ID NO: 4) or a modified RBD region thereof (e.g., the terminal Cys-modified sRBD region, the peptide stretch shown in SEQ ID NO:5 or encoded by the nucleotide molecule shown in SEQ ID NO: 6), or an immunogenic fragment of an RBD region (e.g., the peptide stretch shown in SEQ ID NO:21 or encoded by the nucleotide molecule shown in SEQ ID NO: 22).

In some embodiments, the additional moiety is selected from: immune modulatory sequences, such as IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulatory antibodies, PD-1 and PD-L1 antibodies, CTLA4 antibodies, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and subunits thereof, bacterial flagellin, FimH, HIV p24, HIV gp 41.

In some embodiments, the additional moiety is selected from: enabling the immunogenic peptide to form part of a nanoparticle, such as transferrin (Fn, e.g. a peptide molecule as shown in SEQ ID NO:7 or encoded by a nucleotide molecule as shown in SEQ ID NO: 8).

In some embodiments, the additional moiety is selected from: signal peptides, such as CD33, CD8, CD16, mouse IgG1 antibody.

In some embodiments, the additional moiety is selected from: a transmembrane region enabling the immunogenic peptide to be expressed on the surface of a viral vector, for example, the CD8 transmembrane region (CD8TM, for example, a peptide molecule represented by SEQ ID NO:17 or encoded by a nucleotide molecule represented by SEQ ID NO: 18), the HA2 transmembrane region, the CD4 transmembrane region, the gp41 transmembrane region.

In some embodiments, the additional moiety is selected from: linker peptides, e.g. (G4S)₃、(G4S)_n、 GSAGSAAGSGEF、(Gly)₆、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、 (Gly)₈、EGKSSGSGSESKST。

In some embodiments, the additional moiety is selected from: tags such as His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag, Sof-tag 1, Sof-tag3, Strep-tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag and PNE tag.

In some embodiments, the immunogenic peptide comprises: the RBD region or sRBD region or immunogenic fragments thereof linked to the HA2 region, and optionally other moieties linked to the foregoing.

In some embodiments, the immunogenic peptide comprises: an RBD region or an sRGB region (e.g., a peptide molecule represented by SEQ ID NO:5 or encoded by a nucleotide molecule represented by SEQ ID NO: 6) and an Fn region (e.g., a peptide molecule represented by SEQ ID NO:7 or encoded by a nucleotide molecule represented by SEQ ID NO: 8) linked to an HA2 region; an RBD region or sRBD region linked to the HA2 region, and a CD8 transmembrane region (e.g., a peptide molecule represented by SEQ ID NO:17 or encoded by a nucleotide molecule represented by SEQ ID NO: 18); an immunogenic fragment of the RBD region (e.g., the peptide molecule shown in SEQ ID NO:21 or encoded by the nucleotide molecule shown in SEQ ID NO: 22) linked to the HA2 region and the Fn region.

In some embodiments, the amino acid sequence of the immunogenic peptide is shown as SEQ ID No. 9 or 15 or 25, or the coding sequence of the immunogenic peptide is shown as SEQ ID No. 10 or 16 or 26.

In some embodiments, the present application uses the HA2 region of H7N9 virus linked to the RBD region of the new coronavirus, or an immunogenic fragment thereof, as an immunogen, while further modifying the immunogen, such as adding disulfide bonds, fusion proteins, fusion cytokines to the RBD region, thereby inducing neutralizing antibodies against both influenza virus and new coronavirus.

In some embodiments, the present application provides an immunogenic peptide directed against both influenza and neocorona, comprising the HA2 region of H7N9 viral hemagglutinin HA and the RBD region of SARS-CoV-2 viral spike protein S, which may be further modified with cysteine to form the sRBD region.

In some embodiments, the cysteine modification is the addition of a pair of cysteines at the root of the RBD domain to enable disulfide bond formation.

In one aspect of the disclosure, a nucleotide molecule is provided that encodes an immunogenic peptide herein.

In some embodiments, the coding sequence for the HA2 region comprises the nucleotide sequence set forth in SEQ ID NO. 2; the coding sequence of the immunogen crown comprises a nucleotide sequence shown as SEQ ID NO. 4 or a nucleotide sequence shown as SEQ ID NO. 6 or a nucleotide sequence shown as SEQ ID NO. 22; and/or the coding sequence of said further part comprises the nucleotide sequence shown in SEQ ID NO 8 or SEQ ID NO 18.

In some embodiments, the nucleotide molecule has the sequence shown in SEQ ID NO 10 or 16 or 26.

In one aspect of the present disclosure, a vector is provided comprising a nucleotide molecule herein.

In one aspect of the disclosure, a host cell is provided comprising a nucleotide molecule or vector herein or capable of expressing an immunogenic peptide described herein.

In some embodiments, the host cell is a mammalian cell or an insect cell, such as HEK293, HeLa, K562, 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-672302, Hz2E5 and Ao 38.

In one aspect of the present disclosure, there is provided a vaccine capable of simultaneously inducing an immune response against an influenza virus and another non-influenza virus or a non-alloinfluenza virus, comprising an immunogenic peptide, a nucleotide molecule, a vector and/or a host cell as described herein.

In some embodiments, the vaccine is a nucleic acid vaccine (DNA or RNA vaccine), a recombinant protein subunit vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a virus-like particle vaccine, a nanoparticle vaccine, a cell vector vaccine.

In some embodiments, the vaccine is a viral vector vaccine, the viral vector selected from the group consisting of: poxviruses (e.g., Tiantan strain, North American vaccine strain, Whitman-derived strain, Listeria strain, Ankara-derived strain, Copenhagen strain, and New York strain), adenoviruses (Ad5, Ad11, Ad26, Ad35, AdC68, or modified variants thereof), adeno-associated viruses, herpes simplex viruses, measles viruses, reoviruses, rhabdoviruses, forest encephalitis viruses, influenza viruses, respiratory syncytial viruses, polioviruses.

In some embodiments, the vaccine comprises or is used in combination with an adjuvant, for example selected from the group consisting of: aluminum adjuvant, cholera toxin and its subunit, oligodeoxynucleotide, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21.

In some embodiments, the vaccine is in a form suitable for intramuscular administration, intradermal administration, subcutaneous administration, nasal drip, aerosol inhalation, genital tract, rectal administration, oral administration, or a combination of the different modes of administration described above (e.g., intramuscular injection + nasal drip).

In some embodiments, the vaccine is in a form suitable for combined vaccination (e.g., combined vaccination or sequential vaccination) of 2 or more, such as sequential vaccination before and after with S or S1 vaccine of coronavirus (e.g., SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV), or sequential vaccination before and after with HA or HA2 vaccine of influenza virus (e.g., HA or HA2 derived from any of H1-H18).

In one aspect of the present disclosure, there is provided the use of an immunogenic peptide, nucleotide molecule, vector and/or host cell as described herein in the manufacture of a medicament for the simultaneous prevention or treatment of influenza virus and another non-influenza virus.

In one aspect of the present disclosure, there is also provided an immunogenic peptide, nucleotide molecule, vector and/or host cell of the present disclosure for use in the simultaneous prevention or treatment of influenza and new coronavirus infection.

In one aspect of the present disclosure, there is also provided a method of preventing or treating influenza and new coronavirus infection simultaneously, the method comprising administering to a subject in need thereof an immunogenic peptide, nucleotide molecule, vector, host cell and/or vaccine of the present disclosure.

In some embodiments, the vaccine comprises or is combined with an adjuvant including, but not limited to: aluminum adjuvant, cholera toxin and its subunit, oligodeoxynucleotide, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, SAS adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C 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 is in a form suitable for vaccination as follows: intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drops, aerosol inhalation, reproductive tract, rectal, oral, or any combination thereof.

In some embodiments, vaccination is with one or more of the vaccines, e.g., combined vaccination or sequential vaccination.

In some embodiments, one or more of the vaccines are used to vaccinate against other vaccines against the novel coronavirus or influenza, e.g., the other vaccines include vaccines against coronavirus S or S1, e.g., S or S1 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, etc.; such other vaccines include vaccines against influenza virus HA or HA2, for example HA or HA2 from the list including but not limited to H1-H18.

In some embodiments, the vaccine comprises a nucleic acid vaccine (DNA or RNA vaccine) in combination with an adenoviral vector vaccine.

In some embodiments, the vaccine comprises pcDNA3.1-S in combination with AdC 68-RHAF. In some embodiments, the components of the vaccine combination are administered sequentially, preferably first, before and after administration of the DNA vaccine.

In one aspect of the present disclosure, there is provided a method of preparing a vaccine against both influenza and new coronavirus infection, the method comprising:

(a) providing immunogenic peptides, nucleotide molecules and vectors of the present disclosure;

(b) combining the active substance provided in (a) with an immunologically or pharmaceutically acceptable carrier.

In one aspect of the disclosure, a method is provided for simultaneously inducing immune responses against influenza and other viruses, including coronaviruses, wherein a fusion protein consisting of the HA2 region of influenza virus hemagglutinin HA as an immunogen stem and the membrane proteins of the other viruses or strains as the corona is used as a vaccine immunogen to simultaneously induce immune responses against influenza and other viruses.

In some embodiments, HA2 immunogen is selected from the group including, but not limited to, H1-H18, particularly from the widely circulating human influenza H1, H2, H3 and multiple-occurring human infections with avian influenza H5 and H7;

in some embodiments, the HA2 immunogen is from H1, which was epidemic in 2009, H7, which was epidemic in 2013.

In some embodiments, the HA2 immunogen is from the HA2 sequence of an early strain of 2013 epidemic H7 and is more than 80% full-length or fragment homologous to that sequence. In some embodiments, the HA2 immunogen may comprise the amino acid sequence shown in SEQ ID No. 1 or may be encoded by a coding sequence having SEQ ID No. 2.

In some embodiments, the crown immunogen linked to HA2 is from a group including, but not limited to, coronavirus, hiv, influenza virus, rabies virus, hog cholera virus, porcine reproductive and respiratory syndrome virus, measles virus, ebola virus, herpes virus, arbovirus (zika virus, epidemic encephalitis b virus, forest encephalitis virus, dengue virus, hantavirus, hemorrhagic fever virus).

In some embodiments, the crown immunogen attached to HA2 is selected from coronaviruses including, but not limited to, SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, and the like, particularly the S1 protein comprising the Receptor binding domain of a coronavirus, or the Receptor Binding Domain (RBD), or an engineered Receptor binding domain.

In some embodiments, the crown immunogen linked to HA2 is selected from S1 of coronavirus SARS-CoV-2, particularly the RBD region from S1 or full length and fragments thereof having more than 90% homology thereto. In some embodiments, the RBD region immunogen can comprise the amino acid sequence shown in SEQ ID NO. 3 or can be encoded by a coding sequence having SEQ ID NO. 4. In some embodiments, the RBD region immunogen may comprise the amino acid sequence shown in SEQ ID NO. 5 or may be encoded by a coding sequence having SEQ ID NO. 6.

In one aspect of the disclosure, a method for simultaneously inducing immune responses against influenza and other viruses including coronaviruses is provided, which comprises co-expressing the immunogen of the present application with other sequences, either by fusion or in a single reading frame, to further increase the antiviral species, or to enhance the ability of the immunogen to induce an immune response, thereby preventing multiple viral infections.

In some embodiments, the sequence linked to an immunogen of the present application: from a source including, but not limited to, various coronaviruses, influenza viruses, aids viruses, rabies viruses, hog cholera viruses, blue ear disease viruses, measles viruses, ebola viruses, herpes viruses, arboviruses (zika virus, epidemic encephalitis b virus, forest encephalitis b virus, dengue virus, hantaviruses, hemorrhagic fever viruses), especially co-expressed with immunogens that activate T cell responses, to prepare composite vaccines that are capable of activating both neutralizing antibodies and T cell responses; (iii) are immune modulatory sequences including, but not limited to, IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulatory antibodies, PD-1 and PD-L1 antibodies, CTLA4 antibodies, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and subunits thereof, bacterial flagellin, FimH, and the like; features are provided for forming nanoparticles including, but not limited to, transferrin (Ferritin).

In one aspect of the present disclosure, a method is provided for simultaneously inducing an immune response against influenza and other viruses, including coronaviruses, comprising combining immunogens described herein, alone or in combination with co-expressed molecules described herein, for the preparation of vaccines including, but not limited to, nucleic acid vaccines (DNA or RNA vaccines), recombinant protein subunit vaccines, recombinant viral vector vaccines, recombinant bacterial vector vaccines, virus-like particle vaccines, nanoparticle vaccines, cell vector vaccines, and the like.

In one aspect of the disclosure, a method of simultaneously inducing an immune response against influenza and other viruses, including coronaviruses, is provided, comprising administering an immunogenic peptide, its encoding nucleotide molecule, vector, host cell or vaccine as described herein.

In some embodiments, vaccination with a combination of 2 or more of the vaccines described above is performed, either in combination or sequentially. In some embodiments, the vaccines herein are sequentially inoculated with a vaccine prepared from coronavirus S or S1, said S or S1 being 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, and the like; or the vaccines herein are inoculated sequentially, before and after, with a vaccine prepared from influenza virus HA or HA2, said HA or HA2 being from the group including, but not limited to, H1-H18.

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: the construction of S protein eukaryotic expression vector and sRGB-HA 2-hFn (RHAF) adenovirus and the expression of fusion protein:

a: pcDNA3.1-S and pAdC68XY3-RHAF plasmid construction maps;

b: the pcDNA3.1-S protein is successfully expressed in 293T cells, and the adenovirus AdC68-RHAF can successfully express the RHAF protein.

FIG. 2 immunogenicity of AdC68-RHAF in C57BL/6 mice:

the mice used in the experiment are female C57BL/6 with the age of 6-8 weeks, and the immunogen is plasmid pcDNA3.1-S and adenovirus AdC 68-RHAF.

A: the ELISA method measures the titer of bound antibody in the mouse serum at week 2 after the end of immunization. The abscissa is the immune group and the ordinate is the titer of bound antibody, p <0.01

B: data for bound antibodies to RBD protein before and after second immunization, indicates p < 0.01.

C: 293T-ACE2 cells were tested for the titer of neutralizing antibodies in mouse sera at week 2 after the end of immunization. The abscissa is the immune group and the ordinate is the titer (ID) of neutralizing antibodies₅₀) Denotes p<0.01。

D: neutralizing antibody titers against SARS-Cov-2 pseudovirus before and after immunization of the second needle.

E: the ELISPOT method measures T cell responses in mice at week 2 after completion of immunization. The abscissa is the immunization group and the ordinate is the number of IFN- γ secreting cells per million splenocytes, indicating p < 0.05.

FIG. 3: immunogenicity of AdC68-RHAF in hACE2+ transgenic mice:

the mice used in the experiment are 6-8 weeks old hACE2+ transgenic mice, and the immunogen is adenovirus AdC 68-RHAF.

A: the ELISA method measures the titer of bound antibody in the mouse serum at week 2 after the end of immunization. The abscissa is the immune group and the ordinate is the titer of bound antibody, indicates p < 0.0001.

B: 293T-ACE2 cells were tested for the titer of neutralizing antibodies in mouse sera at week 2 after the end of immunization. The abscissa is the immune group and the ordinate is the titer (ID) of neutralizing antibodies₅₀) Denotes p<0.0001。

FIG. 4: immunogenicity of AdC68-RHAF and AdC68-RBD-HA2-CD8TM in BALB/c mice:

the mice used in the experiment are female BALB/c mice with the age of 6-8 weeks, and the immunogens are adenovirus AdC-RHAF and AdC68-RBD-HA2-CD8 TM.

A: the ELISA method measures the titer of bound antibody in mouse serum 1 week after the completion of immunization. The horizontal axis represents the immunization group, and the vertical axis represents the titer of the bound antibody.

B: neutralizing antibody titers against SARS-CoV-2 pseudovirus detected one week after the end of immunization.

FIG. 5: challenge experiment of new coronavirus:

A: viral load. The abscissa is the immunization group and the ordinate is the viral load.

B: lung tissue H & E staining.

C: body weight changes after challenge in mice. The abscissa is the number of days after challenge, and the ordinate is the weight percentage.

D: survival of mice after challenge. The abscissa is the number of days after challenge, and the ordinate is the percentage of survival.

FIG. 6: different influenza virus challenge experiments:

a: body weight change in mice after H7N9 challenge. The abscissa is the number of days after challenge, and the ordinate is the weight percentage.

B: survival of mice after H7N9 challenge. The abscissa represents days after challenge, and the ordinate represents percentage survival, wherein p is less than 0.05 and p is less than 0.01.

C: body weight change in mice after H3N2 challenge. The abscissa is the number of days after challenge, the ordinate is the weight percentage, p is <0.05, p is < 0.01.

FIG. 7: construction of R545-HA2-IntN-PAB-7XHis eukaryotic expression vector and expression of R545-HA2-IntN-PAB-7XHis protein:

a: constructing a map by pFastBac-Dual-R545-HA2-IntN-PAB-7XHis plasmid;

b: baculovirus successfully expressed the protein R545-HA2-IntN-PAB-7XHis in insect cell line Sf 9.

FIG. 8: purification of R545-HA2-IntN-PAB-7XHis protein and assembly of nanoparticles R545 HAF:

a: purifying R545-HA2-IntN-PAB-7XHis protein;

b: R545-HA2-IntN-PAB-7XHis linked to Intein from gb 1-IntC-Fn; as shown in the figure, the amount of the monomers gb1-IntC-Fn was significantly reduced and cleavage was seen to generate the byproducts IntN-PAB (17kDa) and gb1-IntC (11kDa), indicating the formation of the nanoparticle R545 HAF.

FIG. 9: immunogenicity of nanoparticle R545HAF in ICR mice:

the mice used in the experiment are 6-8 weeks old hACE2+ transgenic mice, and the immunogen is plasmid pcDNA3.1-S and nanoparticle R545 HAF.

A: the ELISA method measures the titer of bound antibody in mouse serum at week 4 after completion of immunization. The horizontal axis represents the immune groups and the vertical axis represents the titers of bound antibodies, indicating that p < 0.05.

B: data for bound antibodies to RBD protein after the first immunization and the third immunization, the left panel is overall boost, the right panel is the corresponding boost of a single mouse, indicating p < 0.05.

C: 293T-ACE2 cell detection of mice at week 4 after immunizationSerum neutralizing antibody titers. The abscissa is the immune group and the ordinate is the titer (ID) of neutralizing antibodies₅₀) Denotes p<0.05。

D: neutralizing antibody titers against SARS-Cov-2 pseudovirus after the first immunization and the third immunization, the left panel is the overall boost, the right panel is the corresponding boost of a single mouse, indicating p < 0.05.

Detailed Description

The present disclosure relates to the field of vaccines, and in particular to a method of simultaneously inducing an immune response against influenza and other viruses, including coronaviruses. 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 influenza and Xinguan.

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, "comprising," having, "or" including "includes" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

Immunogenic peptides and molecules encoding same

As used herein, the term "immunogenic peptide" refers to a peptide capable of simultaneously inducing an immune response against an influenza virus and another non-influenza virus, comprising the following moieties: (a) immunogen stem: it comprises the HA2 region of influenza virus hemagglutinin HA; (b) immunogen crown: comprising a viral membrane protein or an immunogenic fragment thereof, wherein the viral membrane protein is derived from a virus other than the virus from which the HA2 region of (a) was derived; (c) optionally, other moieties attached to the foregoing moieties.

As used herein, the term "linked" has its broadest meaning and can include, for example, fusion, co-expression, covalent linkage, coupling, and the like.

For example, as used herein, the terms "sRBD-HA 2 immunogenic peptide", "immunogenic peptide against influenza virus and novel coronavirus SARS-CoV-2", which are used interchangeably, refer to a peptide that includes the HA2 region of influenza hemagglutinin HA fused to the RBD region of the SARS-CoV-2 virus spike protein S and that HAs the ability to elicit the binding and neutralizing antibody effects and corresponding T cell responses against influenza virus and novel coronavirus SARS-CoV-2.

In some embodiments, the immunogenic peptide can be: (a) has the sequence shown in SEQ ID NO:1 and the amino acid sequence shown in SEQ ID NO 3 or 5 or 21; (b) (ii) a polypeptide which is homologous or similar in sequence to the polypeptide of (a) and has the same or similar immunogenicity, e.g. wherein each segment is identical to the sequence of SEQ ID NO:1 and the amino acid sequence shown in SEQ ID No. 3 or 5 or 21, respectively, have a homology or sequence identity higher than or equal to 80%, higher than or equal to 85%, 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%, or have a homology or sequence identity higher than or equal to 80%, higher than or equal to 85%, 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% with the polypeptide in (a); (c) and (b) protein or polypeptide which is derived from (a) or (b) and has the same or similar immunogenicity through substituting, deleting or adding one or more amino acids in the amino acid sequence defined by (a) or (b).

In some embodiments, the immunogenic peptide can be: (a') has the sequence of SEQ ID NO:9 or 15 or 25; (b') a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO. 10 or 16 or 26; (c ') a polypeptide which is homologous or sequence-similar to the polypeptides described in (a ') and (b ') and has the same or similar immunogenicity, for example has a homology or sequence identity of greater than or equal to 80%, greater than or equal to 85%, 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% to the polypeptides described in (a ') and (b '); (d ') a protein or polypeptide derived from (a ') or (b ') or (c ') by substitution, deletion or addition of one or several amino acids in the amino acid sequence defined in (a ') or (b ') or (c ') and having the same or similar immunogenicity.

In some embodiments, the immunogenic peptide can include other moieties attached to the immunogen stem or crown, for example, to enhance the stability of the immunogen stem or crown or fusion protein, to enhance neutralizing antibody responses, to form multimers, to increase cellular responses, and the like. In some embodiments, other portions may include, but are not limited to: proteins derived from viruses or hosts, transferrin (Fn), IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulating antibody, PD-1 and PD-L1 antibodies, CTLA4 antibodies, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and subunits thereof, bacterial flagellin, FimH, and the like.

In some embodiments, the immunogenic peptide may further include elements such as a signal peptide, a linker, a molecular tag, and the like. For example, a signal peptide element may refer to an amino acid sequence having the function of directing secretion, localization and/or transport of a protein, which is typically 5-30 amino acids in length. In some embodiments, the signal peptide element may be selected from: a protein self signal peptide, a CD33 protein signal peptide, a CD8 protein signal peptide, a CD16 protein signal peptide, a mouse IgG1 antibody signal peptide and an influenza HA protein signal peptide. For example, a linker peptide sequence (or linker) may be referred to as a fusion protein hereinThe length of the short peptide is usually 1-50 (e.g. 5-50, 5-40, 10-40) amino acids. In general, the linker peptide does not affect or seriously affects the formation of the correct fold and spatial conformation of the peptide of the invention. In some embodiments, the linker peptide element may be selected from: (G4S)₃Joint (G4S)_n、GSAGSAAGSGEF、 (Gly)₆、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly)₈、 EGKSSGSGSESKST。

The immunogenic peptide may also include variants thereof, such as deletion, insertion and/or substitution 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 addition of one or several (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.

Immunogenic peptides can be produced by recombinant expression under appropriate circumstances and conditions, e.g., produced by the coding nucleotide molecules, vectors, host cells of the disclosure; it can also be obtained by chemical synthesis or the like, so long as it has the desired amino acid sequence and immunogenicity and reactivity.

As used herein, the terms "immunogenic peptide-encoding molecule", "coding sequence", and the like, are used interchangeably and all refer to a nucleotide molecule that encodes an immunogenic peptide described in the present disclosure.

In some embodiments, the nucleotide molecule may be selected from, for example: (i) has the sequence shown in SEQ ID NO:2 and the nucleotide sequence shown in SEQ ID NO. 4 or 6 or 22; (ii) (ii) a molecule that hybridizes to (i) under stringent conditions; (iii) (ii) a nucleotide molecule homologous or similar to the sequence of (i) and capable of expressing a functional immunogenic peptide, e.g. wherein each segment is identical to the sequence of SEQ ID NO:2 and the nucleotide sequence shown in SEQ ID No. 4 or 6 or 22, respectively, have a homology or sequence identity higher than or equal to 80%, higher than or equal to 85%, 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%, or a nucleotide molecule having a homology or sequence identity higher than or equal to 80%, higher than or equal to 85%, 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% with the sequence in (i) or (ii) and capable of expressing a functional immunogenic peptide; (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 immunogenic peptide.

In some embodiments, the nucleotide molecule may be selected from, for example: (i) has the sequence shown in SEQ ID NO:10 or 16 or 26; (ii) (ii) a molecule that hybridizes to (i) under stringent conditions; (iii) (iii) a nucleotide molecule having greater than or equal to 80%, greater than or equal to 85%, 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 or sequence identity to the sequence in (i) or (ii) and capable of expressing a functional immunogenic peptide; (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 immunogenic peptide.

As used herein, the term "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; 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, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Vectors and host cells

The disclosure also relates to vectors comprising the immunogenic peptide-encoding nucleotide molecules of the present application, 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: 1431). Generally, the following steps are performed:

(1) transferring the coding nucleotide molecules of the present disclosure, or the recombinant expression vectors containing the nucleotide molecules, into suitable host cells;

(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 immunogenic peptide coding sequences 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, AdC68, 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. 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, K562, 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-672302, Hz2E5 and Ao 38.

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 or secreted intracellularly or on the cell membrane to the outside of the cell. 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 immunogenic peptides, nucleotide molecules, vectors, and/or host cells of the disclosure. The vaccine comprises a formulation of immunogenic peptides and/or nucleic acid molecules 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 reduce influenza virus infection and other non-influenza virus infections (e.g., novel coronavirus infections) 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.

The vaccines herein can be recombinant protein vaccines, recombinant DNA vaccines, recombinant viral vector vaccines (e.g., adenoviral vectors, poxvirus vectors, adeno-associated viral vectors, herpes simplex viral vectors, cytomegalovirus vectors), recombinant bacterial vector vaccines, recombinant yeast vector vaccines, or recombinant virus-like particle vaccines. In some embodiments, the vaccine herein is selected from a recombinant DNA vaccine, a recombinant adenoviral vector, or a combination of one or both thereof.

In some embodiments, one or more vaccines or combinations thereof selected from the group consisting of: recombinant plasmid vaccines (DNA), such as DNA vaccines comprising the SARS-CoV2 virus spike protein S coding sequence of the RBD region (e.g., pcDNA3.1-S); recombinant adenoviral vector vaccines, such as AdC 68-RHAF; recombinant protein subunit vaccines, such as nanoparticles, R545 HAF.

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.

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: aluminum adjuvant, cholera toxin and its subunit, oligodeoxynucleotide, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I: C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21, etc.

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 local (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 administration or any combination thereof, e.g., intramuscular injection followed by nasal drops.

In some embodiments, the vaccines herein prevent, eliminate or reduce influenza virus and novel coronavirus infections or at least one symptom thereof in a subject, such as respiratory symptoms (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 an immunogenic peptide, nucleotide molecule, vector, host cell and/or vaccine of the present disclosure, and may further comprise one or more additional substances that contribute to better functioning or enhancing the stability of the aforementioned substances in preventing and/or treating influenza virus infections and other non-influenza virus infections (e.g., novel coronavirus infections) or symptoms thereof. For example, the other substances may include other vaccines against coronavirus S or S1, such as S or S1 vaccines from including, but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV; other vaccines against influenza virus HA or HA2, said HA or HA2 being from the group including but not limited to H1-H18; 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 influenza virus infections and other non-influenza virus infections (e.g., novel coronavirus infections) 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, the primary immunization is performed with a DNA vaccine and the one or more booster immunizations are performed with a recombinant viral vaccine. 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.

Depending on the characteristics of the different vector vaccines, in some preferred embodiments, a systemic prime is performed using a recombinant DNA vaccine to establish a systemic immune response, followed by one or more boosts using other vaccines (e.g., recombinant adenoviral vaccines or recombinant poxvirus vaccines), which may include at least one boost in the respiratory tract (e.g., using an adenoviral 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 packaged in one or more containers, for example sealed containers such as ampoules or sachets, indicating the amount of composition, for example, one or more of the vaccine compositions herein or one or more of its ingredients. 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.

The combination product of the present disclosure can be used for the characteristic of locally inducing high-level antigen-specific CD8+ T cell responses in the respiratory tract, making it promising for the prevention and treatment of respiratory tract pathogen infection, reduction of respiratory tract pathogen pathogenicity, and respiratory tract tumors.

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 protocols, immunogens, pseudoviruses and detection methods involved in the following experiments were as follows:

I. experimental animals:

6-8 week old female C57/BL6 and BALB/C mice, purchased from Shanghai Jihui laboratory animal feeding Co., Ltd.);

6-8 week old female hACE2+ ICR mice, purchased from Beijing Vital rising Tech technologies, Inc.).

Immunization protocol:

intramuscular injection is respectively carried out on the left hind limb and the right hind limb of the mouse; or for nasal drip. Specific dosages are given in the examples.

Selection of immunogen:

the HA2 sequence is from Genebank AGI60292.1, and the S and RBD sequences are from Genebank NC-045512.2; the hFn sequence (human transferrin) is from Genebank: M97164.1, and the specific sequence is shown in a sequence table.

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

2. recombinant adenoviral vector vaccines: AdC68, AdC 68-sRGB-HA 2-hFn (AdC68-RHAF), AdC68-RBD-HA2-CD8 TM.

3. Recombinant protein subunit vaccine (nanoparticle, protein): r545HAF

Immunogen preparation and immunization dose:

preparation of immunogens see example 1

The immunogen immunizing doses used in the examples are as follows:

1. recombinant plasmid vaccine (DNA): 100 mu g/mouse, 100 mu L, dissolved in sterile physiological saline;

2. recombinant adenoviral vector vaccines: 5E10 vp/mouse, 100 μ L (intramuscular injection); 5E10 vp/mouse, 40. mu.L (nasal drip).

3. Recombinant protein subunit vaccine (nanoparticle, protein): the protein (dissolved in sterile PBS) was mixed with aluminum adjuvant (aluminum, InvivoGen, cat # 5200) at a volume ratio of 1:1 and immunized with 20. mu.g/mouse, 100. mu.L;

v. immunization interval:

specific immunization intervals are shown in the table below.

SARS-CoV2 envelope Pseudovirus (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 medium⁶one/mL of cells, 1mL of diluted cells were plated in a 10cm dish at 37 ℃ with 5% CO₂Culturing overnight;

2. sucking SARS-CoV2 membrane protein plasmid pcDNA3.1-S4 μ g and pNL4-3 delta env skeleton plasmid 8 μ g (NIH AIDS Reagent Program,3418) into 500 μ L DMEM without double antibody (serum-free, double antibody is mixed solution of streptomycin), and incubating at room temperature for 5 min;

3. diluting 24 μ L TurboFect (Thermo Fisher Scientific) with DMEM-free medium to a final volume of 500 μ L/sample, and incubating at room temperature for 5 min;

4. the two samples 2 and 3 were mixed well, 1000. mu.L/final sample volume, incubated at room temperature for 20min, and added to 293T cells in a 10cm dish after incubation. After 6h, replacing fresh 15mL of complete culture medium, and continuously culturing in a cell culture box for 48 h;

5. after the culture is finished, collecting cell culture supernatant in a 10cm dish, centrifuging for 10min at 4000g and 4 ℃ in a 15mL centrifuge tube, filtering the cell culture supernatant into a new 15mL centrifuge tube by using a 0.45 mu m filter, freezing the cell culture supernatant at the temperature of minus 80 ℃, and titrating the cell culture supernatant for later use.

Construction of 293T cells stably expressing hACE2 receptor:

1. an artificial synthetic human ACE2(hACE2) sequence (Genebank # NCBI _ NP-001358344.1) as shown in SEQ ID NO:13 with an Age1 cleavage site at the 5 'end and an Xba1 cleavage site at the 3' end, the synthetic fragment cleaved with vector plasmid pHAGE-MCS-puro using Age1 (Thermo Scientific, cat # FD1464) with Xba1 (Thermo Scientific, cat # FD0685) and recovered by gel electrophoresis followed by gel cutting and recovery of the cleaved fragments using Sanprep column DNA gel recovery kit (Promega, cat # A9282).

2. The gene recovery product was ligated to the digested linearized vector using T4 DNA ligase (Thermo Scientific Co., Ltd., 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-hACE2-puro) for the hACE2 gene was successfully cloned.

3. 10cm dishes were taken and inoculated about 5X 10 in each dish⁶293T 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 a purified hACE2 lentivirus.

5. Spread 5X 10 one day in advance⁵293T cells in one well of a 12-well plate in the next dayTo the plated cells, 500. mu.L of the virus concentrated in step 2 was added at 1000g, and centrifuged 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 (capable of binding to S protein) which stably expressed hACE2 were selected by flow screening.

VIII, detection method:

blood collection:

at 2/4 weeks after the last immunization, the mice were decapped before death, whole blood was collected from the periphery of the mice by an eye picking method, collected in 1.5mL EP tubes, allowed to stand at room temperature for natural coagulation, and the coagulated serum was centrifuged at 7000g for 15 min. 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.

ELSIA detection of bound antibodies:

1. the detected antigenic protein (S1, available from Beijing Yiqiao Shenzhou technologies, Inc.; RBD, available from Shanghai, offshore Biotechnologies, Inc.) was diluted with a4 ℃ pre-chilled ELISA coating to a final concentration of 1. mu.g/mL. Add 100. mu.L of the coating antigen solution to each well of the ELISA plate, and keep it at 4 ℃ overnight;

2. the next day, the ELISA plate was removed, the coating solution was discarded, and the plate was washed 3 times with 0.05% PBST buffer, 220. mu.L each time;

3. after washing, patting the mixture on absorbent paper, sealing each hole by using 200 mu L of ELISA sealing solution (0.5% skimmed milk powder, dissolved in PBST), and sealing at room temperature for 2 h;

4. after blocking, wash the plate 3 times with 0.05% PBST, 220 μ L each time;

5. for serum or plasma, dilutions were performed with ELISA sample dilutions (0.5% skim milk powder, PBST lysis) starting from 1:100 and 2-fold dilutions were performed. A negative control was set with non-immunized mouse serum. Setting blank holes, only adding sample diluent, making 2 multiple holes for each sample, wherein the final volume of each hole is 100 mu L, and incubating for 3h at room temperature;

6. after the incubation of the sample is finished, the plate is continuously washed by PBST for 5 times, and each time is 220 mu L;

7. diluting the corresponding proportion of secondary antibody (goat anti-mouse, purchased from Beijing Zhonghua Ching Jinqiao biotechnology Co., Ltd., product No. ZB-2305) with ELISA blocking solution (0.5% skimmed milk powder, PBST dissolved), adding 100 μ L per well, and incubating at room temperature for 1-1.5 h;

8. after the secondary antibody incubation was completed, the plate was washed 5 times with 0.05% PBST, 220 μ L each time;

9. dissolving a pair of gold and silver sheet OPD substrates in 20mL of deionized water, then adding 100 mu L of gold and silver sheet OPD substrates into each hole, and reacting for 5min in a dark place;

10. after the color development was completed, 50. mu.L of 2nM H was used₂SO₄The termination is performed and the OD is read on the microplate reader₄₉₂-OD₆₃₀A value;

11. at the last dilution OD₄₉₂The reciprocal of the serum dilution ratio corresponding to a value of (negative mean + SD) greater than 2-fold was taken as the antibody titer.

293T-ACE2 cells detect neutralizing antibodies:

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 pseudovirus at 200TCID₅₀。

3. Gently shaking and mixing, placing the blackboard with 96 holes bottom in a cell culture box, keeping the temperature at 37 ℃ and 5% CO₂Incubate for 1 h.

4. When the incubation time reached 20min, 293T-hACE2 target cells were initially prepared and diluted to 10 with complete medium⁵Individual 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 10⁴And (4) respectively.

6. Lightly in front, back, left and rightShaking 96-well transparent bottom blackboard to disperse the cells uniformly, and placing the blackboard in a cell culture box at 37 deg.C and 5% CO₂Culturing for 48 h.

7. Culturing for 48h, taking out 96-well transparent bottom blackboard from the cell culture box, sucking off supernatant in the wells, adding 100 μ L PBS to each well for washing, sucking off PBS, adding 50 μ L of 1 × lysis buffer (from Cat # E153A of Promega corporation) to each well, and incubating on a horizontal shaker at room temperature for 30min 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

The luciferase activity is detected by a 96-microplate luminescence-detection instrument.

9. Reading values of fluorescein are derived, neutralization inhibition rates are calculated, and ID is calculated by utilizing Graphpad Prism 5.0 software according to the results of the neutralization inhibition rates₅₀。

ELISPOT detects T cell responses:

mouse spleen single cell isolation:

1) lying the mouse on the back, dissecting the skin of the right abdomen, opening the peritoneum, taking down the spleen of the mouse, and putting the mouse into a small plate added with 5mL of complete RPIM1640 medium;

2) wrapping spleen with sterile gauze with sterile forceps, clamping the gauze with forceps, and lightly grinding spleen to release splenocytes into culture medium;

3) then sucking the spleen cell suspension into a sterile 15mL centrifuge tube through a gauze by using a 5mL pipette, and centrifuging for 5min at 800 g;

4) discarding the centrifuged supernatant, tapping 15mL centrifuge tubes to resuspend cell precipitates, adding 3mL erythrocyte lysate into each centrifuge tube to lyse erythrocytes, reversing and uniformly mixing, and standing at room temperature for 5min to fully lyse erythrocytes without damaging splenocytes;

5) after completion of the flushing, the flushing was stopped with 5 mlpep im1640 medium. 800g, centrifuging for 5 min;

6) discarding the centrifuged supernatant, washing with 5mL of RPIM1640 medium for 1 time, and centrifuging at 800g for 5 min;

7) the supernatant after centrifugation was discarded, and the spleen cells were frozen in a freezing medium (90% FBS and 10% DMSO) for use.

ELISpot experimental procedures were performed according to the Mouse IFN-. gamma./Monkey IFN-. gamma.instructions.

1) Millipore plates provided in a kit (purchased from BD, cat # 551083) were coated with purified IFN- γ antibodies, ratio 1: coating at 250, 4 ℃ overnight;

2) spin off the coated antibody solution in the plate, wash the plate once with 200 μ L RPMI 1640 complete medium, then block the Millipore plate with 200 μ L RPMI 1640 complete medium blocking solution, incubate for 2h at room temperature;

3) the blocking solution was discarded from the well plate and, according to various experimental designs, a pool of stimulating peptides was added to the Millipore plates at 50. mu.L/well and each peptide was at a concentration of 5. mu.g/mL. Add 50. mu.L of RPMI 1640 complete medium to the negative control wells; add 50. mu.L phorbol ester polyclonal stimulator (PMA, purchased from Sigma, cat. FXP012) (final concentration 100ng/mL) and Ionomycin (Ionomycin, final concentration 2. mu.g/mL) RPMI 1640 complete medium to the positive control wells;

wherein, the stimulating peptide library is synthesized by Suzhou Qiangyao biotechnology, Inc., each single peptide is 15 amino acids, covers RBD sequence, and one peptide library is formed by every five single peptides, namely 13 peptide libraries in total. Wherein, the 1 st peptide is MPTESIVRFPNITNL (SEQ ID NO:19), the 2 nd peptide is aa 8-22 in the RBD peptide sequence of SEQ ID NO:3, the following peptides are each shifted backward by four positions compared with other peptide amino acids, namely, the 3 rd peptide is aa 12-26 in the RBD peptide sequence of SEQ ID NO:3, the 4 th peptide is aa 16-30 … … in the RBD peptide sequence of SEQ ID NO:3, and so on until the 64 th peptide is aa 256-270 in the RBD peptide sequence of SEQ ID NO:3, and the 65 th peptide is AVRDPQTLEILDITP (SEQ ID NO: 20).

4) Counting the mouse spleen cells, adjusting the cells to 4 × 10⁶cells/mL, 50. mu.L of cells per well, resulting in a 2X 10 cell count per well⁵And (4) cells. Mixing MillipoThe re plates were placed in a wet box at 37 ℃ 5% CO₂Incubating in incubator for 20-22h without shaking the plate to avoid cell migration;

5) after the culture incubation is finished, taking out the Millipore plate from the incubator, discarding the liquid in the plate, washing twice with precooled deionized water, wherein each time is 220 mu L, and each time is washed and incubated for 3 min;

6) wash the plate 3 times with 0.05% PBST (PBS + 0.05% Tween-20), 200. mu.L each time;

7) the Biotinylated Detection antibody was diluted with 10% FBS in PBS antibody diluent (Biotinylated Detection antibody, ratio 1: 200) adding 100 mu L of the mixture into each hole, and incubating for 2 hours at room temperature;

8) after incubation, wash the plate 3 times with 0.05% PBST, 220 μ L each time;

9) the streptavidin-HRP conjugated antibody was diluted with an antibody diluent (ratio 1: 100) adding 100 mu L of the mixture into each hole, and incubating for 1h at room temperature;

10) after incubation, wash the plate 4 times with 0.05% PBST, 220 μ L each time;

11) washing the plate with PBS for 2 times, 220 μ L each time;

12) a substrate solution (1mL of substrate buffer plus 1 drop of substrate solution) was prepared and 100. mu.L of substrate solution was added to each well. The reaction is carried out for 5-60min, and the incubation time is determined according to the spot formation condition.

13) Washing with deionized water to terminate the reaction, air drying at room temperature, and counting;

14) counting of Spot Forming Cells (SFC) and QC were performed using a champshot type III enzyme linked spot image analyzer.

Example 1: construction of S protein eukaryotic expression vector and RHAF and RBD-HA2-CD8TM adenovirus expression vector and protein expression

To study the function of RBD, we constructed eukaryotic expression vectors for the S protein and expressed the mature protein in vitro in a cell line.

First, we artificially synthesized the S gene (the corresponding amino acid sequence is shown in SEQ ID NO:11, and the gene sequence is shown in SEQ ID NO: 12), the synthesized fragment was digested with vector plasmid pcDNA3.1 (purchased from Youbao, Inc., FD0054) and Not1 (Thermo Scientific Inc., FD0596) using BamHI digestion (Thermo Scientific Inc., FD0054), 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.A 9282).

Subsequently, the gene recovery product was ligated with the digested linearized vector by the method of T4 DNA ligase (Thermo Scientific Co., Ltd., cat. No. 2011A): coli Stable was transformed with the ligation product and grown overnight on ampicillin-containing plates. On day 2, single colony was randomly selected for sequencing, mutation site correction, and after all sequences were verified to be correct, the eukaryotic expression vector pcDNA3.1-S for the S protein was successfully cloned, and the plasmid construction map is shown in FIG. 1A.

We further examined whether the S protein could be expressed in the eukaryotic cell line 293T.

First, 6-well plates were prepared and seeded at approximately 6X 10 in each well⁵293T cells, which ensures that the cell density reaches 90% when the transfection is carried out on the next day; 293T cells (transfection reagent TurboFect) were transfected with pcDNA3.1 and pcDNA3.1-S, respectively. Culturing at 37 deg.C in 5% incubator for about 48 hr, collecting cells according to cell condition, performing Western Blotting (WB) identification, and finding that S protein is expressed in cells and has a size of about 200 kD; whereas the expression of the S protein could not be detected in the pcDNA3.1 control transfected cells (FIG. 1B left panel).

To investigate the function of HA2, we constructed an adenoviral expression vector for RHAF (i.e., sRBD-HA2-hFn fusion protein), and adenovirus AdC68-RHAF, and verified the expression of RHAF in adenovirus.

First, we artificially synthesized the RHAF gene (the specific sequence is shown in SEQ ID NO:10, wherein the HA2 part is based on the sequence shown in SEQ ID NO:2, the sRBD part is based on the sequence shown in SEQ ID NO:6, and the hFn part is based on the sequence shown in SEQ ID NO: 8). sRGB-HA 2 was expressed at the N-terminus of hFn with G between₄And connecting the S joint. The synthetic fragment was ligated with the vector plasmid pAdC68XY3 (constructed according to the method described in patent application No. 201910777937.2, in which the E4 portion of the E1/E3 deleted replication deficient AdC68 vector was replaced with the corresponding E4 portion of AdHu5In addition, the modification can increase the yield of recombinant chimpanzee adenovirus without changing the serological properties of adenovirus), using SrfI restriction enzyme (NEB corporation, # R0629S), and recovering the fragments by gel electrophoresis followed by gel cutting, and recovering the fragments using a Sanprep column DNA gel recovery kit (Promega corporation, cat. No. A9282).

Subsequently, the gene recovery product was ligated with the digested linearized vector by the method of T4 DNA ligase (Thermo Scientific Co., Ltd., cat. No. 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 after all sequences were verified to be correct, the RHAF adenovirus expression vector pAdC68XY3-RHAF was successfully cloned, and the plasmid construction map is shown in FIG. 1A.

In order to research whether RBD-HA2 and hFn are fused and secreted or RBD-HA2 is expressed on the surface of adenovirus through a CD8 transmembrane region (CD8TM) to have better effect, an adenovirus expression vector of RBD-HA2-CD8TM and adenovirus AdC68-RBD-HA2-CD8TM are constructed. The construction method is consistent with that of AdC68-RHAF, and the amino acid sequence is shown as SEQ ID NO: 15 and the coding sequence is shown in SEQ ID NO 16. The plasmid construction map is shown in FIG. 1A.

We further packaged and amplified the recombinant adenovirus.

The constructed recombinant plasmids pAdC68XY3-RHAF and pAdC68XY3-RBD-HA2-CD8TM are respectively linearized for 3.5h in a water bath at 37 ℃ by restriction endonuclease Pac 1, and the endonuclease is inactivated at 65 ℃. The method comprises the steps of paving a six-hole plate with 293A cells, transfecting linearized recombinant plasmids to the 293A cells in the amounts of 2.5 mu g, 2 mu g and 1.5 mu g per hole respectively, culturing for about 11 days to enable plaques to appear, collecting samples after about 14 days to enable the cells to be infected by viruses, collecting cell supernatants together, repeatedly freezing and thawing for three times at-80 ℃, taking a small amount of infected 293A cells, and verifying the expression of target proteins by Western blot to find the expression of proteins with correct sizes (figure 1B).

Infecting the collected adenovirus sample to 293A cells of 1T 175 cell culture bottle, collecting the sample after 24h, placing the sample in a culture bottle for repeated freeze thawing at minus 80 ℃ for three times, infecting the sample to 293A cells of 6T 175 cell culture bottles, carrying out mass amplification in the same way, collecting cell precipitates when the sample is amplified to 36T 175 cell culture bottles, abandoning supernatant, resuspending the cell precipitates by using about 10mL of serum-free and non-resistant DMED culture medium, purifying adenovirus by adopting a cesium chloride density gradient centrifugation method after repeated freeze thawing at minus 80 ℃ for three times, and subpackaging the adenovirus sample for preservation at minus 80 ℃.

Cesium chloride density gradient adenovirus step:

(1) centrifuging the adenovirus subjected to freeze thawing at 4 ℃ and 4000g for 30min, taking the supernatant, and filtering with a 0.8-micron filter membrane;

(2) adding 3mL of 1.2M CsC solution into an ultracentrifuge tube, gently and slowly injecting the 3mL of 1.4CsC solution from the bottom by using an injector, and paving a cesium chloride gradient;

(3) gently adding the filtered adenovirus solution into an ultracentrifuge tube, filling the liquid level with PBS, and placing the solution into an SW41 sleeve after balancing;

(4) selecting an SW41 rotor, centrifuging at 4 ℃, 25000rpm for 2.5h, and reducing the speed to be no brake natural speed reduction;

(5) after the centrifugation is finished, the ultracentrifuge tube is gently taken out, two white virus strips can be seen, the ultracentrifuge tube is poked into the ultrariser tube from the side by using an injector, the lower layer strip is sucked out, and the phenomenon that the upper layer strip is sucked is avoided;

(6) injecting Bio-Gel P-6PG Gel desalting glue into a Liquid chromatography column (Liquid chromatography columns), and adding PBS for washing twice when Liquid falls off and glue of about 2-3cm is left;

(7) adding the virus solution into desalting gel, adding PBS (2 mL) for elution after the virus solution completely falls off, and collecting 3-4 drops of eluent in each 1.5mL EP tube;

(8) nanogrop 2000 determination of OD of eluate from tube₂₆₀Value, OD₂₆₀Mixing the eluates above 2, adding 10% sterile glycerol, and packaging;

(9) nanogrop 2000 determination of OD of Adenoviral solution after subpackaging₂₆₀Value, OD₂₆₀ Value X1.1X 10¹²the/mL is the titer of the purified adenovirus;

(10) the final AdC68-RHAF titer was 1.76X 10¹³The titer of AdC68-RBD-HA2-CD8TM is 1.48X 10¹³/mL。

Example 2: immunogenicity of AdC68-RHAF in C57BL/6 mice

The C57BL/6 mice were immunized with DNA-pcDNA3.1-S and adenovirus AdC68-RHAF, respectively, and the immune combinations were evaluated for induction of the titers of binding antibodies against the RBD protein and H7 protein and neutralizing antibodies against the SARS-CoV-2 pseudovirus 2 after 2 weeks of immunization. The level of T cell responses to the RBD peptide pool was also assessed.

Mice were randomly divided into 2 groups, and named control group and AdC68-RHAF group, respectively, based on the immunogen. The specific immunization combination is shown in table 1, and the immunization mode is intramuscular injection.

The AdC68-RHAF group generated binding antibody titers against RBD protein and H7 protein as shown in FIG. 2A: the titers of binding antibodies to the RBD protein were all above 10,000, up to 50,000; the mean of bound antibody titers against H7 was around 6,000, with the highest being able to reach 10,000. Also, comparing the data of the binding antibodies to the RBD protein before and after the second immunization, it was found that there was a significant increase in the titer of the binding antibodies (fig. 2B).

The AdC68-RHAF group generated neutralizing antibody titers against SARS-Cov-2 pseudovirus as shown in FIG. 2C: most titers were above 1000, the mean was 1360, and there was a neutralizing antibody titer up to 12808 in one mouse, with individual variability between mice. Simultaneously comparing the data of the neutralizing antibody titer against SARS-Cov-2 pseudovirus before and after the second needle immunization, it was found that the neutralizing antibody titer was significantly increased (FIG. 2D).

The AdC68-RHAF group generated T cell responses against the RBD peptide library as shown in FIG. 2E: the number of IFN-gamma secreting cells per million splenocytes is around 3,000.

This experiment demonstrated that AdC68-RHAF was able to induce antibodies against influenza and new corona simultaneously, and could induce neutralizing antibodies against new corona virus and T cell responses against new corona virus in C57BL/6 mice.

TABLE 1 immunization protocol for the immunogenicity test of AdC68-RHAF in C57BL/6 mice

Example 3: immunogenicity of AdC68-RHAF in hACE2+ ICR mice

According to the conclusions drawn in example 2, in order to be able to subsequently verify the protection of mice against challenge with the new coronavirus, we verified the immunogenicity of AdC68-RHAF in hACE2+ ICR mice and evaluated the immunological combination to induce a binding antibody titer against RBD protein, H3 protein and H7 protein and a neutralizing antibody titer against SARS-CoV-2 pseudovirus 2 weeks after completion of the immunization.

The hACE2+ ICR mouse used in this example is a COVID-19 preclinical model, which is a SARS-CoV-2 susceptible human angiotensin converting enzyme 2(hACE2) transgenic mouse constructed in the ICR mouse background, and expresses hACE2 mainly in the lung, heart, kidney and small intestine. The mouse model is an important tool for researching and developing SARS-CoV-2 therapeutic drugs and vaccines.

The experimental procedure was as follows: mice were randomly divided into 2 groups, designated control group and AdC68-RHAF group, respectively, based on immunogen. Specific immunization combinations are shown in table 2, and the immunization regimen was intramuscular injection.

The AdC68-RHAF combination produced binding antibody titers against the RBD, H3 and H7 proteins as shown in fig. 3A: the titers of binding antibodies against RBD proteins are mostly above 10,000, and in part can reach 100,000; the titer of the binding antibody against the H3 protein is mostly above 100, and in part can reach 1000; the binding antibody titer against H7 protein was mostly around 6,400.

2 weeks after the end of the second needle immunization, the AdC68-RHAF group generated neutralizing antibody titers against SARS-CoV-2 pseudovirus as shown in FIG. 3B: most of them are about 1000, and the highest can reach 2253.

The experiment proves that AdC68-RHAF can simultaneously induce the combined antibodies aiming at the new corona and the influenza in an hACE2+ ICR mouse, and can simultaneously induce the combined antibodies aiming at the influenza H3 and H7, thereby verifying the broad spectrum of the HA2 antibody. Meanwhile, the AdC68-RHAF can effectively induce a neutralizing antibody aiming at the new corona, and has good prospect of being developed into anti-influenza and new corona vaccines.

TABLE 2 immunization protocol for immunogenicity testing of AdC68-RHAF in hACE2+ ICR mice

Example 4: immunogenicity of AdC68-RHAF and AdC68-RBD-HA2-CD8TM in BALB/c mice

BALB/c mice were immunized with AdC68-RHAF and AdC68-RBD-HA2-CD8TM, respectively, and the immune combinations were evaluated to induce the titers of binding antibodies against RBD protein and H7 protein and neutralizing antibodies against SARS-CoV-2 pseudovirus 2 after 2 weeks of immunization. The level of T cell responses to the RBD peptide pool was also assessed.

Mice were randomly divided into 3 groups, designated AdC68, AdC68-RHAF and AdC68-RBD-HA2-CD8TM, respectively, based on the immunogen. Specific combinations of immunizations are shown in table 3, and the immunization regimen was intramuscular injection.

Serum collected one week after the end of immunization detected the bound antibody titer to RBD protein as shown in figure 4A: the mean value of the binding antibody titer of the AdC68-RHAF group against the RBD protein is 4031, and the highest energy is 12,800; the mean binding antibody titer against RBD protein of group AdC68-RBD-HA2-CD8TM was 10,160, the highest being able to reach 25,600, when there was no significant difference between group AdC68-RHAF and group AdC68-RBD-HA2-CD8 TM.

Neutralizing antibody titers against SARS-CoV-2 pseudovirus were detected one week after the end of immunization, as shown in figure 4B: the mean value of neutralizing antibody titer of the AdC68-RHAF group is 128, and the highest energy is 274; the mean value of the neutralizing antibody titer of the AdC68-RBD-HA2-CD8TM group was 97, the highest of which was 348, when there was no significant difference between the AdC68-RHAF group and the AdC68-RBD-HA2-CD8 TM.

The titer of bound antibodies against RBD and the titer of neutralizing antibodies against SARS-CoV-2 pseudovirus were then continuously measured two weeks after the end of immunization.

Meanwhile, the experiment further proves that two adenoviruses AdC68-RHAF and AdC68-RBD-HA2-CD8 TM: effective to induce the production of binding antibodies against influenza virus needle H7; can effectively induce neutralizing antibody against SARS-CoV-2 pseudovirus; at the same time, T cell responses against the RBD peptide pool can be induced.

TABLE 3 immunogenicity testing protocol of AdC68-RHAF and AdC68-RBD-HA2-CD8TM in BALB/c mice

Example 5: immunogenicity of AdC68-RHAF in BALB/c mice by different vaccination modes

BALB/c mice were immunized with DNA-pcDNA3.1-S and adenovirus AdC68-RHAF, respectively, and after 2 weeks of immunization, the immune combinations were evaluated for the induction of the titers of binding antibodies against RBD protein and H7 protein and neutralizing antibodies against SARS-CoV-2 pseudovirus. The level of T cell responses to the RBD peptide pool was also assessed.

Mice were randomly divided into 4 groups, and named AdC68 muscle group, AdC68 nasal drip group, AdC68-RHAF muscle group and AdC68-RHAF nasal drip group, respectively, according to immunogen and immunization modes. Specific immunological combinations are shown in table 4.

Collecting serum and lung lavage fluid two weeks after immunization to detect binding antibodies including IgG and IgA against RBD protein and influenza virus H7 protein; neutralizing antibodies against SARS-CoV-2 pseudovirus and T cell responses against the RBD peptide library were simultaneously detected.

TABLE 4 immunogenicity testing protocol generated by different vaccination regimes of AdC68-RHAF in BALB/c mice

Example 6 challenge test for New coronavirus

Mice: nanamo organism ACE 2-transgenic mice (C57BL/6-Tgtn (CAG-human ACE2-IRES-Luciferase-WPRE-polyA) Smoc), females.

Strain: SARS-CoV-2/human/CHN/Shanghai _ CH-02/2020.

Committing second military medical university to challenge

Immunization procedure: mice were randomly divided into 2 groups, designated control group and AdC68-RHAF group, respectively, based on immunogen. Specific combinations of immunizations are shown in table 5, with the immunization being by intramuscular injection.

And (3) toxin counteracting program: performing challenge test on the mice 1-2 weeks after the immunization is finished, observing for 5 days after challenge, and recording the weight change; 3 mice are taken out of each group to be killed on the third day after the challenge, lung tissues are taken, the left half lung tissue (one big leaf) of each mouse is fixed by 4% paraformaldehyde for 48 hours, and pathological sections are made (H & E staining and organizing); right half lung tissue (4 leaflets) of each mouse was ground for viral load qPCR rnaviruses or viral titer PFU/ml; on the fifth day, if there are more mice alive, the mice are weighed out and then sacrificed altogether.

The results show that: from the viral load, the AdC68-RHAF group was 3.7 Logs lower than the control group, with no viral load detectable in two of the three AdC68-RHAF groups (FIG. 5A). Meanwhile, from the results of H & E staining, the lung tissue pathology was significantly better in the AdC68-RHAF group than in the control group, with less damage and less inflammatory infiltration (fig. 5B). From the body weight, the AdC68-RHAF group was unchanged, while the control group showed a sharp drop on the third day (FIG. 5C). From a survival perspective, the AdC68-RHAF group survived one hundred percent, while the control group all died (FIG. 5D). Overall, all data demonstrate excellent protection of the AdC68-RHAF vaccine against new corona.

TABLE 5 AdC68-RHAF immunization protocol in hACE2+ C57BL/6 mice in vivo

Example 7 challenge test for mouse influenza Virus

Mice: hACE2+ ICR mice and BALB/c mice, female.

Strain: H7N9(A/Shanghai/4664T/2013), H3N2(A/Hong Kong/8/68).

The public health clinic centers P2 and P3 in Shanghai city conducted challenge tests.

Immunization procedure: mice were randomly divided into 2 groups, designated control group and AdC68-RHAF group, respectively, based on immunogen. Specific combinations of immunizations are shown in table 6, and the immunization regimen was intramuscular injection.

And (3) toxin counteracting program: and (3) after the immunization is finished, carrying out nasal drop challenge on the mice, continuously observing for 14 days after challenge, weighing every day, recording the weight change, and obtaining a survival curve.

The results showed that for H7N9 challenge, mice in the AdC68-RHAF group had a weight gain at day seven, while the control group had been decreasing (fig. 6A). Whereas from the survival point of view, the AdC68-RHAF group was completely alive, the control group was totally dead on the tenth day (FIG. 6B). For H3N2 challenge, the dose was non-lethal to mice, so that looking at body weight changes alone, it was found that the AdC68-RHAF group had significantly less weight loss than the control group (fig. 6C). In general, AdC68-RHAF can effectively generate protective effect against each test strain, and has broad spectrum.

TABLE 6 AdC68-RHAF immunization protocol in hACE2+ C57BL/6 mice in vivo

Example 8 construction of R545-HA2-IntN-PAB-7XHis eukaryotic expression vector and expression of R545-HA2-IntN-PAB-7XHis protein

From the previous experiments, we found that the adenoviral vector vaccine AdC68-RHAF was able to induce antibodies against both new corona and influenza, and both had protective effects. In subsequent experiments, we tried to replace the carrier, i.e. directly using purified nanoparticles for testing. In order to improve the solubility and renaturation rate of protein, we select a shorter RBD peptide fragment (R545, the corresponding amino acid sequence is shown as SEQ ID NO:21, and the gene sequence is shown as SEQ ID NO: 22), which is more favorable for the inclusion body purification of protein than the RBD in the previous paragraph.

In order to express R545-HA2-IntN protein, donor plasmids of R545-HA2-IntN (R545HA-IntN) are constructed, recombinant baculovirus capable of efficiently expressing R545-HA2-IntN in insect cell line Sf9 is obtained through transposition, and mature protein is expressed in insect cell line Sf9 in a eukaryotic mode.

First, we performed codon optimization of insect Sf9 cells for R545-HA2-IntN gene and added lysotropic tags PAB (protein A B domin) and 7XHis tags, artificially synthesized R545-HA2-IntN-PAB-7XHis gene fragment (department of pronunciations, corresponding amino acid sequence of which is shown in SEQ ID NO:23, gene sequence of which is shown in SEQ ID NO: 24), and inserted between digestion sites BamH I (Thermo Scientific, FD0054) and Hind III (Thermo Scientific, FD0504) downstream of PH promoter of pFastBac-Dual plasmid (Sammer Thermo, 10712024) to obtain donor plasmid pFBD-R545HA-IntN-PAB-7XHIS (FIG. 7A).

We then transformed Bacmid-containing DH10Bac competent (exclusively) with donor plasmid pFBD-R545HA-IntN-PAB-7XHis, plated with X-gal, IPTG, kanamycin and gentamicin resistant plates, cultured for 2 days at 37 ℃, picked white colonies and verified by PCR with M13-F/R primer, and picked positive clones to get bR545HA-IntN-PAB-7 XHis.

We then transfected adherent Sf9(Spodoptera frugiperda clone 9, ATCC) cells with bR545HA-IntN-PAB-7XHis, as specified in the Cellffectin II Reagent (Invitrogen, Cat. 10362100) transfection procedure, to obtain P1 generation recombinant baculovirus vAc^{R545HA-IntN-PAB-7XHis}Substituting P1 for vAc^R545HA ^{-IntN-PAB-7XHis}600mL of Sf9 cell line suspension-cultured in SF-SFM medium (Womei organism, SF10111-2) was infected at a volume ratio of 1:100, with a cell density of 2X 10⁶The expression and distribution of R545HA-IntN-PAB-7XHis protein was examined by harvesting cell cultures after 5 days/mL (FIG. 7B). The results show that the fusion protein is mostly expressed in cells, so the subsequent purification is performed by adopting an inclusion body purification mode.

Example 9 purification of R545-HA2-IntN-PAB-7XHis protein

To obtain nanoparticles displaying R545-HA2, we first purified the R545HA-IntN-PAB-7XHis protein by the following steps:

(1) to convert vAc^{R545HA-IntN-PAB-7XHis} 600mL Sf9 cells 5 days after infection 2000g, centrifuged 10min, and cells were harvestedPrecipitating;

(2) mu.L of 100 Xprotease complex inhibitor (Producer, cat # 600387) was added to 50mL of cell lysate (50mM Tris-HCl pH 8,0.5M NaCl, 1% NP40), sonicated cell disruptor (SCIENTZ-IID), beaten for 15s, stopped for 20s, and microscopic examination was performed until the cells were completely disrupted.

(3)8000g were centrifuged at 4 ℃ for 20min, the pellet was collected and resuspended in 50mL 50mM Tris-HCl pH 8.5, 50. mu.L of a super nuclease (Tiandi and, PE001B) was added and incubated at room temperature for 20 min.

(4)8000g were centrifuged at 4 ℃ for 20min, and the pellet was collected, resuspended in 50mL of 2M urea (50mM Tris-HCl pH 8,0.5M NaCl, 0.5% NP40, 0.05% Tween), and kept on ice for 5 min.

(5)8000g, centrifuging at 4 deg.C for 20min, collecting precipitate, and repeating step (4).

(6)8000g were centrifuged at 4 ℃ for 20min, and the pellet was resuspended in 50mL of 8M urea (50mM Tris-HCl pH 8,0.5M NaCl), and the pellet was thoroughly solubilized by rotating the freezer overnight.

(7)8000g, centrifuging at 4 deg.C for 20min, collecting supernatant, and filtering with 0.22 μm filter membrane.

(8) After 4mL of High Affinity Ni-Charged Resin FF (Kinsery, L00666) was mixed with 10mL of an equilibration solution (8M urea, 50mM Tris-HCl pH 8,0.5M NaCl), 800g was centrifuged for 5min, and Beads was collected and washed 3 times.

(9) The Beads were resuspended in 2mL of equilibration solution, added to 8M urea lysate, spun at 4 ℃ for 4h, centrifuged at 2000g at 4 ℃ for 10min, and the Beads were collected.

(10) The column efficiency was checked by collecting a portion of the flow stream after complete collection of the Beads on the gravity column with 10mL of equilibration fluid.

(11) The column was washed with 20mL of equilibration buffer until complete drainage.

(12) 20mL of column wash (80mM imidazole, 8M urea, 50mM Tris-HCl pH 8,0.5M NaCl) was added to complete run-off.

(13) 11mL of eluent (300mM imidazole, 8M urea, 50mM Tris-HCl pH 8,0.5M NaCl) was added, the first 1mL of flow was discarded, and 10mL of eluent was collected.

(14) dTT was added to a final concentration of 20mM and spun overnight at 4 ℃.

(15) The eluate was diluted 10-fold (protein concentration less than 50. mu.g/mL) and subjected to gradient renaturation as follows:

i.4m urea, 50mM Tris-HCl PH 8,0.5M NaCl, 0.4mM reduced glutathione, 0.2mM oxidised glutathione, 0.1% PEG6000, 5% glycerol, 4 h.

2M Urea, 50mM Tris-HCl pH 8,0.5M NaCl, 0.4mM reduced glutathione, 0.2mM oxidised glutathione, 0.1% PEG6000, 5% glycerol, 4 h.

1M Urea, 50mM Tris-HCl pH 8,0.5M NaCl, 0.4mM reduced glutathione, 0.2mM oxidised glutathione, 0.1% PEG6000, 5% glycerol, 4 h.

Iv.50mm Tris-HCl PH 8,0.5M NaCl, 2mM dTT, 0.1% PEG6000, 5% glycerol, 4 h.

V.50mm Tris-HCl PH 8,0.5M NaCl, 2mM dTT, 5% glycerol, 4 h.

Pbs PH 7.4, 2mM dTT, 5% glycerol 4 h.

(16) The concentration of the renatured protein was measured by BCA (Sermeflier, cat # 23235). Subpackaging and storing at 4 ℃ for later use (growing at-80 ℃).

The purified protein was stained with WB and Coomassie Brilliant blue to show bands of the correct size (about 70kDa) (FIG. 8A).

Example 10 Assembly of nanoparticles R545HAF

Intein (Intein) is a peptide fragment that can self-cleave. As previously described, the N-terminal of the C-terminal fusion intein of R545-HA2 forms the recombinant protein R545HA-IntN-PAB-7 XHis. Fusing the N end of Ferritin (Ferritin, Fn) and the C end of a joint gbl-intein (gb1 is B domin of Protein G and is used as a solubility promoting label) to form a recombinant Protein gbl-intC-fertin, and then self-assembling the recombinant Protein gbl-intC-fertin into 24 polymers to form nanoparticles; specifically recognizing and cutting ferritin exposed on the surface of the nanoparticle by the N-terminal of the intein; the foreign protein and ferritin are covalently cross-linked to obtain R545-HA2-hFn, which is also in the form of 24-mer nanoparticles.

To investigate the effectiveness of this ligation, we first purified 7XHIS-GB1-IntC-Fn (according to patent application No. 201910421408.9) in the following manner:

(1) pET28a 7XHIS-GB1-IntC-Fn was transformed into the BL21 (Prokineticidae organism, cat # CD901-02) expressing strain.

(2) The monoclonal strains were picked, inoculated into 20mL of kanamycin-chloramphenicol double-resistant LB medium, and shake-cultured overnight at 37 ℃.

(3) The next day, 1:25 was inoculated into 500mL of LB medium with kanamycin resistance, 37 ℃ and 250 rpm.

(4) When the culture was carried out until OD was 0.8-1.0, 0.2mM IPTG was added for induction. After induction at 25 ℃ and 250rpm for 5 hours, the cells were collected by centrifugation at 4000rpm and 4 ℃ for 30 minutes, and the supernatant was discarded.

(5) 500mL of LB-cultured bacteria were resuspended in 30mL of lysis buffer (50mM Tris, pH 7.5, 150mM NaCl, 1% Triton X-100) and then sonicated.

(6) After completion of sonication, the mixture was centrifuged at 12000rpm at 4 ℃ for 30 minutes, and the supernatant was collected.

(7) 4.5g of ammonium sulfate was added to each 30mL of the solution, and the mixture was mixed at 4 ℃ for 15 min.

(8) The resulting mixture was centrifuged at 12000rpm at 4 ℃ for 30 minutes, and the precipitate was collected.

(9) 30mL of Tris-HCl solution (0mM Tris, pH 7.5, 150mM NaCl) was added to dissolve overnight at 4 ℃.

(10) BCA (Sammerfei, cat # 23235) was used to measure the concentration of 7XHIS-GB1-IntC-Fn protein, which was dispensed and stored at 4 ℃ until use (stored at-80 ℃).

The purified 7XHIS-GB1-IntC-Fn was ligated to the purified R545HA-IntN-PAB-7XHis (purified in example 9) at a dTT concentration of 2mM, as shown in FIG. 8B. It can be seen that R545HA-IntN-PAB is able to form a ligation product R545HAF (70 kDa band in the left two lanes of FIG. 8B) of about 70kDa (67 kDa before glycosylation) by ligation of Fn and cleavage generates by-products IntN-PAB (17kDa) and gb1-IntC (11 kDa).

Example 10 immunogenicity of nanoparticle R545HAF in hACE2+ mice

We verified the immunogenicity of the nanoparticle vaccine R545HAF in hACE2+ ICR mice and evaluated the immune combination to induce binding antibody titers against the RBD protein and H7 protein and neutralizing antibody titers against SARS-CoV-2 pseudovirus 4 weeks after completion of the immunization.

The experimental procedure was as follows: mice were randomly divided into 2 groups, which were designated as control group and nanoparticle group, respectively, based on the immunogen. Specific combinations of immunizations are shown in table 7, with the immunization being by intramuscular injection.

The titer of the binding antibody against the RBD protein produced by the nanoparticle group, i.e., R545HAF, is as shown in fig. 9A, and the titer of the binding antibody against the RBD protein is mostly over 400,000 and can partially reach 3,000,000 in the fourth week after the completion of immunization; meanwhile, by comparing the data of the binding antibody to the RBD protein after the first immunization with the data of the binding antibody to the RBD protein after the third immunization, it can be found that the titer of the binding antibody is significantly increased (fig. 9B). At the same time, R545HAF was also shown to be effective in inducing binding antibodies against influenza virus H7.

4 weeks after the end of immunization, the nanoparticle group produced neutralizing antibody titers against SARS-CoV-2 pseudovirus as shown in FIG. 9C: most of the total content is about 10,000, and the highest content can reach 92,172; meanwhile, by comparing the data of the binding antibody against the RBD protein after the first immunization with the data of the binding antibody against the RBD protein after the third immunization, it can be found that the titer of the binding antibody is significantly increased (fig. 9D).

This experiment demonstrates that the nanoparticle vaccine R545HAF is able to simultaneously induce binding antibodies against neocorona and influenza in hACE2+ ICR mice. Meanwhile, the nanoparticle vaccine R545HAF can effectively induce a neutralizing antibody aiming at the new corona, and has good prospects in development of anti-influenza and new corona vaccines.

TABLE 7 immunization protocol for immunogenicity testing of R545HAF in hACE2+ ICR mice

All documents referred to in this disclosure are incorporated by reference into this application as if each were 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.

Appendix: sequence Listing information

Sequence listing

<110> Shanghai city public health clinic center

<120> method for simultaneously inducing immune responses against various viruses

<130> 21A805 1CNCN

<160> 26

<170> PatentIn version 3.3

<210> 1

<211> 186

<212> PRT

<213> Influenza Virus (Influenza virus)

<400> 1

Met Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu

1 5 10 15

Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala Gln Gly

20 25 30

Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile Asp Gln

35 40 45

Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln Gln Phe

50 55 60

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

65 70 75 80

Val Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser Tyr Asn

85 90 95

Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp Leu Ala

100 105 110

Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg Gln Leu Arg

115 120 125

Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe His Lys

130 135 140

Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr Asp His

145 150 155 160

Ser Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile Asp Pro

165 170 175

Val Lys Leu Ser Ser Gly Tyr Lys Asp Val

180 185

<210> 2

<211> 558

<212> DNA

<213> Influenza Virus (Influenza virus)

<400> 2

atgggcctgt tcggcgccat tgccggcttc atcgagaacg gctgggaggg cctcattgac 60

ggctggtacg gcttcaggca ccagaacgcc cagggcgagg gcacagccgc cgactacaag 120

tccacccagt ccgccattga ccagatcaca ggcaagctga acagactcat tgaaaaaaca 180

aaccagcagt tcgagctgat tgacaacgag ttcaacgagg tggagaagca gattggcaac 240

gtgattaact ggacacggga ctctattaca gaggtgtggt cttacaacgc cgagttactc 300

gtggcaatgg agaaccagca cacaattgac ctcgccgact ctgagatgga caagctgtac 360

gagcgggtga agagacagct cagagagaac gccgaggagg acggcacagg ctgcttcgag 420

atattccaca agtgcgacga cgactgcatg gccagcatcc ggaacaacac atacgaccac 480

tctaagtacc gggaggaggc catgcagaac cgcatccaga ttgacccagt gaagctgtct 540

agcggctaca aggacgtg 558

<210> 3

<211> 272

<212> PRT

<213> novel coronaviruses (COVID-19)

<400> 3

Met Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr

1 5 10 15

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu

130 135 140

Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr

245 250 255

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

260 265 270

<210> 4

<211> 816

<212> DNA

<213> novel coronaviruses (COVID-19)

<400> 4

atgagggtgc agcctaccga gtccatcgtg cgctttccca atatcacaaa cctgtgccct 60

tttggcgagg tgttcaacgc aacccgcttc gcaagcgtgt acgcctggaa taggaagcgc 120

atctccaact gcgtggccga ctattctgtg ctgtacaaca gcgcctcctt ctctaccttt 180

aagtgctatg gcgtgagccc cacaaagctg aatgacctgt gctttaccaa cgtgtacgcc 240

gattccttcg tgatcagggg cgacgaggtg cgccagatcg caccaggaca gacaggcaag 300

atcgcagact acaattataa gctgcctgac gatttcaccg gctgcgtgat cgcctggaac 360

tctaacaatc tggatagcaa agtgggcggc aactacaatt atctgtaccg gctgtttaga 420

aagtctaatc tgaagccatt cgagagggac atctccacag aaatctacca ggccggctct 480

accccctgca atggcgtgga gggctttaac tgttatttcc ctctgcagag ctacggcttc 540

cagccaacaa acggcgtggg ctatcagccc taccgcgtgg tggtgctgtc ttttgagctg 600

ctgcacgcac ctgcaacagt gtgcggacca aagaagagca ccaatctggt gaagaacaag 660

tgcgtgaact tcaacttcaa cggactgacc ggcacaggcg tgctgaccga gtccaacaag 720

aagttcctgc cttttcagca gttcggcagg gacatcgcag ataccacaga cgccgtgcgc 780

gaccctcaga ccctggagat cctggatatc acacca 816

<210> 5

<211> 272

<212> PRT

<213> Artificial sequence

<400> 5

Met Val Cys Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys

260 265 270

<210> 6

<211> 816

<212> DNA

<213> Artificial sequence

<400> 6

atggtgtgtc ctaccgagtc catcgtgcgc tttcccaata tcacaaacct gtgccctttt 60

ggcgaggtgt tcaacgcaac ccgcttcgca agcgtgtacg cctggaatag gaagcgcatc 120

tccaactgcg tggccgacta ttctgtgctg tacaacagcg cctccttctc tacctttaag 180

tgctatggcg tgagccccac aaagctgaat gacctgtgct ttaccaacgt gtacgccgat 240

tccttcgtga tcaggggcga cgaggtgcgc cagatcgcac caggacagac aggcaagatc 300

gcagactaca attataagct gcctgacgat ttcaccggct gcgtgatcgc ctggaactct 360

aacaatctgg atagcaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 420

tctaatctga agccattcga gagggacatc tccacagaaa tctaccaggc cggctctacc 480

ccctgcaatg gcgtggaggg ctttaactgt tatttccctc tgcagagcta cggcttccag 540

ccaacaaacg gcgtgggcta tcagccctac cgcgtggtgg tgctgtcttt tgagctgctg 600

cacgcacctg caacagtgtg cggaccaaag aagagcacca atctggtgaa gaacaagtgc 660

gtgaacttca acttcaacgg actgaccggc acaggcgtgc tgaccgagtc caacaagaag 720

ttcctgcctt ttcagcagtt cggcagggac atcgcagata ccacagacgc cgtgcgcgac 780

cctcagaccc tggagatcct ggatatcaca ccatgc 816

<210> 7

<211> 183

<212> PRT

<213> Artificial sequence

<400> 7

Met Thr Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp

1 5 10 15

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

20 25 30

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

35 40 45

Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg

50 55 60

Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg

65 70 75 80

Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser

85 90 95

Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn

100 105 110

Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro

115 120 125

His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys

130 135 140

Ala Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly

145 150 155 160

Ala Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu

165 170 175

Gly Asp Ser Asp Asn Glu Ser

180

<210> 8

<211> 549

<212> DNA

<213> Artificial sequence

<400> 8

atgacgaccg cgtccacctc gcaggtgcgc cagaactacc accaggactc agaggccgcc 60

atcaaccgcc agatcaacct ggagctctac gcctcctacg tttacctgtc catgtcttac 120

tactttgacc gcgatgatgt ggctttgaag aactttgcca aatactttct tcaccaatct 180

catgaggaga gggaacatgc tgagaaactg atgaagctgc agaaccaacg aggtggccga 240

atcttccttc aggatatcaa gaaaccagac tgtgatgact gggagagcgg gctgaatgca 300

atggagtgtg cattacattt ggaaaaaaat gtgaatcagt cactactgga actgcacaaa 360

ctggccactg acaaaaatga cccccatttg tgtgacttca ttgagacaca ttacctgaat 420

gagcaggtga aagccatcaa agaattgggt gaccacgtga ccaacttgcg caagatggga 480

gcgcccgaat ctggcttggc ggaatatctc tttgacaagc acaccctggg agacagtgat 540

aatgaaagc 549

<210> 9

<211> 684

<212> PRT

<213> Artificial sequence

<400> 9

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

1 5 10 15

Ala Ser Arg Gly Ser Tyr Tyr His His His His His His Val Cys Pro

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser

210 215 220

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

225 230 235 240

Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu

245 250 255

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

260 265 270

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

275 280 285

Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Val Asp Glu

290 295 300

Leu Thr Ser Arg Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile

305 310 315 320

Glu Asn Gly Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg His

325 330 335

Gln Asn Ala Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln

340 345 350

Ser Ala Ile Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys

355 360 365

Thr Asn Gln Gln Phe Glu Leu Ile Asp Asn Glu Phe Asn Glu Val Glu

370 375 380

Lys Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu

385 390 395 400

Val Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His

405 410 415

Thr Ile Asp Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg Val

420 425 430

Lys Arg Gln Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe

435 440 445

Glu Ile Phe His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn

450 455 460

Asn Thr Tyr Asp His Ser Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg

465 470 475 480

Ile Gln Ile Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val Val

485 490 495

Asp Gly Gly Gly Gly Ser Thr Thr Ala Ser Thr Ser Gln Val Arg Gln

500 505 510

Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu

515 520 525

Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp

530 535 540

Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln

545 550 555 560

Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn

565 570 575

Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys

580 585 590

Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu

595 600 605

Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr

610 615 620

Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu

625 630 635 640

Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His Val Thr Asn

645 650 655

Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe

660 665 670

Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser

675 680

<210> 10

<211> 2052

<212> DNA

<213> Artificial sequence

<400> 10

atggttttca cacctcagat acttggactt atgctttttt ggatttcagc ctccagaggt 60

tcgtactacc atcaccatca ccatcacgtg tgtcctaccg agtccatcgt gcgctttccc 120

aatatcacaa acctgtgccc ttttggcgag gtgttcaacg caacccgctt cgcaagcgtg 180

tacgcctgga ataggaagcg catctccaac tgcgtggccg actattctgt gctgtacaac 240

agcgcctcct tctctacctt taagtgctat ggcgtgagcc ccacaaagct gaatgacctg 300

tgctttacca acgtgtacgc cgattccttc gtgatcaggg gcgacgaggt gcgccagatc 360

gcaccaggac agacaggcaa gatcgcagac tacaattata agctgcctga cgatttcacc 420

ggctgcgtga tcgcctggaa ctctaacaat ctggatagca aagtgggcgg caactacaat 480

tatctgtacc ggctgtttag aaagtctaat ctgaagccat tcgagaggga catctccaca 540

gaaatctacc aggccggctc taccccctgc aatggcgtgg agggctttaa ctgttatttc 600

cctctgcaga gctacggctt ccagccaaca aacggcgtgg gctatcagcc ctaccgcgtg 660

gtggtgctgt cttttgagct gctgcacgca cctgcaacag tgtgcggacc aaagaagagc 720

accaatctgg tgaagaacaa gtgcgtgaac ttcaacttca acggactgac cggcacaggc 780

gtgctgaccg agtccaacaa gaagttcctg ccttttcagc agttcggcag ggacatcgca 840

gataccacag acgccgtgcg cgaccctcag accctggaga tcctggatat cacaccatgc 900

tccgtcgacg agctcactag tcgcggccgc ggcctgttcg gcgccattgc cggcttcatc 960

gagaacggct gggagggcct cattgacggc tggtacggct tcaggcacca gaacgcccag 1020

ggcgagggca cagccgccga ctacaagtcc acccagtccg ccattgacca gatcacaggc 1080

aagctgaaca gactcattga aaaaacaaac cagcagttcg agctgattga caacgagttc 1140

aacgaggtgg agaagcagat tggcaacgtg attaactgga cacgggactc tattacagag 1200

gtgtggtctt acaacgccga gttactcgtg gcaatggaga accagcacac aattgacctc 1260

gccgactctg agatggacaa gctgtacgag cgggtgaaga gacagctcag agagaacgcc 1320

gaggaggacg gcacaggctg cttcgagata ttccacaagt gcgacgacga ctgcatggcc 1380

agcatccgga acaacacata cgaccactct aagtaccggg aggaggccat gcagaaccgc 1440

atccagattg acccagtgaa gctgtctagc ggctacaagg acgtggtcga cggcggaggc 1500

gggagcacga ccgcgtccac ctcgcaggtg cgccagaact accaccagga ctcagaggcc 1560

gccatcaacc gccagatcaa cctggagctc tacgcctcct acgtttacct gtccatgtct 1620

tactactttg accgcgatga tgtggctttg aagaactttg ccaaatactt tcttcaccaa 1680

tctcatgagg agagggaaca tgctgagaaa ctgatgaagc tgcagaacca acgaggtggc 1740

cgaatcttcc ttcaggatat caagaaacca gactgtgatg actgggagag cgggctgaat 1800

gcaatggagt gtgcattaca tttggaaaaa aatgtgaatc agtcactact ggaactgcac 1860

aaactggcca ctgacaaaaa tgacccccat ttgtgtgact tcattgagac acattacctg 1920

aatgagcagg tgaaagccat caaagaattg ggtgaccacg tgaccaactt gcgcaagatg 1980

ggagcgcccg aatctggctt ggcggaatat ctctttgaca agcacaccct gggagacagt 2040

gataatgaaa gc 2052

<210> 11

<211> 1281

<212> PRT

<213> novel coronaviruses (COVID-19)

<400> 11

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> 12

<211> 3843

<212> DNA

<213> novel coronaviruses (COVID-19)

<400> 12

atgttcgtgt ttctggtgct gctgcctctg gtgagctccc agtgcgtgaa cctgaccaca 60

aggacccagc tgccccctgc ctataccaat tccttcacac ggggcgtgta ctatcccgac 120

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

gtc 3843

<210> 13

<211> 805

<212> PRT

<213> Artificial sequence

<400> 13

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

<210> 14

<211> 2415

<212> DNA

<213> Artificial sequence

<400> 14

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 ccttt 2415

<210> 15

<211> 602

<212> PRT

<213> Artificial sequence

<400> 15

Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala

1 5 10 15

Asp Tyr Lys Asp Asp Asp Asp Lys Ser Leu Gln Val Cys Pro Thr Glu

20 25 30

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

35 40 45

Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys

50 55 60

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

65 70 75 80

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

85 90 95

Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly

100 105 110

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

115 120 125

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

130 135 140

Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu

145 150 155 160

Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile

165 170 175

Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu

180 185 190

Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr

195 200 205

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

210 215 220

Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn

225 230 235 240

Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly

245 250 255

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

260 265 270

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

275 280 285

Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Gly Gly Gly Gly Ser

290 295 300

Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly

305 310 315 320

Leu Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala Gln Gly Glu

325 330 335

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

340 345 350

Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln Gln Phe Glu

355 360 365

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

370 375 380

Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser Tyr Asn Ala

385 390 395 400

Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp Leu Ala Asp

405 410 415

Ser Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg Gln Leu Arg Glu

420 425 430

Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe His Lys Cys

435 440 445

Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr Asp His Ser

450 455 460

Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile Asp Pro Val

465 470 475 480

Lys Leu Ser Ser Gly Tyr Lys Asp Val Ala Ser Thr Thr Thr Pro Ala

485 490 495

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

500 505 510

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

515 520 525

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

530 535 540

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

545 550 555 560

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

565 570 575

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

580 585 590

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

595 600

<210> 16

<211> 1806

<212> DNA

<213> Artificial sequence

<400> 16

atgaaaacaa ttattgccct gtcttacatt ttctgcctcg tgttcgccga ctacaaggac 60

gacgacgaca agtccctgca ggtgtgccct accgagtcta tcgtgcgctt cccaaacatc 120

acaaacctgt gccctttcgg cgaggtgttc aacgccaccc ggttcgcctc tgtgtacgcc 180

tggaaccgga agcggatttc taactgcgtg gccgactact ccgtgctgta caactctgcc 240

tctttctcca cattcaagtg ctacggcgtg tcccctacca agctgaacga cctgtgcttc 300

accaacgtgt acgccgactc tttcgtgatt aggggcgacg aggtgagaca gattgcccct 360

ggccagacag gcaagatcgc cgactacaac tacaagctcc ctgacgactt cacaggctgc 420

gtgattgcct ggaactctaa caacctggac tctaaggtgg gcggcaacta caactacctg 480

tacagactgt tccggaagtc taacctcaag ccattcgagc gcgacattag caccgagatt 540

taccaggccg gcagcacccc atgcaacggc gtggagggct tcaactgcta cttcccactt 600

caatcttacg gcttccagcc aacaaacggc gtgggctacc agccataccg ggtggtggtg 660

ctgtccttcg agctactcca cgccccagcc acagtgtgcg gcccaaagaa gagcaccaac 720

ctcgtgaaga acaagtgcgt gaacttcaac ttcaacggcc tgacaggcac aggcgtgctc 780

accgagtcta acaagaagtt cctccctttc cagcagttcg gcagggacat cgccgacacc 840

accgacgccg tgcgcgaccc tcagacactc gaaattctgg acatcacccc ttgctctggc 900

ggcggcggct ctggcctgtt cggcgccatt gccggcttca tcgagaacgg ctgggagggc 960

ctcattgacg gctggtacgg cttcaggcac cagaacgccc agggcgaggg cacagccgcc 1020

gactacaagt ccacccagtc cgccattgac cagatcacag gcaagctgaa cagactcatt 1080

gaaaaaacaa accagcagtt cgagctgatt gacaacgagt tcaacgaggt ggagaagcag 1140

attggcaacg tgattaactg gacacgggac tctattacag aggtgtggtc ttacaacgcc 1200

gagttactcg tggcaatgga gaaccagcac acaattgacc tcgccgactc tgagatggac 1260

aagctgtacg agcgggtgaa gagacagctc agagagaacg ccgaggagga cggcacaggc 1320

tgcttcgaga tattccacaa gtgcgacgac gactgcatgg ccagcatccg gaacaacaca 1380

tacgaccact ctaagtaccg ggaggaggcc atgcagaacc gcatccagat tgacccagtg 1440

aagctgtcta gcggctacaa ggacgtggct agcaccacca cacccgcccc tagacctcct 1500

acccccgccc caacaattgc ctctcagcca ctgtctctca gacctgaggc gtgcaggccc 1560

gccgccggcg gcgccgtgca cacacggggc ctcgacttcg cctgcgacat ttacatttgg 1620

gccccactcg ccggcacatg cggcgtgctc ctcctgtctc tggtgatcac actgtactgc 1680

aagcggggca gaaagaagct cctgtacatt ttcaagcagc ctttcatgag acccgtgcag 1740

accacccagg aggaggacgg ctgctcttgc aggttccctg aggaggagga gggcggctgc 1800

gaacta 1806

<210> 17

<211> 111

<212> PRT

<213> Artificial sequence

<400> 17

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

65 70 75 80

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

85 90 95

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

100 105 110

<210> 18

<211> 333

<212> DNA

<213> Artificial sequence

<400> 18

accaccacac ccgcccctag acctcctacc cccgccccaa caattgcctc tcagccactg 60

tctctcagac ctgaggcgtg caggcccgcc gccggcggcg ccgtgcacac acggggcctc 120

gacttcgcct gcgacattta catttgggcc ccactcgccg gcacatgcgg cgtgctcctc 180

ctgtctctgg tgatcacact gtactgcaag cggggcagaa agaagctcct gtacattttc 240

aagcagcctt tcatgagacc cgtgcagacc acccaggagg aggacggctg ctcttgcagg 300

ttccctgagg aggaggaggg cggctgcgaa cta 333

<210> 19

<211> 15

<212> PRT

<213> Artificial sequence

<400> 19

Met Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

1 5 10 15

<210> 20

<211> 15

<212> PRT

<213> Artificial sequence

<400> 20

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

1 5 10 15

<210> 21

<211> 227

<212> PRT

<213> Artificial sequence

<400> 21

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

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Phe Asn Gly

225

<210> 22

<211> 681

<212> DNA

<213> Artificial sequence

<400> 22

cgtgtccaac ctaccgagtc catcgtgcgc ttccctaaca tcaccaacct gtgccctttc 60

ggtgaagtgt tcaacgctac ccgcttcgct tctgtgtacg cttggaaccg caagcgcatc 120

tccaactgcg tggctgacta ctctgtgctc tacaactccg cctccttctc caccttcaag 180

tgttacggcg tgtcccctac caagttgaac gatctgtgct tcaccaacgt ctacgctgac 240

tccttcgtga tccgtggcga cgaggtccgc caaatcgctc ctggtcagac cggtaagatc 300

gccgactaca actacaagct gcctgacgac ttcaccggtt gcgtgatcgc ttggaactcc 360

aacaacctgg actccaaggt gggtggtaac tacaactacc tgtacaggct gttccgcaag 420

agcaacctca agcccttcga aagggacatc tccactgaga tctaccaggc tggctccaca 480

ccctgcaacg gtgtggaagg tttcaactgc tacttccccc tccagtccta cggtttccag 540

cccaccaacg gtgtgggata ccagccctac cgcgtggtgg tgctctcctt cgagctgctg 600

cacgcccctg ctaccgtctg cggccctaag aagtccacca acctggtcaa gaacaagtgc 660

gtgaacttca acttcaacgg t 681

<210> 23

<211> 640

<212> PRT

<213> Artificial sequence

<400> 23

Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr

1 5 10 15

Ser Lys Met Val Ser Ala Thr Val Leu Tyr Val Leu Leu Ala Ala Ala

20 25 30

Ala His Ser Ala Phe Ala Thr Ser Tyr Tyr Glu Phe Arg Val Gln Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser

225 230 235 240

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

245 250 255

Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Gly

260 265 270

Gly Gly Ser Gly Gly Gly Gly Ser Gly Leu Phe Gly Ala Ile Ala Gly

275 280 285

Phe Ile Glu Asn Gly Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe

290 295 300

Arg His Gln Asn Ala Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser

305 310 315 320

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

325 330 335

Glu Lys Thr Asn Gln Gln Phe Glu Leu Ile Asp Asn Glu Phe Asn Glu

340 345 350

Val Glu Lys Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile

355 360 365

Thr Glu Val Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn

370 375 380

Gln His Thr Ile Asp Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu

385 390 395 400

Arg Val Lys Arg Gln Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly

405 410 415

Cys Phe Glu Ile Phe His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile

420 425 430

Arg Asn Asn Thr Tyr Asp His Ser Lys Tyr Arg Glu Glu Ala Met Gln

435 440 445

Asn Arg Ile Gln Ile Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp

450 455 460

Val Val Asp Gly Gly Gly Gly Ser Thr Arg Ser Gly Tyr Cys Leu Asp

465 470 475 480

Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser Asn

485 490 495

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

500 505 510

Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr Leu

515 520 525

Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro Thr

530 535 540

Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met Cys

545 550 555 560

Leu Tyr Val Lys Glu Gly Ser Ser Arg Gly Ser Leu Met Thr Ala Asp

565 570 575

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

580 585 590

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

595 600 605

Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys

610 615 620

Leu Asn Asp Gly Gly Gly Gly Ser Gly His His His His His His His

625 630 635 640

<210> 24

<211> 1920

<212> DNA

<213> Artificial sequence

<400> 24

atgctgctgg tgaaccagtc ccaccagggt ttcaacaagg agcacacctc caagatggtc 60

tccgccaccg tgttgtacgt gctgttggct gccgccgccc actccgcttt cgctacctcc 120

tactacgaat tccgtgtcca acctaccgag tccatcgtgc gcttccctaa catcaccaac 180

ctgtgccctt tcggtgaagt gttcaacgct acccgcttcg cttctgtgta cgcttggaac 240

cgcaagcgca tctccaactg cgtggctgac tactctgtgc tctacaactc cgcctccttc 300

tccaccttca agtgttacgg cgtgtcccct accaagttga acgatctgtg cttcaccaac 360

gtctacgctg actccttcgt gatccgtggc gacgaggtcc gccaaatcgc tcctggtcag 420

accggtaaga tcgccgacta caactacaag ctgcctgacg acttcaccgg ttgcgtgatc 480

gcttggaact ccaacaacct ggactccaag gtgggtggta actacaacta cctgtacagg 540

ctgttccgca agagcaacct caagcccttc gaaagggaca tctccactga gatctaccag 600

gctggctcca caccctgcaa cggtgtggaa ggtttcaact gctacttccc cctccagtcc 660

tacggtttcc agcccaccaa cggtgtggga taccagccct accgcgtggt ggtgctctcc 720

ttcgagctgc tgcacgcccc tgctaccgtc tgcggcccta agaagtccac caacctggtc 780

aagaacaagt gcgtgaactt caacttcaac ggtggtggtg gtagtggtgg tggtggttct 840

ggtttgttcg gtgctatcgc tggtttcatc gagaacggtt gggagggtct gatcgatggt 900

tggtacggtt tccgtcacca gaacgctcag ggtgagggta ctgctgctga ttacaagtct 960

acccagagtg ctatcgacca gatcaccgga aagttgaaca ggctgatcga gaagactaac 1020

cagcagttcg aactcatcga caacgagttc aacgaggtgg agaagcagat cggcaacgtg 1080

atcaactgga ccagggacag catcacagag gtgtggtcct acaacgccga gctgctggtg 1140

gctatggaaa accagcacac catcgacctg gctgatagcg agatggacaa gctgtacgag 1200

cgcgtgaaga ggcagttgcg tgagaacgct gaggaggacg gaaccggttg cttcgaaatc 1260

ttccacaagt gcgacgacga ctgcatggct tccatcagga acaacacata cgaccactcc 1320

aagtacaggg aggaggctat gcagaacagg attcaaatcg accctgtgaa gctgtcatcc 1380

ggttacaagg acgtggtcga cggtggtggt ggttctactc gttctggtta ctgcttggat 1440

ttgaagactc aggtgcagac tcctcagggt atgaaggaga tctctaacat ccaggtgggt 1500

gacttggtgc tgagcaacac tggttacaac gaggtgctga acgtgttccc taagtccaag 1560

aagaagagct acaagatcac tctcgaggac ggcaaggaga tcatctgctc cgaggagcac 1620

ctgttcccca cacaaaccgg cgagatgaac atctccggcg gcctgaagga aggtatgtgc 1680

ctgtacgtga aggagggcag ctctagaggt tctttgatga ctgctgataa caagttcaac 1740

aaggagcagc agaacgcttt ctacgagatc ttgcacttgc ctaacttgaa cgaggagaac 1800

aggaacggtt tcatccagtc tctgaaggac gaccctagcc agtccgccaa cctgctggct 1860

gaggctaaga agctgaacga cggtggtggt ggttccggtc accaccacca ccaccaccac 1920

<210> 25

<211> 654

<212> PRT

<213> Artificial sequence

<400> 25

Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr

1 5 10 15

Ser Lys Met Val Ser Ala Thr Val Leu Tyr Val Leu Leu Ala Ala Ala

20 25 30

Ala His Ser Ala Phe Ala Thr Ser Tyr Tyr Glu Phe Arg Val Gln Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser

225 230 235 240

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

245 250 255

Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Gly

260 265 270

Gly Gly Ser Gly Gly Gly Gly Ser Gly Leu Phe Gly Ala Ile Ala Gly

275 280 285

Phe Ile Glu Asn Gly Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe

290 295 300

Arg His Gln Asn Ala Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser

305 310 315 320

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

325 330 335

Glu Lys Thr Asn Gln Gln Phe Glu Leu Ile Asp Asn Glu Phe Asn Glu

340 345 350

Val Glu Lys Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile

355 360 365

Thr Glu Val Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn

370 375 380

Gln His Thr Ile Asp Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu

385 390 395 400

Arg Val Lys Arg Gln Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly

405 410 415

Cys Phe Glu Ile Phe His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile

420 425 430

Arg Asn Asn Thr Tyr Asp His Ser Lys Tyr Arg Glu Glu Ala Met Gln

435 440 445

Asn Arg Ile Gln Ile Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp

450 455 460

Val Val Asp Gly Gly Gly Gly Ser Thr Thr Ala Ser Thr Ser Gln Val

465 470 475 480

Arg Gln Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile

485 490 495

Asn Leu Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser Tyr Tyr

500 505 510

Phe Asp Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe Leu

515 520 525

His Gln Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys Leu

530 535 540

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

545 550 555 560

Asp Cys Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala Leu

565 570 575

His Leu Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys Leu

580 585 590

Ala Thr Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His

595 600 605

Tyr Leu Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His Val

610 615 620

Thr Asn Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala Glu Tyr

625 630 635 640

Leu Phe Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser

645 650

<210> 26

<211> 1962

<212> DNA

<213> Artificial sequence

<400> 26

atgctgctgg tgaaccagtc ccaccagggt ttcaacaagg agcacacctc caagatggtc 60

tccgccaccg tgttgtacgt gctgttggct gccgccgccc actccgcttt cgctacctcc 120

tactacgaat tccgtgtcca acctaccgag tccatcgtgc gcttccctaa catcaccaac 180

ctgtgccctt tcggtgaagt gttcaacgct acccgcttcg cttctgtgta cgcttggaac 240

cgcaagcgca tctccaactg cgtggctgac tactctgtgc tctacaactc cgcctccttc 300

tccaccttca agtgttacgg cgtgtcccct accaagttga acgatctgtg cttcaccaac 360

gtctacgctg actccttcgt gatccgtggc gacgaggtcc gccaaatcgc tcctggtcag 420

accggtaaga tcgccgacta caactacaag ctgcctgacg acttcaccgg ttgcgtgatc 480

gcttggaact ccaacaacct ggactccaag gtgggtggta actacaacta cctgtacagg 540

ctgttccgca agagcaacct caagcccttc gaaagggaca tctccactga gatctaccag 600

gctggctcca caccctgcaa cggtgtggaa ggtttcaact gctacttccc cctccagtcc 660

tacggtttcc agcccaccaa cggtgtggga taccagccct accgcgtggt ggtgctctcc 720

ttcgagctgc tgcacgcccc tgctaccgtc tgcggcccta agaagtccac caacctggtc 780

aagaacaagt gcgtgaactt caacttcaac ggtggtggtg gtagtggtgg tggtggttct 840

ggtttgttcg gtgctatcgc tggtttcatc gagaacggtt gggagggtct gatcgatggt 900

tggtacggtt tccgtcacca gaacgctcag ggtgagggta ctgctgctga ttacaagtct 960

acccagagtg ctatcgacca gatcaccgga aagttgaaca ggctgatcga gaagactaac 1020

cagcagttcg aactcatcga caacgagttc aacgaggtgg agaagcagat cggcaacgtg 1080

atcaactgga ccagggacag catcacagag gtgtggtcct acaacgccga gctgctggtg 1140

gctatggaaa accagcacac catcgacctg gctgatagcg agatggacaa gctgtacgag 1200

cgcgtgaaga ggcagttgcg tgagaacgct gaggaggacg gaaccggttg cttcgaaatc 1260

ttccacaagt gcgacgacga ctgcatggct tccatcagga acaacacata cgaccactcc 1320

aagtacaggg aggaggctat gcagaacagg attcaaatcg accctgtgaa gctgtcatcc 1380

ggttacaagg acgtggtcga cggtggtggt ggttctacga ccgcgtccac ctcgcaggtg 1440

cgccagaact accaccagga ctcagaggcc gccatcaacc gccagatcaa cctggagctc 1500

tacgcctcct acgtttacct gtccatgtct tactactttg accgcgatga tgtggctttg 1560

aagaactttg ccaaatactt tcttcaccaa tctcatgagg agagggaaca tgctgagaaa 1620

ctgatgaagc tgcagaacca acgaggtggc cgaatcttcc ttcaggatat caagaaacca 1680

gactgtgatg actgggagag cgggctgaat gcaatggagt gtgcattaca tttggaaaaa 1740

aatgtgaatc agtcactact ggaactgcac aaactggcca ctgacaaaaa tgacccccat 1800

ttgtgtgact tcattgagac acattacctg aatgagcagg tgaaagccat caaagaattg 1860

ggtgaccacg tgaccaactt gcgcaagatg ggagcgcccg aatctggctt ggcggaatat 1920

ctctttgaca agcacaccct gggagacagt gataatgaaa gc 1962

Claims

1. An immunogenic peptide comprising the following moieties:

(c) optionally, other moieties attached to the foregoing moieties.

2. The immunogenic peptide of claim 1, wherein,

the source of the HA2 region is selected from the group consisting of: any of H1 to H18, particularly from the widely prevalent human influenza H1, H2, H3 and the multiple-appearing human infections with avian influenza H5 and H7, for example from 2009 pandemic H1(H1N1), 2013 pandemic H7(H7N 9);

for example, the amino acid sequence of the HA2 region is shown in SEQ ID NO. 1, or is encoded by a nucleotide molecule having the sequence shown in SEQ ID NO. 2.

3. The immunogenic peptide of claim 1, wherein the immunogenic crown comprises: an influenza virus of a different origin from the HA2 region in (a); a membrane protein of a non-influenza virus or other influenza strain or an immunogenic fragment thereof;

the source of the immunogen crown is selected from the group consisting of: coronavirus, aids virus, influenza virus (e.g., the HA1 region of influenza virus hemagglutinin HA), rabies virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, measles virus, ebola virus, herpes virus, arbovirus (e.g., zika virus, epidemic encephalitis b virus, forest encephalitis virus, dengue virus, hantavirus, hemorrhagic fever virus);

for example, the source of the immunogen crown is selected from: coronavirus SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, e.g., an S1 protein comprising the receptor binding domain of a coronavirus, or a Receptor Binding Domain (RBD), or an engineered receptor binding domain (e.g., the RBD region is modified with a terminal cysteine to form an sRBD region), or an immunogenic fragment thereof;

for example, the immunogenic crown is selected from S1 of coronavirus SARS-CoV-2, particularly the RBD region from S1 (e.g., the sequence of which is shown in SEQ ID NO:3 or encoded by the nucleotide molecule of SEQ ID NO: 4), or a modified RBD region thereof (e.g., the region of sRBD modified with a terminal Cys, the peptide stretch shown in SEQ ID NO:5 or encoded by the nucleotide molecule of SEQ ID NO: 6), or an immunogenic fragment of the RBD region (e.g., the peptide stretch shown in SEQ ID NO:21 or encoded by the nucleotide molecule of SEQ ID NO: 22).

4. The immunogenic peptide of claim 1, wherein the additional moiety is selected from the group consisting of:

immune modulatory sequences, such as IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulatory antibodies, PD-1 and PD-L1 antibodies, CTLA4 antibodies, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and subunits thereof, bacterial flagellin, FimH, HIV p24, HIV gp 41;

a moiety that enables the immunogenic peptide to form a nanoparticle, such as transferrin (Fn, e.g., a peptide molecule as shown in SEQ ID NO:7 or encoded by a nucleotide molecule as shown in SEQ ID NO: 8);

signal peptides such as CD33, CD8, CD16, mouse IgG1 antibody;

a transmembrane region enabling the immunogenic peptide to be expressed on the surface of a viral vector, such as the CD8 transmembrane region (CD8TM, e.g., a peptide molecule as set forth in SEQ ID NO:17 or encoded by a nucleotide molecule as set forth in SEQ ID NO: 18), the HA2 transmembrane region, the CD4 transmembrane region, the gp41 transmembrane region;

linker peptides, e.g. (G4S)₃、(G4S)_n、GSAGSAAGSGEF、(Gly)₆、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly)₈、EGKSSGSGSESKST；

Tags such as His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag, Sof-tag 1, Sof-tag3, Strep-tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag and PNE tag.

5. The immunogenic peptide of any one of claims 1-4, wherein the immunogenic peptide comprises: the RBD region or sRBD region or immunogenic fragment thereof linked to the HA2 region, and optionally other moieties linked to the foregoing;

for example, the immunogenic peptide comprises: an RBD region or sRGB region (e.g., a peptide molecule represented by SEQ ID NO:5 or encoded by a nucleotide molecule represented by SEQ ID NO: 6) and an Fn region (e.g., a Fn peptide molecule represented by SEQ ID NO:7 or encoded by a nucleotide molecule represented by SEQ ID NO: 8) linked to the HA2 region; an RBD region or sRBD region linked to the HA2 region, and a CD8 transmembrane region (e.g., the CD8 transmembrane region peptide molecule shown in SEQ ID NO:17 or encoded by the nucleotide molecule shown in SEQ ID NO: 18); an immunogenic fragment of an RBD region (e.g., a peptide molecule represented by SEQ ID NO:21 or encoded by a nucleotide molecule represented by SEQ ID NO: 22) linked to the HA2 region and an Fn region;

for example, the amino acid sequence of the immunogenic peptide is shown as SEQ ID NO 9 or 15 or 25, or the coding sequence of the immunogenic peptide is shown as SEQ ID NO 10 or 16 or 26.

6.A nucleotide molecule encoding the immunogenic peptide of any one of claims 1-5;

for example, the coding sequence of the HA2 region comprises the nucleotide sequence shown in SEQ ID NO. 2; the coding sequence of the immunogen crown comprises a nucleotide sequence shown as SEQ ID NO. 4 or a nucleotide sequence shown as SEQ ID NO. 6 or a nucleotide sequence shown as SEQ ID NO. 22; and/or the coding sequence of said further part comprises the nucleotide sequence shown in SEQ ID NO 8 or SEQ ID NO 18;

for example, the nucleotide molecule has a sequence shown in SEQ ID NO 10 or 16 or 26.

7. A vector comprising the nucleotide molecule of claim 6.

8. A host cell comprising the nucleotide molecule of claim 6 or the vector of claim 7 or capable of expressing the immunogenic peptide of any one of claims 1-5,

for example, the host cell is a mammalian cell or an insect cell, such as HEK293, HeLa, K562, 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-672302, Hz2E5 and Ao 38.

9. A vaccine capable of simultaneously inducing an immune response against an influenza virus and another non-influenza virus, comprising the immunogenic peptide of any one of claims 1-5, the nucleotide molecule of claim 6, the vector of claim 7, and/or the host cell of claim 8;

for example, the vaccine is a nucleic acid vaccine (DNA or RNA vaccine), a recombinant protein subunit vaccine, a recombinant viral vector vaccine, a recombinant bacterial vector vaccine, a virus-like particle vaccine, a nanoparticle vaccine, a cell vector vaccine;

for example, the non-influenza virus is selected from: coronavirus, aids virus, rabies virus, hog cholera virus, porcine reproductive and respiratory syndrome virus, measles virus, ebola virus, herpes virus, arbovirus (e.g., zika virus, epidemic encephalitis b virus, forest encephalitis virus, dengue virus, hantavirus, hemorrhagic fever virus);

for example, the vaccine is a viral vector vaccine, the viral vector selected from the group consisting of: poxviruses (e.g., Tiantan strain, North American vaccine strain, Huishi derived strain, Listeria strain, Ankara derived strain, Copenhagen strain, and New York strain), adenoviruses (Ad5, Ad11, Ad26, Ad35, AdC68), adeno-associated viruses, herpes simplex viruses, measles viruses, reoviruses, rhabdoviruses, forest encephalitis viruses, influenza viruses, respiratory syncytial viruses, poliovirus;

for example, the vaccine comprises or is used in combination with an adjuvant, for example selected from: aluminum adjuvant, cholera toxin and subunits thereof, oligodeoxynucleotide, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, PolyI C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21;

for example, the vaccine is in a form suitable for intramuscular administration, intradermal administration, subcutaneous administration, nasal drops, aerosol inhalation, genital tract, rectal administration, oral administration, or a combination of the different modes of administration described above (e.g. intramuscular injection + nasal drops);

for example, the vaccine is in a form suitable for combined vaccination (e.g., combined or sequential vaccination) of 2 or more species, such as sequential vaccination before and after an S or S1 vaccine against a coronavirus (e.g., SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV), or sequential vaccination before and after an HA or HA2 vaccine against an influenza virus (e.g., HA or HA2 from any of H1-H18).

10. Use of the immunogenic peptide according to any one of claims 1 to 5, the nucleotide molecule according to claim 6, the vector according to claim 7 and/or the host cell according to claim 8 for the preparation of a medicament for the simultaneous prevention or treatment of an influenza virus and another non-influenza virus,

for example, the non-influenza virus is selected from: coronavirus, aids virus, rabies virus, hog cholera virus, porcine reproductive and respiratory syndrome virus, measles virus, ebola virus, herpes virus, arbovirus (e.g., zika virus, epidemic encephalitis b virus, forest encephalitis virus, dengue virus, hantavirus, hemorrhagic fever virus).