A vaccine composition against betacoronavirus and its application

Abstract

The present invention discloses a vaccine composition against betacoronavirus and its application, belonging to the field of biotechnology. Among them, the vaccine composition against betacoronavirus is inoculated through the mucosal route and contains the nucleocapsid protein of the novel coronavirus, phosphodiester backbone CpG oligodeoxynucleotide, and pharmaceutical excipients. At the same time, the present invention also relates to the application of the vaccine composition in the production of drugs against the novel coronavirus. And a method for inducing humoral immune response and cell-mediated immune response against betacoronavirus by inoculating the vaccine composition is further provided. By inoculating through the mucosal route, especially after intranasal administration, under the regulated Th1 immune response pattern, it can induce a cell-mediated immune cross-response against the N protein and induce cross-antibodies against the N protein. At the same time, the vaccine composition preparation of the present application has potential safety.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a vaccine composition against betacoronavirus and its application. Background Art

[0002] At present, the novel coronavirus (SARS-CoV-2)-induced coronavirus disease 2019 (COVID-19) has brought unprecedented trauma to humanity. Laboratories around the world are working on developing different SARS-CoV-2 candidate vaccines. At present, several vaccines based on different technical platforms have completed emergency use registration. Inactivated vaccines are effective against SARS-CoV-2 variants, with an effectiveness rate of 50%-78% according to the regional and variant transmission situation (MBBS Nawal Al Kaabi et al, 2021). The main advantages of inactivated vaccines include safety, suitable storage conditions, and easy distribution. However, compared with other types of vaccines, the intensity of the humoral immune response against SARS-CoV-2 in the body is still relatively low after two doses of inactivated vaccines are administered (Lim W et al, 2021); especially the detection after 6 months shows that the intensity of the cell-mediated immune response is very limited (Cao Y, 2021). The innovative mRNA vaccines are very effective against the first batch of SARS-CoV-2 variants, but are limited in effectiveness against the Omicron variant (Lu L et al, 2021). mRNA vaccines can induce humoral and cell-mediated immune responses against SARS-CoV-2 variants, but also cause adverse reactions at different levels, such as myocarditis in the young population (Oster M.E et al, 2022). In addition, mRNA vaccines require ultra-low temperature cold chain transportation, which greatly limits the possibility of transportation to developing countries. Currently, the developed vaccines also include non-replicating viral vector vaccines, which can simultaneously induce humoral and cell-mediated immune responses against SARS-CoV-2. And, there are literature reports that non-replicating viral vector vaccines have a good protection level (Voysey M et al, 2020). Their disadvantage is that they can cause a certain degree of adverse reactions. Such as the coagulation events induced by the AstraZeneca AZD1222 vaccine (MacIntyre C.R et al, 2021). In addition, for adenovirus type 5 (Ad5) vector vaccines, the background immunity of Ad5 will affect the immune response against SARS-CoV-2 antigens (Zhu F-C et al, 2020). The main advantage of subunit vaccines lies in their safety, especially subunit vaccines adjuvanted with alum (Hernández-Bernal Fet al, 2021). At the same time, subunit vaccines have suitable storage conditions and are easy to distribute. The main disadvantage is that they cannot induce a persistent humoral and cell-mediated immune response.

[0003] In addition to the above-mentioned drawbacks, the above-mentioned SARS-CoV-2 vaccines also have two common limitations: First, the designs of the above-mentioned SARS-CoV-2 vaccines are all based on the primary transmission lineages of SARS-CoV-2, inducing anti-SARS-CoV-2 immune responses, especially those against low-mutation branches; Second, the above-mentioned SARS-CoV-2 vaccines are unable to induce mucosal immune responses and cannot block the transmission of the virus.

[0004] Single-stranded RNA viruses mutate rapidly. It is estimated that the SARS-CoV-2 lineage accumulates nucleotide mutations at a rate of about one to two times per month. The emergence of the highly contagious Omicron variant recently has posed new challenges to vaccine development. For those who have been infected with SARS-CoV-2, reinfection is possible. Moreover, following the severe acute respiratory syndrome coronavirus (SARS-CoV-1) that caused severe acute respiratory syndrome in 2002 and the Middle East respiratory syndrome coronavirus (MERS-CoV) that caused the Middle East respiratory syndrome in 2012, the COVID-19 pandemic caused by SARS-CoV-2 is the third zoonotic disease associated with human-lethal coronaviruses. Also, among the coronavirus samples collected in East Asia and Southeast Asia, about 50 SARS-related coronaviruses were detected in 10 bat species. Some studies have found that the SARS-related coronaviruses transmitted by bats pose a huge threat to the pandemic because several co-distributed species of the Rhinolophus bat host have significant characteristics of viral genetic diversity (Ravelomanantsoa NAF et al, 2021).

[0005] Based on the above factors, the newly emerging SARS-CoV-2 variants (as well as potential "pre-pandemic" zoonotic virus strains that may emerge in the future) have led the scientific community to start thinking about developing a new generation of vaccines with broad-spectrum protection capabilities, namely, coronavirus broad-spectrum vaccines.

[0006] Two methods are currently being used to develop a broad-spectrum coronavirus vaccine: screening multiple immunogenic antigens / regions based on the spike (S) protein (multivalent); identifying and designing proteins based on highly conserved regions of coronaviruses. Multivalent vaccines based on the S protein require multiple immunodominant regions, and the variants are limited to those within the immunodominant regions. The method based on conserved antigens can achieve broad-spectrum effects according to the conservation level of the antigen and its proper presentation. There have been research reports published based on the S2 subunit / fragment. For example, after mice were inoculated with a DNA vaccine containing the SARS-CoV-2 S2 protein region, antibodies against the S2 protein could be induced. The antibodies against the S2 protein could neutralize multiple human and animal betacoronaviruses in vitro and protect against SARS-CoV-2 attack in vivo (Ng K.W et al, 2021). The conserved antigen N protein was detected using different vaccine platforms and combinations (Dangi T et al, 2021). The N protein could induce humoral and cell-mediated immune responses against SARS-CoV-2, and the protective ability of the N protein against SARS-CoV-2 was verified using the Ad5 vector vaccine platform (Matchett W.E et al, 2021). However, although the above-mentioned candidate vaccines based on conserved regions have all obtained good results, these candidate vaccines have an important drawback, that is, there is a lack of data on the intensity of the induced cell-mediated immune response, especially against coronaviruses of the Sarbe coronavirus subgenus. In addition, the above-mentioned candidate vaccines also have a common limitation, that is, they cannot induce functional mucosal immune cross-response because most candidate vaccines are not administered through the intranasal route. Therefore, the preventive ability of the above-mentioned candidate vaccines against future coronavirus transmission is limited. Summary of the Invention

[0007] In view of the above-mentioned defects in the prior art, the present invention aims to provide a vaccine composition administered through the mucosal route.

[0008] In a first aspect of the present invention, there is provided a vaccine composition against betacoronavirus, which is administered through the mucosal route and comprises: 1) the nucleocapsid protein of the novel coronavirus; 2) a nucleic acid having a nucleotide sequence of SEQ ID NO: 1; and 3) a pharmaceutical excipient.

[0009] Further, the amino acid sequence of the nucleocapsid protein of the novel coronavirus is the sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 2.

[0010] Further, the mucosal route is the intranasal inoculation route.

[0011] Further, the vaccine composition further comprises a protein that induces neutralizing antibodies against betacoronavirus.

[0012] Furthermore, the protein that induces anti-β genus coronavirus neutralizing antibodies is the receptor-binding domain protein of the spike protein of the novel coronavirus.

[0013] Furthermore, the receptor-binding domain protein is the receptor-binding domain protein of the Delta variant of the novel coronavirus.

[0014] Furthermore, the amino acid sequence of the receptor-binding domain protein is the sequence of SEQ ID NO: 3.

[0015] In the second aspect of the present invention, there is provided the use of a vaccine composition in the production of a medicament against the novel coronavirus, wherein the vaccine composition induces a humoral immune response and a cell-mediated immune response against β genus coronavirus.

[0016] Furthermore, the amino acid sequence of the nucleocapsid protein of the novel coronavirus is the sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 2.

[0017] Furthermore, the vaccine composition is a formulation administered by the mucosal route.

[0018] Furthermore, the mucosal route is the intranasal administration route.

[0019] Furthermore, the vaccine composition further comprises a protein that induces anti-β coronavirus neutralizing antibodies.

[0020] Furthermore, the protein that induces anti-β coronavirus neutralizing antibodies is the receptor-binding domain protein of the spike protein of the novel coronavirus.

[0021] Furthermore, the receptor-binding domain protein is the receptor-binding domain protein of the Delta variant of the novel coronavirus.

[0022] Furthermore, the vaccine composition is a formulation administered by both the mucosal route and the parenteral route.

[0023] Furthermore, the vaccine composition is used as a booster vaccine for booster immunization of individuals who have been vaccinated against the novel coronavirus.

[0024] In the third aspect of the present invention, there is provided a method for inoculating the vaccine composition to induce a humoral immune response and a cell-mediated immune response against β genus coronavirus, wherein the vaccine composition comprises the nucleocapsid protein of the novel coronavirus and the nucleic acid with the nucleotide sequence of SEQ ID NO: 1.

[0025] Furthermore, the amino acid sequence of the novel coronavirus nucleocapsid protein is the sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity with the sequence of SEQ ID NO: 2.

[0026] Furthermore, the vaccine composition is administered by the mucosal route.

[0027] Furthermore, the mucosal route is the intranasal administration route.

[0028] Furthermore, the vaccine composition further comprises a protein that induces anti-β-coronavirus neutralizing antibodies.

[0029] Furthermore, the protein that induces anti-β-coronavirus neutralizing antibodies is the receptor-binding domain protein of the novel coronavirus spike protein.

[0030] Furthermore, the receptor-binding domain protein is the receptor-binding domain protein of the novel coronavirus Delta variant.

[0031] Furthermore, the vaccine composition is administered simultaneously by the mucosal route and the parenteral route.

[0032] Furthermore, the vaccine composition is used as a booster vaccine for booster immunization of individuals who have been vaccinated against the novel coronavirus.

[0033] Compared with existing marketed vaccines and vaccines under research, the N protein + ODN-39M vaccine composition provided by the present invention, combining a single vaccine component, has the following advantages:

[0034] 1. Inducing a cell-mediated immune cross-response against the N protein. The cell-mediated immune response has a protective effect against coronavirus infections including SARS-CoV-2, and the cell-mediated immune response induced by SARS-CoV infection can be maintained for up to 17 years. The anti-N protein cell-mediated immune cross-response induced by the N protein + ODN-39M vaccine composition has potential protective ability against at least coronavirus infections of the Sarbe coronavirus subgenus.

[0035] 2. Inducing cross-antibodies against the N protein and regulating the Th1 immune response pattern. Based on a non-neutralizing mechanism, anti-N protein antibodies have a potential protective effect against coronavirus infections. Therefore, the N protein + ODN-39M vaccine composition induces cross-antibodies against the N protein, which have a protective effect against potential coronavirus infections.

[0036] 3. The N protein + ODN-39M vaccine composition has an adjuvant effect on the RBD recombinant protein. Adding the N protein + ODN-39M vaccine composition to the vaccine preparation containing the inducible neutralizing antibody protein and administering it via the intranasal route can increase the local and systemic neutralizing antibody levels, thereby enhancing the cross-reactivity of anti-N protein antibodies. The new preparation has a wider protection range in the two major immune systems, namely the mucosal system and the systemic system.

[0037] 4. Administering the preparation containing the N protein + ODN-39M vaccine composition via the intranasal route is beneficial for inducing mucosal immune responses. Based on the important role of mucosal immune responses in blocking virus transmission, the mucosal immune responses induced by the bivalent antigen preparation provided by the present invention have significant advantages over existing vaccines. In addition, the intranasal administration route is more feasible and can avoid the related problems caused by injection administration. Therefore, it is particularly suitable for developing countries and large-scale vaccination.

[0038] 5. Potential safety. The N protein + ODN-39M vaccine composition preparation has potential safety. First, the N protein + ODN-39M vaccine composition preparation is developed based on a subunit vaccine platform (producing recombinant N protein through recombinant DNA technology). Second, the oligodeoxynucleotide adjuvant is relatively safe, and some have been approved for human use. Animal studies including non-human primates have verified the safety of ODN-39M. In addition, the oligodeoxynucleotide has the key characteristics of a fully phosphorothioate oligodeoxynucleotide, avoiding the related safety problems caused by chemical modifications of the nucleotide backbone (thiolate), and such safety problems are related to the side effects of oligodeoxynucleotides in different treatment regimens. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained based on the provided drawings.

[0040] The structures, ratios, sizes, etc. depicted in this specification are only used to cooperate with the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the limiting conditions under which the present invention can be implemented. Therefore, they do not have technical substance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the efficacy and purpose that the present invention can achieve, should still fall within the scope covered by the technical content disclosed in the present invention.

[0041] Figure 1a: Under reducing conditions, the expression of the cloned N protein was analyzed by 12% SDS-PAGE electrophoresis, where 1) molecular weight marker (MWM), 2) BL21(DE3) whole cells (negative control), 3) BL21(DE3) whole cells expressing the N protein, 4) purified N protein.

[0042] Figure 1b : Under reducing conditions, Western blot analysis was performed using a polyclonal antibody against the N protein (40588-T62, purchased from Sino Biological Inc., Beijing, China), where 1) MWM, 2) BL21(DE3) whole cells (negative control), 3) BL21(DE3) whole cells expressing the N protein, 4) purified N protein.

[0043] Figure 2 : By ELISA assay, the recognition of recombinant N protein by anti-SARS-CoV-2 human sera from healthy populations vaccinated with inactivated vaccines was detected. Human sera: HD1 - HD6.

[0044] Figure 3 : Schematic diagram of the aggregation (precipitation) curves of the N protein + ODN-39M vaccine composition at different mass ratios; Two independent experiments were carried out.

[0045] Figure 4a : The aggregation process of different samples with N protein + ODN-39M at a mass ratio of 0.66:1 was analyzed by 2% agarose gel electrophoresis stained with ethidium bromide, where 1: N protein + ODN-39M process 1; 2: N protein + ODN-39M process 2; 3: N protein; 4: control protein; 5: ODN-39M; 6: N protein + ODN-39M process 3; 7: control protein + ODN-39M; 8: MWM.

[0046] Figure 4b : The aggregation process of different samples with N protein + ODN-39M at a mass ratio of 0.66:1 was analyzed by 2% agarose gel electrophoresis stained with Coomassie Brilliant Blue, where 1: N protein + ODN-39M process 1; 2: N protein + ODN-39M process 2; 3: N protein; 4: control protein; 5: ODN-39M; 6: N protein + ODN-39M process 3; 7: control protein + ODN-39M; 8: MWM.

[0047] Figure 5a: Under non-reducing conditions, the aggregation state of the sample with N protein + ODN-39M at a mass ratio of 0.66:1 was analyzed by 2% agarose gel, where 1: N protein + ODN-39M (0.66:1) + 1% FA; 2: ODN-39M + 0.5% FA; 3: N + 0.5% FA; 4: N protein + ODN-39M (0.66:1) + 0.5% FA; 5: N protein + ODN-39M (0.66:1); 6: N protein; 7: ODN-39M; 8: MWM (DNA). FA refers to formaldehyde, and all FA reactions were quenched.

[0048] Figure 5b : Under non-reducing conditions, the aggregation state of the sample with N protein + ODN-39M at a mass ratio of 0.66:1 was analyzed by 10% SDS-PAGE, where 1: N protein + ODN-39M (0.66:1) + 1% FA; 3: N + 0.5% FA; 4: N protein + ODN-39M (0.66:1) + 0.5% FA; 5: N protein + ODN-39M (0.66:1); 6: N protein; 9: MWM (protein); 10: N protein + 1% FA. FA refers to formaldehyde, and all FA reactions were quenched.

[0049] Figure 6 : Schematic diagram for evaluating the immunogenicity of N protein preparation in the sera of immunized Balb / C mice by anti-N protein IgG ELISA assay; G1: N protein + alum, subcutaneous injection; G2: N protein + ODN-39M + alum, subcutaneous injection; G3: N protein (PBS), intranasal inoculation; G4: N protein + ODN-39M, intranasal inoculation; G5: N protein + ODN-39M, subcutaneous injection; G6: PBS + alum, subcutaneous injection; G7: PBS, intranasal inoculation; After logarithmic transformation of the titer, one-way analysis of variance (One Way Anova) and Tukey's multiple comparison method were used for statistical analysis.

[0050] Figure 7a : Schematic diagram for evaluating the humoral mucosal immune response in the BALF samples (undiluted) of immunized Balb / C mice by anti-N protein IgA ELISA assay, where G1: N protein + alum, subcutaneous injection; G2: N protein + ODN-39M + alum, subcutaneous injection; G3: N protein (PBS), intranasal inoculation; G4: N protein + ODN-39M, intranasal inoculation; G5: N protein + ODN-39M, subcutaneous injection; G6: PBS + alum, subcutaneous injection; G7: PBS, intranasal inoculation; After logarithmic transformation of the titer, one-way analysis of variance (One Way Anova) and Tukey's multiple comparison method were used for statistical analysis.

[0051] Figure 7b : Schematic diagram for evaluating the humoral mucosal immune response in BALF samples (undiluted) of immunized Balb / C mice by anti-N protein IgG ELISA test. Among them, G1: N protein + alum, subcutaneous injection; G2: N protein + ODN-39M + alum, subcutaneous injection; G3: N protein (PBS), intranasal inoculation; G4: N protein + ODN-39M, intranasal inoculation; G5: N protein + ODN-39M, subcutaneous injection; G6: PBS + alum, subcutaneous injection; G7: PBS, intranasal inoculation. After logarithmic transformation of the titer, one-way analysis of variance (One Way Anova) and Tukey's multiple comparison method were used for statistical analysis.

[0052] Figure 8a : Schematic diagram for evaluating IgG subclass antibodies in the sera of immunized Balb / C mice by anti-N protein IgG1 ELISA test. Among them, G1: N protein + alum, subcutaneous injection; G2: N protein + ODN-39M + alum, subcutaneous injection; G3: N protein (PBS), intranasal inoculation; G4: N protein + ODN-39M, intranasal inoculation; G5: N protein + ODN-39M, subcutaneous injection; G6: PBS + alum, subcutaneous injection; G7: PBS, intranasal inoculation. After logarithmic transformation of the titer, one-way analysis of variance (One Way Anova) and Tukey's multiple comparison method were used for statistical analysis.

[0053] Figure 8b : Schematic diagram for evaluating IgG subclass antibodies in the sera of immunized Balb / C mice by anti-N protein IgG2a ELISA test. Among them, G1: N protein + alum, subcutaneous injection; G2: N protein + ODN-39M + alum, subcutaneous injection; G3: N protein (PBS), intranasal inoculation; G4: N protein + ODN-39M, intranasal inoculation; G5: N protein + ODN-39M, subcutaneous injection; G6: PBS + alum, subcutaneous injection; G7: PBS, intranasal inoculation. After logarithmic transformation of the titer, one-way analysis of variance (One Way Anova) and Tukey's multiple comparison method were used for statistical analysis.

[0054] Figure 9:Schematic diagram of analyzing the IgG1 / IgG2a ratio (titer) in the sera of immunized Balb / C mice by anti-N protein ELISA test; G1: N protein + alum, subcutaneous injection; G2: N protein + ODN-39M + alum, subcutaneous injection; G3: N protein (PBS), intranasal inoculation; G4: N protein + ODN-39M, intranasal inoculation; G5: N protein + ODN-39M, subcutaneous injection; G6: PBS + alum, subcutaneous injection; G7: PBS, intranasal inoculation.

[0055] Figure 10 :Analysis of the cell-mediated immune response induced by stimulating mouse splenocytes with the conserved peptide N through IFNγ-ELISPOT test 351-365 :Schematic diagram of analyzing the cell-mediated immune response induced by stimulating mouse splenocytes with the N protein of the Delta variant through IFNγ-ELISPOT test; G1: N protein + alum, subcutaneous injection; G2: N protein + ODN-39M + alum, subcutaneous injection; G3: N protein (PBS), intranasal inoculation; G4: N protein + ODN-39M, intranasal inoculation; G5: N protein + ODN-39M, subcutaneous injection; G6: PBS + alum, subcutaneous injection; G7: PBS, intranasal inoculation; Statistical analysis was performed using the Kruskal-Wallis non-parametric test and Dunns multiple comparison test.

[0056] Figure 11 :Schematic diagram of analyzing the cell-mediated immune response induced by stimulating mouse splenocytes with the N protein of the Delta variant through IFNγ-ELISPOT test; G1: N protein + alum, subcutaneous injection; G2: N protein + ODN-39M + alum, subcutaneous injection; G3: N protein (PBS), intranasal inoculation; G4: N protein + ODN-39M, intranasal inoculation; G5: N protein + ODN-39M, subcutaneous injection; G6: PBS + alum, subcutaneous injection; G7: PBS, intranasal inoculation; Statistical analysis was performed using the Kruskal-Wallis non-parametric test and Dunns multiple comparison test.

[0057] Figure 12a:Schematic diagram for evaluating the immunogenicity of N protein and RBD protein preparations in the sera of immunized Balb / C mice by anti-N protein IgG ELISA test. Among them, G1: N protein (PBS), intranasal inoculation; G2: N protein + ODN-39M, intranasal inoculation; G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G4: N protein + RBD protein (PBS), intranasal inoculation; G5: RBD protein (PBS), intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G7: N protein + RBD protein + alum, subcutaneous injection; G9: PBS, intranasal inoculation; G10: PBS + alum, subcutaneous injection; After logarithmic transformation of the titer, one-way ANOVA and Tukey's multiple comparison method were used for statistical analysis.

[0058] Figure 12b :Schematic diagram for evaluating the immunogenicity of N protein and RBD protein preparations in the sera of immunized Balb / C mice by anti-RBD protein IgG ELISA test. Among them, G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G4: N protein + RBD protein (PBS), intranasal inoculation; G5: RBD protein (PBS), intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G7: N protein + RBD protein + alum, subcutaneous injection; G8: RBD protein + alum, subcutaneous injection; G9: PBS, intranasal inoculation; G10: PBS + alum, subcutaneous injection; After logarithmic transformation of the titer, one-way ANOVA and Tukey's multiple comparison method were used for statistical analysis.

[0059] Figure 13a :Schematic diagram for evaluating the humoral mucosal immune response in the BALF samples (undiluted) of immunized Balb / C mice by anti-N protein IgA ELISA test. Among them, G1: N protein (PBS), intranasal inoculation; G2: N protein + ODN-39M, intranasal inoculation; G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G4: N protein + RBD protein (PBS), intranasal inoculation; G5: RBD protein (PBS), intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G7: N protein + RBD protein + alum, subcutaneous injection; G8: RBD protein + alum, subcutaneous injection; G9: PBS, intranasal inoculation; G10: PBS + alum, subcutaneous injection; Kruskal-Wallis non-parametric test and Dunn's multiple comparison test were used for statistical analysis.

[0060] Figure 13b:Schematic diagram for evaluating the humoral mucosal immune response in BALF samples (undiluted) of immunized Balb / C mice by anti-RBD protein IgA ELISA test. Among them, G1: N protein (PBS), intranasal inoculation; G2: N protein + ODN-39M, intranasal inoculation; G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G4: N protein + RBD protein (PBS), intranasal inoculation; G5: RBD protein (PBS), intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G7: N protein + RBD protein + alum, subcutaneous injection; G8: RBD protein + alum, subcutaneous injection; G9: PBS, intranasal inoculation; G10: PBS + alum, subcutaneous injection; Statistical analysis was performed using the Kruskal-Wallis non-parametric test and Dunns multiple comparison test.

[0061] Figure 14a :Schematic diagram for evaluating IgG subclass antibodies in the sera of immunized Balb / C mice by anti-N protein IgG1 ELISA test. Among them, G2: N protein + ODN-39M, intranasal inoculation; G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G7: N protein + RBD protein + alum, subcutaneous injection.

[0062] Figure 14b :Schematic diagram for evaluating IgG subclass antibodies in the sera of immunized Balb / C mice by anti-N protein IgG2a ELISA test. Among them, G2: N protein + ODN-39M, intranasal inoculation; G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G7: N protein + RBD protein + alum, subcutaneous injection.

[0063] Figure 15a :Schematic diagram for evaluating IgG subclass antibodies in the sera of immunized Balb / C mice by anti-RBD protein IgG1 ELISA test. Among them, G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G4: N protein + RBD protein, intranasal inoculation; G5: RBD protein, intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G7: N protein + RBD protein + alum, subcutaneous injection; G8: RBD protein + alum, subcutaneous injection.

[0064] Figure 15b:Schematic diagram for evaluating IgG subclass antibodies in the sera of immunized Balb / C mice by anti-RBD protein IgG2a ELISA test. Among them, G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G4: N protein + RBD protein, intranasal inoculation; G5: RBD protein, intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G7: N protein + RBD protein + alum, subcutaneous injection; G8: RBD protein + alum, subcutaneous injection.

[0065] Figure 16 :By IFNγ-ELISPOT test, analyze the cell-mediated immune response induced after stimulating mouse splenocytes with the conserved peptide N 351-365 Schematic diagram of the cell-mediated immune response induced after stimulating mouse splenocytes with the conserved peptide N; G1: N protein (PBS), intranasal inoculation; G2: N protein + ODN-39M, intranasal inoculation; G3: N protein + ODN-39M + RBD protein, intranasal inoculation;

[0066] G4: N protein + RBD protein, intranasal inoculation; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; Statistical analysis was performed using one-way analysis of variance (One Way Anova) and Tukey's multiple comparison method.

[0067] Figure 17 :Schematic diagram of the cell-mediated immune response induced after stimulating mouse splenocytes with the RBD protein of the Delta variant by IFNγ-ELISPOT test; G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G4: N protein + RBD protein, intranasal inoculation; G5: RBD protein, intranasal inoculation; G9: PBS (negative control), intranasal inoculation.

[0068] Figure 18a :Schematic diagram for evaluating the immunogenicity of the N protein + ODN-39M preparation in the sera of immunized Balb / C mice (G2: N protein + ODN-39M, intranasal inoculation) by anti-N protein IgG antibody ELISA test coated with SARS-CoV-1 N protein and SARS-CoV-2 N protein.

[0069] Figure 18b :Schematic diagram for evaluating the immunogenicity of the N protein + ODN-39M preparation in the sera of immunized Balb / C mice (G3: N protein + ODN-39M + RBD protein, intranasal inoculation) by anti-N protein IgG antibody ELISA test coated with SARS-CoV-1 N protein and SARS-CoV-2 N protein.

[0070] Figure 19a:Schematic diagram of humoral mucosal immune response induced in BALF samples (undiluted) of Balb / C mice immunized with G2 (N protein + ODN-39M, intranasal administration) evaluated by anti-N protein IgA antibody ELISA test coating SARS-CoV-1 N protein and SARS-CoV-2 N protein.

[0071] Figure 19b :Schematic diagram of humoral mucosal immune response induced in BALF samples (undiluted) of Balb / C mice immunized with G3 (N protein + ODN-39M + RBD protein, intranasal administration) evaluated by anti-N protein IgA antibody ELISA test coating SARS-CoV-1 N protein and SARS-CoV-2 N protein.

[0072] Figure 20a :Evaluation of humoral mucosal immune response against RBD protein in serum by anti-RBD protein IgG antibody ELISA test for primary virus strain and Delta variant, where G3: N protein + ODN-39M + RBD protein, intranasal administration; G6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection; G8: RBD protein + alum, subcutaneous injection.

[0073] Figure 20b :Evaluation of humoral mucosal immune response against RBD protein in BALF by anti-RBD protein IgA antibody ELISA test for Delta variant, where G3: N protein + ODN-39M + RBD protein, intranasal administration; G9: PBS, intranasal administration.

[0074] Figure 20c :Evaluation of humoral mucosal immune response against RBD protein in BALF by anti-RBD protein IgA antibody ELISA test for primary virus strain, where G3: N protein + ODN-39M + RBD protein, intranasal administration; G9: PBS, intranasal administration.

[0075] Figure 21a :Analysis of cell-mediated immune response induced after stimulation of mouse splenocytes with SARS-CoV-2 Delta variant N protein, SARS-CoV-1 N protein and conserved peptide N 351-365 :by IFNγ-ELISPOT test, G2: N protein + ODN-39M, intranasal administration; G9: PBS (negative control).

[0076] Figure 21b :Analysis of cell-mediated immune response induced after stimulation of mouse splenocytes with SARS-CoV-2 Delta variant N protein, SARS-CoV-1 N protein and conserved peptide N 351-365Schematic diagram of cell-mediated immune response induced after stimulating mouse splenocytes, G3: N protein + ODN-39M + RBD protein, intranasal inoculation; G9: PBS (negative control), intranasal inoculation. Detailed implementation mode

[0077] The following specific embodiments illustrate the implementation modes of the present invention. Those skilled in this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0078] The vaccine composition comprises: 1) SARS-CoV-2 nucleocapsid (N) protein with the amino acid sequence of SEQ ID NO: 2; 2) a nucleic acid with the nucleotide sequence of SEQ ID NO: 1. The vaccine composition can solve the following two problems: 1. Induce a broad functional immune response against non-spike (S) proteins; 2. Induce a mucosal immune response.

[0079] To solve the above two problems, the present invention proposes a technical solution: using the SARS-CoV-2 N recombinant protein to induce cross-immune, mucosal immune and systemic immune responses against coronaviruses of the Sarbe coronavirus subgenus.

[0080] The SARS-CoV-2 N protein is obtained by incubating Escherichia coli, and the obtained protein is purified to a purity of more than 90%. In addition, the SARS-CoV-2 N protein can be detected in the sera of volunteers who have been vaccinated with the inactivated vaccine in China, indicating the existence of conformational epitopes. In one embodiment of the present invention, the SARS-CoV-2 N protein has the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2.

[0081] To develop a suitable vaccine formulation, the SARS-CoV-2 N protein was combined with a phosphodiester backbone CpG oligodeoxynucleotide (ODN-39M) having the sequence of SEQ ID NO: 1. The SARS-CoV-2 Delta variant N protein was mixed and incubated with different amounts of ODN-39M. When the ratio of ODN-39M to N protein was 3:1, aggregation (precipitation) reached its maximum, yielding a typical saturation curve. This ratio corresponds to the molar ratio of the two molecules, which is the first proof of their interaction. Since the N protein remained soluble after mixing with ODN-39M, screening for supersaturated conditions of ODN and conducting in-depth research are key factors in vaccine formulation research. After characterizing ODN-39M and the N protein using agarose gel at room temperature, a common region where both the N protein and ODN-39M were present was detected, which also proved their interaction. Through cross-linker experiments, it was demonstrated that this interaction could form soluble high-molecular-weight N protein aggregates. Different from other viral N proteins that form particles by naturally encapsulating viral RNA, the SARS-CoV-2 N protein adheres to long-chain RNA through a histone-like conformation, and its structure is completely different from the capsid-encapsulating structure. Therefore, the formation of the soluble high-molecular-weight N protein aggregates was almost unexpected. In addition, the ODN-39M used has 39 nucleotides, which is significantly shorter than the long-chain structure of viral RNA. The aggregate structure can enhance the immunogenicity of the protein. Therefore, the formation of soluble N protein aggregates in the presence of ODN-39M is an important feature of the N protein + ODN-39M vaccine composition.

[0082] Mice were inoculated with the N protein + ODN-39M vaccine composition via the intranasal administration route, and high levels of anti-N protein IgG antibodies were induced in both serum and bronchoalveolar lavage fluid (BALF). At the same time, IgA antibodies were also induced, which was an unexpected result. In the research reports of the prior art, after the dengue virus capsid protein was combined with ODN-39M, no humoral immune response could be induced by intranasal inoculation of mice (Lazo L. et al, 2017). On the other hand, the vaccine composition of the present invention could induce a typical Th1 immune response pattern. After stimulating splenocytes with the N protein, the cell-mediated immune response was measured by the ELISPOT IFNγ assay, and the result showed a 100% response level. In the group inoculated with the N protein alone via the intranasal administration route, neither humoral immune response nor cell-mediated immune response was detected. This clearly demonstrated the correlation between the intranasal administration of the N protein + ODN-39M vaccine composition and the immunogenicity of the Delta variant N protein.

[0083] In another embodiment of the present invention, after inoculating mice with the N protein + ODN-39M vaccine composition via the intranasal administration route, a cross-response of humoral immunity and cell-mediated immunity was induced. Using the conserved peptide N 351-365 After stimulating splenocytes with the SARS-CoV-1 N protein, a significant cell-mediated immune response can be induced, and high levels of anti-SARS-CoV-1 N protein antibodies are induced in BALF and serum. SARS-CoV-1 is a representative coronavirus in the Sarbe coronavirus subgenus. The conserved peptide N 351-365 is a conserved peptide shared by coronaviruses in the Sarbe coronavirus subgenus and also shows protective effects in SARS-CoV-2-infected animal models. Therefore, the immune response against the conserved peptide N 351-365 has a significant correlation. And intranasal administration of the N protein + ODN-39M vaccine composition can induce a cell-mediated immune cross-response against the conserved peptide N 351-365 Whether with or without alum as an adjuvant, administering the N protein + ODN-39M vaccine composition via the subcutaneous injection route cannot induce a cell-mediated immune cross-response against the conserved peptide N 351-365 even when the immune response against the N protein of the Delta variant in the animal model reaches 100%. Intranasal administration of the N protein + ODN-39M vaccine composition can selectively induce a cell-mediated immune cross-response and induce a humoral immune cross-response in the mucosa and serum. Therefore, the N protein + ODN-39M vaccine composition becomes a promising component for future broad-spectrum coronavirus vaccines.

[0084] In a specific embodiment of the present invention, the N protein + ODN-39M vaccine composition is combined with a second-generation antigen of SARS-CoV-2, and neutralizing antibodies are induced by intranasal inoculation. Neutralizing antibodies are an important group of protective antibodies. In a preferred embodiment, the second-generation antigen for inducing neutralizing antibodies is the receptor-binding domain (RBD) protein of the SARS-CoV-2 Delta variant, and its amino acid sequence is the sequence of SEQ ID NO: 3. In the Th1 immune response mode, the N protein + ODN-39M vaccine composition has a significant adjuvant effect on inducing mucosal and systemic immune responses against the RBD protein of the Delta variant. The anti-RBD antibodies induced in BALF and serum can recognize the RBD proteins of the primary virus and the Delta variant. The antibodies in serum have an ACE-2 binding inhibitory effect on the two RBD proteins and can also induce an anti-N protein cross-immune response. Administering a single RBD via the intranasal route has no immunogenicity at all.

[0085] Based on the above results, the N protein + ODN-39M vaccine composition can be a promising component of a future broad-spectrum coronavirus vaccine, which can induce cross-immune responses against the N protein, and humoral and cell-mediated immune responses through the mucosal and systemic systems. By intranasal administration of a bivalent antigen preparation of N protein + ODN-39M + RBD, cross-immune responses against the N protein, anti-RBD IgA antibodies in the mucosa, and neutralizing antibodies against the two RBD proteins in the serum can be induced. The above results provide a theoretical basis for the bivalent antigen preparation as a booster by intranasal administration, especially after vaccination with inactivated vaccines. Since the inactivated vaccines contain N and S proteins, they can successfully activate the immune system against N and S proteins.

[0086] Example 1

[0087] Cloning, Expression, Purification and Antigen Characterization of the Nucleoprotein (N Protein) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta Variant

[0088] The DNA sequence of the SARS-CoV-2 Delta variant N protein (YP_009724397.2, corresponding to the amino acid sequence SEQ ID NO: 2) was cloned into the PET-28 vector and then expressed in Escherichia coli to obtain the protein. The protein accounted for 11% of the total protein of Escherichia coli ( Figure 1a ). First, the protein was precipitated by ammonium sulfate fractionation, and then semi-purified by hydrophobic interaction chromatography. Subsequently, the semi-purified protein was further purified by anion exchange chromatography to obtain the purified protein. After eluting the purified protein, the protein buffer was changed to phosphate buffer (PBS) by Sephadex G-25 gel filtration chromatography, so that the purity of the purified protein reached over 95%. The purified protein was immunologically identified by Western blot (WB) and a polyclonal anti-N protein antibody preparation (Beijing Sino Biological Inc.) ( Figure 1b ).

[0089] By administering the recombinant protein, the recognition of the recombinant protein by the sera of volunteers vaccinated with inactivated vaccines (vaccines produced by Sinovac and Sinopharm in Beijing, China) was detected to complete antigen characterization. The results showed that the sera of vaccinated volunteers were positive for the N protein, indicating the folding and exposure of the relevant antigenic epitopes ( Figure 2 ).

[0090] Example 2

[0091] Characterization of the N Protein + ODN-39M Vaccine Composition

[0092] Aggregate the N protein at different N protein / oligodeoxynucleotide 39M (ODN-39M) mass ratios. ODN-39M is a 39-mer fully phosphorodiester conjugate with the sequence of SEQ ID NO: 1 and was synthesized by Sangon Biotech (Shanghai) Co., Ltd., China.

[0093] To obtain the aggregation curve, mix 40 μg of N protein with different amounts of ODN-39M to obtain a 100 μL reaction mixture. The concentration range of ODN-39M is 0.04 μg - 80 μg ODN-39M / 10 mM Tris (pH = 8). Incubate the reaction mixture at 30 °C for 30 minutes, then store it in a 4 °C refrigerator for 4 hours and centrifuge at 10,000 x g. Extract the supernatant and measure the protein concentration. As Figure 3 shown, when the mass ratio of N protein to ODN-39M is 5:1 - 2.5:1, the maximum aggregation (precipitation) of the reaction mixture is obtained, and the corresponding molar ratio of N protein to ODN-39M is 1:3, indicating that this molar ratio is the potential saturation point (theoretically, 3 ODN-39M molecules are required to neutralize 1 N protein molecule). The resulting curve clearly shows the interaction between the two molecules.

[0094] Since solubility is one of the key characteristics of vaccine formulations, screen the conditions of N protein and ODN-39M at a mass ratio of 0.66:1 (under the condition of ODN-39M supersaturation) for further characterization. To verify the interaction between N protein and ODN-39M at a mass ratio of 0.66:1, load the reaction mixture onto a 2% agarose gel and observe the co-localization of the two molecules. The 2% agarose gel was stained with ethidium bromide to detect ODN-39M and with Coomassie Brilliant Blue to detect N protein. As Figure 4a shown in 4b the figure, in the samples of the aggregation process, a small amount of ODN-39M migrates slowly, and the N protein migrates more compared to the N protein without ODN-39M. This indicates that there is a common region where both N protein and ODN-39M are present (in the indicated box). The change in the migration patterns of N protein and ODN-39M and the detected common region directly prove the interaction between the two molecules.

[0095] Finally, to evaluate the true effect of ODN-39M on the conformation of N protein, under non-reducing conditions, the N protein + ODN-39M vaccine composition fixed with the cross-linking agent formaldehyde and the N protein were subjected to SDS-PAGE electrophoresis and quenching. The resulting image clearly demonstrated that the mixing of N protein and ODN-39M could produce high-molecular-weight N protein aggregates, because the N protein + ODN-39M vaccine composition fixed with the cross-linking agent formaldehyde was completely retained in the gel wells. When analyzing the cross-linked N protein + ODN-39M vaccine composition using 2% agarose gel, it had the same ODN-39M retention rate ( Figure 5a and 5b ).

[0096] Example 3

[0097] Design the first immunization protocol for mice to conduct immunological evaluation of different N protein-based formulations

[0098] On days 0, 7, and 21, 7 groups of Balb / C mice (female, 6 - 8 weeks old) were immunized according to the following design, with an inoculation dose of 10 μg.

[0099] Group 1: N protein + alum, subcutaneous injection (100 μL)

[0100] Group 2: N protein + ODN-39M + alum, subcutaneous injection (100 μL)

[0101] Group 3: N protein, intranasal inoculation (50 μL)

[0102] Group 4: N protein + ODN-39M, intranasal inoculation (50 μL)

[0103] Group 5: N protein + ODN-39M, subcutaneous injection (100 μL)

[0104] Group 6: PBS + alum (negative control), subcutaneous injection (100 μL)

[0105] Group 7: PBS (negative control), intranasal inoculation (50 μL)

[0106] The mice were sacrificed on days 12 and 19 after the last dose of immunization to evaluate the induced humoral immune response and cell-mediated immune response.

[0107] Example 4

[0108] Evaluate the humoral immune response of immunized mice

[0109] On the 12th day after the last dose of immunization, sera were collected from the blood samples of the mice and analyzed by anti-Delta variant N protein (SAR-CoV-2 Delta variant N protein (40588-V07E29), purchased from Sino Biological Inc., Beijing, China) IgG ELISA assay. As Figure 6 shown, all groups injected with N protein subcutaneously induced high levels of anti-N protein antibodies. In contrast, only the group inoculated intranasally with the N protein + ODN-39M vaccine composition induced high levels of IgG antibodies (p<0.05).

[0110] The IgA antibodies against the Delta variant N protein in bronchoalveolar lavage fluid (BALF) were detected. The results are as Figure 7a shown in 7b As expected, group 4 induced a higher level of anti-N protein IgA antibody response. The antigen-specific IgG antibody response detected in BALF was the same as that detected in serum because IgG antibodies can penetrate mucosal tissues through systemic tissues.

[0111] To detect the Th1 immune response patterns induced by each preparation, the IgG1 and IgG2a subclasses of anti-N protein in serum were measured using Sigma antibodies. As Figure 8a shown in 8b compared with groups 1, 2, and 5 (all subcutaneously inoculated), the IgG1 level induced by group 4 (N protein + ODN-39M, intranasally inoculated) was significantly lower. In contrast, the IgG2a levels induced by groups 4 and 5 were significantly increased. Therefore, it was concluded that the IgG1 / IgG2a ratio of each group was close to 1, indicating that a typical Th1 immune response pattern was induced in the mice inoculated only with the N protein + ODN-39M preparation (without alum) ( Figure 9 ). An important feature of the induced Th1 immune response pattern is that the level of IgG2a subclass antibodies reaches or even exceeds the level of IgG1 subclass antibodies. The Th1 immune response pattern implies the induction of cell-mediated immune responses.

[0112] Example 5

[0113] Evaluating the cell-mediated immune response of immunized mice

[0114] To evaluate the cell-mediated immune response, after in vitro stimulation with two reagents, the conserved peptide N 351-365 and the Delta variant N protein, the frequency of IFNγ production by splenocytes was detected using Mabtech antibodies and microtiter plates. The conserved peptide N 351-365(ILLNKHIDAYKTFPP), synthesized by Zhejiang Peptide Biotech Co., Ltd., China, with a purity of over 97%; the Delta variant N protein is the N protein of the Delta variant of SARS-CoV-2 (40588-V07E29), purchased from Sino Biological Inc., Beijing, China. On the 18th day after the last dose of immunization, the conserved peptide N was used. 351-365 and the Delta variant N protein were used to stimulate splenocytes. As Figure 10 shown, in response to the stimulation of the conserved peptide N 351-365 , IFNγ-secreting cell responses were detected in all mice in Group 4 (N protein + ODN-39M, intranasal inoculation), while IFNγ-secreting cell responses were not detected in mice in Group 3 (N protein, intranasal inoculation). In addition, among Group 1 and Group 2 by subcutaneous injection, and Group 5 (N protein + ODN-39M, subcutaneous injection) with the same inoculation preparation as Group 4, only 1 or 2 mice in each group of 6 mice showed responses ( Figure 10 ). The results indicate that the intranasal inoculation route is crucial for inducing cell-mediated immune responses against the conserved peptide N 351-365 .

[0115] Different results were obtained for the cell-mediated immune responses induced using the N protein as the stimulating antigen. 100% responses were obtained in Groups 1, 2, 4, and 5, with no statistical differences between the groups, and Group 4 had the highest response level ( Figure 11 ).

[0116] Example 6

[0117] Design a second immunization protocol for mice and conduct immunological evaluation of the intranasal inoculation bivalent antigen preparation

[0118] The experimental objective was to detect the immune responses of the bivalent antigen preparation. The bivalent antigen preparation contains the Delta variant N protein that induces cell-mediated immune cross-responses and the Delta variant RBD protein (40592-V08H90, whose sequence is the sequence of SEQ ID NO: 3, purchased from Sino Biological Inc., Beijing, China) that induces neutralizing antibodies. At the same time, different control groups were used to explore the effects of the two immunization routes. Ten groups of Balb / C mice (female, 6-8 weeks old) were immunized according to the following design on days 0, 7, and 21, with an inoculation dose of 10 μg.

[0119] Group 1: N protein, intranasal inoculation (50 μL)

[0120] Group 2: N protein + ODN-39M, intranasal inoculation (50 μL)

[0121] Group 3: N protein + ODN-39M + receptor binding domain (RBD) protein, intranasal inoculation (50 μL)

[0122] Group 4: N protein + RBD protein, intranasal inoculation (50 μL)

[0123] Group 5: RBD protein, intranasal inoculation (50 μL)

[0124] Group 6: N protein + ODN-39M + RBD protein + alum, subcutaneous injection (100 μL)

[0125] Group 7: N protein + RBD protein + alum, subcutaneous injection (100 μL)

[0126] Group 8: RBD protein + alum, subcutaneous injection (100 μL)

[0127] Group 9: PBS (negative control), intranasal inoculation (50 μL)

[0128] Group 10: PBS + alum (negative control), subcutaneous injection (100 μL)

[0129] On the 12th and 26th days after the last dose of immunization, blood was collected from the mice to evaluate the induced humoral immune response and cell-mediated immune response.

[0130] Example 7

[0131] Evaluation of the humoral immune response in immunized mice

[0132] On the 12th day after the last dose of immunization was completed, serum was collected from the blood samples of the mice and analyzed by IgG ELISA assay against the N protein of the Delta variant (SARS-CoV-2 Delta variant N protein (40588-V07E29), purchased from Sino Biological Inc., Beijing, China). As Figure 12a shown, compared with the control group (p < 0.05), Groups 2, 3, 6, and 7 significantly induced antibodies against the N protein of the Delta variant. Among the groups inoculated intranasally, only the group inoculated with the N protein + ODN-39M vaccine composition induced a high level of antibodies against the N protein. The level of antibodies against the N protein induced by Group 3 (N protein + ODN-39M + RBD protein, the bivalent antigen preparation) was lower than that of Groups 2, 6, and 7, possibly due to the effect of the RBD protein in the preparation. Nevertheless, the level of antibodies against the N protein induced by Group 3 was still relatively high.

[0133] Meanwhile, the IgG antibody levels against the RBD protein of the Delta variant (SARS-CoV-2 Delta variant RBD protein (40592-V08H90), purchased from Sino Biological Inc., Beijing, China) were detected. No significant induction of anti-Delta variant RBD protein antibodies was observed in the sera of mice in Group 5 (RBD protein, intranasal inoculation) and Group 4 (N protein + RBD protein, intranasal inoculation). In contrast, Group 3 (N protein + ODN-39M + RBD protein bivalent antigen preparation, intranasal inoculation) induced a higher level of anti-Delta variant RBD protein antibodies, which was similar to the level of anti-Delta variant RBD protein antibodies induced by Group 6 (N protein + ODN-39M + RBD protein + alum, subcutaneous injection). As Figure 12b shown.

[0134] The IgA antibodies against the N protein of the Delta variant and the RBD protein of the Delta variant in BALF were detected. The results are as Figure 13a shown 13b in. In the groups inoculated intranasally, the level of IgA antibodies induced in BALF was consistent with that in serum. The groups inoculated with the preparation containing N protein + ODN-39M induced anti-N protein IgA antibodies. Among the intranasal inoculation groups, only the group inoculated with the N protein + ODN-39M + RBD protein bivalent antigen preparation induced an anti-RBD protein response (as Figure 13b shown).

[0135] Regarding the IgG subclass antibodies against the N protein of the Delta variant in serum, the levels of IgG1 antibodies induced by Group 2 and Group 3 were significantly lower than those of Group 6 and Group 7 (subcutaneous inoculation with the preparation containing N protein) ( Figure 14a ). Compared with Group 6 and Group 7, the levels of IgG2a antibodies induced by Group 2 and Group 3 were significantly higher ( Figure 14b ). In the mice inoculated only through the intranasal route with the N protein + ODN-39M vaccine composition and the N protein + ODN-39M + RBD protein preparation, the level of IgG2a antibodies increased while the level of IgG1 antibodies decreased, which was caused by the regulation of the typical Th1 immune response pattern.

[0136] The IgG subclass antibodies against the RBD protein of the Delta variant in serum are as Figure 15a shown 15b in. Except for Group 4 and Group 5, the IgG1 titers were similar in all groups inoculated with the preparation containing RBD protein ( Figure 15a ). Compared with other groups (p < 0.05), the IgG2a titer against the RBD protein of the Delta variant in Group 3 (N protein + ODN-39M + RBD protein, the bivalent antigen preparation, intranasal inoculation) was higher (Figure 15b )。In the third group, the level of IgG2a antibody against the RBD protein of the Delta variant increased, indicating that the Th1 immune response pattern can resist the RBD protein of the Delta variant.

[0137] Example 8

[0138] Evaluating the cell-mediated immune response of immunized mice

[0139] To evaluate the cell-mediated immune response, after in vitro stimulation with two reagents, the conserved peptide N 351-365 and the RBD protein of the Delta variant, the frequency of IFNγ-producing splenocytes was detected. On the 12th day after the last dose of immunization, the splenocytes of mice in groups 1, 2, 3, 4 and 9 (negative control) of the intranasal vaccination group were stimulated with the conserved peptide N 351-365 .

[0140] As Figure 16 shown, IFNγ-secreting cell responses were detected in both groups 2 and 3 of mice, while IFNγ-secreting cells were not detected in the splenocytes of all the remaining groups. The IFNγ-secreting cell response was similar to the response detected in the group vaccinated with the N protein + ODN-39M vaccine composition in the mouse experiment of Example 3. There was no statistical difference compared with group 2. The positive response detected in the group vaccinated with the bivalent antigen preparation indicated that the addition of the RBD protein to the N protein + ODN-39M vaccine composition did not affect the induction of the cell-mediated immune response against the conserved peptide N 351-365 .

[0141] No immune response was induced in group 1, confirming the immunogenicity of the intranasal vaccination with the N protein + ODN-39M vaccine composition in the mouse experiment of Example 3.

[0142] Meanwhile, mice in groups 3, 4, 5 and 9 (negative control) were stimulated with the RBD protein of the Delta variant. The above groups were intranasally vaccinated with preparations containing the RBD protein. The results were as Figure 17 shown, and a positive response was detected only in the group vaccinated with the bivalent preparation containing ODN-39M (2 / 5 mice).

[0143] Example 9

[0144] Evaluating the cross-immune response of immunized mice

[0145] Humoral immune responses against SARS-CoV-1 N protein and RBD protein of the primary virus strain of SARS-CoV-2

[0146] In order to determine the range of immune response induced by intranasal vaccination containing the N protein + ODN-39M vaccine composition, on the 26th day after the last dose of immunization, the mouse sera of Group 2 and Group 3 in the mouse experiment of Example 6 were collected, and ELISA tests against SARS-CoV-2Delta variant N protein, SARS-CoV-2Omicron variant N protein (40588-V07E34, purchased from Sino Biological Technology Co., Ltd., Beijing, China) and SARS-CoV-1N protein (40143-V08B, purchased from Sino Biological Technology Co., Ltd., Beijing, China) were carried out. The results are as follows Figure 18a and 18b As shown, both groups induced IgG antibodies against the N proteins of the three variants in the preparations studied.

[0147] IgA and IgG ELISA tests were performed on BALF samples from groups 2 and 3 against the N proteins of the three variants, and the results were consistent with the serum results ( Figure 19a and 19b ). This indicates that the N protein + ODN-39M preparation as a monovalent preparation or a bivalent preparation combined with the RBD protein can induce IgA antibodies against the SARS-CoV-2 (Delta variant and Omicron variant) N protein and against the SARS-CoV-1 N protein.

[0148] At the same time, the mouse sera from groups 3, 6, and 8 were tested for IgG antibodies against the RBD protein of the primary SARS-CoV-2 virus strain (40592-V08H, purchased from Sino Biological Technology Co., Ltd., Beijing, China) and antibodies against the RBD protein of the Delta variant. Figure 20a As shown, it shows that cross-reactive antibodies were induced in groups 3, 6 and 8. Based on the above results, the neutralizing activity of the RBD protein of the primary virus strain of SARS-CoV-2 and the RBD protein of the Delta variant in the sera of groups 3 and 6 was detected by alternative virus neutralization test. As shown in Table 1, antibodies with inhibitory activity against the RBD protein of the primary virus strain of SARS-CoV-2 and the RBD protein of the Delta variant were induced in all mice in groups 3 and 6. Moreover, the antibody level was higher in group 6, which was vaccinated by subcutaneous injection. However, after intranasal inoculation of the bivalent antigen preparation of N protein + ODN-39M + RBD protein, the level of inhibitory antibodies in the serum increased, indicating that vaccination of vaccinated individuals with the bivalent antigen preparation can provide systemic neutralizing antibody levels against other virus strains.

[0149] Table 1 Inhibitory titers of the RBD protein of the primary strain of SARS-CoV-2 and the Delta variant in the serum of mice in groups 3 and 6 detected by the alternative virus neutralization test

[0150]

[0151]

[0152] Finally, an ELISA test was used to detect the IgA antibodies against the RBD protein of the SARS-CoV-2 primary virus strain and the RBD protein of the Delta variant in the BALF samples of Group 3. The results were as Figure 20b shown in 20c Among the 5 mice in Group 3, 3 were detected with positive responses, indicating that antibodies against the RBD protein of the primary virus strain and the RBD protein of the Delta variant can be induced in mucosal tissues.

[0153] Cell-mediated immune response against the SARS-CoV-1N protein

[0154] On the 26th day after the last immunization, splenocytes from the mice in Groups 2, 3, and 9 (negative control) were stimulated with the SARS-CoV-2 Delta variant N protein, the SARS-CoV-1N protein, and the conserved peptide N 351-365 Consistent with the results of the humoral immune response detection, positive responses against the SARS-CoV-2 Delta variant N protein, the SARS-CoV-1N protein, and the conserved peptide N 351-365 were detected in both Group 2 and Group 3. Therefore, verified by the Sarbe coronavirus subgenus member SARS-CoV-1, the cell-mediated immune response has the characteristic of cross-reactivity ( Figure 21a shown in 21b ).

[0155] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed. Sequence Listing <110> Yongzhou Zhonggu Biotechnology Co., Ltd., Genetic Engineering and Biotechnology Center <120> A vaccine composition against betacoronavirus and its application <130> FD220850 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 39 <212> DNA <213> Artificial Sequence <400> 1 atcgactctc gagcgttctc gggggacgat cgtcggggg 39 <210> 2 <211> 420 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile 1 5 10 15 Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu 20 25 30 Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn 35 40 45 Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Gly 50 55 60 Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser 65 70 75 80 Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg 85 90 95 Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr 100 105 110 Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys 115 120 125 Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys 130 135 140 Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu 145 150 155 160 Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly 165 170 175 Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg 180 185 190 Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Met Gly Thr Ser Pro 195 200 205 Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu 210 215 220 Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln 225 230 235 240 Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser 245 250 255 Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr 260 265 270 Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly 275 280 285 Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln 290 295 300 Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg 305 310 315 320 Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly 325 330 335 Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile 340 345 350 Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu 355 360 365 Pro Lys Lys Asp Lys Lys Lys Lys Ala Tyr Glu Thr Gln Ala Leu Pro 370 375 380 Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp 385 390 395 400 Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp 405 410 415 Ser Thr Gln Ala 420 <210> 3 <211> 223 <212> PRT <213> Artificial Sequence <400> 3 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 Arg 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 Lys 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 210 215 220

Claims

1. A vaccine composition against betacoronavirus, administered by the mucosal route, characterized in that, Comprising: 1) Nucleocapsid protein of novel coronavirus; 2) Nucleic acid with nucleotide sequence of SEQ ID NO: 1; and 3) Pharmaceutical excipients.

2. The vaccine composition according to claim 1, characterized in that, The amino acid sequence of the nucleocapsid protein of the novel coronavirus is the sequence of SEQ ID NO:

2.

3. The vaccine composition according to claim 1, wherein The mucosal route is the intranasal inoculation route.

4. The vaccine composition according to claim 1, wherein The vaccine composition further comprises a protein that induces neutralizing antibodies against betacoronavirus, and the protein that induces neutralizing antibodies against betacoronavirus is the receptor-binding domain protein of the spike protein of the novel coronavirus.

5. The vaccine composition according to claim 4, wherein The receptor-binding domain protein is the receptor-binding domain protein of the Delta variant of the novel coronavirus.

6. The vaccine composition according to claim 5, wherein The amino acid sequence of the receptor-binding domain protein is the sequence of SEQ ID NO:

3.

7. Use of the vaccine composition according to any one of claims 1-6 in the production of an anti-SARS-CoV-2 drug, characterized in that, The vaccine composition induces a humoral immune response and a cell-mediated immune response against betacoronavirus.

8. The application according to claim 7, wherein The amino acid sequence of the nucleocapsid protein of the novel coronavirus is the sequence of SEQ ID NO:

2.

9. The application according to claim 7, wherein The vaccine composition is a preparation administered by the mucosal route.

10. The application according to claim 9, characterized in that, The mucosal route is the intranasal inoculation route.

11. The application according to claim 7, characterized in that, The vaccine composition further comprises a protein that induces neutralizing antibodies against the betacoronavirus, and the protein that induces neutralizing antibodies against the betacoronavirus is the receptor-binding domain protein of the spike protein of the novel coronavirus.

12. The application according to claim 11, characterized in that, The receptor-binding domain protein is the receptor-binding domain protein of the Delta variant of the novel coronavirus.

13. The application according to claim 7, wherein The vaccine composition is a preparation administered by both the mucosal route and the parenteral route.

14. The application according to claim 7, characterized in that, The vaccine composition is used as a booster vaccine for booster immunization of individuals who have been vaccinated against the novel coronavirus.

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