Recombinant polyvalent vaccine

Abstract

The present invention relates to a recombinant multivalent vaccine. The recombinant multivalent vaccine comprises a recombinant protein, which comprises, from the N-terminus to the C-terminus, a first antigenic peptide, an N-polypeptide (SEQ ID NO.1), a second antigenic peptide, a C-polypeptide (SEQ ID NO.3) and a third antigenic peptide, the N-polypeptide and the C-polypeptide being polypeptides formed with an intramolecular scaffold, which supports and stabilizes the intramolecular scaffold NC of the conformation of the first antigenic peptide, the second antigenic peptide and the third antigenic peptide. The present invention provides recombinant multivalent vaccines 3Ro-NC (SEQ ID NO.17) and 3Rs-NC (SEQ ID NO.19) against SARS-CoV-2 mutant strains. The present invention can provide protection against Omicron infection in the upper and lower respiratory tracts by using 3Ro-NC and KFD as a prophylactic mucosal SARS-CoV-2 vaccine.

Description

[Technical Field]

[0001] The present invention relates to recombinant polyvalent vaccines, and more specifically to recombinant polyvalent vaccines against cancer and infectious bacteria and viruses, particularly against infectious bacteria and viruses having multiple serological subtypes or variants, such as coronaviruses and influenza viruses, and belongs to the field of biopharmaceuticals. [Background technology]

[0002] Because cancer is a cause of death, many cancer-specific or related antigens have been discovered and are being used in the development of cancer vaccines.

[0003] Infectious pathogens, including bacteria and viruses, particularly those with multiple serological subtypes or variants, pose a significant threat to public health.

[0004] The disease caused by the novel coronavirus SARS-CoV-2, named COVID-19, has seen the successive emergence of variants such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) following the original SARS-CoV-2 strain, raising serious questions about the nature, extent, and consequences of antigenic drift in SARS-CoV-2. The unpredictable emergence of SARS-CoV-2 variants of concern (VOCs) with differing antigenicity has increased the risk of global transmission, leading to waves of the COVID-19 pandemic.

[0005] In addition to non-pharmacological interventions and strict border control measures, several different vaccines based on different platforms, including mRNA, adenovirus, and inactivated virus, are being clinically deployed worldwide. However, new infection cases around the world are still increasing from time to time.

[0006] Therefore, there is an urgent need to develop safe and effective vaccines against cancer and infectious pathogens (e.g., coronaviruses and influenza viruses), particularly effective mucosal vaccines, to explore new ways to prevent initial viral infection and potential transmission in the respiratory tract. [Overview of the Initiative]

[0007] The present invention aims to provide safe and effective vaccines against cancer and infectious pathogens.

[0008] To achieve the above objective, the technical means used in the present invention is to provide a recombinant polyvalent vaccine. The recombinant polyvalent vaccine comprises a recombinant protein, the recombinant protein comprising a first antigen peptide, an N-polypeptide, a second antigen peptide, a C-polypeptide, and a third antigen peptide, from the N-terminus to the C-terminus. The N-polypeptide has the amino acid sequence shown in SEQ ID NO.1 or its variant, The C-polypeptide has the amino acid sequence shown in SEQ ID NO.3 or its variant, The N-polypeptide and C-polypeptide are polypeptides formed with an intramolecular skeleton, which support and stabilize the intramolecular skeleton NC of the conformations of the first antigen peptide, second antigen peptide, and third antigen peptide.

[0009] In some embodiments of the present invention, the variant of the N-polypeptide has the same function as SEQ ID NO.1 and at least 95% sequence homology, and the variant of the C-polypeptide has the same function as SEQ ID NO.3 and at least 95% sequence homology.

[0010] In some embodiments of the present invention, the first antigen peptide, the second antigen peptide, and the third antigen peptide are polypeptides consisting of 10 to 900 amino acids, and when the recombinant polyvalent vaccine is used to immunize a host subject, it induces an antigen peptide-specific humoral and / or cellular immune response.

[0011] In some embodiments of the present invention, the first antigen peptide, the second antigen peptide, and the third antigen peptide are derived from an infectious pathogen, a variant of an infectious pathogen, or a cancer / tumor-specific antigen.

[0012] In some examples of the present invention, the first antigen peptide, the second antigen peptide, and the third antigen peptide are derived from SARS-CoV-2 variants.

[0013] In some embodiments of the present invention, the antigen peptide is the receptor-binding domain-RBD region aa.319-527 of the spike S protein of the SARS-CoV-2 variant.

[0014] In some embodiments of the present invention, the RBD is derived from one of the following: SARS-CoV-2 prototype, Delta, Omicron, Gamma mutants, and SHC014 and WIV1 SARS-related coronavirus strains. Delta RBD has the amino acid sequence shown in SEQ ID NO. 5 and the nucleotide sequence shown in SEQ ID NO. 6; Omicron RBD has the amino acid sequence shown in SEQ ID NO. 7 and the nucleotide sequence shown in SEQ ID NO. 8; SARS-CoV-2 prototype RBD has the amino acid sequence shown in SEQ ID NO. 9 and the nucleotide sequence shown in SEQ ID NO. 10; Gamma RBD has the amino acid sequence shown in SEQ ID NO. 11 and the nucleotide sequence shown in SEQ ID NO. 12; SHC014 RBD has the amino acid sequence shown in SEQ ID NO. 13 and the nucleotide sequence shown in SEQ ID NO. 14; and WIV1 RBD has the amino acid sequence shown in SEQ ID NO. 15 and SEQ ID It has the nucleotide sequence shown in NO.16.

[0015] In some embodiments of the present invention, the recombinant protein comprises an Omicron RBD, an N-polypeptide, a Delta RBD, a C-polypeptide, and an Omicron RBD, and has the amino acid sequence shown in SEQ ID NO. 17, from the N-terminus to the C-terminus, or the recombinant protein comprises an Omicron RBD, an N-polypeptide, a SHC014 RBD, a C-polypeptide, and a WIV1 RBD, from the N-terminus to the C-terminus, and has the amino acid sequence shown in SEQ ID NO. 19.

[0016] In some embodiments of the present invention, the recombinant polyvalent vaccine further comprises an adjuvant, the adjuvant being selected from at least one of flagellin, polyinosine / polycytidic acid, MF59, AS01, AS03, AS04, CpG, MPL, CT, CTB, IL-1α, IL-2, IL-12, IL-18, GM-CSF, PIKA, and BFA03.

[0017] In some embodiments of the present invention, the adjuvant is recombinant flagellin KFD, whose amino acid sequence is shown in SEQ ID NO. 21 or has at least 95% sequence homology to SEQ ID NO. 21 and retains TLR-5 agonist activity.

[0018] The present invention further provides nucleic acid molecules encoding recombinant proteins for recombinant polyvalent vaccines. The nucleic acid molecule comprises, from 5' to 3', a first polynucleotide encoding a first antigen peptide, a polynucleotide encoding an N-polynucleotide, a second polynucleotide encoding a second antigen peptide, a polynucleotide encoding a C-polynucleotide, and a third polynucleotide encoding a third antigen peptide. The polynucleotide encoding the N-polypeptide is shown in SEQ ID NO. 2, or has a nucleotide sequence having at least 95% sequence homology to SEQ ID NO. 2. The polynucleotide encoding the C-polypeptide is shown in SEQ ID NO. 4, or has a nucleotide sequence having at least 95% sequence homology to SEQ ID NO. 4. When a recombinant protein encoded by the nucleic acid molecule is expressed, the N-polypeptide and C-polypeptide are polypeptides formed with an intramolecular skeleton, forming the intramolecular skeleton NC that supports the first antigen peptide, the second antigen peptide, and the third antigen peptide.

[0019] Compared to the prior art, the present invention has the following beneficial effects.

[0020] (1) The present invention provides a recombinant polyvalent vaccine based on an intramolecular scaffold, the intramolecular scaffold maintaining the conformation of the antigen peptide and being able to present the antigen peptide at three different positions on the intramolecular scaffold, and the intramolecular scaffold is suitable for antigen peptides of viral, bacterial, or cancer origin. Accordingly, the present invention solves at least two problems in vaccine research and development. First, by maintaining the conformation of the antigen peptide, it is ensured that the antigen peptide can induce humoral and cellular immune responses, particularly effective neutralizing antibody responses, and second, by presenting the polyvalent antigen peptide, it is possible to induce humoral and cellular immune responses against multiple cancers or multiple infectious pathogen variants.

[0021] (2) Furthermore, the present invention provides a recombinant polyvalent vaccine against SARS-CoV-2, utilizing RBDs of different viral strains to design an immunogen having a tandem RBD chimeric RBD-dimer, and SARS-CoV-2 RBD conjugated to a human IgG Fc fragment as an immunostimulant can induce potent antibody neutralizing activity against SARS-CoV-2 infection in mice, overcoming the limitations of the immunogenicity of viral RBD antigens, inducing a broad immune response, and overcoming the drawback that RBD-based subunit vaccines do not strongly induce a mucosal immune response in the airways to prevent SARS-CoV-2 nasal infection and asymptomatic transmission.

[0022] (3) The recombinant multivalent vaccine against SARS-CoV-2 provided by the present invention has high immunogenicity because the included recombinant protein 3Ro-NC or 3Rs-NC has the native conformation of RBD, and can effectively induce a broad defensive immune response against SARS-CoV-1 and SARS-CoV-2 variants, especially against the immune-evasive Omicron variant.

[0023] (4) Furthermore, the recombinant multivalent vaccine against SARS-CoV-2 provided by the present invention contains recombinant flagellin KFD as a mucosal adjuvant. Nasal immunization with 3Ro-NC and recombinant flagellin protein KFD as a mucosal adjuvant can induce synergistic mucosal immunity and systemic immunity against SARS-CoV-1 and SARS-CoV-2 variants in mice. The antibodies induced by nasal immunization with 3Ro-NC and KFD show higher specificity and neutralizing activity against the Omicron variant. In human ACE2 gene-modified mice infected with the Omicron variant, the immunity induced by nasal immunization with 3Ro-NC and KFD significantly reduces the viral RNA copy number in the lungs and turbinates, and also detects a significant decrease in histopathology in the lungs of mice inoculated nasally.

Brief Description of the Drawings

[0024] [Figure 1] KF, KFD and 3Ro-NC are named, and the schematic domain composition of the chimeric protein of three RBDs is shown. [Figure 2] Schematic diagram of the 3D structure of 3Ro-NC predicted by Alpha Fold 2. [Figure 3] Western blot image of the purified 3Ro-NC protein. [Figure 4] Shows the TLR5 agonist activity of recombinant proteins such as RBD of SARS-CoV-2 prototype strain, RBD of Omicron strain, P-KFD and 3Ro-NC. [Figure 5]This is a 3D schematic diagram of the binding of SARS-CoV-2 RBD to four neutralizing monoclonal antibodies: B38, M-S309, CR3022, and COVA2-39. [Figure 6] This graph shows the results of ELISA detection of the binding affinity between Delta strain RBD(Rd), Omicron strain (B.1.1.529) RBD(Ro), and 3Ro-NC and four representative neutralizing monoclonal antibodies: B38, M-S309, CR3022, and COVA2-39. [Figure 7] This is a schematic diagram of the immunization and sampling scheme (5 mice / group). [Figure 8] This graph shows the RBD-specific IgG response in serum. Solid triangles represent the 3Ro-NC group, solid squares represent the RBD dimer group, and solid circles represent the control. Data are expressed as mean ± SEM and represent at least two independent experiments. The two immunization groups are compared using an unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 9] This is a dot plot of neutralizing antibody titers against SARS-CoV-2 variants after the second and third immunizations using pseudotyped virus assays. Solid triangles represent the 3Ro-NC group, solid squares represent the RBD dimer group, and solid circles represent the control group. Data are expressed as mean ± SEM and represent at least two independent experiments. One-way ANOVA is used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 10] This is a dot plot of the results of neutralizing antibody titers against SARS-CoV-1 after the second and third immunizations using pseudotyped virus measurement. Solid triangles represent the 3Ro-NC group, solid squares represent the RBD dimer group, and solid circles represent the control. Data are expressed as mean ± SEM and represent at least two independent experiments. One-way ANOVA is used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 11]This is a dot plot of the results of neutralizing antibody titers against Omicron and the prototype strain after the first immunization using pseudotyped virus measurement. Solid triangles represent the 3Ro-NC group, solid squares represent the RBD dimer group, and solid circles represent the control. Data are expressed as mean ± SEM and represent at least two independent experiments. One-way ANOVA is used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 12] This is a geometric mean titer (GMT) diagram of neutralizing antibodies against different mutant strains. [Figure 13] This is a dot plot of the ratio of neutralizing antibody titers against the Omicron strain (50% neutralizing titer, NT 50) to neutralizing antibody titers against the SARS-CoV-2 prototype or Delta strain. Data are expressed as mean ± SEM and represent at least two independent experiments. Two immunization groups are compared using an unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 14] These antigen maps were generated from second (square) or third (triangle) vaccinated serum samples of the RBD dimer (white) and 3Ro-NC (gray) groups. Each symbol corresponds to Alpha, Beta, Gamma, Delta, Omicron, SARS-CoV-2 prototype, and SARS-CoV-1, respectively, and each grid corresponds to a 2x dilution in neutralization testing. [Figure 15] This is a schematic diagram (5 mice / group) of the immunization and sampling scheme used to generate an antibody response in BALB / c mice by nasal immunization with 3Ro-NC using KFD as an adjuvant. [Figure 16] This is a dot plot of the results of Delta, Gamma, or Omicron RBD-specific IgG responses in serum. Data are expressed as mean ± SEM and represent at least two independent experiments. One-way ANOVA is used to compare differences between groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 17]This graph shows the neutralizing antibody titers against SARS-CoV-2 variants or SARS-CoV-1, measured by pseudotyped virus assay, in serum after the second and third immunizations in the 3Ro-NC+KFD nasal spray group and the 3Ro-NC+AL intramuscular injection group. Each connecting line represents the neutralizing titer of individual serum samples against different virus strains. [Figure 18] This is a geometric mean titer diagram of neutralizing antibodies against different mutant strains in serum after the second and third immunizations in the 3Ro-NC+KFD nasal spray group and the 3Ro-NC+AL intramuscular injection group. [Figure 19] This is a dot plot of the ratio of neutralizing antibody titers against omicron to neutralizing antibody titers against Delta or SARS-CoV-2 prototype strains. Data are expressed as mean ± SEM and represent at least two independent experiments. One-way ANOVA is used to compare differences between groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 20] This antigen map was generated from serum samples from the 3Ro-NC+KFD nasal spray group after the second (square) or third (triangular) doses of vaccination. Each symbol corresponds to Alpha, Beta, Gamma, Delta, Omicron, SARS-CoV-2 prototype, and SARS-CoV-1, respectively. Each grid corresponds to a 2-fold dilution in the neutralization test, and the antigen distance can be interpreted in any direction. [Figure 21] These are antigen maps generated by the 3Ro-NC+KFD nasal spray group (brown) and the 3Ro-NC+AL intramuscular injection group (gray). Each code corresponds to Alpha, Beta, Gamma, Delta, Omicron, SARS-CoV-2 prototype, and SARS-CoV-1, respectively. Each grid corresponds to a 2x dilution in the neutralization test, and the antigen distance can be interpreted in any direction. [Figure 22] This shows a dot plot of RBD-specific mucosal IgA response in saliva. Data are expressed as mean ± SEM and represent at least two independent experiments. One-way ANOVA is used to compare differences between groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 23]This dot plot shows the RBD-specific mucosal IgA response to vaginal douche solutions. Data are expressed as mean ± SEM and represent at least two independent experiments. One-way ANOVA is used to compare differences between groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 24] This is a dot plot of RBD-specific mucosal IgA response in nasal lavage solution. Data are expressed as mean ± SEM and represent at least two independent experiments. One-way ANOVA is used to compare differences between groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 25] This shows the correlation between RBD-specific IgA in nasal lavage fluid and RBD-specific IgA in saliva (triangles represent Delta, and squares represent Omicron). [Figure 26] This shows the correlation between RBD-specific IgG in nasal lavage fluid and RBD-specific IgG in serum (triangles represent the Delta, and squares represent the Omicron). [Figure 27] This study shows the correlation between RBD-specific IgA and IgG in nasal lavage fluid and the neutralizing antibody response of BALB / c mice immunized with 3Ro-NC using a pseudotyped virus system. [Figure 28] This diagram shows a schematic of the immune and viral attack scheme (6-8 mice / group) for studying the protective effect of 3Ro-NC immunized hACE2 mice against infection by SARS-CoV-2 Omicron variants. [Figure 29] The dot plots of omicron and delta RBD-specific serum IgG titers are shown. The meaning of the shape symbols used is the same as in Figure 28. One-way ANOVA is used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 30] The dot plots of Delta and Omicron RBD-specific salivary IgA titers are shown. The meaning of the shape symbols used is the same as in Figure 28. One-way ANOVA is used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 31] The dot plots of Delta and Omicron RBD-specific vaginal douche IgA titers after the third immunization are shown. The meaning of the shape symbols used is the same as in Figure 28. One-way ANOVA is used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 32] The dot plots of RNA copy numbers in the nasal turbinates and lungs of SARS-CoV-2 RBD, as determined by qPCR, and the measurement of infectious viral plaques in the lungs 3 days after infection are shown. One-way ANOVA is used to compare differences between groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 33] Images of lung sections stained with hematoxylin and eosin (H&E) are shown (scale 100 μm). [Figure 34] This figure shows dot plots (6-8 mice / group) of pathological and infiltration scores for immune cell aggregation around bronchioles, pulmonary vessels, and interstitial pneumonia based on H&E stained sections. The meaning of the shape symbols used is the same as in Figure 28. One-way ANOVA is used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 35] The correlation diagrams of RNA copy numbers in the lungs and nasal turbinates are shown. 95% confidence intervals are indicated by dashed lines. One-way ANOVA is used to compare differences between groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 36] This diagram shows the correlation between RNA copy number in the lungs and Omicron RBD-specific serum IgG and salivary IgA. 95% confidence intervals are indicated by dashed lines. One-way ANOVA is used to compare differences between groups. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 37]This diagram shows the correlation between RNA copy number in the nasal turbinates and Omicron RBD-specific serum IgG and salivary IgA. 95% confidence intervals are indicated by dashed lines. One-way ANOVA is used to compare group differences. *p<0.05, **p<0.01, ***p<0.001, ns: not significant. [Figure 38] This is a dot plot showing the serum RBD-specific IgG response after the second immunization. Triangles represent the Ro+Rs+Rw group (1.33 μg Ro(Omicron RBD) + 1.33 μg Rs(SHC014 RBD) + 1.33 μg Rw(WIV1 RBD) with 200 μg aluminum adjuvant), squares represent the 4 μg 3Rs-NC group with 200 μg aluminum adjuvant, and circles represent the control group. Data are expressed as mean ± SEM and represent at least two independent experiments. Two immunization groups are compared using an unpaired t-test. **p<0.01, ***p<0.001. [Figure 39] This is a dot plot of neutralizing antibody titers after the second immunization against the SARS-CoV-2 variant Omicron or SARS-like viruses SHC014 and WIV1. Data are represented by mean ± SEM. One-way ANOVA is used to compare groups. *p<0.05, ***p<0.001. [Modes for carrying out the invention]

[0025] To make the present invention easier to understand, preferred embodiments will be described in detail with reference to the drawings.

[0026] Unless otherwise specified, the implementation of this invention utilizes prior art in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are within the scope of the skills of the art. These techniques are fully described in literature such as Molecular Cloning: Laboratory Specification, 3rd Edition (Sambrook and Russell, 2001). In this invention, “cancer” is a general term for tumors and cancers of any type or form.

[0027] Many types of vaccines have been developed for COVID-19, including attenuated live viruses, inactivated viral particles, or subunit vaccines. Recombinant protein subunit vaccines containing purified viral proteins are the safest type of immunogen for vaccine research and development. Because such subunit vaccines contain only purified viral protein fragments, they do not cause COVID-19. Protein subunit vaccines have been used for other diseases such as hepatitis B (HBV) and cervical cancer (HPV). However, how to preserve the natural conformation of recombinant subunit proteins in subunit vaccines has always been a serious challenge. Current methods include human Fc peptides, clover structure technology, and self-assembling nanoparticles (e.g., ferritin).

[0028] The present invention solves at least two problems in vaccine research and development. First, by maintaining the conformation of the antigen peptide, it ensures that the antigen peptide can induce humoral and cellular immune responses, particularly effective neutralizing antibody responses. Second, by presenting a multivalent antigen peptide, it is possible to induce humoral and cellular immune responses against multiple cancers or multiple variants of infectious pathogens. The present invention provides an intramolecular scaffold that maintains the conformation of the antigen peptide and can present the antigen peptide at three different positions on the intramolecular scaffold. The intramolecular scaffold is suitable for antigen peptides derived from viruses, bacteria, or cancer. The following multivalent COVID-19 vaccine fully illustrates the practical applications of the present invention.

[0029] The present invention provides a recombinant polyvalent vaccine comprising a recombinant protein, the recombinant protein comprising a first antigen peptide, an N-polypeptide, a second antigen peptide, a C-polypeptide, and a third antigen peptide, arranged from the N-terminus to the C-terminus, wherein the N-polypeptide and C-polypeptide are intramolecular backbone-forming polypeptides that form an intramolecular backbone NC that stabilizes the conformations of the first, second, and third antigen peptides. In some examples, linkers are inserted into one or more binding sites between the first antigen peptide and the N-polypeptide, between the N-polypeptide and the second antigen peptide, between the second antigen peptide and the C-polypeptide, and between the C-polypeptide and the third antigen peptide. The linker consists of 4 to 40 amino acids, one exemplary linker being (GSSS)n, where n is 1 to 10.

[0030] The N-polypeptides and C-polypeptides of the intramolecular backbone NC were modified from flagellin of Escherichia coli strain K12 (KF). In some examples, the N-polypeptide of the intramolecular backbone NC has the amino acid sequence shown in SEQ ID NO. 1 or its variant, and the variant has at least 95% sequence homology with SEQ ID NO. 1. In some examples, the N-polypeptide of the intramolecular backbone NC is encoded by the nucleotide sequence shown in SEQ ID NO. 2 or its variant, and the variant has at least 95% sequence homology with SEQ ID NO. 2. In some examples, the C-polypeptide of the intramolecular backbone NC has the amino acid sequence shown in SEQ ID NO. 3 or its variant, and the variant has at least 95% sequence homology with SEQ ID NO. 3. In some examples, the C-polypeptide of the intramolecular backbone NC is encoded by the nucleotide sequence shown in SEQ ID NO. 4 or its variant, and the variant has at least 95% sequence homology with SEQ ID NO. 4.

[0031] The antigenic peptide may be a polypeptide, and when a recombinant polyvalent vaccine is used to immunize host subjects such as humans, cattle, cats, dogs, horses, and pigs, the polypeptide can induce a fluid and / or T-cell epitope-based cellular immune response. In some examples, the first, second, and third antigenic peptides are all derived from a single mutant strain of an infectious pathogen or a single cancer-specific antigen. In some examples, the first, second, and third antigenic peptides are derived from one or more mutant strains of an infectious pathogen or one or more cancer-specific antigens of cancer. In some examples, the antigenic peptides are derived from one or more infectious pathogens or from one or more types of cancer. Tumor-specific antigens include, for example, the MARA antigen in melanoma and Ras oncogenic variants in gastrointestinal and lung cancer. In this application, "cancer-specific antigen" includes cancer / tumor-specific antigens, cancer / tumor-associated antigens, and cancer-associated pathogens. For example, tumor-specific antigens include the MARA antigen in melanoma and Ras oncogenic variants in gastrointestinal and lung cancers, while cancer / tumor-associated antigens include CD19 and CD20, and cancer-associated pathogens include EB virus, HPV, and HBV. Antigen peptides contain 10-900, 10-600, and 10-300 amino acids, respectively.

[0032] This invention provides a recombinant polyvalent vaccine against SARS-CoV-2 mutant strains.

[0033] Clinical findings indicate that SARS-CoV-2 infection and replication typically begin in the ciliated cells of the nasal cavity. However, these ciliated cells have little contact with serum IgG antibodies, which are usually induced by the COVID-19 vaccine administered by injection and are therefore less effective in preventing initial viral entry and upper respiratory tract infection. The highly transmissible variant Omicron is vaccine-evading and causes a breakthrough infection. Furthermore, more than 90% of Omicron infections are asymptomatic or mild cases, but in reality, replicated SARS-CoV-2 viruses are present in the nasopharyngeal mucosa and can be transmitted to others.

[0034] The spike (S) protein on the surface of the SARS-CoV-2 virus mediates viral attachment and entry via receptor-binding domains (RBDs) (aa.319-527), which have become a potential target for COVID-19 vaccine design. RBD-based antigens have been widely used in the development of coronavirus vaccines. However, with the emergence of COVID-19 variants, the protective efficacy of SARS-CoV-2 vaccines has decreased. To overcome the limitations of the immunogenicity of viral RBD antigens and induce a broad immune response, we designed immunogens with chimeric RBD-dimers of tandem RBDs by utilizing RBDs from different viral strains. SARS-CoV-2 RBDs conjugated to the Fc fragment of human IgG as an immunoenhancing agent can induce potent antibody-neutralizing activity against SARS-CoV-2 infection in mice. However, these RBD-based subunit vaccines do not strongly induce a mucosal immune response in the airways to prevent SARS-CoV-2 nasal infection and asymptomatic transmission.

[0035] In some examples, the recombinant polyvalent vaccine against SARS-CoV-2 mutants comprises a recombinant protein, the recombinant protein comprising a first RBD, an N-polypeptide, a second RBD, a C-polypeptide, and a third RBD, from the N-terminus to the C-terminus, wherein the N-polypeptide and C-polypeptide are as described above. In some examples, the first, second, and third RBDs are derived from one SARS-CoV-2 mutant. In some examples, the first, second, and third RBDs are derived from one or more SARS-CoV-2 mutants.

[0036] In some examples, RBDs are derived from SARS-CoV-2 prototypes, Delta, Omicron, Gamma variants, and SARS-related coronavirus strains such as SHC014 and WIV1. Delta RBD has the amino acid sequence shown in SEQ ID NO. 5 and the corresponding nucleotide sequence shown in SEQ ID NO. 6. Omicron RBD has the amino acid sequence shown in SEQ ID NO. 7 and the corresponding nucleotide sequence shown in SEQ ID NO. 8. SARS-CoV-2 prototype RBD has the amino acid sequence shown in SEQ ID NO. 9 and the corresponding nucleotide sequence shown in SEQ ID NO. 10. Gamma RBD has the amino acid sequence shown in SEQ ID NO. 11 and the corresponding nucleotide sequence shown in SEQ ID NO. 12. SHC014 RBD has the amino acid sequence shown in SEQ ID NO. 13 and the corresponding nucleotide sequence shown in SEQ ID NO. 14. WIV1 RBD has the amino acid sequence shown in SEQ ID NO. 15 and the corresponding nucleotide sequence shown in SEQ ID NO. 16.

[0037] In some examples, recombinant polyvalent vaccines against SARS-CoV-2 mutants include a recombinant protein comprising, from N-terminus to C-terminus, an Omicron RBD, an N-polypeptide, a Delta RBD, a C-polypeptide, and an Omicron RBD (designated as 3Ro-NC). Because 3Ro-NC has the natural conformation of the RBD, it is highly immunogenic and can effectively induce a broad protective immune response against SARS-CoV-1 and SARS-CoV-2 mutants, particularly against immune-evading Omicron mutants. The 3Ro-NC used in the experiments in the examples has an amino acid sequence (SEQ ID NO. 17) and a nucleotide sequence (SEQ ID NO. 18).

[0038] In some examples, recombinant polyvalent vaccines against SARS-CoV-2 mutants include a recombinant protein comprising, from N-terminus to C-terminus, an Omicron RBD, an N-polypeptide, an SHC014 RBD, a C-polypeptide, and a WIV1 RBD (designated as 3Rs-NC). Because 3Rs-NC has the natural conformation of the RBD, it is highly immunogenic and can effectively induce a broad protective immune response against SARS-CoV-1 and SARS-CoV-2 mutants. The 3Rs-NC used in the experiments in the examples has an amino acid sequence (SEQ ID NO. 19) and a nucleotide sequence (SEQ ID NO. 20).

[0039] In some embodiments, recombinant polyvalent vaccines further include adjuvants. Adjuvants include pathogen-associated molecular patterns such as flagellin and poly I:C, AS series such as MF59 (Novartis), AS01, AS03, AS04 (GSK), Matrix-M (Novavax), CpG (e.g., CpG1018) (Dynavax), MPL (Lipid A-based), cytokines (e.g., IL-2, IL-12, GM-CSF), PIKA (Lycoplasma), and BFA03 (Jiangsu Ruike).

[0040] In some embodiments, the mucosal adjuvant includes CT, CTB, IL-1α, IL-12, IL-18, CpG (e.g., CpG1018) (Dynavax), cytokines (e.g., IL-2, IL-12, GM-CSF), PIKA (parasitic organism), and flagellin.

[0041] In some embodiments, the mucosal adjuvant is recombinant flagellin KFD, which has been proven safe and effective for nasal immunization in our previous work. KFD has the amino acid sequence shown in SEQ ID NO. 21 or a variant thereof, the variant having at least 95% sequence homology with SEQ ID NO. 21, and KFD also has the nucleotide sequence shown in SEQ ID NO. 22 or a variant thereof, the variant having at least 95% sequence homology with SEQ ID NO. 22.

[0042] KFD and its variants exert their immunomodulatory activity by activating the TLR5 pathway in nasal epithelial cells and enhancing local and distal mucosal IgA responses. Nasal immunization with 3Ro-NC and recombinant flagellin protein KFD as a mucosal adjuvant can induce synergistic mucosal and systemic immunity against SARS-CoV-1 and SARS-CoV-2 mutants in mice. Compared to intramuscular injection immunization using 3Ro-NC and aluminum adjuvant or RBD-dimer and aluminum adjuvant, antibodies induced by nasal immunization with 3Ro-NC and KFD show higher specificity and neutralizing activity against Omicron mutants. In human ACE2 gene-modified mice infected with Omicron mutants, immunization induced by nasal immunization with 3Ro-NC and KFD significantly reduces the viral RNA copy number in the lungs and nasal turbinates. Furthermore, a significant reduction in histopathology is detected in the lungs of mice inoculated into the nasal cavity. As correlation analysis clearly shows, the degree of viral control in the nasal cavity is highly correlated with the response level of RBD-specific secretory IgA antibodies.

[0043] The amount of recombinant protein in a recombinant polyvalent vaccine necessary to induce a sufficient immune response against infectious pathogens or cancer can be determined by standard clinical trials. In some examples, the amount of recombinant protein in the vaccine is in the range of 1–1000 μg, 10–500 μg, or 50–250 μg.

[0044] The present invention further provides a nucleic acid molecule encoding a recombinant protein for recombinant polyvalent vaccines, the nucleic acid molecule comprising, from 5' to 3', a first polynucleotide encoding a first antigen peptide, a polynucleotide encoding an N-polynucleotide, a second polynucleotide encoding a second antigen peptide, a polynucleotide encoding a C-polynucleotide, and a third polynucleotide encoding a third antigen peptide, wherein the polynucleotide encoding the N-polynucleotide has the nucleotide sequence shown in SEQ ID NO.2 or its variant, and the polynucleotide encoding the C-polynucleotide has the nucleotide sequence shown in SEQ ID NO.4 or its variant, and when the recombinant protein encoded by the nucleic acid molecule is expressed, the N-polynucleotide and C-polynucleotide are polypeptides formed by an intramolecular skeleton, forming the intramolecular skeleton NC that supports the antigen peptide. "Variant" means having at least 95% sequence homology with SEQ ID NO.2 or 4. In some examples, the antigen peptide consists of 10 to 900 amino acids. In some examples, the antigen peptide is the receptor-binding domain (RBD) region (aa.319-527) of the spike (S) protein of the SARS-CoV-2 mutant. In some examples, the RBD region is the polypeptide shown in sequence ID number 5 (Delta), sequence ID number 7 (Omicron), sequence ID number 9 (SARS-CoV-2 prototype), sequence ID number 11 (Gamma), sequence ID number 13 (SHC014), and sequence ID number 15 (WIV1).

[0045] Nucleic acid molecules may be present in mRNA, expression vectors, or viral vectors (e.g., adenoviruses and adeno-associated viruses). The number of nucleic acid molecules capable of inducing a sufficient immune response against infectious pathogens or cancer can be determined by standard clinical studies.

[0046] Recombinant polyvalent vaccines further contain any pharmaceutically acceptable components, such as excipients (e.g., saline solution, PBS, etc.).

[0047] The following examples are provided solely for the purpose of illustrating the principles of the present invention and are not intended to limit the scope of the invention.

[0048] (Examples) 1. Materials and Methods 1.1 Mice and Ethics Six-to-eight-week-old female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HFH4-hACE2 genetically modified mice with a C57BL / 6 background were obtained from Dr. Ralph Baric of the University of North Carolina at Chapel Hill and reared at the Animal Center of the Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS). The mice were randomly assigned to groups. Under conditions free from specific pathogens (SPFs), all mice were reared in individual ventilated cages (IVCs). Infection experiments were conducted in the CAS WIV's Animal Biosafety Level 3 (ABSL-3) laboratory. The animal research was approved by the Wuhan Institute of Virology Animal Welfare and Ethics Review Committee and conducted in accordance with the "Regulations on the Management of Laboratory Animals in China" (Research No. WIVA09202101).

[0049] 1.2. Preparation of vaccines The KFD gene (SEQ ID NO. 21) was constructed by ligating the N-terminal and C-terminal regions of the D0-D1 gene of flagellin KF (E. coli strain K12 MG1655), and then cloned into the pET-28a plasmid vector (Invitrogen). The pET-28a-KFD recombinant plasmid was used to transform E. coli strain BL21 DE3, and the transformed bacteria were cultured overnight at 37°C in Luria-Bertani broth containing 50 mg / ml kanamycin. Logarithmic-phase bacteria were induced with IPTG and lactose for KFD expression. To improve the yield of soluble protein, 1% ethanol was added 1 hour before induction, the culture temperature was lowered to 18°C, and the cells were grown for 16 hours.

[0050] The NC skeleton was designed based on the 3D structure of KFD to retain the conserved domains D0 and D1. The amino acid (aa.) homology between the N-polypeptide (SEQ ID NO.1) and C-polypeptide (SEQ ID NO.3) and KFD (SEQ ID NO.21) is approximately 72%. The 3Ro-NC gene (SEQ ID NO.18) was constructed by sequentially joining polynucleotide sequences encoding one RBD (aa.319-527) (SEQ ID NO.8) and N-polypeptide (SEQ ID NO.2) from the SARS-CoV-2 Omicron (B.1.1.529) mutant BA.1, one RBD (aa.319-527) (SEQ ID NO.6) and C-polypeptide (SEQ ID NO.4) from the Delta mutant, and one RBD (aa.319-527) (SEQ ID NO.8) from the Omicron mutant. The 3Rs-NC gene (SEQ ID NO.20) was constructed by sequentially joining polynucleotide sequences encoding one RBD (aa.319-527) (SEQ ID NO.8) and N-polypeptide (SEQ ID NO.2) from the SARS-CoV-2 Omicron mutant BA.1 (B.1.1.529), one RBD (aa.319-527) (SEQ ID NO.14) and C-polypeptide (SEQ ID NO.4) from the SHC014 mutant, and one RBD (aa.319-527) (SEQ ID NO.16) from the WIV1 mutant. The RBD dimer gene was constructed by joining two polynucleotide sequences encoding RBD (aa.319-527) (SEQ ID NO.10) from the SARS-CoV-2 prototype. When the signal peptide tPA is present in the 5' region, the following genes were encoded: SARS-CoV-2 prototype RBD (aa.319-527), Gamma mutant RBD (SEQ ID NO.12), Delta mutant RBD (Rd), Omicron mutant RBD (Ro), and the RBD dimer gene, as well as the 3Ro-NC gene, and each was cloned into a pcDNA3.1 plasmid vector. 293F cells from Thermo Fisher Scientific were transfected using recombinant plasmids in the presence of polyethyleneimine (PEI).After culturing at 37°C, 5% CO2, and 130 rpm with shaking for 72 hours, the culture supernatant was collected.

[0051] For each construct, a 6×His tag or an 8×His tag was added to the C-terminus to accelerate protein purification. The recombinant proteins were purified by affinity chromatography using a Ni-NTA column (QIAGEN), and any contaminating lipopolysaccharide (LPS) was removed. The residual LPS content was measured using the Limulus assay (Cape Cod Association) and was found to be less than 0.01 EU / μg of protein.

[0052] 1.3. Caco-2 cells, culture, and TLR5 agonist activity measurement TLR5 agonist activity was measured using Caco-2 cells (HTB-37) obtained from the American Center for Typical Cultures Depository (ATCC). Caco-2 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (standard medium) supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin / streptomycin at 37°C under 5% carbon dioxide conditions. Cell counts were 2 × 10⁶ 5 The cells were inoculated into 24-well plates at a rate of one well per well and maintained in standard medium at 37°C under 5% CO2 conditions for 5 days to form a tightly bound monolayer of cells. Subsequently, they were cultured overnight in serum-free DMEM and then stimulated with recombinant protein in a continuous concentration gradient for 8 hours. The supernatant was then collected, and interleukin-8 (IL-8) was detected by enzyme-linked immunosorbent assay (ELISA) kit (BD Bioscience).

[0053] 1.4 Manufacturing of SARS-CoV-2 inactivated vaccine (IAV) Vero cells were infected with SARS-CoV-2 virus (prototype strain). The supernatant was collected on day 3 or 4, when cytopathic effects (CPE) were observed. Subsequently, β-propiolactone was added to the supernatant at a ratio of 1:4000 (v / v), and the virus was inactivated at 2°C to 8°C for 48 hours. Cell fragments were then removed, and the mixture was concentrated by ultrafiltration. Inactivation was verified by passage the treated samples through three generations without causing CPE. After gel chromatography, ion exchange chromatography, and sterile filtration, virus particles were prepared with buffer.

[0054] 1.5. Vaccination Female BALB / c mice aged 6-8 weeks or hACE2 mice aged 12-16 weeks were immunized either by intramuscular injection after mixing with Imject™ (Thermo Fisher) (aluminum AL-adjuvant) in the hind limbs, or by nasal immunization (KFD) three times at 3-week intervals after mixing with flagellin-derived KFD protein adjuvant. They were anesthetized with pentobarbital sodium (50 mg / kg) prior to nasal immunization.

[0055] 1.6 Schematic diagram of the binding of SARS-CoV-2 RBD to neutralizing antibodies The simulated structures of the SARS-CoV-2 receptor-binding domain and the neutralizing antibodies B38, COV2-39, CR3022, and REGN10987 complexes were downloaded from the PDB database (PDB IDs: 7BZ5, 7JMP, 6W41, 6XDG) and imported using the Pymol software. The four RBD domains were docked to create 3D structures of SARS-CoV-2 RBDs bound to four different neutralizing antibodies.

[0056] 1.7 Enzyme-linked immunosorbent assay (ELISA) Antibody response was evaluated by ELISA. In short, the target protein (2 μg / mL) in carbonate-bicarbonate buffer was coated onto a 96-well plate overnight at 4°C, followed by blocking with 1% BSA at 37°C for 2 hours. Subsequently, samples diluted 4-fold in series were added to the well plates and left at 37°C for 2 hours. After washing, alkaline phosphatase-labeled secondary antibodies (goat anti-mouse IgG, human ads-AP antibody, or goat anti-mouse IgA-AP antibody, Southern Biotech) were added to the well plates, washed, and then colored with a substrate (p-nitrophenyl phosphate, Sigma). OD values ​​were read at 405 nm using an ELISA plate reader (Thermo Labsystems).

[0057] 1.8. Production and neutralization testing of pseudotyped viruses To produce SARS-CoV-2 spike pseudotype viruses, 60 μg of plasmid pNL4-3.luc.RE and 20 μg of different SARS-CoV-2 spike mutants were transfected with PEI into 15 cm cell culture dishes containing HEK293T cells. The supernatant was collected 72 hours after transfection, centrifuged at 1000 rpm, and stored at -80°C for use. To evaluate serum neutralization efficiency, samples were sequentially diluted from 1:10 to 1:3200 in DMEM supplemented with 10% FBS to a total volume of 50 μl, and then incubated at 37°C in 200×TCID in 20 μl of solution. 50 Incubated with SARS-CoV-2 pseudotype virus for 1 hour. Approximately 3 x 10⁶ wells were incubated in each well. 5 30 μl of complete medium containing ACE2-293T cells was added and incubated at 37°C under 5% CO2 conditions for 48 hours. Luciferase activity was analyzed using a luciferase assay system (Promega). Inhibition against SARS-CoV-2 pseudotyped virus is expressed as an inhibition percentage. The 50% neutralizing titer (NT50) was determined by 4-parameter logistic regression analysis using GraphPad Prism 8.0 (GraphPad Software Inc.).

[0058] 1.9, Antigen Mapping Using serum neutralizing titers for SARS-CoV-2 pseudoviruses (Alpha, Beta, Gamma, Delta, Omicron variants and Wuhan-Hu-1 strain) and SARS-CoV-1 pseudotype viruses, R pakages Racmacs ( https: / / acorg.github.io / Racmacs / index.html An antigen map was created using the following method. Antigen distance is measured in antigen units (AU). One AU corresponds to a 2-fold dilution of the antibody in the neutralization test. Each block in the mapping represents one AU. Antigen distance can be measured from any direction.

[0059] 1.10. Production and inoculation of SARS-CoV-2 Omicron variant strains SARS-CoV-2 Omicron strain BA.1 (IVCAS 6.7600) was provided by the National Center for Virus Resources (Wuhan, China). The virus was propagated in Cercopithecus aethiops kidney cells (Vero-E6, ATCC CRL-1586) and titrated. For vaccine protection experiments, 28 days after the final immunization, HFH4-hACE2 mice were given 50 μl of SARS-CoV-2 Omicron strain (5 × 10) under abertin (250 mg / kg) anesthesia. 4 TCID 50 The drug was administered intranasally. Three days after infection, the mice were euthanized, and lung and nasal turbinate tissue was collected. All procedures related to infectious SARS-CoV-2 were performed in a biosafety level 3 laboratory.

[0060] 1.11. Tissue collection and viral titration One day prior to use, Vero-E6 cells were inoculated into 24-well plates. Infected lung and nasal turbinate tissues were homogenized with DMEM and serially diluted 10-fold. Cells were inoculated with tissue diluent for 1 hour. The inoculation was then removed, and the cells were incubated with 0.9% methylcellulose for 5 days. After crystal violet staining, plaque counts were performed, and viral titers were calculated.

[0061] 1.12, Histology Mouse lungs were resected, fixed in 4% paraformaldehyde at room temperature for one week, and then embedded in paraffin. After sectioning, tissue sections were stained with hematoxylin and eosin (H&E). The entire section image was observed using a Panoramic MIDI (3DHISTECH) section scanner. Pathological changes were evaluated and scored on a severity scale from 1 to 4. The scoring criteria for inflammatory cell aggregation and interstitial pneumonia are as follows: Around the bronchioles, pulmonary vessels, or air gaps in lung sections, 1 represents normal, 2 represents mild and occasional cell aggregation, 3 represents moderate cell infiltration, and 4 represents moderate to severe and multifocal cell aggregation.

[0062] 1.13, Statistical analysis Mean, standard deviation, neutralizing titer, and correlation were calculated using GraphPad Prism 8.0 software. Significance tests were applied as shown in the legend for each figure. Unless otherwise specified, statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test. To analyze the difference between two groups, an independent Student's t-test was used for normally distributed data with variance, and the Mann-Whitney U test was used for non-normally distributed data. Simple linear regression was used for correlation analysis. Analysis was performed using GraphPad Prism 8.0 software. Significance values ​​are: * P<0.05, ** P<0.01, *** P<0.001 is expressed as ns, meaning it is not significant, and P<0.05 is considered significant.

[0063] 2. Experimental results 2.1 Neutralization epitope retention of the three RBD chimeric protein 3Ro-NC Figure 1 shows the schematic domain structure of three RBD chimeric proteins named KF, KFD, and 3Ro-NC. As shown in Figure 1, 3Ro-NC incorporates three immunodominant RBDs from Omicron and Delta, which are SARS-CoV-2 volatile organic compounds (VOCs), into NC. The chimeric protein 3Ro-NC contains NC, one RBD from the Delta mutant incorporated into the N-polypeptide and C-polypeptide binding sites of NC, and two RBDs from the Omicron mutant BA.1 (B.1.1.529) bound to the N-terminus and C-terminus of NC, respectively.

[0064] Figure 2 shows the 3D structure of 3Ro-NC predicted by Alpha Fold 2. As shown in Figure 2, the three RBDs exist in a stable conformation.

[0065] Figure 3 shows a Western blot image of the purified 3Ro-NC protein. Immunogen 3Ro-NC was expressed in 293F cells and purified to a single band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) verified that its molecular weight was approximately 120 kDa and its purity exceeded 95%.

[0066] Figure 4 shows the TLR5 agonist activity of recombinant proteins such as SARS-CoV-2 prototype RBD, Omicron RBD, P-KFD, and 3Ro-NC. IL8 production in cultured Caco-2 cell supernatant stimulated by recombinant proteins was detected by ELISA in three repeat samples from each group. P-KFD is a flagellin fusion protein expressed in an E. coli strain, as previously reported by the inventors (B. Zhao et al., A safe and effective mucosal RSV vaccine in mice consisting of RSV phosphoprotein and flagellin variant. Cell reports 36, 109401 (2021)). Unlike the previously produced E. coli strain flagellin fusion protein P-KFD(7), 3Ro-NC does not possess Toll-like receptor 5 (TLR5) activating activity.

[0067] Figure 5 shows a 3D schematic diagram of the binding of SARS-CoV-2 RBD to four types of neutralizing monoclonal antibodies. The four monoclonal antibodies (mAbs) B38, M-S309, CR3022, and COVA2-39 represent four RBD-specific neutralizing antibodies (B. He et al., Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq. Signal transduction and targeted therapy 6, 195 (2021).).

[0068] Figure 6 shows three ELISA results illustrating the binding affinity of Delta strain RBD (Rd), Omicron (B.1.1.529)BA.1 strain RBD (Ro), and 3Ro-NC to four representative neutralizing monoclonal antibodies: B38, M-S309, CR3022, and COVA2-39. As shown in Figure 6, the monomeric RBD of the Delta mutant (Rd) showed effective binding affinity to all four monoclonal antibodies, while the monomeric RBD of the Omicron mutant (Ro) showed significantly reduced binding affinity to mAbs CR3022 and M-S309, and lost binding affinity to B38 and COVA2-39. In contrast, 3Ro-NC showed high binding affinity to all four mAbs and higher affinity than Rd. These results indicate that the chimeric protein 3Ro-NC retains a good native RBD conformation.

[0069] Figure 7 shows the immunization and sampling scheme (5 mice / group). To analyze the immunogenicity of 3Ro-NC, BALB / c mice were immunized three times by intramuscular injection with 4 μg / dose of 3Ro-NC or SARS-CoV-2 protozoan (5.L. Dai et al., A Universal Design of Betacoronavirus Vaccines against COVID-19, MERS, and SARS. Cell 182, 722-733.e711 (2020)) RBD dimer and aluminum adjuvant (AL-adjuvant). Physiological saline alone was administered as a control. Mouse serum samples were collected 14 days after each immunization, and the fluid response was detected using ELISA.

[0070] Figure 8 shows a graph of the RBD-specific IgG response in serum. After the first and second immunizations, 3Ro-NC (3Ro-NC + AL im) induced a higher RBD-specific IgG response compared to RBD dimers (RBD dimer + AL im) not only against Omicron and Delta mutant RBDs but also against SARS-CoV-2 Gamma and prototype RBDs. Notably, after the first immunization, only 3Ro-NC induced a significant Omicron RBD-specific IgG response.

[0071] Figure 9 shows dot plots of neutralizing antibody titers after the second and third immunizations against SARS-CoV-2 mutants, as measured by pseudotyped virus assay. Neutralizing antibodies against SARS-CoV-2 VOCs were evaluated by pseudotyped virus assay. After the second and third immunizations, serum from 3Ro-NC immunized mice (3Ro-NC+AL im) was able to effectively neutralize all tested SARS-CoV-2 mutant pseudotyped viruses, and its neutralizing titer was higher than that of RBD dimer immunized mice (RBD dimer+AL im), although not statistically significant at all time points.

[0072] Figure 10 shows a dot plot of the results of neutralizing antibody titers after second and third immunizations against SARS-CoV-1, as detected by pseudotyped virus. After second and third immunizations, serum from 3Ro-NC immunized mice (3Ro-NC+AL im) was able to effectively neutralize SARS-CoV-1 pseudotyped virus, and its neutralizing titer was higher than that of RBD dimer immunized mice (RBD dimer+AL im), although not statistically significant at all time points.

[0073] Figure 11 shows a dot plot of the neutralizing antibody titers against Omicron and the original strain after the first immunization using pseudotyped virus detection. Note that only 3Ro-NC could induce significant neutralizing antibodies against Omicron and the original strain after the first immunization.

[0074] Figure 12 shows the geometric mean titer (GMT) diagram of neutralizing antibodies against different mutant strains. The geometric mean titers of the neutralizing antibodies further demonstrate that 3Ro-NC can induce an effective neutralizing antibody response against all tested SARS-CoV-2 mutants and SARS-CoV-1.

[0075] Figure 13 shows a dot plot of the ratio of the neutralizing antibody titer against Omicron (50% neutralizing titer, NT50) to the neutralizing antibody titer against the original strain or Delta. In 3Ro-NC immunized mice, the ratio of the neutralizing titer against the Omicron mutant to the neutralizing titers against the original strain and Delta mutant was significantly higher than in RBD dimmer immunized mice (6.1-fold and 10.7-fold, respectively, after the third immunization). This indicates that the antibody response induced by 3Ro-NC can easily neutralize Omicron, which may otherwise escape neutralization.

[0076] Figure 14 shows antigen maps generated from serum samples of the second (square) or third (triangle) vaccination groups of the RBD dimer (white) and 3Ro-NC (gray) groups. The inventors also investigated how serum samples distinguish between different spike antigens using the antigen maps. Antigen maps were created using neutralizing antibody titers after the second or third immunization. For the RBD dimer + AL im group, serum samples after the second and third vaccinations (Figure 14, top image) showed more tight agglutination around the prototype and Delta strains. Consistent with the neutralizing titers (Figures 9, 11, 12, and 13), the antigenic distance between Omicron and the prototype or Delta strain was larger for the RBD dimer-immunized serum of these two groups. In the 3Ro-NC+AL im group, serum samples after the second vaccination (Figure 14, lower left) still showed tight agglutination around the prototype and Delta strains, but the antigenic distance between Omicron and the prototype strain (a 9.2-fold difference) was closer than in the RBD dimer+AL im group (a 25.1-fold difference). Furthermore, after the third 3Ro-NC vaccination, the distance between Omicron and Delta decreased significantly from a 26.9-fold difference to a 3.5-fold difference (Figure 14, bottom). Moreover, after the third vaccination, the antigenic distances between Omicron and the prototype or Delta in the 3Ro-NC+AL im group (6.1-fold and 3.5-fold differences, respectively) were closer than in the RBD dimer+AL im group (a 55.7-fold and 48.5-fold difference) (Figure 14, right). The change in antigenic distance from Omicron indicates that the proportion of neutralizing antibodies against Omicron increased after the third immunization in the 3Ro-NC+AL im group. As these results clearly show, 3Ro-NC has excellent immunogenicity and can induce an effective and broad RBD-specific IgG response against SARS-CoV-2 mutants, and consequently against SARS-CoV-1, particularly highly immune-evading mutants.

[0077] Figure 15 shows the immunization and sampling scheme (n=5 mice per group) for antibody response in BALB / c mice immunized via nasal immunization with 3Ro-NC using KFD as an adjuvant. When developing effective mucosal vaccines, a suitable mucosal adjuvant is usually essential for subunit protein immunogens. In short, BALB / c mice were immunized via nasal immunization with 4 μg of 3Ro-NC and 1 μg of KFD adjuvant (3Ro-NC + KFD in), or three intramuscular injections with 4 μg of 3Ro-NC and 200 μg of AL-adjuvant (3Ro-NC + AL im). Saline alone was administered as a control. Mouse serum and mucosal samples were collected 14 days after each immunization, and the fluid response was detected using ELISA.

[0078] Figure 16 is a dot plot of the Delta, Gamma, or Omicron RBD-specific IgG response results in serum. As is clear from the results, the RBD-specific IgG response levels induced in the 3Ro-NC+KFD in immunization group after the second and third immunizations were similar to those of the 3Ro-NC+AL imization group, but significantly lower antibody titers were observed in the 3Ro-NC+KFD in group after the first immunization.

[0079] Figure 17 shows graphs of neutralizing antibody titers against SARS-CoV-2 variants or SARS-CoV-1, measured by pseudotyped virus, in serum after the second and third immunizations of the 3Ro-NC+KFD nasal spray group and the 3Ro-NC+AL intramuscular injection group. Each connecting line represents the neutralizing titer of individual serum samples against different virus strains. Serum neutralizing antibodies were evaluated using pseudotyped viruses. Generally, after the second and third immunizations, serum from 3Ro-NC+KFD-immunized mice can effectively neutralize all tested SARS-CoV-2 variants and, consequently, SARS-CoV-1, although the neutralizing titer against most variants is slightly lower than that of 3Ro-NC+AL-immunized mice. Notably, only in 3Ro-NC+KFD-immunized mice was the neutralizing titer against the Omicron variant equivalent to that against the Delta variant, and even higher than that against the original strain.

[0080] Figure 18 shows the geometric mean titers of neutralizing antibodies against different mutants in serum after the second and third immunizations of the 3Ro-NC+KFD nasal spray group and the 3Ro-NC+AL intramuscular injection group. Interestingly, the geometric mean neutralizing titer (GMT) against Omicron in the serum of 3Ro-NC+KFD in-immunized mice was higher than that of 3Ro-NC+AL im-immunized mice, particularly at the point after the second immunization.

[0081] Figure 19 shows a dot plot of the ratio of neutralizing antibody titers against Omicron to neutralizing antibody titers against Delta or the original strain. In 3Ro-NC+KFD in-immunized mice, the neutralizing titer ratios between the Omicron mutant and the original and Delta mutants were significantly higher than in 3Ro-NC+AL im-immunized mice (42-fold and 32-fold, respectively, after the second immunization).

[0082] Figure 20 shows antigen maps generated from serum samples of the second (square) or third (triangle) vaccination of the 3Ro-NC+KFD nasal spray group. Figure 21 shows antigen maps generated by the 3Ro-NC+KFD nasal spray group (brown) and the 3Ro-NC+AL intramuscular injection group (gray). Antigen maps are used to investigate how the serum of 3Ro-NC nasal-immunized mice distinguishes between different spike antigens. After the second or third immunization, antigen maps (Figures 20 and 21) were created using neutralizing antibody titers alone or together. For the 3Ro-NC+KFDi.n group, serum samples after both the second and third inoculations agglutinated tightly around the Delta and Omicron strains (Figures 20 and 21), while serum samples from the 3Ro-NC+AL im group all agglutinated tightly around the Delta strain (Figure 21). After the second vaccination, the antigenic distance between Omicron and Delta in the 3Ro-NC+KFD in group (a 6.1-fold difference) was closer than in the 3Ro-NC+AL im group (a 26.9-fold difference) (Figure 20, left panel). After the third vaccination, the antigenic distance between Omicron and the original strain or Delta in the 3Ro-NC+KFD in group (differences of 2.8-fold and 1.7-fold, respectively) was still closer than in the 3Ro-NC+AL im group (differences of 6.1-fold and 3.5-fold, respectively) (Figure 20, right panel). The difference in antigenic distance to Omicron reflects the tendency for 3Ro-NC+KFD in immunization to induce a stronger neutralizing antibody response against the Omicron variant than 3Ro-NC+AL im immunization. These findings clearly demonstrate that changes in the immune pathway and adjuvant type alter the specificity of the antibody response.

[0083] Figures 22, 23, and 24 show dot plots of RBD-specific mucosal IgA responses in saliva, vaginal douche, and nasal turbinate lavage, respectively. As expected, the evaluation of mucosal immune responses was as predicted, with only nasal immunization (3Ro-NC + KFD in) inducing specific mucosal IgA antibody responses to Omicron and Delta RBD in saliva (Figure 22). Similar levels of IgA responses were also detected in vaginal douche (Figure 23) and nasal lavage (Figure 24, right panel) after a third intranasal immunization. Comparing different vaccination routes, similar titers of RBD-specific IgG (approximately 102) were detected in nasal lavage from intramuscular (3Ro-NC + AL im) and nasal (3Ro-NC + KFD in) immunized mice (Figure 24, left panel).

[0084] Figures 25, 26, and 27 show the correlation between RBD-specific IgA in nasal lavage fluid and RBD-specific IgA in saliva (triangles represent Delta, squares represent Omicron), the correlation between RBD-specific IgG in nasal lavage fluid and RBD-specific IgG in serum (triangles represent Delta, squares represent Omicron), and the correlation between RBD-specific IgA and IgG in nasal lavage fluid and the neutralizing antibody response of BALB / c mice immunized with 3Ro-NC as measured by a pseudotyped virus system. A high correlation was observed between RBD-specific IgA titer in nasal lavage fluid and titer in saliva (Figure 25), indicating that the salivary IgA response can reflect the IgA response in the upper respiratory tract. Such detectable low-titer RBD-specific mucosal IgG is shown to be highly correlated with high-titer RBD-specific serum IgG (Figure 26). Interestingly, the serum neutralizing titer of Omicron was shown to be significantly correlated with the Omicron RBD-specific nasal IgA response or nasal IgA and IgG levels, but unrelated to the nasal IgG response (Figure 27). These data suggest that RBD-specific antibodies, particularly mucosal IgA, may play a crucial role in preventing initial SARS-CoV-2 infection in the nasal mucosa.

[0085] Overall, 3Ro-NC exhibits high immunogenicity through nasal immunization with KFD as an adjuvant, and can induce coordinated systemic and local mucosal immune responses against different SARS-CoV-2 variants, particularly Omicron.

[0086] Figure 28 shows the immunization and viral attack scheme (6-8 mice / group) for studying the protective effect of 3Ro-NC-immunized hACE2 mice against infection by SARS-CoV-2 Omicron variants. To further investigate the protective effect of the 3Ro-NC vaccine, the mucosal protective effect against infection by SARS-CoV-2 Omicron variants was evaluated using human ACE2 gene-modified mice (hACE2). In short, mice were immunized either via the intranasal route (3Ro-NC + KFD in) with 3Ro-NC and KFD, or via the intramuscular route (3Ro-NC + AL im) with 3Ro-NC and AL-adjuvant, or via intramuscular injection route (IAV + AL im) with 2.5 μg of inactivated SARS-CoV-2 and 200 μg of AL adjuvant, with a control group inoculated with physiological saline.

[0087] Figures 29, 30, and 31 show dot plots of Omicron, Delta, and Gamma RBD-specific serum IgG titers, dot plots of Delta and Omicron RBD-specific salivary IgA, and dot plots of Delta and Omicron RBD-specific vaginal douche IgA titers after a third immunization, respectively. Consistent with the results for BALB / c mice, nasal immunization (3Ro-NC+KFD in) and intramuscular injection immunization (3Ro-NC+AL im) induced strong serum IgG antibody responses against Omicron, Delta, and Gamma RBD in mice. Of the three immunization groups, the IAV+AL im group produced the lowest RBD-specific serum IgG, while the 3Ro-NC+KFD in group produced the highest Omicron RBD-specific serum IgG (Figure 29). In saliva (Figure 30) and vaginal douche (Figure 31), RBD-specific IgA was induced and produced only in mice in the 3Ro-NC+KFD in group.

[0088] Figure 32 shows dot plots of the RNA copy numbers of the SARS-CoV-2 RBD gene in the turbinate and lung by qPCR assay, and plaque measurements of infectious virus in the lung 3 days after infection, respectively. 28 days after the third immunization, 5 × 10 4 individuals of TCID 50 mice were intranasally challenged with the Omicron variant. After exposure to the Omicron variant for 3 days, the viral loads in the turbinate tissue and lung were measured by qPCR and plaque assay. Compared with the saline group or the other two intramuscular injection immunization groups, only the 3Ro-NC+KFD i.n group showed a significant decrease in virus in the turbinate. In the lung tissue, the viral copy numbers in both the IAV+AL i.m group and the 3Ro-NC+KFD i.n group were significantly decreased (Figure 32, middle figure). In the 3Ro-NC+AL i.m group, only some mice showed a decrease in the Omicron virus amount in the lung tissue, but the Omicron RBD-specific serum IgG titer in this group was even higher than that in the IAV+AL i.m group. Compared with the saline control group, almost no infectious virus was detected in the lung tissue of all three immunization groups except for one mouse in the 3Ro-NC+AL i.m group (Figure 32, right figure).

[0089] Figure 33 shows hematoxylin and eosin (H&E) stained images of lung sections (scale 100 μm). Figure 34 shows dot plots of pathological and infiltration scores of immune cell aggregation around bronchioles, pulmonary blood vessels, and interstitial pneumonia based on H&E stained sections (6-8 mice / group). Histopathological examination was also performed to analyze infection and immune-related inflammation in the lungs after viral attack (Figure 33). As a result, widespread inflammatory cell infiltration was observed in the lungs of IAV-immunized (IAV+AL im) mice, particularly in the perivascular areas (Figure 33). Conversely, inflammatory cell infiltration in the lungs of 3Ro-NC+KFD in or 3Ro-NC+AL im immunized mice was significantly reduced. As these results clearly show, nasal immunization with 3Ro-NC and KFD adjuvant can limit infection and pathology in the lungs, demonstrating that 3Ro-NC and KFD can provide protection against omicron infection in the upper and lower respiratory tracts as a prophylactic mucosal SARS-CoV-2 vaccine.

[0090] Figure 35 shows the correlation diagram of RNA copy numbers in the lungs and nasal turbinates. In all vaccinated groups, a protective effect against Omicron infection in the alveolar or vascular portions (Figure 35) could be observed to a greater or lesser extent. However, protective effects against infection in the lungs and nasal turbinates (Figure 32) could only be observed in mice that underwent nasal immunization, which produced protective serum IgG and mucosal IgA. This raised the question of whether mucosal IgA and IgG exert different protective effects in the upper and lower respiratory tracts. Therefore, no significant correlation between viral copy numbers and lung tissue and nasal turbinates was observed in the experiment (Figure 36). Because nasal or bronchoalveolar lavage fluid was insufficient before the mice were sacrificed, the inventors selected salivary IgA and serum IgG, which correlated highly with nasal IgA and IgG levels, respectively, as representatives of the humoral immune response. In lung tissue, viral RNA copy number shows a negative correlation with serum IgG titer but is unrelated to salivary IgA titer (Figure 36). On the other hand, viral RNA copy number in nasal turbinate tissue shows a negative correlation only with salivary IgA titer and is unrelated to serum IgG titer (Figure 37). These findings indicate that protection to the lungs and nasal turbinates is mainly provided by the RBD-specific IgG response and mucosal IgA, respectively.

[0091] Figure 38 is a dot plot showing the serum RBD-specific IgG response after the second immunization. Figure 39 is a dot plot showing the neutralizing antibody titers after the second immunization against the SARS-CoV-2 variant Omicron or SARS-like viruses SHC014 and WIV1.

[0092] The foregoing are merely preferred embodiments of the present invention and do not limit the present invention in any form or substantially. Those skilled in the art can make some improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any equivalent transformations of some modifications, modifications and evolutions that can be made by those skilled in the art using the technical content disclosed herein without departing from the spirit and scope of the present invention are all equivalent embodiments of the present invention. Furthermore, any equivalent modifications, modifications and evolutions of the above embodiments based on the essential art of the present invention are all within the scope of the technical means of the present invention.

Claims

1. A recombinant polyvalent vaccine comprising a recombinant protein, wherein the recombinant protein comprises a first antigen peptide, an N-polypeptide, a second antigen peptide, a C-polypeptide, and a third antigen peptide, arranged from the N-terminus to the C-terminus. The first, second, and third antigen peptides are the receptor-binding domain-RBD region aa. 319-527 of the spike S protein of the SARS-CoV-2 variant, and the RBD is derived from one of the following: SARS-CoV-2 prototype, Delta, Omicron, Gamma variants, SHC014, and WIV1 SARS-related coronavirus strains. The N-polypeptide has an amino acid sequence shown in SEQ ID NO. 1 or a variant that has the same function as SEQ ID NO. 1 and exhibits at least 95% sequence identity. The C-polypeptide has an amino acid sequence shown in SEQ ID NO. 3 or a variant that has the same function as SEQ ID NO. 3 and at least 95% sequence identity. The N-polypeptide and C-polypeptide form an intramolecular skeleton NC, which supports and stabilizes the conformations of the first antigen peptide, the second antigen peptide, and the third antigen peptide. A recombinant polyvalent vaccine characterized by the following features.

2. Delta RBD has the amino acid sequence shown in SEQ ID NO. 5, Omicron RBD has the amino acid sequence shown in SEQ ID NO. 7, SARS-CoV-2 prototype RBD has the amino acid sequence shown in SEQ ID NO. 9, Gamma RBD has the amino acid sequence shown in SEQ ID NO. 11, SHC014 RBD has the amino acid sequence shown in SEQ ID NO. 13, and WIV1 RBD has the amino acid sequence shown in SEQ ID NO.

15. The recombinant polyvalent vaccine according to claim 1.

3. The recombinant protein comprises Omicron RBD, N-polypeptide, Delta RBD, C-polypeptide, and Omicron RBD from the N-terminus to the C-terminus, and has the amino acid sequence shown in SEQ ID NO. 17, or the recombinant protein comprises Omicron RBD, N-polypeptide, SHC014 RBD, C-polypeptide, and WIV1 RBD from the N-terminus to the C-terminus, and has the amino acid sequence shown in SEQ ID NO.

19. The recombinant polyvalent vaccine according to claim 1.

4. The present invention further comprises an adjuvant, the adjuvant being selected from at least one of flagellin, polyinosine / polycytidic acid, MF59, AS01, AS03, AS04, CpG, MPL, CT, CTB, IL-1α, IL-2, IL-12, IL-18, GM-CSF, PIKA, and BFA03. The recombinant polyvalent vaccine according to claim 1.

5. A nucleic acid molecule encoding a recombinant protein for a recombinant polyvalent vaccine, wherein the nucleic acid molecule comprises, from 5' to 3', a first polynucleotide encoding a first antigen peptide, a polynucleotide encoding an N-polynucleotide, a second polynucleotide encoding a second antigen peptide, a polynucleotide encoding a C-polynucleotide, and a third polynucleotide encoding a third antigen peptide. The first, second, and third polynucleotides are the receptor-binding domain-RBD region aa. 319-527 of the spike S protein of the SARS-CoV-2 mutant, and the RBD is derived from one of the following: SARS-CoV-2 prototype, Delta, Omicron, Gamma, SHC014, and WIV1 SARS-related coronavirus strain mutants. The polynucleotide encoding the N-polypeptide has a nucleotide sequence shown in SEQ ID NO. 2 or a variant that is functionally identical to SEQ ID NO. 2 and has at least 95% sequence identity. The polynucleotide encoding the C-polypeptide has a nucleotide sequence shown in SEQ ID NO. 4 or a variant that is functionally identical to SEQ ID NO. 4 and has at least 95% sequence identity. When a recombinant protein encoded by the nucleic acid molecule is expressed, the N-polypeptide and C-polypeptide form an intramolecular backbone NC, which supports and stabilizes the conformations of the first antigen peptide, the second antigen peptide, and the third antigen peptide. A nucleic acid molecule characterized by the following features.

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