CN115089700A

CN115089700A - Vaccine composition for resisting beta coronavirus and application thereof

Info

Publication number: CN115089700A
Application number: CN202210587491.9A
Authority: CN
Inventors: H·C·利塞特; L·M·亚迪拉; S·P·伊迪丝; 杨科; P·N·亚西尔; 陈榕; 艾攀超; S·R·里卡多; 蒋胜男; H·G·薇薇安; 谭昌源; M·L·亚历克西斯; 傅文博; E·N·G·杰拉尔多; 谭满元; L·V·劳拉; 戴凌锋; M·M·D·亚历杭德罗; 刘俊杰; 胡丽丽
Original assignee: Yongzhou Zhonggu Biotechnology Co ltd; Centro de Ingenieria Genetica y Biotecnologia CIGB
Current assignee: Yongzhou Zhonggu Biotechnology Co ltd; Centro de Ingenieria Genetica y Biotecnologia CIGB
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2022-09-23

Abstract

The invention discloses a vaccine composition for resisting beta coronavirus and application thereof, belonging to the technical field of biology. The vaccine composition for resisting the beta coronavirus is inoculated through a mucosal route and comprises novel coronavirus nucleocapsid protein, phosphodiester backbone CpG oligodeoxynucleotide and pharmaceutic adjuvants. Meanwhile, the invention also relates to the application of the vaccine composition in the production of a medicine for resisting the novel coronavirus. And further provides methods of vaccinating the compositions to induce both humoral and cell-mediated immune responses against the beta coronavirus. By vaccination via the mucosal route, particularly after intranasal administration, cell-mediated immune cross-responses against N protein can be induced, and cross-antibodies against N protein can be induced, in a modulated Th1 immune response pattern. Meanwhile, the vaccine composition preparation has potential safety.

Description

Vaccine composition for resisting beta coronavirus and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a vaccine composition for resisting beta coronavirus and application thereof.

Background

At present, the pandemic of new coronary pneumonia (COVID-19) caused by the new coronavirus (SARS-CoV-2) is a global public health crisis, which has caused millions of people to die and has brought unprecedented trauma to human beings. Laboratories around the world are working on developing different SARS-CoV-2 vaccine candidates. Currently, several vaccines based on different technology platforms have already completed registration for emergency use. The inactivated vaccine is effective on SARS-CoV-2 variant, and the effective rate is 50-78% according to the region and the propagation condition of the variant (MBBS Nawal Al Kaabi et Al, 2021). The main advantages of inactivated vaccines include safety, suitable storage conditions and ease of distribution. However, the magnitude of the humoral immune response against SARS-CoV-2 in vivo is still low after two inactivated vaccine doses compared to other types of vaccine (Lim W et al, 2021); in particular, measurements after 6 months show that the magnitude of the cell-mediated immune response is very limited (Cao Y, 2021). The innovative mRNA vaccine was very effective against the first SARS-CoV-2 variant strain, but only against the Onckrojon variant strain (Lu L et al, 2021). The mRNA vaccine is capable of inducing both humoral and cell-mediated immune responses against the SARS-CoV-2 variant, but also causes different levels of adverse effects such as myocarditis in the young population (Oster M.E et al, 2022). In addition, mRNA vaccines require ultra-low temperature cold chain transport, greatly limiting the possibility of transport to developing countries. Currently, vaccines are being developed that also include non-replicating viral vector vaccines, which are capable of simultaneously inducing both a humoral immune response and a cell-mediated immune response against SARS-CoV-2. Furthermore, non-replicating viral vector vaccines have been reported in the literature to have a good level of protection (Voysey M et al, 2020). The disadvantage is that it causes some degree of adverse reactions. Such as the clotting event induced by the astrazene AZD1222 vaccine (MacIntyre c.r et al, 2021). In addition, for adenovirus type 5 (Ad5) vector vaccines, background immunity to Ad5 affects the immune response against the SARS-CoV-2 antigen (Zhu F-C et al, 2020). The main advantage of subunit vaccines is their safety, especially those adjuvanted with alum (Hern lndez-Bernal F et al, 2021). Meanwhile, the subunit vaccine has proper storage conditions and is easy to distribute. The main disadvantage is the inability to induce a long lasting humoral immune response and a cell mediated immune response.

In addition to the above disadvantages, the above SARS-CoV-2 vaccine has two common limitations: firstly, the SARS-CoV-2 vaccine is designed based on SARS-CoV-2 primary generation spreading branch system, and induced anti-SARS-CoV-2 immune response, especially anti-low mutation branch immune response; secondly, the SARS-CoV-2 vaccine can not induce mucosal immune response and can not block the spread of virus.

Single-stranded RNA viruses mutate rapidly. Preliminary estimates suggest that the SARS-CoV-2 lineage accumulates nucleotide mutations at a rate of about one to two times per month. The recent emergence of the variant strains of Ormcken, which are highly contagious, presents new challenges for vaccine development. Reinfection is possible for populations already infected with SARS-CoV-2. Furthermore, following the severe acute respiratory syndrome coronavirus (SARS-CoV-1) in 2002 and the respiratory syndrome in the middle east in 2012 caused by the respiratory syndrome coronavirus (MERS-CoV), the COVID-19 pandemic caused by SARS-CoV-2 is a third zoonotic disease associated with human fatal coronavirus. Furthermore, coronavirus samples collected from east and south east Asia regions revealed that about 50 SARS-associated coronaviruses were detected in 10 bats. It has been found that bat-transmitted SARS-associated coronavirus is a great threat to pandemics, as several co-regional species of the horseshoe bat host have significant features of viral genetic diversity (Ravelomanantsoa NAF et al, 2021).

Based on the above factors, the emerging SARS-CoV-2 variant (and the possible future emergence of "pre-epidemic" zoonotic virus strains) has led the scientific community to consider: a new generation of vaccines with broad-spectrum protection capability, namely coronavirus broad-spectrum vaccines, is developed.

Coronavirus broad-spectrum vaccines are currently being developed by two approaches: screening for a variety of immunogenic antigens/regions based on spike (S) proteins (multivalent); proteins based on highly conserved regions of coronaviruses were identified and designed. Multivalent S protein-based vaccines require multiple immunodominant regions and are limited in scope to variants of the immunodominant regions. Conservative antigen-based approaches can achieve a broad spectrum of effects depending on the level of conservation of the antigen and its appropriate presentation. Studies based on the S2 subunit/fragment have been reported. For example, after mice are inoculated with DNA vaccine containing SARS-CoV-2S2 protein region, antibodies against S2 protein can be induced. The antibody against the S2 protein can neutralize various human and animal beta coronaviruses in vitro and resist the attack of SARS-CoV-2 in vivo (Ng K.W et al, 2021). Conserved antigen N protein was detected using different vaccine platforms and combinations (Dangi T et al, 2021). The N protein is capable of inducing both humoral and cell-mediated immune responses against SARS-CoV-2, and the Ad5 vector vaccine platform demonstrated the protective ability of the N protein against SARS-CoV-2 (Matchett W.E et al, 2021). However, although the above-mentioned vaccine candidates based on conserved regions all achieved benign results, these vaccine candidates have a major drawback, namely lack of data on the strength of the induced cell-mediated immune response, in particular against coronaviruses of the Sarbe coronavirus subgenus. Furthermore, the above-mentioned vaccine candidates have a common limitation in that functional mucosal immune cross-responses cannot be induced, since most vaccine candidates are not administered by the intranasal route. Therefore, the preventive ability of the candidate vaccine is limited against the spread of future coronaviruses.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to propose a vaccine composition for administration by the mucosal route.

In a first aspect of the invention, there is provided a vaccine composition against a β genus coronavirus, for vaccination via the mucosal route, comprising: 1) novel coronavirus nucleocapsid proteins; 2) has a nucleotide sequence of SEQ ID NO: 1; and 3) a pharmaceutical excipient.

Further, the amino acid sequence of the novel coronavirus nucleocapsid protein is SEQ ID NO: 2, or a sequence identical to SEQ ID NO: 2 sequences having at least 90% identity.

Further, the mucosal route is the intranasal vaccination route.

Further, the vaccine composition further comprises a protein that induces neutralizing antibodies against the beta coronavirus.

Further, the protein inducing neutralizing antibodies against the genus betacoronavirus is a receptor binding domain protein of a novel coronavirus spike protein.

Further, the receptor binding domain protein is a receptor binding domain protein of a novel coronavirus Delta variant.

Further, the amino acid sequence of the receptor binding domain protein is SEQ ID NO: 3 sequence (b).

In a second aspect of the invention, there is provided the use of a vaccine composition for the manufacture of a medicament against a novel coronavirus, said vaccine composition inducing both a humoral immune response and a cell-mediated immune response against a beta coronavirus.

Further, the amino acid sequence of the novel coronavirus nucleocapsid protein is SEQ ID NO: 2 or a sequence substantially identical to SEQ ID NO: 2 sequences having at least 90% identity.

Further, the vaccine composition is a formulation for vaccination via the mucosal route.

Further, the mucosal route is the intranasal vaccination route.

Further, the protein inducing neutralizing antibodies against the β -coronavirus is a receptor binding domain protein of a novel coronavirus spike protein.

Further, the vaccine composition is a formulation for simultaneous mucosal and parenteral vaccination.

Further, the vaccine composition is used as a booster vaccine for boosting immunization of individuals who have been vaccinated against the novel coronavirus vaccine.

In a third aspect of the invention, there is provided a method of vaccinating said vaccine composition to induce a humoral immune response and a cell-mediated immune response against said beta coronavirus, said vaccine composition comprising said novel coronavirus nucleocapsid protein and said nucleotide sequence is SEQ ID NO: 1 sequence of seq id no.

Further, the amino acid sequence of the novel coronavirus nucleocapsid protein is SEQ ID NO: 2 or a sequence identical to SEQ ID NO: 2 sequence having at least 90% identity.

Further, the vaccine composition is administered by mucosal route.

Further, the mucosal route is the intranasal vaccination route.

Further, the protein that induces neutralizing antibodies against the betagenus coronavirus is a receptor binding domain protein of a novel coronavirus spike protein.

Further, the vaccine composition is administered by both mucosal and parenteral routes.

Compared with the existing vaccine on the market and the vaccine under research, the N protein + ODN-39M vaccine composition provided by the invention combines a single vaccine component, and has the following advantages:

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

2. Induces cross-antibodies against the N protein, and modulates the Th1 immune response pattern. Based on a non-neutralizing mechanism, anti-N protein antibodies potentially protect against coronavirus infection. Thus, the N protein + ODN-39M vaccine composition induces cross-antibodies against the N protein, and has a protective effect against potential coronavirus infection.

The N protein + ODN-39M vaccine composition has an auxiliary effect on the RBD recombinant protein. The N protein + ODN-39M vaccine composition is added to a vaccine formulation containing an induced neutralizing antibody protein and administered by intranasal route to increase the level of neutralizing antibodies locally and systemically, thereby enhancing the cross-reactivity of the anti-N protein antibodies. The novel preparation has wider protection range in two immune systems, such as a mucous membrane system, a systemic system and the like.

4. Intranasal administration of formulations containing the N protein + ODN-39M vaccine composition is beneficial for inducing mucosal immune responses. Based on the important effect of mucosal immune response on blocking virus transmission, the mucosal immune response induced by the bivalent antigen preparation provided by the invention has obvious advantages compared with the existing vaccine. In addition, the intranasal route of administration is more feasible and can avoid the problems associated with administration by injection. Thus, it is particularly suitable for both developing countries and mass vaccination.

5. Potential security. The N protein + ODN-39M vaccine composition preparation has potential safety. First, N protein + ODN-39M vaccine composition formulations were developed based on a subunit vaccine platform (recombinant N protein production by recombinant DNA technology). Second, oligodeoxynucleotide adjuvants are safer and some have been approved for use in humans. Animal studies, including non-human primates, have demonstrated the safety of ODN-39M. In addition, the oligodeoxynucleotide has the key characteristics of full-phosphodiester oligodeoxynucleotides, and related safety problems caused by chemical modification of nucleotide skeletons (thioates) are avoided, wherein the safety problems are related to side effects of the oligodeoxynucleotides in different treatment schemes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary and that other implementation drawings may be derived from the provided drawings by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used for limiting the conditions of the present invention, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the functions and purposes of the present invention, shall fall within the scope of the present invention.

FIG. 1 a: the cloned N protein expression was analyzed by 12% SDS-PAGE electrophoresis under reducing conditions, wherein 1) molecular weight standard reference (MWM), 2) BL21(DE3) whole cells (negative control), 3) BL21(DE3) whole cells expressing N protein, 4) purified N protein.

FIG. 1 b: western blot analysis was performed under reducing conditions using an anti-N protein polyclonal antibody (40588-T62, available from kyoto technologies, inc., beijing, yi, china) in which 1) MWM, 2) BL21(DE3) whole cells (negative control), 3) BL21(DE3) whole cells expressing N protein, and 4) purified N protein.

FIG. 2: the recognition of recombinant N protein by anti-SARS-CoV-2 human serum inoculated with an inactivated vaccine healthy population was examined by ELISA assay. Human serum: HD1-HD 6.

FIG. 3: schematic of aggregation (precipitation) curves of the N protein + ODN-39M vaccine composition at different mass ratios; two independent experiments were performed.

FIG. 4 a: the aggregation process of different samples with N protein + ODN-39M in a mass ratio of 0.66:1 was analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide, where 1: n protein + ODN-39M Process 1; 2: n protein + ODN-39M Process 2; 3: an N protein; 4: a control protein; 5: ODN-39M; 6: n protein + ODN-39M Process 3; 7: control protein + ODN-39M; 8: MWM.

FIG. 4 b: the aggregation process of the different samples with N protein + ODN-39M in a mass ratio of 0.66:1 was analyzed by 2% agarose gel electrophoresis stained with coomassie blue, wherein 1: n protein + ODN-39M Process 1; 2: n protein + ODN-39M Process 2; 3: an N protein; 4: a control protein; 5: ODN-39M; 6: n protein + ODN-39M Process 3; 7: control protein + ODN-39M; 8: MWM.

FIG. 5 a: the aggregation state of the sample and the N protein + ODN-39M in a mass ratio of 0.66:1 was analyzed by 2% agarose gel under non-reducing conditions, wherein 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: an N protein; 7: ODN-39M; 8: MWM (DNA). FA refers to formaldehyde and all FA reactions are quenched.

FIG. 5 b: the aggregation state of the sample and the N protein + ODN-39M at a mass ratio of 0.66:1 was analyzed by 10% SDS-PAGE under non-reducing conditions, wherein 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: an N protein; 9: MWM (protein); 10: n protein + 1% FA. FA refers to formaldehyde and all FA reactions are quenched.

FIG. 6: schematic representation of the assessment of immunogenicity of the N protein preparation in the serum of immunized Balb/C mice by anti-N protein IgG ELISA assay; g1: protein N + alum, injected subcutaneously; 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, injected subcutaneously; g6: PBS + alum, subcutaneous injection; g7: PBS, intranasal inoculation; after conversion of the log of titer, statistical analysis was performed using One-Way Anova (One Way Anova) and Tukeys multiple comparison.

FIG. 7 a: schematic diagram for assessment of the humoral mucosal immune response in immunized Balb/C mouse BALF samples (undiluted) by anti-N protein IgA ELISA assay, where G1: protein N + alum, injected subcutaneously; 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, injected subcutaneously; g6: PBS + alum, subcutaneous injection; g7: PBS, intranasal inoculation; after conversion of the log of titer, statistical analysis was performed using One-Way Anova (One Way Anova) and Tukeys multiple comparison.

FIG. 7 b: schematic diagram for assessment of the humoral mucosal immune response in immunized Balb/C mouse BALF samples (undiluted) by anti-N protein IgG ELISA assay, where G1: protein N + alum, injected subcutaneously; g2: n protein + ODN-39M + alum, and is injected subcutaneously; g3: n Protein (PBS), intranasal inoculation; g4: n protein + ODN-39M, intranasal inoculation; g5: protein N + ODN-39M, injected subcutaneously; g6: PBS + alum, subcutaneous injection; g7: PBS, intranasal inoculation; after conversion of the log of titer, statistical analysis was performed using One-Way Anova (One Way Anova) and Tukeys multiple comparison.

FIG. 8 a: schematic representation of the evaluation of IgG subclass antibodies in sera of immunized Balb/C mice by an anti-N protein IgG1 ELISA assay, wherein G1: protein N + alum, injected subcutaneously; 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, injected subcutaneously; g6: PBS + alum, subcutaneous injection; g7: PBS, intranasal inoculation; after the conversion of the logarithm of the titer, statistical analysis was performed by One-Way Anova (One Way Anova) and Tukeys multiple comparison.

FIG. 8 b: schematic representation of the evaluation of IgG subclass antibodies in sera of immunized Balb/C mice by an anti-N protein IgG2a ELISA assay, wherein G1: protein N + alum, injected subcutaneously; 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, injected subcutaneously; g6: PBS + alum, subcutaneous injection; g7: PBS, intranasal inoculation; after the conversion of the logarithm of the titer, statistical analysis was performed by One-Way Anova (One Way Anova) and Tukeys multiple comparison.

FIG. 9: schematic representation of IgG1/IgG2a ratio (titer) in sera of immunized Balb/C mice analyzed by anti-N protein ELISA assay; g1: protein N + alum, injected subcutaneously; 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, injected subcutaneously; g6: PBS + alum, subcutaneous injection; g7: PBS, intranasal inoculation.

FIG. 10: analysis of conserved peptide N by IFN γ -ELISPOT assay _351-365 Schematic representation of cell-mediated immune response induced after stimulation of mouse splenocytes; g1: protein N + alum, injected subcutaneously; g2: n protein + ODN-39M + alum, subcutaneous injection; g3: n Protein (PBS), intranasal inoculation; g4: n protein + ODN-39M, intranasal inoculation; g5: protein N + ODN-39M, injected subcutaneously; g6: PBS + alum, subcutaneous injection; g7: PBS, intranasal inoculation; statistical analysis was performed using Kruskal-Wallis nonparametric test and Dunns multiple comparative test.

FIG. 11: analyzing a cell-mediated immune response schematic diagram induced after the Delta variant N protein stimulates splenocytes of the mouse through an IFN gamma-ELISPOT test; g1: protein N + alum, injected subcutaneously; g2: n protein + ODN-39M + alum, subcutaneous injection; g3: n Protein (PBS), intranasal inoculation; g4: n protein + ODN-39M, intranasal inoculation; g5: protein N + ODN-39M, injected subcutaneously; g6: PBS + alum, subcutaneous injection; g7: PBS, intranasal inoculation; statistical analysis was performed using Kruskal-Wallis nonparametric test and Dunns multiple comparative test.

FIG. 12 a: immunogenicity profiles of the N protein and RBD protein preparations in sera of immunized Balb/C mice were evaluated by anti-N protein IgG ELISA assay, where 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 vaccination; g6: n protein + ODN-39M + RBD protein + alum, and subcutaneous injection; g7: n protein + RBD protein + alum, subcutaneous injection; g9: PBS, intranasal inoculation; g10: PBS + alum, subcutaneous injection; after the conversion of the logarithm of the titer, statistical analysis was performed by One-Way Anova (One Way Anova) and Tukeys multiple comparison.

FIG. 12 b: a schematic representation of the immunogenicity of the N and RBD protein preparations in the sera of immunized Balb/C mice evaluated by an anti-RBD protein IgG ELISA assay, wherein G3: n protein + ODN-39M + RBD protein, intranasal inoculation; g4: n protein + RBD Protein (PBS), intranasal inoculation; g5: RBD Protein (PBS), intranasal vaccination; g6: n protein + ODN-39M + RBD protein + alum, and subcutaneous injection; g7: n protein + RBD protein + alum, subcutaneous injection; g8: RBD protein + alum, injected subcutaneously; g9: PBS, intranasal inoculation; g10: PBS + alum, subcutaneous injection; after the conversion of the logarithm of the titer, statistical analysis was performed by One-Way Anova (One Way Anova) and Tukeys multiple comparison.

FIG. 13 a: schematic representation of assessment of the humoral mucosal immune response in immunized Balb/C mouse BALF samples (undiluted) by anti-N protein IgA ELISA assay, wherein G1: n Protein (PBS), intranasal inoculation; g2: n protein + ODN-39M, intranasal inoculation; g3: n protein + ODN-39M + RBD protein, inoculating intranasally; g4: n protein + RBD Protein (PBS), intranasal inoculation; g5: RBD Protein (PBS), intranasal vaccination; g6: n protein + ODN-39M + RBD protein + alum, and is injected subcutaneously; g7: n protein + RBD protein + alum, subcutaneous injection; g8: RBD protein + alum, injected subcutaneously; g9: PBS, intranasal inoculation; g10: PBS + alum, subcutaneous injection; statistical analysis was performed using Kruskal-Wallis nonparametric test and Dunns multiple comparative test.

FIG. 13 b: schematic diagram for assessment of the humoral mucosal immune response in immunized Balb/C mouse BALF samples (undiluted) by anti-RBD protein IgA ELISA assay, wherein G1: n Protein (PBS), intranasal inoculation; g2: n protein + ODN-39M, intranasal inoculation; g3: n protein + ODN-39M + RBD protein, inoculating intranasally; g4: n protein + RBD Protein (PBS), intranasal inoculation; g5: RBD Protein (PBS), intranasal inoculation; g6: n protein + ODN-39M + RBD protein + alum, and subcutaneous injection; g7: n protein + RBD protein + alum, subcutaneous injection; g8: RBD protein + alum, injected subcutaneously; g9: PBS, intranasal inoculation; g10: PBS + alum, subcutaneous injection; statistical analysis was performed using Kruskal-Wallis nonparametric test and Dunns multiple comparative test.

FIG. 14 a: schematic representation of the evaluation of IgG subclass antibodies in sera of immunized Balb/C mice by anti-N protein IgG1 ELISA assay, wherein G2: n protein + ODN-39M, intranasal inoculation; g3: n protein + ODN-39M + RBD protein, intranasal inoculation; g6: n protein + ODN-39M + RBD protein + alum, and subcutaneous injection; g7: n protein + RBD protein + alum, injected subcutaneously.

FIG. 14 b: schematic representation of the evaluation of IgG subclass antibodies in sera of immunized Balb/C mice by an anti-N protein IgG2a ELISA assay, wherein G2: n protein + ODN-39M, intranasal inoculation; g3: n protein + ODN-39M + RBD protein, intranasal inoculation; g6: n protein + ODN-39M + RBD protein + alum, and subcutaneous injection; g7: n protein + RBD protein + alum, injected subcutaneously.

FIG. 15 a: schematic representation of the evaluation of IgG subclass antibodies in sera of immunized Balb/C mice by anti-RBD protein IgG1 ELISA assay, wherein G3: n protein + ODN-39M + RBD protein, inoculating intranasally; g4: n protein + RBD protein, intranasal vaccination; g5: RBD protein, intranasal vaccination; g6: n protein + ODN-39M + RBD protein + alum, and subcutaneous injection; g7: n protein + RBD protein + alum, subcutaneous injection; g8: RBD protein + alum, injected subcutaneously.

FIG. 15 b: schematic representation of the evaluation of IgG subclass antibodies in sera of immunized Balb/C mice by anti-RBD protein IgG2a ELISA assay, wherein G3: n protein + ODN-39M + RBD protein, inoculating intranasally; g4: n protein + RBD protein, intranasal vaccination; g5: RBD protein, intranasal vaccination; g6: n protein + ODN-39M + RBD protein + alum, and subcutaneous injection; g7: n protein + RBD protein + alum, subcutaneous injection; g8: RBD protein + alum, injected subcutaneously.

FIG. 16: analysis of conserved peptide N by IFN γ -ELISPOT assay _351-365 Schematic representation of cell-mediated immune response induced after stimulation of mouse splenocytes; g1: n Protein (PBS), intranasal inoculation; g2: n protein + ODN-39M, intranasal inoculation; g3: n protein + ODN-39M + RBD protein, inoculating intranasally; g4: n protein + RBD protein, intranasal vaccination; g6: n protein + ODN-39M + RBD protein + alum, and subcutaneous injection; statistical analysis was performed using One-Way Anova (One Way Anova) and Tukeys multiple comparison.

FIG. 17: analyzing a cell-mediated immune response schematic diagram induced after the RBD protein of the Delta variant stimulates splenocytes of the mice through an IFN gamma-ELISPOT test; g3: n protein + ODN-39M + RBD protein, inoculating intranasally; g4: n protein + RBD protein, intranasal vaccination; g5: RBD protein, intranasal vaccination; g9: PBS (negative control), intranasal inoculation.

FIG. 18 a: a schematic presentation of the immunogenicity of N protein + ODN-39M preparations in the sera of G2 immunized Balb/C mice (N protein + ODN-39M, intranasal vaccination) was evaluated by an anti-N protein IgG antibody ELISA assay coated with SARS-CoV-1N protein and SARS-CoV-2N protein.

FIG. 18 b: a schematic presentation of the immunogenicity of the N protein + ODN-39M preparation in the serum of Balb/C mice immunized with G3(N protein + ODN-39M + RBD protein, intranasal inoculation) was evaluated by an anti-N protein IgG antibody ELISA assay coated with SARS-CoV-1N protein and SARS-CoV-2N protein.

FIG. 19 a: a schematic representation of the humoral mucosal immune response induced in Balb/C mouse BALF samples (undiluted) immunized with G2(N protein + ODN-39M, intranasal inoculation) was evaluated by an anti-N protein IgA antibody ELISA assay coated with SARS-CoV-1N protein and SARS-CoV-2N protein.

FIG. 19 b: a schematic representation of the humoral mucosal immune response induced in Balb/C mouse BALF samples (undiluted) immunized with G3(N protein + ODN-39M + RBD protein, intranasal inoculation) was evaluated by an anti-N protein IgA antibody ELISA assay coated with SARS-CoV-1N protein and SARS-CoV-2N protein.

FIG. 20 a: and (2) evaluating the humoral mucosal immune response of the anti-RBD protein in the serum by an IgG antibody test of the anti-RBD protein of the primary virus strain and the Delta variant, wherein G3: n protein + ODN-39M + RBD protein, inoculating intranasally; g6: n protein + ODN-39M + RBD protein + alum, and is injected subcutaneously; g8 RBD protein + Alum, injected subcutaneously.

FIG. 20 b: the humoral mucosal immune response against RBD proteins in BALF was assessed by anti-Delta variant RBD protein IgA antibody ELISA assay, where G3: n protein + ODN-39M + RBD protein, inoculating intranasally; g9: PBS, intranasal inoculation.

FIG. 20 c: the humoral mucosal immune response against RBD proteins in BALF was assessed by the RBD protein IgA antibody ELISA assay against the primary strain, where G3: n protein + ODN-39M + RBD protein, intranasal inoculation; g9: PBS, intranasal inoculation.

FIG. 21 a: analysis of SARS-CoV-2Delta variant N protein and SARS-CoV-1N protein and conserved peptide N by IFN γ -ELISPOT assay _351-365 Schematic of cell-mediated immune responses induced after stimulation of mouse splenocytes, G2: n protein + ODN-39M, intranasal inoculation; g9: PBS (negative control).

FIG. 21 b: analysis of SARS-CoV-2Delta variant N protein and SARS-CoV-1N protein and conserved peptide N by IFN γ -ELISPOT assay _351-365 Schematic of cell-mediated immune responses induced after stimulation of mouse splenocytes, G3: n protein + ODN-39M + RBD protein, inoculating intranasally; g9: PBS (negative control), intranasal inoculation.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and benefits of the present invention will become apparent to those skilled in the art from the description herein, and it is understood that the described embodiments are intended to be illustrative of some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The vaccine composition comprises: 1) SARS-CoV-2 nucleocapsid (N) protein, amino acid sequence is SEQ ID NO: 2, a sequence; 2) a nucleic acid having the nucleotide sequence of SEQ ID NO: 1 sequence (c). The vaccine composition can solve the following two problems: 1. induces a broad range of functional immune responses against non-spike (S) proteins; 2. inducing a mucosal immune response.

In order to solve the two problems, the invention provides a technical scheme that: uses SARS-CoV-2N recombinant protein to induce cross immunity, mucosa immunity and systemic immune response for resisting Sarbe coronavirus of subfamily coronavirus.

The SARS-CoV-2N protein is obtained by incubating escherichia coli, and the obtained protein is purified to ensure that the purity reaches more than 90%. In addition, SARS-CoV-2N protein could be detected in the serum of volunteers vaccinated with Chinese inactivated vaccine, indicating the presence of conformational epitopes. In one embodiment of the invention, the SARS-CoV-2N protein has the amino acid sequence of SEQ ID NO: 2 or a sequence substantially identical to SEQ ID NO: 2, an amino acid sequence having at least 90% identity to the sequence.

To develop a suitable vaccine formulation, the SARS-CoV-2N protein is compared to a protein having the sequence of SEQ ID NO: 1 sequence (1) is combined with a phosphodiester backbone CpG oligodeoxynucleotide (ODN-39M). The SARS-CoV-2Delta variant N protein was incubated with different amounts of ODN-39M, and when the ratio of ODN-39M to N protein was 3:1, aggregation (precipitation) reached a maximum, resulting in a typical saturation curve. The ratio corresponds to the molar ratio of the two molecules, which is the first proof of the interaction between the two. Since ODN-39M was mixed with N protein, the N protein was soluble. Therefore, screening of ODN supersaturation conditions for in-depth studies is a key factor in vaccine formulation research. After characterization of ODN-39M and N protein using agarose gel at room temperature, the presence of a common region between the N protein and ODN-39M was detected, which also demonstrates the interaction between the two. This interaction was shown by cross-linker experiments to form soluble high molecular weight N protein aggregates. Because of its difference from other viral N proteins that naturally wrap viral RNA into particles, the SARS-CoV-2N protein is attached to long-chain RNA via a histone-like conformation, and its structure is completely different from the capsid wrapping structure. Therefore, the formation of soluble high molecular weight N protein aggregates is almost unexpected. In addition, the ODN-39M used had 39 nucleotides, which were significantly shorter than the long-chain structure of the viral RNA. The aggregate structure may contribute to the immunogenicity of the protein. Thus, N protein soluble aggregate formation in the presence of ODN-39M is an important feature of N protein + ODN-39M vaccine compositions.

Mice were vaccinated by the intranasal route with the N protein + ODN-39M vaccine composition and induced high levels of anti-N protein IgG antibodies in both serum and bronchoalveolar lavage (BALF). At the same time, IgA antibodies could also be induced, an unexpected result. In prior art studies, it was reported that mice were not able to induce humoral immune responses by intranasal inoculation after binding of dengue virus capsid protein to ODN-39M (Lazo l.et al, 2017). In another aspect, the vaccine compositions of the present invention induce a typical Th1 immune response pattern. After stimulation of spleen cells with N protein, cell mediated immune responses were measured by ELISPOT IFN γ assay, showing 100% response levels. Whereas the group vaccinated with the N protein alone by the intranasal route of administration did not detect both humoral and cell-mediated immune responses. This clearly demonstrates the relevance of the intranasal N protein + ODN-39M vaccine composition to the immunogenicity of the Delta variant N protein.

In another embodiment of the present invention, cross-responses between humoral and cell-mediated immunity are induced after mice are vaccinated with the N protein + ODN-39M vaccine composition via the intranasal route of administration. Using conserved peptide N _351-365 After stimulating spleen cells with SARS-CoV-1N protein, significant cell-mediated immune response can be induced, and high levels of anti-SARS-CoV-1N protein antibodies are induced in BALF and serum. SARS-CoV-1 is a representative coronavirus from the sub-genus Sarbe coronavirus. The conserved peptide N _351-365 Is a conserved peptide shared by the Sarbe coronavirus subgenus coronavirus, and also has protective effect in SARS-CoV-2 infected animal model. Thus, against the conserved peptide N _351-365 Has significant relevance to the immune response of (c). The N protein + ODN-39M vaccine composition for intranasal administration can induce the resistance to the conserved peptide N _351-365 The cell-mediated immune cross-response. Administration of the N protein + ODN-39M vaccine composition by the subcutaneous route, with or without alum as an adjuvant, failed to induce N-directed resistance to the conserved peptide _351-365 Even if the immune response to the stimulation of the Delta variant N protein in the animal model reaches 100 percent. Intranasal administration of the N protein + ODN-39M vaccine composition selectively induces cell-mediated immune cross-responses, and humoral immune cross-responses in the mucosa and serum. Therefore, the N protein + ODN-39M vaccine composition becomes a prospect component of the future coronavirus broad-spectrum vaccine.

In one embodiment of the invention, the N protein + ODN-39M vaccine composition is conjugated to a second generation antigen of SARS-CoV-2, and neutralizing antibodies are induced by intranasal vaccination. Neutralizing antibodies are an important group of antibodies with protective effects. In a preferred embodiment, the second generation antigen of the neutralizing antibody is the Receptor Binding Domain (RBD) protein of the SARS-CoV-2Delta variant having the amino acid sequence of SEQ ID NO: 3 sequence (b). Under the Th1 immune response mode, the N protein + ODN-39M vaccine composition has a remarkable auxiliary effect on inducing mucosal immune response and systemic immune response of the RBD protein of the Delta variant. anti-RBD antibodies induced in BALF and serum recognize primary viral RBD proteins and Delta variant RBD proteins. The antibody in the serum has ACE-2 binding inhibition effect on the two RBD proteins, and can also induce anti-N protein cross immune response. Administration of a single RBD by the intranasal route is completely non-immunogenic.

Based on the above results, the N protein + ODN-39M vaccine composition can become a promising component of future coronavirus broad-spectrum vaccines, can induce cross immune response against N protein, and induce humoral immune response and cell-mediated immune response through a mucosal system and a systemic system. Cross-immune responses against N protein, anti-RBD IgA antibodies in the mucosa, and neutralizing antibodies against both RBD proteins in serum can be induced by intranasal administration of a bivalent antigen formulation of N protein + ODN-39M + RBD. The above results provide theoretical support for the bivalent antigen formulation as a booster needle by intranasal administration, especially after vaccination with inactivated vaccine. Because the inactivated vaccine contains both N and S proteins, it is able to successfully mount the immune system against both N and S proteins.

Example 1

Cloning, expression, purification and antigen characterization of novel coronavirus (SARS-CoV-2) Delta variant nucleoprotein (N protein)

The DNA sequence (YP _009724397.2, corresponding to the amino acid sequence SEQ ID NO: 2) of the SARS-CoV-2Delta variant N protein was cloned into a PET-28 vector, and then expressed in E.coli to obtain a protein. The protein accounted for 11% of total protein in E.coli (FIG. 1 a). The proteins were first precipitated by ammonium sulphate fractional precipitation and then semi-purified by hydrophobic interaction chromatography. Subsequently, the semi-purified protein is further purified by anion exchange chromatography to obtain a purified protein. And after eluting the purified protein, replacing a protein buffer solution with a Phosphate Buffer Solution (PBS) by Sephadex G-25 gel filtration chromatography so that the purity of the purified protein reaches more than 95%. The purified protein was immunoblotted (WB) and polyclonal anti-N protein antibody preparation (beijing yiqiao shenzhou science ltd) (fig. 1 b).

Through the administration of the recombinant protein, the condition that the serum of a volunteer inoculated with an inactivated vaccine (a vaccine produced by Beijing Kexing and national drug group in China) identifies the recombinant protein is detected, so that the antigen characterization is completed. The results showed that sera from vaccinated volunteers reacted positively to N protein, indicating protein folding and exposure of the relevant epitope (figure 2).

Example 2

Characterization of N protein + ODN-39M vaccine compositions

The N proteins were aggregated at different N protein/oligodeoxynucleotide 39M (ODN-39M) mass alignments. ODN-39M is a 39-mer holodiester phosphate conjugate having the sequence of SEQ ID NO: 1 sequence, synthesized by the company of bioengineering (Shanghai) of China.

To obtain an aggregation curve, 40. mu. g N protein was mixed with different amounts of ODN-39M at concentrations ranging from 0.04. mu.g to 80. mu.g ODN-39M/10mM Tris (pH 8) to give 100. mu.L of reaction mixture, which was incubated at 30 ℃ for 30 minutes and then stored in a refrigerator at 4 ℃ for 4 hours and centrifuged at 10000 Xg. The supernatant was extracted and the protein concentration was determined. As shown in FIG. 3, when the mass ratio of N protein to ODN-39M was 5:1 to 2.5:1, the maximum aggregation (precipitation) of the reaction mixture was obtained, and the corresponding molar ratio of N protein to ODN-39M was 1:3, indicating that the molar ratio is a potential saturation point (theoretically, 3 ODN-39M molecules are required for neutralizing 1N protein molecule). The resulting curves clearly demonstrate the interaction between the two molecules.

Since solubility is one of the key characteristics of vaccine formulations, conditions were screened for N protein to ODN-39M at a mass ratio of 0.66:1 (conditions where ODN-39M is supersaturated) for further characterization. To verify the interaction of the N protein with ODN-39M at a mass ratio of 0.66:1, the reaction mixture was loaded on a 2% agarose gel and co-localization of the two molecules was observed. The 2% agarose gel was stained with ethidium bromide to detect ODN-39M, and stained with Coomassie blue to detect N protein. As shown in FIGS. 4a and 4b, in the samples of the aggregation process, a small amount of ODN-39M delayed the migration, and the N protein migrated more than the N protein without ODN-39M. This indicates that there is a common region (in the box) where both the N protein and ODN-39M are present. The alteration of the mode of migration of the N protein and ODN-39M and the detected common regions directly demonstrate the interaction between these two molecules.

Finally, to evaluate the true effect of ODN-39M on the conformation of the N protein, the crosslinker formaldehyde-fixed N protein + ODN-39M vaccine composition and the N protein were subjected to SDS-PAGE electrophoresis and quenching under non-reducing conditions. The resulting images clearly demonstrate that mixing of the N protein with ODN-39M can produce high molecular weight N protein aggregates because the crosslinker formaldehyde-fixed N protein + ODN-39M vaccine composition is completely retained in the gel pores. The cross-linked N protein + ODN-39M vaccine composition was analyzed on a 2% agarose gel and showed the same retention rate of ODN-39M (FIGS. 5a and 5 b).

Example 3

A first immunization protocol for mice was designed and immunological evaluation was performed on different formulations based on N protein

7 groups of Balb/C mice (female, 6-8 weeks old) were immunized on

days

0, 7, and 21 with the following schedule, and the dose of the immunization was 10. mu.g.

Group 1: n protein + alum, subcutaneous injection (100 μ L)

Group 2: n protein + ODN-39M + Alum, subcutaneous injection (100. mu.L)

Group 3: n protein, intranasal inoculation (50. mu.L)

Group 4: n protein + ODN-39M, intranasal inoculation (50. mu.L)

Group 5: n protein + ODN-39M, subcutaneous injection (100. mu.L)

Group 6: PBS + Alum (negative control), subcutaneous injection (100. mu.L)

Group 7: PBS (negative control), intranasal inoculation (50. mu.L)

Mice were sacrificed on days 12 and 19 after completion of the last dose of immunization and the induced humoral and cell-mediated immune responses were evaluated.

Example 4

Assessment of humoral immune response in immunized mice

On day 12 after completion of the last immunization dose, serum was collected from the blood sample from the mice and analyzed by an anti-Delta variant N protein (SAR-CoV-2Delta variant N protein (40588-V07E29) purchased from Hibiscus technologies, Beijing, China) IgG ELISA assay. As shown in fig. 6, all groups injected subcutaneously with N protein induced high levels of anti-N protein antibodies. In contrast, only the group intranasally vaccinated with the N protein + ODN-39M vaccine composition induced high levels of IgG antibodies (p < 0.05).

IgA antibodies against the Delta variant N protein were detected in bronchoalveolar lavage fluid (BALF). The results are shown in FIGS. 7a and 7 b. As expected, group 4 induced a higher level of anti-N protein IgA antibody response. The antigen specific IgG antibody response detected in BALF is the same as the result detected in serum, since IgG antibodies can penetrate through systemic tissues to mucosal tissues.

To examine the Th1 immune response pattern induced by each preparation, IgG1 and IgG2a subclasses of anti-N protein in serum were assayed using Sigma antibody. As shown in FIGS. 8a and 8b, group 4 (N protein + ODN-39M, intranasal inoculation) induced significantly lower levels of IgG1 compared to

groups

1, 2 and 5 (all inoculated subcutaneously). In contrast,

groups

4 and 5 induced significantly higher levels of IgG2 a. It was therefore concluded that the ratio of IgG1/IgG2a was close to 1 for each group, indicating that a typical Th1 immune response pattern was induced in mice vaccinated with only the N protein + ODN-39M preparation (without alum) (FIG. 9). An important feature of inducing a Th1 immune response pattern is that IgG2a subclass antibody levels reach IgG1 subclass antibody levels or even higher. The Th1 immune response pattern implies the induction of a cell-mediated immune response.

Example 5

Assessing cell-mediated immune response in immunized mice

To evaluate cell-mediated immune responses, the immune response was evaluated byCantor peptide N _351-365 And Delta variant N protein, the frequency of IFN γ production by splenocytes was measured using a Mabtech antibody and well plates after completion of the in vitro stimulation. The conserved peptide N _351-365 (ILLNKHIDAYKTFPP), synthesized by Zhejiang surge peptide biology of China Limited, with purity up to more than 97%; the Delta variant N protein is a Delta variant N protein (40588-V07E29) of SARS-CoV-2, and is purchased from Beijing Yiqian Shenzhou science and technology corporation in China. On day 18 after completion of the last dose of immunization, the conserved peptide N was used _351-365 And the Delta variant N protein stimulates splenocytes. As shown in FIG. 10, in the conserved peptide N _351-365 Upon stimulation, an IFN γ secreting cell response was detected in all mice of group 4 (N protein + ODN-39M, intranasal inoculation), while no IFN γ secreting cell response was detected in mice of group 3(N protein, intranasal inoculation). In addition, in

groups

1 and 2 injected subcutaneously, and group 5 (N protein + ODN-39M, injected subcutaneously) in which the inoculated formulation was the same as in group 4, only 1 or 2 mice in each group were tested for response (FIG. 10). The results show that the intranasal route of inoculation is directed to induce resistance to the conserved peptide N _351-365 Is crucial for cell-mediated immune responses.

The results obtained with the use of the N protein as a stimulating antigen induced cell mediated immune response were different.

Groups

1, 2, 4 and 5 achieved 100% response, with no statistical difference between groups, while group 4 achieved the highest level of response (figure 11).

Example 6

A second immunization protocol for mice was designed and immunological evaluation of intranasal bivalent antigen preparations was performed

The purpose of the experiment was to examine the immune response profile of the bivalent antigen preparation. The bivalent antigen preparation comprises the Delta variant N protein for inducing cell-mediated immune cross response and the Delta variant RBD protein (40592-V08H90 with the sequence of SEQ ID NO: 3, purchased from Beijing-Yi-Qianzhou science and technology GmbH in China) for inducing neutralizing antibody. At the same time, different control groups were used to explore the effects of the two immune pathways. 10 groups of Balb/C mice (female, 6-8 weeks old) were immunized on

days

0, 7, and 21 with the following schedule, and the inoculation dose was 10. mu.g.

Group 1: n protein, intranasal inoculation (50. mu.L)

Group 2: n protein + ODN-39M, intranasal inoculation (50. mu.L)

Group 3: n protein + ODN-39M + Receptor Binding Domain (RBD) protein, intranasal inoculation (50. mu.L)

Group 4: n protein + RBD protein, intranasal inoculation (50. mu.L)

Group 5: RBD protein, intranasal inoculation (50. mu.L)

Group 6: n protein + ODN-39M + RBD protein + Alum, subcutaneous injection (100. mu.L)

Group 7: n protein + RBD protein + Alum, subcutaneous injection (100 μ L)

Group 8: RBD protein + Alum, subcutaneous injection (100 μ L)

Group 9: PBS (negative control), intranasal inoculation (50. mu.L)

Group 10: PBS + Alum (negative control), subcutaneous injection (100. mu.L)

On days 12 and 26 after the last dose of immunization, mice were bled and the induced humoral and cell-mediated immune responses were evaluated.

Example 7

Assessment of humoral immune response in immunized mice

On day 12 after completion of the last immunization dose, serum was collected from the blood sample from the mice and analyzed by an IgG ELISA test against the Delta variant N protein (SARS-CoV-2Delta variant N protein (40588-V07E29), purchased from Hibiscus technologies, Beijing, Chiense, China). As shown in FIG. 12a,

groups

2, 3, 6 and 7 induced significant anti-Delta variant N protein antibodies compared to the control group (p < 0.05). While the group vaccinated intranasally, the group vaccinated with only the N protein + ODN-39M vaccine composition induced high levels of anti-N protein antibodies. The level of anti-N protein antibodies induced by group 3(N protein + ODN-39M + RBD protein, the bivalent antigen preparation) was lower than in

groups

2, 6 and 7, probably due to the effect of RBD protein in the preparation. Nevertheless, the level of anti-N protein antibodies induced by group 3 was still high.

Meanwhile, the level of IgG antibody against the Delta variant RBD protein (SARS-CoV-2Delta variant RBD protein (40592-V08H90) purchased from Beijing Yi Qianzhou science and technology, Inc., China) was tested. anti-Delta variant RBD protein antibodies were not significantly induced in the sera of mice in groups 5 (RBD protein, intranasal inoculation) and 4 (N protein + RBD protein, intranasal inoculation). In contrast, group 3(N protein + ODN-39M + RBD protein bivalent antigen formulation, intranasal inoculation) induced higher levels of anti-Delta variant RBD protein antibody, similar to group 6 (N protein + ODN-39M + RBD protein + Alum, subcutaneous injection). As shown in fig. 12 b.

And detecting IgA antibodies resisting the Delta variant N protein and the Delta variant RBD protein in the BALF. The results are shown in FIGS. 13a and 13 b. In the group vaccinated intranasally, the IgA antibody levels induced in BALF were consistent with serum. Vaccination with the N protein + ODN-39M containing preparation group induced anti-N protein IgA antibodies. In the intranasal vaccination group, vaccination with only the N protein + ODN-39M + RBD protein bivalent antigen formulation group induced an anti-RBD protein response (as shown in FIG. 13 b).

With respect to the IgG subclass antibodies against the N protein of the Delta variant in serum,

groups

2 and 3 induced significantly lower levels of IgG1 antibody than groups 6 and 7 (inoculated with N-containing protein preparations by subcutaneous injection) (FIG. 14 a).

Groups

2 and 3 induced significantly higher levels of IgG2a antibody compared to groups 6 and 7 (FIG. 14 b). The increased levels of IgG2a antibody, along with decreased levels of IgG1 antibody, in mice vaccinated with the N protein + ODN-39M vaccine composition and the N protein + ODN-39M + RBD protein formulation via the intranasal route alone were the result of modulation of the typical Th1 immune response pattern.

IgG subclass antibodies against the Delta variant RBD protein in serum are shown in FIGS. 15a and 15 b. IgG1 titers were similar in all groups vaccinated with the RBD protein-containing preparations, except groups 4 and 5 (FIG. 15 a). Group 3(N protein + ODN-39M + RBD protein, the bivalent antigen preparation, intranasal inoculation) had higher IgG2a titers against the Delta variant RBD protein than the other groups (p <0.05) (FIG. 15 b). In group 3, the increased levels of IgG2a antibodies against the Delta variant RBD protein indicates that a Th1 immune response pattern may protect against the Delta variant RBD protein.

Example 8

Assessing cell-mediated immune response in immunized mice

To evaluate cell-mediated immune responses, the peptide N was conserved _351-365 And the Delta variant RBD protein, and detecting the frequency of IFN gamma splenocytes generated after the in vitro stimulation is finished. On day 12 after completion of the last dose of immunization, the conserved peptide N was used _351-365 Mice splenocytes from

groups

1, 2, 3, 4 and 9 (negative control) of the intranasal inoculation group were stimulated.

As shown in fig. 16, IFN γ -secreting cell responses were detected in both group 2 and group 3 mice, while IFN γ -secreting cells were not detected in the remaining splenocytes of all groups. The IFN γ -secreting cell response was similar to that detected in the panel inoculated with the N protein + ODN-39M vaccine composition in the mouse experiment of example 3. Compared with the group 2, the statistical difference is not generated, and the positive response detected in the group inoculated with the bivalent antigen preparation indicates that the addition of the RBD protein in the N protein + ODN-39M vaccine composition does not influence the resistance of the conserved peptide N _351-365 Induction of cell-mediated immune responses.

Group 1 did not induce an immune response, confirming the immunogenicity of the N protein + ODN-39M vaccine composition by intranasal vaccination in the mouse experiment of example 3.

Meanwhile, mice from

groups

3, 4, 5 and 9 (negative controls) were stimulated with Delta variant RBD protein, and the groups were inoculated intranasally with a formulation containing RBD protein. As shown in FIG. 17, positive responses were detected only in the group inoculated with the bivalent ODN-39M preparation (2/5 mice).

Example 9

Assessment of Cross-immune response in immunized mice

Humoral immune response against SARS-CoV-1N protein and SARS-CoV-2 primary virus strain RBD protein

To determine the range of immune responses induced by intranasal vaccination with the N protein + ODN-39M vaccine composition, on day 26 after completion of the last dose of vaccination, sera from mice from

groups

2 and 3 of the mouse experiment of example 6 were collected and ELISA tests were performed against the SARS-CoV-2Delta variant N protein, the SARS-CoV-2 Omicron variant N protein (40588-V07E34, available from Hibiscus technologies, Beijing Y., China) and the SARS-CoV-1N protein (40143-V08B, available from Hibiscus technologies, Beijing Y., China). Results as shown in fig. 18a and 18b, IgG antibodies against the three variant N proteins were induced in both groups of the preparations studied.

IgA and IgG ELISA tests against the three variant N proteins were performed on

group

2 and 3 mouse BALF samples with results consistent with serum results (fig. 19a and 19 b). It was shown that the N protein + ODN-39M preparation as a monovalent preparation or a bivalent preparation binding to RBD protein was able to induce IgA antibodies against SARS-CoV-2(Delta variant and Omicron variant) N protein and SARS-CoV-1N protein.

Simultaneously, the sera of mice in

groups

3, 6 and 8 were tested for IgG antibodies against the RBD protein of SARS-CoV-2 primary strain (40592-V08H, available from Beijing Yi Qianzhou science and technology, Inc., China) and antibodies against the RBD protein of Delta variant. The results are shown in FIG. 20a, indicating that

groups

3, 6 and 8 induced cross-reactive antibodies. Based on the above results, the neutralizing activity against the RBD protein of the SARS-CoV-2 primary strain and against the RBD protein of the Delta variant strain in the sera of

groups

3 and 6 was examined by a substitution virus neutralization test. As shown in Table 1, all mice in

groups

3 and 6 induced antibodies having inhibitory activity against the RBD protein of the SARS-CoV-2 primary strain and the RBD protein of the Delta variant. Also, the antibody level was higher in group 6 inoculated by the subcutaneous injection route. However, the level of inhibitory antibodies in serum increased by intranasal inoculation of a bivalent antigen formulation of N protein + ODN-39M + RBD protein, indicating that vaccination of vaccinated individuals with the bivalent antigen formulation provides systemic levels of neutralizing antibodies against other strains.

TABLE 1 alternative Virus neutralization assay for the detection of inhibitory titers in sera of

groups

3 and 6 mice against the RBD protein of the SARS-CoV-2 Primary Virus Strain and the RBD protein of the Delta variant

Finally, the IgA antibodies against the RBD protein of the SARS-CoV-2 primary virus strain and the RBD protein of the Delta variant strain in the BALF sample of the group 3 were detected by ELISA assay, and the results are shown in FIGS. 20b and 20c, and 3 of the 5 mice of the group 3 detected positive responses, indicating that the antibodies against the RBD protein of the primary virus strain and the RBD protein of the Delta variant strain can be induced in mucosal tissues.

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

On the 26 th day after the last immunization, the N protein of the SARS-CoV-2Delta variant, the SARS-CoV-1N protein and the conserved peptide N were used _351-365 Splenocytes were stimulated in

groups

2, 3 and 9 (negative control) mice. Consistent with the result of the humoral immune response test, the group 2 and the group 3 test the anti-SARS-CoV-2 Delta variant N protein, SARS-CoV-1N protein and the conservative peptide N _351-365 A positive response. Thus, the cell-mediated immune response was characterized by cross-reactivity as demonstrated by SARS-CoV-1, a member of the Sarbe subfamily of coronaviruses (fig. 21a and 21 b).

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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370 375 380

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Claims

1. A vaccine composition against a beta coronavirus, vaccinated via a mucosal route, comprising:

1) novel coronavirus nucleocapsid proteins;

2) has a nucleotide sequence of SEQ ID NO: 1; and

3) and (3) a medicinal auxiliary material.

2. The vaccine composition of claim 1, wherein the amino acid sequence of the novel coronavirus nucleocapsid protein is SEQ ID NO: 2, or a sequence identical to SEQ ID NO: 2 sequences having at least 90% identity.

3. The vaccine composition according to claim 1, characterized in that the mucosal route is an intranasal vaccination route.

4. The vaccine composition of claim 1, further comprising a protein that induces neutralizing antibodies against the beta coronavirus.

5. The vaccine composition of claim 4, wherein the protein that induces neutralizing antibodies against the genus Beta coronavirus is a receptor binding domain protein of a novel coronavirus spike protein.

6. The vaccine composition of claim 5, wherein the receptor binding domain protein is a receptor binding domain protein of a novel coronavirus Delta variant.

7. The vaccine composition of claim 6, wherein the amino acid sequence of the receptor binding domain protein is SEQ ID NO: 3 sequence (b).

8. Use of a vaccine composition according to any one of claims 1 to 7 in the manufacture of a medicament against a novel coronavirus, wherein the vaccine composition induces a humoral immune response and a cell-mediated immune response against a beta coronavirus.

9. The use according to claim 8, wherein the amino acid sequence of the novel coronavirus nucleocapsid protein is SEQ ID NO: 2 or a sequence identical to SEQ ID NO: 2 sequences having at least 90% identity.

10. Use according to claim 8, characterized in that the vaccine composition is a preparation to be administered by mucosal route.

11. Use according to claim 10, wherein the mucosal route is the intranasal vaccination route.

12. The use according to claim 8, wherein the vaccine composition further comprises a protein that induces neutralizing antibodies against the β -coronavirus.

13. The use according to claim 12, wherein said protein inducing neutralizing antibodies against said β -coronavirus is a receptor binding domain protein of a novel coronavirus spike protein.

14. The use according to claim 13, wherein the receptor binding domain protein is that of a novel coronavirus Delta variant.

15. Use according to claim 8, wherein the vaccine composition is a formulation for simultaneous mucosal and parenteral vaccination.

16. Use according to claim 8, wherein the vaccine composition is used as a booster vaccine for boosting vaccination of individuals who have been vaccinated against a novel coronavirus vaccine.

17. A method of vaccinating said vaccine composition to induce a humoral immune response and a cell-mediated immune response against said beta coronavirus, said vaccine composition comprising said novel coronavirus nucleocapsid protein and said nucleotide sequence is SEQ ID NO: 1, or a nucleic acid having the sequence of 1.

18. The method of claim 17, wherein the amino acid sequence of the novel coronavirus nucleocapsid protein is SEQ ID NO: 2 or a sequence identical to SEQ ID NO: 2 sequences having at least 90% identity.

19. The method of claim 17, wherein the vaccine composition is administered by a mucosal route.

20. The method of claim 19, wherein the mucosal route is an intranasal vaccination route.

21. The method of claim 17, wherein the vaccine composition further comprises a protein that induces neutralizing antibodies against the beta coronavirus.

22. The method of claim 21, wherein the protein that induces neutralizing antibodies against the betacoronavirus is a receptor binding domain protein of a novel coronavirus spike protein.

23. The method of claim 22 wherein the receptor binding domain protein is a receptor binding domain protein of a novel coronavirus Delta variant.

24. The method of claim 17, wherein the vaccine composition is administered by both a mucosal route and a parenteral route.

25. The method of claim 17, wherein the vaccine composition is used as a booster vaccine for boosting vaccination of individuals who have been vaccinated against the novel coronavirus.