Broad-spectrum Anti-covid-19 vaccine immunogen composition, and preparation and use thereof

Abstract

Provided are a broad-spectrum anti-COVID-19 vaccine immunogen composition, and the preparation and a use thereof. Specifically, provided is an immunogen composition, comprising an RBD recombinant chimeric antigen, wherein the chimeric antigen comprises S protein RBD domains from two or more COVID-19 subtypes, or functional fragments thereof. The immunogen composition can further comprise a multimerization domain and a T-cell immunogen domain. Further provided are a use of the immunogen composition, a corresponding encoding molecule therefor, a vector, and / or a host cell in preparing an anti-COVID-19 vaccine. The vaccine can induce a broad-spectrum protective effect against a prototype strain and various currently prevalent variants, and can also elicit a potent cross-protective T-cell response, thereby achieving effective and broad-spectrum prevention against COVID-19 and achieving a protective effect against other coronaviruses.

Description

A broad-spectrum anti-COVID-19 vaccine immunogen composition, its preparation and application TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biotechnology and vaccines, especially the field of genetically engineered drugs and vaccine manufacturing. Specifically, the present disclosure relates to a broad-spectrum anti-COVID-19 vaccine immunogen composition that can simultaneously induce humoral immune response and cellular immune response, its preparation and application in the prevention and treatment thereof against COVID-19. BACKGROUND

[0002] So far, three highly pathogenic human coronaviruses (CoVs) have been identified, including Middle East Respiratory Syndrome (MERS) coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS) coronavirus (SARS-CoV) and novel coronavirus (SARS-CoV-2, referred to as new coronavirus), among which the human-to-human transmission rate of the new coronavirus has exceeded that of SARS-CoV and MERS-CoV.

[0003] The disease caused by the new coronavirus has brought great challenges to public health. Achieving herd immunity through mass vaccination is the most effective method to prevent the infectious disease. Therefore, developing vaccines and drugs to prevent and treat the new coronavirus is a key problem to be solved.

[0004] More and more evidence shows that although vaccination can induce certain humoral response, effective neutralizing antibodies will rapidly weaken over time. In addition, with the frequent mutation of the Spike protein, a series of newly emerging Omicron variants have been discovered recently, which have the characteristics of evading humoral immunity induced by vaccination or natural infection, which may lead to a decrease or disappearance of the preventive effect of existing vaccines. Therefore, it is of great significance to develop effective new COVID-19 vaccines.

[0005] Coronaviruses contain four structural proteins, including spike protein (S protein), envelope protein, membrane protein and nucleocapsid protein. Among them, S protein plays the most important role in the attachment, fusion and entry process of the virus, and is also the main target of antibodies, entry inhibitors and vaccines. S protein mediates viral entry into host cells, first by the receptor binding domain (RBD) of the S1 subunit binding to host receptors, and then by the S2 subunit fusing the virus and host cell membranes.

[0006] Currently authorized vaccines are all based on Spike (S) protein or its receptor binding domain (RBD) to induce potent neutralizing antibodies to prevent virus entry into host cells. Compared with S protein, RBD antigen structure is less complex, and the epitopes shielded in S protein are better exposed in the form of RBD. Therefore, RBD is the most commonly used immunogen for SARS-CoV-2 vaccine in addition to full-length S sequence. Because the RBD monomer is small, its immunogenicity is poor. To improve its immunogenicity, a large number of studies have increased the size of RBD molecules through multimerization strategies. These include dimer or trimer vaccines of tandem RBD; dimer vaccines of RBD and Fc sequence fusion; multimer vaccines of RBD and different trimerization domains fusion; RBD nanoparticle vaccines, etc.

[0007] An ideal vaccine response should be able to activate both neutralizing antibodies and T cell immune responses, especially long-lasting memory B / T cell responses. Studies have shown that a strong T cell response can provide effective immune protection, and even in the absence of neutralizing antibodies, effective virus-specific T cells are essential for viral clearance. However, the currently marketed or in development COVID-19 vaccines all focus on inducing broad-spectrum neutralizing antibody responses. Some studies have reported that vaccines designed with mutant sequences as targets can induce strong immune responses to variant strains, but have weak cross-reactivity to other strains, new variants or prototype strains.

[0008] Therefore, in order to cope with the current emerging SARS-CoV-2 variants causing epidemic diseases and possibly further increasing the types of human coronavirus, there is an urgent need to develop a broad-spectrum COVID-19 coronavirus vaccine that can cope with current or future multiple epidemic variants.

[0009] SUMMARY

[0010] A broad-spectrum COVID-19 coronavirus immunogenic composition capable of coping with current or future multiple epidemic variants is provided in the present application. A SARS-CoV-2 immunogenic composition capable of inducing both neutralizing antibodies and T cell immune responses, its preparation method and application are further provided in the present application. The immunogen inducing neutralizing antibodies in the present application is a RBD multimerization chimeric antigen derived from different variants of SARS-CoV-2; the T cell immunogen inducing cellular responses is a recombinant highly conserved antigen CoV-T derived from endogenous proteins of SARS-CoV-2 and designed by immunoinformatics method. The neutralizing antibody immunogen and T cell immunogen in the present application can be loaded into the same carrier, so as to achieve both broad-spectrum protection effect against prototype strains and various variants currently prevalent, and strong T cell response, thereby achieving effective prevention and / or treatment of COVID-19.

[0011] In some aspects of the present application, there is provided an immunogenic composition comprising a RBD recombinant chimeric antigen, wherein the chimeric antigen comprises S protein RBD domains or functional fragments thereof from two or more SARS-CoV-2 variants.

[0012] In some embodiments, the immunogenic composition further comprises one or more domains selected from the group consisting of: multimerization domains; T cell immunogens.

[0013] In some aspects of the present application, there is provided a polynucleotide molecule encoding the immunogenic composition of the present application, preferably codon-optimized.

[0014] In some aspects of the present application, there is provided a vector and / or host cell comprising the polynucleotide molecule of the present application.

[0015] In some aspects of the present application, there is provided a vaccine against the novel coronavirus SARS-CoV-2 comprising the immunogenic composition, polynucleotide molecule, vector and / or host cell described herein.

[0016] In some aspects of the present application, there is provided the use of the immunogenic composition, polynucleotide molecule, vector and / or host cell described herein in the manufacture of a vaccine for the prevention or treatment of the novel coronavirus SARS-CoV-2.

[0017] In some aspects of the present application, there is provided a method of manufacturing a vaccine against the novel coronavirus SARS-CoV-2, the method comprising:

[0018] (a) providing the immunogenic composition, polynucleotide molecule, vector and / or host cell described herein;

[0019] (b) combining the active substance provided in (a) with an immunologically or pharmaceutically acceptable carrier.

[0020] The person skilled in the art can combine the aforementioned technical solutions and technical features in any manner without departing from the inventive concept and the scope of protection of the present disclosure. Other aspects of the present disclosure will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0021] The present disclosure is further described below in conjunction with the accompanying drawings, which are shown only for the purpose of illustrating embodiments of the present disclosure and not for limiting the scope of the present disclosure.

[0022] Figure 1: Construction of multimeric chimeric antigens for Omicron variants, including:

[0023] Figure 1A: SARS-CoV-2 Omicron variant BA.2-BA.2.75-BA.4 / 5-foldon construct (abbreviated as BA.2 trimer);

[0024] Figure 1B: BA.2-BA.2.75-BA.4 / 5-foldon-CoV-T construct (abbreviated as BA.2 trimer-CoV-T);

[0025] Figure 1C: BA.2-XBB.1.5-BA.4 / 5-foldon-CoV-T construct (abbreviated as XBB.1.5 trimer-CoV-T).

[0026] Figure 2: Expression verification of BA.2 trimer (Figure 2A), BA.2 trimer-CoV-T (Figure 2B), XBB.1.5 trimer-CoV-T mRNA (Figure 2C) after transfection of HEK293T cells.

[0027] Figure 3: Immunogenicity evaluation of BA.2 trimer, BA.2 trimer-CoV-T, XBB.1.5 trimer-CoV-T mRNA vaccines in BALB / c mice:

[0028] The mice used in the experiment were 6-8 week old female BALB / c mice, and the immunogens were 3 kinds of mRNA vaccines. Two weeks after the first boost (Boost-1 2w) and two weeks after the second boost (Boost-2 2W), the ELISA method was used to detect the specific binding antibody titers of different subtypes of RBD in the serum of mice, Figure 3A (BA.2 trimer), Figure 3B (BA.2 trimer-CoV-T), Figure 3C (XBB.1.5 trimer-CoV-T).

[0029] ns or no label: indicates not significant; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0030] Figure 4: BA.2 trimer-CoV-T mRNA vaccine was inoculated in 6-8 week old female C57BL / 6 mice, and 10 days after the second boost, the T cell response level in the spleen of mice was detected by ELISpot:

[0031] The vertical coordinate is the number of IFN-γ secreting cells per million spleen cells, and the horizontal coordinate is respectively: the immunization group (Figure 4A), the name of the single peptide pool of RBD protein (Figure 4B), and the name of the single peptide pool of CoV-T protein (Figure 4C);

[0032] ns or no label means not significant; * means p<0.05; ** means p<0.01.

[0033] Figure 5: Immunogenicity of BA.2 trimer-CoV-T mRNA vaccine and protection test against SARS-CoV-2 virus and different SARS-CoV-2 mutant strains including BA.2, BF.7, XBB.1 in k18-hACE2 transgenic mice:

[0034] Figure 5A: Vaccination and challenge procedure;

[0035] Figure 5B: Binding antibody titers in Empty-LNP group and BA.2 trimer-CoV-T mRNA vaccine group;

[0036] Figure 5C: Neutralizing antibody titers in Empty-LNP group and BA.2 trimer-CoV-T mRNA vaccine group;

[0037] Figure 5D: Body weight changes of mice after challenge in Empty-LNP group and BA.2 trimer-CoV-T mRNA vaccine group;

[0038] Figure 5E: Survival rate of mice after challenge in Empty-LNP group and BA.2 trimer-CoV-T mRNA vaccine group.

[0039] ns or no label means not significant; * means p<0.05; ** means p<0.01; *** means p<0.001; **** means p<0.0001. DETAILED DESCRIPTION

[0040] The present disclosure relates to the field of vaccines, and provides an anti-COVID-19 immunogenic composition capable of inducing both humoral and cellular immune responses, a preparation method and application thereof. Specifically, the immunogen for inducing neutralizing antibodies in the present disclosure is a RBD multimerization chimeric antigen derived from different mutant strains of SARS-CoV-2, and the T cell immunogen for inducing cellular responses is a recombinant sequence derived from the conserved region of the endogenous protein of SARS-CoV-2. The neutralizing antibody immunogen and T cell immunogen in the present disclosure can be loaded into the same vector, thereby achieving not only broad-spectrum protection against the prototype strain and the currently popular various mutant strains, but also strong and effective cross-protective T cell responses, so as to not only achieve broad-spectrum and effective prevention of COVID-19, but also have a protective effect on other coronaviruses.

[0041] All numerical ranges herein are intended to expressly include all values and ranges subsumed therein. The features or embodiments of the disclosure mentioned can be combined. All features disclosed in the specification may be used in any combination, each feature disclosed in the specification may be used in place of an alternative feature disclosed in the specification, to provide an equivalent or similar purpose. Thus, unless specifically noted, the features disclosed are merely exemplary of the generic features that can be used in the disclosure.

[0042] As used herein, "about" in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed.

[0043] It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the application. For example, "0.1-2.5 mg / day" includes 0.1 mg / day, 0.2 mg / day, 0.3 mg / day, etc. up to 2.5 mg / day.

[0044] As used herein, "containing", "having", or "including" encompasses "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of", "consisting essentially of", and "consisting of" are subsumed by "containing", "having", or "including".

[0045] RBD immunogenic peptides, recombinant chimeric antigens and multimerization thereof

[0046] As used herein, the term "RBD domain or functional fragment thereof" refers to a peptide comprising the RBD region of the SARS-CoV-2 viral spike protein S or a modified RBD region (e.g. cysteine modification, also referred to herein as sRBD region) and having the effect of eliciting binding and neutralizing antibodies.

[0047] In some embodiments of the disclosure, the RBD domain or functional fragment thereof can be from the SARS-CoV-2 prototype or different variants, including but not limited to: SARS-CoV-2 prototype strain, SARS-CoV-2 variant strain Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1), Delta (B.1.617.2), Omicron subtypes BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, BA.4, BA.5, XBB, XBB.1.5, XBB.1.6, BQ.1, BQ.1.1, BF.7, EG.5, BA.2.86.

[0048] In some embodiments, the RBD domain or functional fragment thereof is selected from the group consisting of: (a) a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4; (b) a homologous polypeptide of the polypeptide described in (a), for example, having a homology higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than or equal to 99% to SEQ ID NO: 1, 2, 3, or 4; (c) a protein or polypeptide derived from (a) with one or several amino acids substituted, deleted or added in the amino acid sequence defined in (a) and having immunogenicity.

[0049] The RBD recombinant chimeric antigen is obtained by recombining RBD domains or functional fragments thereof from different SARS-CoV-2 subtypes of viruses. As used herein, the terms “RBD recombinant chimeric antigen” and “RBD chimeric antigen” are used interchangeably to refer to a chimeric antigen comprising two or more, for example, three, four, five, six, etc., RBD domains or functional fragments thereof from different SARS-CoV-2 subtypes of viruses.

[0050] In some embodiments, the RBDs in the chimeric antigen can be directly connected or connected through a connecting peptide, for example, one or more connecting peptides selected from the group consisting of: (G4S) n (n = 1-8, for example, (G4S)3, G4S, GSAGSAAGSGEF, (Gly)6, EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly)8, EGKSSGSGSESKST. In some embodiments, the connecting peptide used comprises an amino acid sequence set forth in SEQ ID NO: 5, 6, or 7.

[0051] In some embodiments, the RBD recombinant chimeric antigen comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 4, for example, a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1, 2, and 4 or SEQ ID NO: 1, 3, and 4. In some embodiments, the RBD recombinant chimeric antigen comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1, 2, and 4 or SEQ ID NO: 1, 3, and 4, wherein the different RBD sequences are connected with a connecting peptide set forth in SEQ ID NO: 5 or 6.

[0052] In some preferred embodiments, the RBD recombinant chimeric antigen of the present application is further linked to a multimerization domain, thereby forming a RBD multimerization chimeric antigen. In some embodiments, the multimerization is a trimer, hexamer, or nonamer, etc. formed by multimerization. In some embodiments, the multimerization domain comprises Fc domain derived from antibody, different trimerization motif such as foldon domain of T4 phage fibritin protein, GCN4IZ leucine zipper domain, human collagen trimerization domain or cartilage matrix protein domain and mutants of these domains, and further preferably foldon domain, such as the amino acid sequence shown in SEQ ID NO: 8.

[0053] In some preferred examples of the present application, an anti-COVID-19 immunogenic composition is provided, which induces neutralizing antibodies, and the immunogen is derived from RBD multimerization chimeric antigens of different variants of SARS-CoV-2. Wherein the multimerization chimeric antigen is arranged in the order of “A-Linker1-B / C-Linker2-D-Linker3-multimerization domain” by appropriate linker sequences.

[0054] Specifically, A represents the amino acid sequence of the S protein RBD domain of the novel coronavirus Omicron variant BA.2 strain or a part thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity as it, and further preferably the amino acid sequence shown in SEQ ID NO: 1;

[0055] B represents the amino acid sequence of the S protein RBD domain of the novel coronavirus Omicron variant BA.2.75 strain or a part thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity as it, and further preferably the amino acid sequence shown in SEQ ID NO: 2

[0056] C represents the amino acid sequence of the S protein RBD domain of the novel coronavirus Omicron variant XBB.1.5 strain or a part thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity as it, and further preferably the amino acid sequence shown in SEQ ID NO: 3;

[0057] D represents an amino acid sequence of the S protein RBD domain of the new coronavirus Omicron variant BA.4 or BA.5 strain or a part thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity as the same, and is further preferably an amino acid sequence as shown in SEQ ID NO: 4;

[0058] Optionally, the length of the linker is 1-24 amino acids. The linker 1, the linker 2 and the linker 3 are the same or different, wherein glycine and serine which can provide softness and / or glutamic acid and lysine which can improve water solubility, etc. are mainly derived, such as the following sequences including EGKSSGSGSESKST, GSAGSAAGSGEF, KESGSVSSEQLAQFRSLD, (GGGS) n an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity as the same; wherein n = 0, 1, 2, 3, 4 or 5;

[0059] Further, the linker 1 and the linker 2 are preferably sequences containing glycine, serine, glutamic acid and lysine, and further preferably the amino acid sequence EGKSSGSGSESKST as shown in SEQ ID NO: 5, the amino acid sequence GSAGSAAGSGEF as shown in SEQ ID NO: 6, and the order of the linker 1 and the linker 2 can be in front or in back;

[0060] Further, the linker 3 is preferably a glycine and serine amino acid sequence which can provide softness, further preferably n = 1, and preferably the amino acid sequence GGGS as shown in SEQ ID NO: 7;

[0061] Optionally, the multimerization domain includes an Fc domain derived from an antibody, a different trimer motif such as the foldon domain of the T4 phage fibritin protein, a GCN4IZ leucine zipper domain, a human collagen trimerization domain or a cartilage matrix protein domain, and mutants of these domains, and is further preferably a foldon domain, such as the amino acid sequence as shown in SEQ ID NO: 8;

[0062] Further, in the preferred specific embodiments, the order of A, B / C, D can be changed to the following sequences, A-B-D, A-C-D, D-B-A, D-C-A;

[0063] Still more preferably, the chimeric antigens of the Omicron variant multimerization are arranged in the order of A-B-D and / or A-C-D, including the amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10.

[0064] T cell immunogens and their combinations with RBD recombinant chimeric antigens

[0065] In order to enable the immunogen compositions of the present application to further elicit T cell responses against COVID-19, T cell immunogens can also be included in the immunogen compositions of the present application.

[0066] T cell immunogens that can be used in the immunogen compositions of the present application can be obtained by a method comprising:

[0067] i) performing big data analysis on one or more proteins of a coronavirus (such as the virus causing COVID-19) selected from the group consisting of coronavirus early expression protein polyprotein (ORF lab), coronavirus membrane protein (M), coronavirus nucleocapsid (N) protein, coronavirus envelope (E) protein and coronavirus spike (S) protein, extracting their shared peptide sequence information by, for example, sequence similarity alignment;

[0068] ii) performing T cell epitope prediction on the resulting shared peptide sequence information to obtain information of conserved regions with immunogenicity;

[0069] iii) designing and preparing immunogenic peptides or their coding sequences according to the information of conserved regions with immunogenicity;

[0070] iv) optionally, combining the resulting immunogenic peptides or their coding sequences or combining them with other sequences co-expressed (such as fusion expression or separate reading frame expression).

[0071] In the method, step i) can comprise aligning different coronavirus types (such as SARS-CoV, MERS-CoV and SARS-CoV-2 viruses) and / or different subtypes of the same coronavirus (such as

[0072] In the method, step ii) can comprise predicting polypeptides that can be strongly bound by HLA-I supertype molecules, i.e. polypeptides that can be presented, in different proteins by using online tools.

[0073] In the method, step iii) can comprise mapping the shared peptides into the SARS-CoV-2 virus and further analyzing the enrichment region to find regions recommended by T cell epitope prediction results to have high immunogenicity, and truncating them to form recombinant amino acid sequences, such as the CoV-T amino acid sequence as shown in SEQ ID NO: 11.

[0074] In the method, the other sequences in step iv) can include, but are not limited to, RBD recombinant chimeric immunogens in the present application, other polypeptides for expanding the antiviral spectrum, improving the ability of immune response induction, and the like.

[0075] Further, the T cell epitopes involved in the method can be a plurality of different shared sequences, which are mainly connected in a tandem form, and a flexible linker or a rigid linker can be added. The T cell epitopes can be predicted by bioinformatics methods such as artificial neural network (ANN), and the prediction software is known in the art and includes but is not limited to the tools provided by http: / / www.bio.med.ucm.es / episopt.html, http: / / www.ddg-pharmfac.net / mhcpred / MHCPred, http: / / www.syfpeithi.de / software, etc.

[0076] In some embodiments, the T cell immunogen used in the present application, CoV-T, comprises an amino acid sequence as shown in SEQ ID NO: 11, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto and has T cell immunogenicity. In some embodiments, the CoV-T used can be further connected with a tag (such as a flag tag) to facilitate subsequent purification, detection, localization, etc., for example, the CoV-T amino acid sequence with a flag tag can be as shown in SEQ ID NO: 21.

[0077] In some cases, the immunogen composition can include other moieties to, for example, enhance the stability of the RBD region, increase the neutralizing antibody response, form a multimer, increase the cellular response, etc. The moieties connected to the RBD region, which can be modified or unmodified, include but are not limited to: proteins of viral or host origin, transferrin (Fn), HIV p24, the stem of a capsid virus, such as influenza HA2, HIV gp41, antibody Fc fragment, GM-CSF, IL-21, CD40L or CD40 antibody, etc.

[0078] In some embodiments, a connecting peptide can be used to connect CoV-T with other units or elements in the immunogen composition, such as RBD recombinant chimeric antigens and / or multimerization domains. For example, one or more connecting peptides selected from the group consisting of: (G4S) n(n = 1-8, e.g., (G4S)3, G4S, GSAGSAAGSGEF, (Gly)6, EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly)8, EGKSSGSGSESKST, IRES, P2A, T2A. In some embodiments, the linker peptide used comprises an amino acid sequence set forth in SEQ ID NO: 5, 6, 7, or 12. In some embodiments, a T2A self-cleaving peptide is used to link the CoV-T domain to the multimerization domain Foldon linked to the RBD recombinant chimeric antigen. In some embodiments, the linker sequence is preferably a 2A self-cleaving peptide, including but not limited to F2A derived from Foot-and-mouth disease virus, E2A derived from Equine rhinitis A virus, P2A derived from Porcine teschovirus, T2A peptide derived from Thosea asigna virus, and more preferably a T2A peptide cleavage sequence, with an amino acid sequence set forth in SEQ ID NO: 12.

[0079] In some embodiments, the preferred immunogenic composition in the present application has an amino acid sequence set forth in SEQ ID NO: 13 or 14, comprising the RBD recombinant chimeric antigen-foldon multimerization domain-CoV-T domain.

[0080] In some embodiments, the immunogen inducing neutralizing antibodies and the T cell immunogen inducing cellular responses are arranged in tandem in a single-cistronic sequence, so that translation is from the same transcript. Preferably, the immunogen inducing neutralizing antibodies is located before the T cell immunogen.

[0081] Immunogenic peptides can also include variants thereof, such as deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids, as well as additions of one or several (generally within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or the N-terminus. For example, substitutions with similar or identical properties in the art do not generally change the function of a protein or polypeptide. For another example, additions of one or several amino acids at the C-terminus and / or the N-terminus also generally do not change the function of a protein or polypeptide.

[0082] The immunogenic peptides can be produced by recombinant expression in appropriate environments and conditions, for example, from the encoding nucleotide molecules, vectors, host cells of the present disclosure; or can be obtained by chemical synthesis and the like, as long as they have the desired amino acid sequence and immunogenicity and reactivity.

[0083] Encoding molecules and templates

[0084] As used herein, the term "immunogenic peptide-encoding molecule" refers to a polynucleotide molecule encoding the full length or part of the immunogenic peptide of the present disclosure. The polynucleotide molecule can be selected from, for example: (i) a nucleotide molecule having a sequence as shown in SEQ ID NO: 15, 16, 17, 18 or 19; (ii) a molecule hybridizing to (i) under stringent conditions; (iii) a nucleotide molecule having a homology higher than or equal to 90%, higher than or equal to 95%, higher than or equal to 96%, higher than or equal to 97%, higher than or equal to 98%, higher than or equal to 99% to the sequence in (i) or (ii); (iv) a nucleotide molecule having one or several nucleotides substituted, deleted or added in the nucleotide sequence defined in (i) or (ii) and capable of expressing the full length or part of the functional immunogenic peptide.

[0085] As used herein, the term "stringent conditions" refers to: (1) hybridization and elution under lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, more preferably more than 95%.

[0086] The full length nucleotide sequence of the present disclosure or its fragments can generally be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present disclosure, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each amplification in the correct order.

[0087] In another aspect of the present application, a DNA template for preparing mRNA is also provided. In some embodiments, the DNA template comprises a nucleotide sequence as set forth in SEQ ID NO: 22, 23, or 24, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto and has an activity of being transcribed into a desired mRNA sequence.

[0088] Expression vectors and host cells

[0089] The present disclosure also relates to a polynucleotide molecule encoding the immunogen composition herein, a vector comprising the polynucleotide molecule, and a host cell genetically engineered with the vector.

[0090] The coding sequences of the present disclosure can be used to express or produce recombinant immunogenic peptides by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are involved:

[0091] (1) introducing the coding polynucleotide molecule of the present disclosure, or a recombinant expression vector containing the polynucleotide molecule, into a suitable host cell;

[0092] (2) culturing the host cell in a suitable medium;

[0093] (3) isolating and purifying the protein or polypeptide from the medium or the cell.

[0094] In the present disclosure, the term "vector" and "recombinant expression vector" are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and a translation control element.

[0095] In some embodiments, the recombinant adenovirus vector includes, but is not limited to, adenovirus types 5, 11, 26, 35, 63, 68, etc.; the recombinant poxvirus vector includes, but is not limited to, the Tian Tan strain, the North American vaccine strain, the Wyeth derivative strain, the Lister strain, the Ankara derivative strain, the Copenhagen strain, and the New York strain, etc., especially the Tian Tan strain.

[0096] In some embodiments, the immunogen of the present application that induces neutralizing antibodies and the T cell immunogen can be loaded into an mRNA expression vector. In some embodiments, the mRNA expression vector further comprises the basic elements required for transcription from the 5' end to the 3' end, specifically the T7 promoter region, the 5' untranslated region, the coding sequence, the 3' non-coding region, and the polyadenylation tail (polyA) sequence. The coding sequence includes, but is not limited to, the immunogen composition of the immunogen that can induce neutralizing antibodies and the T cell immunogen that can induce cellular responses.

[0097] Expression vectors containing the immunogenic peptide-encoding sequences and appropriate transcriptional / translational control sequences can be constructed from plasmids or vectors employing methods known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequences described can be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Expression systems such as pcDNA3.1 vector, pIRES2-EGFP vector, AdMax™, and the like can be employed in the present disclosure.

[0098] In addition, the expression vector can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance for eukaryotic and prokaryotic cell culture, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli.

[0099] Vectors containing the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable them to express the protein or polypeptide. The host cells can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as animal cells. Representative examples of which are: E. coli, Streptomyces, Agrobacterium, fungal cells such as yeast, animal cells, and the like. In the present disclosure, host cells selected from the group consisting of HEK293, HeLa, CHO, K562, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38 can be employed.

[0100] The nucleotide molecules of the present disclosure, when expressed in higher eukaryotic cells, will be transcribed more efficiently if an enhancer sequence is inserted in the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, which act on a promoter to increase the transcription of a gene. Those of ordinary skill in the art will be aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0101] The recombinant polypeptides in the above methods can be expressed intracellularly or on the cell membrane or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0102] In some embodiments, for the RBD multimerization chimeric antigen, T cell antigen CoV-T and anti-COVID-19 immunogenic composition of the present application, some or all of the uracil and / or cytosine nucleosides can be replaced when transcribing the corresponding mRNA. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uracil nucleosides in the mRNA are replaced with one or more selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, or 5-methoxyuridine and 2'-O-methyluridine, preferably pseudouridine or N1-methylpseudouridine or N1-ethylpseudouridine, further preferably N1-methylpseudouridine.

[0103] Vaccines and immunoconjugates

[0104] Also provided herein is a vaccine, or immunological composition, comprising the immunogenic composition, polynucleotide molecule, vector and / or host cell of the present disclosure. The vaccine comprises a formulation of the immunogenic peptide and / or nucleic acid molecule of the present disclosure in a form capable of being administered to a vertebrate, preferably a mammal, and which induces a protective immune response that enhances immunity to prevent and / or alleviate the novel coronavirus and / or at least one symptom thereof.

[0105] The term "protective immune response" or "protective response" refers to an immune response mediated by an immunogen against an infectious agent or disease exhibited by a vertebrate (e.g., a human) that prevents or alleviates infection or reduces at least one symptom of the disease.

[0106] The term "vertebrate" or "subject" or "patient" refers to any member of the subphylum chordata, including, without limitation: humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic animals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The terms "mammal" and "animal" are included in this definition and are intended to encompass adult, young, and newborn individuals.

[0107] The vaccines herein can be a recombinant protein vaccine, a recombinant DNA vaccine, a recombinant viral vector vaccine (e.g., adenovirus vector, poxvirus vector, adeno-associated virus vector, herpes simplex virus vector, cytomegalovirus vector), a recombinant bacterial vector vaccine, a recombinant yeast vector vaccine, or a recombinant virus-like particle vaccine. In some embodiments, the vaccines herein are selected from a recombinant DNA vaccine, a recombinant adenovirus vector, a recombinant poxvirus vector, or a combination of one or two or three thereof.

[0108] In some embodiments, one or more vaccines selected from the group consisting of: a recombinant plasmid vaccine (DNA), such as a DNA vaccine comprising a sequence encoding a cysteine-modified RBD region fused to human recombinant Ferritin or HA2 (e.g., pcDNA3.1-sRBD-hFn); a recombinant protein subunit vaccine (protein), such as an RBD protein (without disulfide bond modification), a cysteine-modified RBD protein fused to human recombinant Ferritin or HA2 (sRBD-hFn protein, sRBD-HA2 protein); a recombinant human cell carrier vaccine, such as K562-HA2-sRBD, or a combination thereof can be employed.

[0109] In some embodiments, the induced neutralizing antibody immunogens and T cell immunogens of the present application can be inserted into different expression vectors to constitute different vector vaccines. The vectors used include, but are not limited to, mRNA vectors, DNA plasmid vectors, recombinant viral vectors, recombinant bacterial vectors, etc.; wherein the mRNA vectors include, but are not limited to, linear, circular and self-replicating vectors; the recombinant viral vectors include, but are not limited to, poxvirus, adenovirus, adeno-associated virus, herpes simplex virus, measles virus, reovirus, rhabdovirus, forest encephalitis virus, influenza virus, respiratory syncytial virus, poliovirus, etc.

[0110] In some embodiments, the induced neutralizing antibody immunogens and T cell immunogens can be inserted into multiple vectors of the same type to constitute a multivalent vaccine, or inserted into different vectors to constitute a combination vaccine, both of which can activate T cell and antibody responses against COVID-19 through sequential or combined immunization, achieving combined protection.

[0111] In some embodiments, the vaccine or anti-COVID-19 immunogen composition is a novel coronavirus mRNA vaccine comprising:

[0112] i) a codon-optimized Omicron variant multimerization chimeric antigen sequence as described in the first aspect above, the codon-optimized nucleic acid sequence is set forth in SEQ ID NO: 15, SEQ ID NO: 16;

[0113] ii) a codon-optimized T cell antigen CoV-T as described in the second aspect above, the codon-optimized nucleic acid sequence is set forth in SEQ ID NO: 17;

[0114] iii) a codon-optimized anti-COVID-19 immunogen composition loaded into the same mRNA vector, the codon-optimized nucleic acid sequence is set forth in SEQ ID NO: 18, SEQ ID NO: 19.

[0115] In some embodiments, the vaccine can be prepared using cationic liposomes, polymers, proteins, or lipid nanoparticles, preferably cationic liposomes and cationic lipid nanoparticles, more preferably cationic lipid nanoparticles, which are mixed with the mRNA vaccine to form the formulation.

[0116] The vaccine compositions herein comprise an effective amount of the immunogens herein. The vaccine compositions of the present disclosure comprise an amount of immunogen sufficient to achieve a desired biological effect. The term “effective amount” generally refers to an amount of immunogen that can induce a protective immune response sufficient to induce immunity to prevent and / or reduce an infection or disease and / or to reduce at least one symptom of an infection or disease.

[0117] The vaccines herein can also comprise an adjuvant. Adjuvants known to those of ordinary skill in the art can be used, such as those described in Vogel et al., “A Compendium of Vaccine Adjuvants and Excipients” (2ndEdition), which is incorporated herein by reference in its entirety. Examples of known adjuvants include, but are not limited to, complete Freund’s adjuvant, incomplete Freund’s adjuvant, aluminum hydroxide adjuvant, lipopolysaccharide (LPS), RIBI adjuvant, MF-59, and the like.

[0118] The vaccine compositions herein can also include pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, emulsifiers, and the like excipients. For example, pharmaceutically acceptable carriers are known and include, but are not limited to, water for injection, saline solution, buffered saline, dextrose, water, glycerol, sterile isotonic buffered water solutions, and combinations thereof. Pharmaceutically acceptable carriers, diluents, and other excipients can be found, for example, in Remington’s Pharmaceutcal Sciences.

[0119] The vaccine compositions herein can be in a form suitable for systemic or local (especially intrapulmonary) administration. Methods of administering the vaccine compositions include, but are not limited to, intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal instillation, aerosol inhalation, reproductive tract, rectal, oral, or any combination thereof.

[0120] In some embodiments, the vaccine herein prevents, eliminates, or alleviates a novel coronavirus infection or at least one symptom thereof, such as respiratory symptoms (e.g., nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, rales, wheezing, dyspnea, pneumonia caused by infection, severe acute respiratory syndrome, kidney failure, and the like, in a subject.

[0121] Also contemplated herein is an immunoconjugate (also known as an immunoconjugate) comprising an immunogen herein and another substance coupled thereto. The other substance can be a targeting substance (e.g., a moiety that specifically recognizes a particular target), a therapeutic substance (e.g., a drug, a toxin, a cytotoxic agent), a labeling substance (e.g., a fluorescent label, a radioisotope label).

[0122] Also provided in the present disclosure is a combination product comprising an immunogenic peptide, a nucleotide molecule, a vector, a host cell, and / or a vaccine of the present disclosure, and can further comprise one or more other substances that facilitate better prevention and / or treatment of a novel coronavirus infection or symptoms thereof or enhance the stability of the aforementioned substances. For example, the other substances can include other vaccines against coronavirus S or S1, such as S or S1 vaccines from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, and the like; other active substances that benefit from T cell activation and / or memory immune response with T cells.

[0123] Immunization methods

[0124] Also provided herein is a method for preventing and / or treating SARS-CoV-2 infection and / or symptoms thereof, comprising: administering at least once a prophylactically and / or therapeutically effective amount of one or more vaccines of the present disclosure. The administration can be performed by any suitable means, including but not limited to: systemic immunization, such as intramuscular injection, subcutaneous injection, intradermal injection, etc.; and intranasal immunization, such as nebulization, nose drop, etc. In some embodiments, the initial immunization is performed by systemic administration or intranasal administration, preferably systemic administration.

[0125] In some embodiments of the present disclosure, the interval between each two administrations is at least 1 week, such as 2 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer.

[0126] In some embodiments, the initial immunization is performed by DNA vaccine, and one or more booster immunizations are performed by recombinant viral vaccines. The immunization methods of the present disclosure can be performed by “prime-boost” or “prime-boost-boost”, and can be performed by a single systemic immunization or intranasal immunization, or a combination of the two.

[0127] Depending on the characteristics of the different vector vaccines, in some preferred embodiments, the initial systemic immunization is performed by a recombinant DNA vaccine, to establish a systemic immune response, and one or more booster immunizations are performed by other vaccines, such as recombinant adenovirus vaccines or recombinant poxvirus vaccines, which can include at least one intranasal booster immunization (e.g., by adenovirus vaccine).

[0128] The immune responses established by the immunization methods herein, both locally in the respiratory tract and systemically, can help to enhance the effectiveness of the vaccine protection.

[0129] The combination products herein can be provided in the form of a pharmaceutical pack or kit, for example, one or more vaccine compositions or components thereof herein can be packaged in one or more containers, such as a sealed container with an amount of the composition indicated, such as an ampoule or a sachette. The vaccine compositions can be provided in the form of a liquid, a sterile lyophilized powder, or an anhydrous concentrate, etc., which can be diluted, reconstituted, and / or formulated with an appropriate liquid (e.g., water, saline, etc.) before use to obtain an appropriate concentration and form for administration to a subject.

[0130] Compared with the prior art, the present application provides an anti-COVID-19 immunogen composition which can induce neutralizing antibodies and T cell immune response at the same time. The immunogen for inducing neutralizing antibodies in the present application is a multimeric RBD chimeric antigen, which can produce potent broad-spectrum neutralizing antibodies; the T cell immunogen CoV-T for inducing cell response covers the conserved T cell epitopes of coronavirus and can induce broad-spectrum and potent cell response. The neutralizing antibody immunogen and the T cell immunogen in the present application can be loaded into the same carrier, when applied to the vaccine, can be simply, conveniently and quickly prepared, have high immunogenicity, can induce high level of neutralizing antibodies to the prototype strain and various strains of the current epidemic, and can also stimulate strong T cell response, and can be used as a broad-spectrum vaccine for SARS-CoV-2 to cope with various epidemic strains.

[0131] Preferred embodiments

[0132] The following preferred embodiments are provided in the present application. It should be understood that these embodiments are only used to illustrate the present disclosure and are not used to limit the scope of the present disclosure. Those skilled in the art can make appropriate modifications and changes to the present disclosure, and these modifications and changes are all within the scope of the present disclosure.

[0133] 1. A vaccine immunogen composition which can induce broad-spectrum anti-COVID-19 neutralizing antibodies, containing:

[0134] a) the immunogen composition is a chimeric antigen trimerized from RBDs from three different strains of SARS-CoV-2, including the amino acid sequence of the RBD domain of the S protein from the Omicron variant BA.2 strain or a portion thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity thereto, such as the amino acid sequence set forth in SEQ ID NO: 1; the amino acid sequence of the RBD domain of the S protein from the Omicron variant BA.2.75 strain or a portion thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity thereto, such as the amino acid sequence set forth in SEQ ID NO: 2; and the amino acid sequence of the RBD domain of the S protein from the Omicron variant XBB.1.5 strain or a portion thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity thereto, such as the amino acid sequence set forth in SEQ ID NO: 3. Alternatively, the RBD sequence of XBB.1.5 is replaced with the amino acid sequence of the RBD domain of the S protein from the Omicron variant BA.4 or BA.5 strain or a portion thereof, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto and having the same or substantially the same immunogenicity thereto, in particular the amino acid sequence set forth in SEQ ID NO: 4;

[0135] b) the trimerized RBD chimeric antigens are arranged between each other by appropriate linking sequences, including sequences of glycine, serine, glutamic acid, and lysine, in particular the amino acid sequence EGKSSGSGSESKST set forth in SEQ ID NO: 5, or the amino acid sequence GSAGSAAGSGEF set forth in SEQ ID NO: 6, and the order of different linking sequences can be in front or in back;

[0136] c) the trimerization of the RBD chimeric antigens is in the form of an oligomer after fusion with a multimerization domain, which is the domain foldon of the T4 bacteriophage fibritin protein, in particular the amino acid sequence set forth in SEQ ID NO: 8;

[0137] d) the SARS-CoV-2 prototype and its Omicron variant RBD multimerization chimeric antigen sequence is specifically the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, or its nucleic acid sequence is optimized for codon and RNA secondary structure, specifically the full-length DNA sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with the nucleic acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16;

[0138] e) and / or one or more pharmaceutically acceptable carriers or excipients comprising the above immunogenic composition, preferably suitable for oral, intradermal, subcutaneous, intramuscular or intranasal administration.

[0139] 2. The immunogen of embodiment 1, which can be used in combination with a T cell immunogen including but not limited to the following: endogenous gene ORF1ab from SARS-CoV-2 and all or part of the amino acid sequence encoded by it, ORF1ab-derived sequences in combination with S, M, E, N, etc. gene-derived sequences, or conserved sequences and shared sequences from different coronaviruses;

[0140] 3. The T cell immunogen of embodiment 2, which is a recombinant sequence derived from the conserved region of the endogenous protein of SARS-CoV-2 coronavirus, specifically the full-length recombinant protein amino acid sequence shown in SEQ ID NO: 11, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with the amino acid sequence shown in SEQ ID NO: 11; or its nucleic acid sequence is optimized for codon and RNA secondary structure, specifically the full-length DNA sequence shown in SEQ ID NO: 17, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with the nucleic acid sequence shown in SEQ ID NO: 17;

[0141] 4. The RBD trimerization immunogen of embodiment 1, wherein the RBD is a recombinant chimeric antigen selected from the COVID-19 novel coronavirus Omicron variant, including Omicron subtypes BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, BA.4, BA.5, XBB, XBB.1.5, XBB.1.6, BQ.1, BQ.1.1, BF.7, EG.5, BA.2.86; optionally, the RBD trimerization immunogen is selected from three non-heterotypic RBD strains, including BA.2, BA.2.75, BA.4 / BA.5 Omicron variant, and / or BA.2, XBB.1.5, BA.4 / BA.5 Omicron variant.

[0142] 5. The immunogen of embodiments 1-4, inserted into different expression vectors to constitute different vector vaccines. The vectors used include but are not limited to mRNA vectors, DNA plasmid vectors, nanoparticle vectors, recombinant virus vectors, recombinant bacterial vectors, etc.; wherein the mRNA vectors include but are not limited to linear, circular and self-replicating vectors; the recombinant virus vectors include but are not limited to poxvirus, adenovirus, adeno-associated virus, herpes simplex virus, measles virus, enterovirus, rhabdovirus, forest encephalitis virus, influenza virus, respiratory syncytial virus, poliovirus, etc.

[0143] 6. The immunogen of embodiments 1-4, which can be inserted into the same type of vector or different types of vectors in embodiment 3. Inserted into multiple vectors of the same type to constitute a multivalent vaccine, or inserted into different vectors to constitute a combination vaccine, both of which can activate a broad spectrum of neutralizing antibody responses and cross-protective T cell responses against COVID-19 coronavirus through sequential or combined immunization, achieving combined protection; or activate a broad spectrum of neutralizing antibody responses against COVID-19 coronavirus and cross-protective T cells against different coronaviruses, achieving more broad-spectrum immune protection against SARS-CoV-2 and other different coronaviruses at the same time.

[0144] 7. The viral vector of embodiment 5, wherein the recombinant adenovirus vector includes but is not limited to adenovirus types 5, 11, 26, 35, 63, 68, etc.; the recombinant poxvirus vector includes but is not limited to Tian Tan strain, North American vaccine strain, Wyeth derivative strain, Lister strain, Ankara derivative strain, Copenhagen strain and New York strain, etc., especially Tian Tan strain.

[0145] 8. The use according to embodiments 1, 2, 3, 5 and 6, wherein the neutralizing antibody inducing immunogen and the T cell immunogen are loaded into the same mRNA expression vector, and the neutralizing antibody inducing immunogen and the T cell immunogen inducing cellular response are arranged in a single-cistronic sequence in tandem with a linker, so as to be translated from the same transcript.

[0146] 9. The immunogen composition according to embodiment 8, wherein the linker sequence connecting the neutralizing antibody inducing immunogen and the T cell immunogen is a 2A self-cleavage peptide, in particular the amino acid sequence of SEQ ID NO: 12.

[0147] 10. The anti-COVID-19 immunogen composition according to embodiments 1, 2, 3, 8, 9 and the construction method thereof, which is the sequence of the trimerized RBD chimeric antigen-2A self-cleavage peptide-T cell immunogen (CoV-T) for SARS-CoV-2 mutant strains, in particular the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14; or the nucleic acid sequence thereof is codon and RNA secondary structure optimized, in particular the full-length DNA sequence of the anti-COVID-19 immunogen composition of SEQ ID NO: 18, SEQ ID NO: 19, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology with the nucleic acid sequence of SEQ ID NO: 18, SEQ ID NO: 19.

[0148] 11. A method for obtaining the T cell immunogen CoV-T as described in embodiment 3, the method comprising:

[0149] a) performing sequence similarity alignment on the amino acid sequences of the polyprotein ORF1ab, membrane protein (M), nucleocapsid (N) protein, and spike protein (S) of SARS-CoV-2;

[0150] b) performing T cell epitope prediction on the obtained similarity sequence information to obtain information of conserved regions with immunogenicity. Including predicting polypeptides that can be strongly bound to HLA-I supertype molecules, i.e. polypeptides that can be presented, in different proteins by using online tools;

[0151] c) Design and prepare different immunogen sequences or polypeptide sequences according to the conserved region information. Including mapping these polypeptides to SARS-CoV-2, and further comprehensive analysis of the enrichment region, finding the T cell epitope prediction result recommended region with high immunogenicity, and truncating it to form a recombinant amino acid sequence, such as the CoV-T amino acid sequence shown in SEQ ID NO: 11;

[0152] 12. The vaccine administration mode according to embodiment 1e, which can be administered once or multiple times; when the vaccine is administered multiple times, it can be administered multiple times by the same inoculation route, or sequentially inoculated by different inoculation routes; in particular, the sequential inoculation mode of intranasal / respiratory aerosol inhalation and intramuscular administration, in which the sequential inoculation mode can be intranasal / respiratory aerosol inhalation first and intramuscular inoculation second, or intramuscular inoculation first and intranasal / respiratory aerosol inhalation second.

[0153] 13. In the sequential administration mode of the vaccine according to embodiment 12, the vaccines with different carriers or excipients can be sequentially inoculated by the most suitable inoculation mode, in particular, the nanoparticle vaccine prepared in embodiment 5 or the adenovirus vector vaccine prepared in embodiment 7 is used for intranasal / respiratory aerosol inhalation, and the mRNA vaccine prepared in embodiment 8 is used for intramuscular inoculation.

[0154] 14. The vaccine prepared according to embodiments 1-11 can be used in combination with other vaccines, in particular, respiratory vaccines, including but not limited to influenza vaccine, tuberculosis vaccine, RSV vaccine, etc.

[0155] Examples

[0156] The present disclosure will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present disclosure and not to limit the scope of the present disclosure. Those skilled in the art can make appropriate modifications and changes to the present disclosure, and these modifications and changes are within the scope of the present disclosure.

[0157] The experimental methods in the following examples without specific conditions can use conventional methods in the art, for example, refer to Molecular Cloning: A Laboratory Manual (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or follow the conditions suggested by the supplier. The sequencing method of DNA is the conventional method in the art, which can also be provided by commercial companies.

[0158] Unless otherwise indicated, percentages and parts are by weight. As used herein, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Furthermore, any method and material similar or equivalent to those described herein can be used in the practice of the disclosed methods. The preferred materials and methods are described herein.

[0159] Materials, Methods and Animals

[0160] The mRNA preparation, mRNA vaccine preparation, animal immunization protocol, and detection methods involved in the experiments in the examples are as follows:

[0161] Biosafety and Ethical Statement

[0162] The mice involved in this study and related experiments were raised in the Shanghai Public Health Clinical Center Affiliated to Fudan University. Routine immunization was performed in the relevant laboratory in the specific pathogen free (SPF) area, and virus challenge experiments were performed in the biosafety level 3 laboratory. All experimental operation procedures were approved by the "Ethics Committee" of the Shanghai Public Health Clinical Center and implemented after approval, and the experimental process was strictly in accordance with the established procedures.

[0163] Experimental Animals

[0164] The experimental mice were all specific pathogen free (SPF) level, among which BALB / c mice / C57BL / 6 (female, 6-8 weeks old) were purchased from Suzhou Huachang Biological Co., Ltd., and H11-K18-hACE2 (Strain NO. T037657) was purchased from Jiangsu Jicui Yekang Biological Technology Co., Ltd., and were all raised in the SPF experimental area of the Shanghai Public Health Clinical Center.

[0165] The virus challenge experiments against SARS-CoV-2 prototype strain WT and Omicron (BA.2, BF.7, XBB.1) mutant strains of experimental animals were performed in the animal biosafety level 3 (ABSL3) facility of the Navy Medical University.

[0166] Cells and Viruses

[0167] HEK-293T and Vero E6 cell lines (human embryonic kidney 293) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. SARS-CoV-2 prototype strain WT, SARS-CoV-2 Omicron mutant strains BA.2, BF.7, XBB.1 were obtained from the Navy Medical University. SARS-CoV-2 virus and its mutant strains were all propagated in Vero E6 cells.

[0168] I. Design of Immunogens

[0169] The RBD protein involved in the present application is selected from amino acid sequences at positions 320 to 542 of a novel coronavirus S protein or S protein mutant sequence.

[0170] The various Omicron variant multimerization chimeric antigens involved in the present application are respectively constructed in the following manner:

[0171] 1) BA.2-BA.2.75-BA.4 / 5-foldon construct (referred to as BA.2 trimer)

[0172] The adopted RBD protein is the amino acid sequence shown in the RBD domain of Omicron variant BA.2 (SEQ ID NO: 1), the RBD domain of BA.2.75 (SEQ ID NO: 2), and the RBD domain of BA.4 / 5 (SEQ ID NO: 4), and is sequentially connected in series with linker 1 shown in SEQ ID NO: 5 and linker 2 shown in SEQ ID NO: 6, respectively, to obtain a chimeric RBD protein amino acid sequence; a signal peptide (MKTIIALSYIFCLVFA, SEQ ID NO: 20) is connected at the N-terminus thereof, and a polymeric foldon domain (shown in SEQ ID NO: 8) is connected at the C-terminus thereof with the amino acid sequence GGGGS shown in SEQ ID NO: 7, to obtain a construct of Omicron variant RBD trimer BA.2 trimer;

[0173] Among them, the basic composition of BA.2-BA.2.75-BA.4 / 5-foldon construct (referred to as BA.2 trimer, SEQ ID NO: 9) is: RBD domain of Omicron variant BA.2 (SEQ ID NO: 1) + linker (SEQ ID NO: 5) + RBD domain of BA.2.75 (SEQ ID NO: 2) + linker (SEQ ID NO: 6) + RBD domain of BA.4 / 5 (SEQ ID NO: 4) + GGGGS (SEQ ID NO: 7) + polymeric foldon domain (SEQ ID NO: 8);

[0174] 2) BA.2-BA.2.75-BA.4 / 5-foldon-CoV-T construct (referred to as BA.2 trimer-CoV-T):

[0175] In the construct of Omicron variant RBD trimer BA.2 trimer, a T2A self-cleavage peptide (amino acid sequence as shown in SEQ ID NO: 12) is added at the C-terminus, which is connected with the T cell immunogen sequence CoV-T (amino acid sequence as shown in SEQ ID NO: 11), to obtain the BA.2-BA.2.75-BA.4 / 5-foldon-CoV-T construct (amino acid sequence as shown in SEQ ID NO: 13);

[0176] 3) BA.2-XBB.1.5-BA.4 / 5-foldon-CoV-T construct (abbreviated as XBB.1.5 trimer-CoV-T):

[0177] On the basis of the BA.2-BA.2.75-BA.4 / 5-foldon-CoV-T construct (amino acid sequence as shown in SEQ ID NO: 13), the RBD domain (SEQ ID NO: 2) of BA.2.75 is replaced with the RBD domain (SEQ ID NO: 3) of XBB.1.5 to obtain the BA.2-XBB.1.5-BA.4 / 5-foldon-CoV-T construct (amino acid sequence as shown in SEQ ID NO: 14);

[0178] The T cell immunogen CoV-T sequence in the present application is designed by the following method:

[0179] i) Perform sequence similarity alignment on the amino acid sequences of the polyprotein ORF1ab, membrane protein (M), nucleocapsid (N) protein, and spike protein (S) of SARS-CoV-2;

[0180] ii) Perform T cell epitope prediction on the obtained similarity sequence information to obtain information on conserved regions with immunogenicity. Including predicting polypeptides that can strongly bind to HLA-I supertype molecules, i.e., polypeptides that can be presented, in different proteins by using online tools;

[0181] iii) Design and prepare different immunogen sequences or polypeptide sequences according to the conserved region information. Including mapping these polypeptides to the SARS-CoV-2 virus, and further comprehensive analysis of the enrichment region, finding the T cell epitope prediction result recommended region with high immunogenicity, and truncating it to form a recombinant amino acid sequence, such as the CoV-T amino acid sequence shown in SEQ ID NO: 11, and fusing a flag tag at the C-terminal end to facilitate subsequent experimental detection, obtaining an amino acid sequence as shown in SEQ ID NO: 21. Different immunogens BA.2 trimer, BA.2 trimer-CoV-T, XBB.1.5 trimer-CoV-T, their corresponding nucleotide sequences are optimized for eukaryotic cells, and the codon-optimized nucleic acid sequences are shown in SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; synthesized by Shanghai Jeery Bioengineering Co., Ltd., and inserted into a high-expression mRNA vector (CN 117721129 A, embodiment of the disclosed vector IV-eGFP (alpha-globin+Mit) (250A)) to prepare the mRNA DNA template sequence, which is shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively.

[0182] II. Preparation of mRNA vaccine

[0183] The different immunogen BA.2 trimer, BA.2 trimer-CoV-T, XBB.1.5 trimer-CoV-T expression vectors constructed in I above are linearized by XbaI restriction endonuclease, and candidate mRNA is prepared by in vitro transcription by co-transcriptional capping method. According to the T7 in vitro transcription kit (E131-01A) of Haicai, N1-methyl pseudouridine triphosphate (Nanjing Shenji Biological, N1-Me-pUTP, 100 mM, NMPUTP001) is completely replaced with uridine triphosphate during in vitro transcription. Add CAP GAG(30Me) (Nanjing Shenji Biological, CAP3111) to produce natural Cap1 structure by one-step co-transcriptional capping. According to the instruction manual, the corresponding modified mRNA is synthesized and purified by lithium chloride precipitation, washed with 75% ethanol for 3 times, and then air-dried. Dissolve in RNAase-free water, determine the concentration by Nano drop, and send the prepared mRNA molecules to Jiangsu Yaohai Biopharmaceutical Co., Ltd. to prepare mRNA liposome nanoparticle vaccine for subsequent mouse experiments.

[0184] III. Animal immunization scheme

[0185] 1. The first group of immune experiments is to evaluate the immunogenicity of BA.2 trimer, BA.2 trimer-CoV-T, XBB.1.5 trimer-CoV-T mRNA vaccine in BALB / c mice:

[0186] BALB / c mice aged 6-8 weeks were selected, and the "prime-boost" immunization strategy was used, with 3 injections at 3-week intervals, 5-6 mice per group. Each mouse was injected intramuscularly with 10 μg of vaccine, and the control group was injected with an equal volume of Empty-LNP, with a volume of 100 μL. Blood samples were collected 2 weeks after the first boost (Boost-1 2W) and 2 weeks after the second boost (Boost-2 2W), respectively. The binding ability to the original strain and Omicron (BA.2, BA.4 / 5, BF.7, BQ.1, XBB.1, XBB.1.5) mutant strains of the new coronavirus was detected by enzyme-linked immunosorbent assay (ELISA).

[0187] 2. The second group of immune experiments is to evaluate the T cell response level of BA.2 trimer-CoV-T mRNA vaccine:

[0188] According to the "prime-boost" immunization strategy, female C57 mice were immunized with 3 injections at 3-week intervals, with 5 mice per group in the experimental group and 4 mice per group in the control group. Each mouse was injected intramuscularly with 10 μg of vaccine, and the control group was injected with an equal volume of Empty-LNP. Ten days after the second boost, the T cell response level in the mouse spleen was detected by ELISpot method.

[0189] 3. The third group of immune experiments is to evaluate the broad-spectrum protection effect of BA.2 trimer-CoV-T mRNA against different coronaviruses in K18-hACE2 transgenic mice:

[0190] K18-hACE2 transgenic mice aged 6-8 weeks were used as the attack model, and the "prime-boost" immunization strategy was used, with blood samples collected 2 weeks after the third injection, followed by nasal challenge 3 weeks after the third injection. The original strain SARS-CoV-2 virus and different mutants including BA.2, BF.7, and XBB.1 were all at a dose of 1E4 PFU, with a volume of 20 μL for each virus. The body weight changes and survival of mice in different groups were observed for 14 days.

[0191] IV. Detection method

[0192] 1. Western blotting was used to detect the expression of the target protein:

[0193] a) According to the size of the target protein, we select the corresponding concentration of the separation gel to perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE);

[0194] b) According to the sample to be verified, add 20 μL of prepared sample in sequence; after half an hour at 80 V, adjust the voltage to 100 V and continue electrophoresis for 1.5 h; determine whether to end the electrophoresis according to the position of the bromophenol blue strip;

[0195] c) Membrane transfer: use the wet transfer method, first activate the PVDF membrane in methanol for 30 s, then soak the sponge, filter paper and PVDF membrane with the transfer solution, and arrange them in the order of "cathode plate (black) - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - anode plate (white)"; constant current 200 mA, 1.5-2 h;

[0196] d) After the end, seal the PVDF membrane in 5% skimmed milk powder for 1-2 h;

[0197] e) Primary antibody incubation: add specific antibodies at 1:1000, shake on the shaker at room temperature for 2 h, and then wash with PBST for 5 times, 3 min each time;

[0198] f) Secondary antibody incubation: add HRP-labeled IgG antibodies at 1:5000, incubate at room temperature for 1 h, and then wash with PBST for 5 times, 3 min each time;

[0199] g) Color development, add color developing solution and incubate for 1 min, use the analyzer to expose for 2 min, record and analyze the color development results.

[0200] 2. Enzyme-linked immunosorbent assay (ELISA) for detecting RBD-specific binding antibody titers

[0201] a) Dilute different subtypes of RBD proteins to 1 ng / μL with coating buffer solution (50 mM carbonate buffer, pH = 9.6), 100 μL / well, and incubate at 4°C overnight;

[0202] b) Wash once with PBST (containing 0.05% Tween-20), pat dry, and then add 200 μL / well of 5% skimmed milk for room temperature blocking for 2 h;

[0203] c) Use 5% skimmed milk as the sample diluent, dilute the serum sample 100 times in the first well, perform 2-fold gradient dilution, and add to the reaction well, and react at 37°C for 1 h;

[0204] d) After washing 5 times with PBST, pat dry, add 100 μL / well of HRP-labeled goat anti-mouse IgG antibody (1:5000), and react at room temperature for 1 h;

[0205] e) After the reaction is completed, wash 6 times with PBST, pat dry, and then add gold and silver sheet OPD solutions (1 sheet of gold and 1 sheet of silver, dissolved in 20 mL of deionized water) in the dark, and react for 5-10 min;

[0206] f) Add 2M sulfuric acid to terminate the reaction. Read the value at 490 nm on the enzyme label instrument;

[0207] g) Analyze the data, take 2.1 times the value of the negative control group as the cut-off value, calculate the RBD-specific binding antibody dilution endpoint, i.e., the corresponding titer, and express it as the geometric mean (GMT), and plot it using Graphpad Prism 9 software.

[0208] 3. Mouse IFN-γ ELISPot detection

[0209] a) Take out the antibody package of the Elispot mouse kit of Biodot Medical Biotechnology Co., Ltd.: according to the number of wells required for the experiment, dilute the anti-IFN-γ antibody by 1:200 with sterile PBS, and add 100 μL / well to the Elispot plate. (Antibody dilution volume calculation: number of experimental wells x 110 μL) Incubate at 4°C overnight;

[0210] b) Take out the Elispot plate incubated overnight, discard the liquid in the wells, add 200 μL of R10 and wash once, discard after 3 min. Add 200 μL of fresh R10 and block at room temperature for 2 h;

[0211] c) After blocking, discard the culture medium, add 50 μL of stimulant (stimulant peptide library, RBD peptide library includes single peptides R1-R13 as shown in FIG. 4B, which correspond to the peptide segments in aa320-aa591 of the wild-type RBD shown in SEQ ID NO: 25, respectively; CoV-T peptide library 1-31 is established as shown below; final concentration of single peptide 5 μg / mL), negative control R10, positive control [PMA (50 ng / mL) + Ionomycin (1 μg / mL)], and then adjust the cell concentration to 4 x 10 6 cells / mL, add 50 μL of cells to each well in order. After gentle mixing, incubate in a humidified box in a carbon dioxide incubator for about 20 h. The first three steps need to be performed in a biological safety cabinet, and the following steps do not need to be operated under sterile conditions. Freeze the remaining cells, 500g, 5 min, discard the supernatant, add 2 mL of commercial freezing solution, and divide each sample into 2 branches;

[0212] d) After the incubation, discard the culture. Wash twice with 200 μL pre-cooled distilled water per well, and wash three times with 200 μL PBST per well, each time for 3-5 min, then discard, and pat the residual liquid dry on the absorbent paper (keep the plate in a wet state during the washing process, do not dry it);

[0213] e) Dilute the biotin-coupled detection antibody in the antibody diluent (PBS + 10% FBS) at 1:250, add 100 μL per well, and incubate at room temperature for 2 h. Discard the liquid, and wash four times with 200 μL PBST per well. Discard after each time for 1-2 min, and pat the residual liquid dry on the absorbent paper;

[0214] f) Dilute Streptavidin-HRP in the antibody diluent (PBS + 10% FBS) at 1:100, add 100 μL per well, and incubate at room temperature for 1 h. Discard the reaction liquid, and wash five times with 200 μL PBST per well, generally for 1-2 min each time. Finally, wash three times with 200 μL PBS per well, and discard;

[0215] g) Color development: prepare the AEC color developing liquid immediately before use. Add 1 drop (about 20 μL) of color developing substrate to 1 mL of the color developing liquid, mix thoroughly, and add 100 μL per well. React at room temperature for 5-60 min in the dark. Pay attention to the color development degree, and when a clear red spot appears, wash the plate gently with tap water for 5 min to stop the reaction. If the color development time is short, the color of the positive spot is light, and if the time is too long, a deep background will appear. You can use the positive and negative wells as a reference. Dry the plate naturally or with a fan; read the data. Place the 96-well plate in the Elispot plate reader to read the positive spots.

[0216] Establishment of CoV-T peptide library:

[0217] — Single peptide 1~Single peptide 308: 15 amino acids for each single peptide (except single peptide 307 is 13 amino acids), covering the entire CoV T sequence (SEQ ID NO: 11), a total of 308 single peptides (i.e. n = 1~308), wherein: from single peptide 1~306 (i.e. n = 1~306), the corresponding amino acid position of CoV T (SEQ ID NO: 11) conforms to the following rules: starting position = n x 4-2, end position = n x 4+12. For example, single peptide 1 (i.e. n = 1), the starting position is the 2nd amino acid of SEQ ID NO: 11, and the end position is the 16th amino acid of SEQ ID NO: 11; single peptide 2 (i.e. n = 2), is the 6th to 20th amino acid of SEQ ID NO: 11; and so on, single peptide 306 (i.e. n = 306), is the 1222nd to 1236th amino acid of SEQ ID NO: 11. Single peptide 307 and single peptide 308 have reached the end of SEQ ID NO: 11, and their sequences correspond to the 1226th to 1238th and the 1224th to 1238th of SEQ ID NO: 11, respectively.

[0218] — Peptide library: 31 peptide libraries are established in turn with the above single peptides, each of the peptide libraries 1~30 contains 10 single peptides, and the peptide library 31 contains 8 single peptides (i.e. single peptides 301~308).

[0219] 4. SARS-CoV-2 and variant pseudovirus packaging

[0220] SARS-CoV-2 envelope pseudovirus packaging (WT, BA.2, BA.5, BF.7, BQ.1, XBB.1, XBB.1.5)

[0221] a) Preparation of cells: 293T cells are plated in 10em dishes, each containing 5x106 cells, with a total volume of 10mL, cultured in a 37°C, 5% CO2 incubator to ensure that the cells are 75%-85% confluent before transfection the next day;

[0222] b) Transfection of plasmid: Add 1 mL double DMEM medium to SARS-CoV-2 membrane protein plasmid pcDNA3.1-S 4 μg and pNL4-3Δenv backbone plasmid 8 μg (NIH AIDS Reagent Program, 3418), vortex well, then add 20 μL transfection reagent PEI, vortex well, incubate at room temperature for 15-20 min, then slowly drop the mixed solution into the 10 cm dish, replace with fresh 15 mL complete medium after 6 h, and incubate at 37°C for 48 h;

[0223] c) After the end of the culture, the cell culture supernatant was collected, placed in a 15 mL centrifuge tube, centrifuged at 4,000 g at 4°C for 10 min, filtered with a 0.45 μm filter, and stored at -80°C after freezing. Titration was performed and the sample was stored for later use.

[0224] SARS-CoV-2 and its variant pseudovirus neutralization experiment

[0225] a) Take a 96-well transparent bottom white plate for the neutralization experiment, set the cell control (CC) (150 μL D10 / well) in the first column, the virus control (VC) (100 μL D10 / well) in the second column, and the sample wells in the other columns. Dilute the serum sample by the ratio, and the final volume in the well is 100 μL;

[0226] b) Add 50 μL of pseudovirus diluent to each well except the cell control group, so that the final amount of pseudovirus in each well is 200 TCID 50 ;

[0227] c) Mix gently, place the above-mentioned plate in the cell culture incubator, and incubate for 1 h; when the incubation time is 30 min, prepare the target cells and dilute them to 10 5 cells / mL with D10; when the incubation time is 1 h, add 100 μL of target cells to the well, so that the number of cells in each well is 10 4 ; place the plate in the cell culture incubator and culture for 48 h;

[0228] d) Take out the plate, observe the growth state of the cells, aspirate the cell supernatant in the well, wash each well with 100 μL of PBS, then add 50 μL of 1x cell lysis buffer to each well, and incubate on a shaking platform at room temperature for 30 min to lyse the cells;

[0229] e) Add 30 μL of luciferase substrate to the well, and detect the luciferase activity with a 96 Microplate Luminometer instrument; export the fluorescence reading, calculate the neutralization inhibition rate, inhibition rate = [1-(sample well luminescence intensity mean-cell control mean) / (virus control luminescence intensity mean-cell control value mean)]x100%, and calculate the ID 50 using Graphpad Prism 5.0 software.

[0230] The present patent will be further described in detail below in conjunction with the drawings and specific experiments. Unless otherwise specified, the reagents, instruments, equipment and methods used in the present patent are conventional commercially available reagents, instruments, equipment and methods in the technical field.

[0231] Example 1. Construction of multimeric chimeric antigen of Omicron variant

[0232] The BA.2 trimer, BA.2 trimer-CoV-T, XBB.1.5 trimer-CoV-T were constructed according to the experimental method I described above. The structure schematic diagram of the construct is shown in FIG. 1A, 1B, 1C, respectively.

[0233] Example 2. Expression verification of BA.2 trimer, BA.2 trimer-CoV-T, XBB.1.5 trimer-CoV-T mRNA transfected HEK293T cells

[0234] The BA.2 trimer, BA.2 trimer-CoV-T, XBB.1.5 trimer-CoV-T mRNA were prepared according to the experimental method II, and the Lipofectamine TM 3000 After mixing the reagent transfection reagent and mRNA molecules respectively, transfect HEK293T for verification. 12h before transfection, seed HEK293T cells into 12-well plates at a density of 200000 cells / well, the culture medium is DMEM complete medium, dilute with Opti-MEM (invitrogen), transfect 3ug mRNA per well plate, mix with Lipofectamine TM 3000 Prepare the mixture according to the mass volume ratio of 1:2, add dropwise to the well plate, incubate in 37℃ incubator for 24h, collect sample for detection. Western blot verification was performed, and the primary antibodies were RBD specific antibody and anti-flag tag antibody, respectively.

[0235] Results as shown in Figure 2, all candidate mRNA molecules can express a large amount after transfecting HEK293T cells; among them, as shown in Figure 2A, after transfection of BA.2 trimer sample, the expression of target band BA.2 trimer-foldon monomer can be detected at the position of 100 kDa, the size is correct, and the multimerization band RBD oligomers of the three variant RBD trimers in BA.2 trimer can be detected at about 220 kDa above 180 kDa. Similarly, as shown in Figure 2B and 2C, after transfection of BA.2 trimer-CoV-T construct and XBB.1.5 trimer-CoV-T construct coupled with T cell immunogen CoV-T, the expression was identified with RBD specific antibody, and the monomer band of BA.2 RBD-trimer-foldon and XBB.1.5 RBD-trimer-foldon can be seen at about 100 kDa; but only the sample of XBB.1.5 trimer-CoV-T construct detected the full-length band of XBB.1.5 RBD-trimer-foldon-CoV-T at about 220 kDa above 180 kDa, and the multimer band XBB.1.5 RBD oligomers above 220 kDa can also be seen; similarly, the expression of CoV-T was identified with anti-flag specific antibody, in Figure 2B and Figure 2C, the CoV-T gene band can be seen at about 130 kDa, and the full-length band of BA.2 RBD-trimer-foldon-CoV-T (Figure 2B right) and XBB.1.5 RBD-trimer-foldon-CoV-T (Figure 2C right) can be detected at about 220 kDa, and the multimer band (BA.2 RBD oligomers and XBB.1.5 RBD oligomers) above 220 kDa can also be observed.

[0236] The above results show that the constructed constructs can successfully express a variety of multimerization chimeric antigens with the required structure in host cells.

[0237] Example 3. Immunogenicity evaluation of BA.2 trimer, BA.2 trimer-CoV-T, XBB.1.5 trimer-CoV-T mRNA vaccine in BALB / c mice

[0238] This example selects the mRNA that has been verified to express correctly in Example 2, and prepares it into mRNA cationic lipid nanoparticle vaccine, and evaluates the animal experiment immunization. The RBD-specific antibody response of the vaccinated mice is detected according to the method described in Experimental Method III. The serum is collected 2 weeks after the second vaccination and 2 weeks after the third vaccination, respectively, and the antibody response level of different vaccine groups to the prototype strain and different mutant strains of SARS-CoV-2 virus is analyzed by enzyme-linked immunosorbent assay (ELISA).

[0239] The results show that the three vaccines can all induce specific binding antibodies against RBD, including the prototype strain WT, Omicron mutant strains BA.2, BA.4 / 5, BF.7, BQ.1, XBB.1, XBB.1.5, wherein the geometric mean titer (GMT) of the binding antibodies ranges from 10 4 to 10 5 (FIGS. 3A-3C). After immunization with the multimeric chimeric antigen BA.2 trimer without T cell immunogen, the RBD-specific binding antibody titer of 2 needles 2 weeks can reach 10 4 , while the specific binding antibody titer of the serum of the two chimeric antigen mRNA vaccines BA.2 trimer-CoV-T and XBB.1.5 trimer-CoV-T with fusion T cell immunogen is significantly higher than 10 4 , close to 2x10 5 levels. The addition of T cell immunogen can improve the level of induced RBD-specific binding antibodies to a certain extent.

[0240] These experimental results show that the heterologous multimeric vaccine with fusion T cell immunogen in the present application has better immunogenicity and broad-spectrum.

[0241] Example 4. T cell response level induced by BA.2 trimer-CoV-T mRNA vaccine

[0242] This example further explores the T cell response level induced by BA.2 trimer-CoV-T mRNA vaccine in animals. Studies have reported that effective virus-specific T cells are essential for the clearance of viruses. According to the second group of immunization experiments described in the animal immunization scheme in Experimental Method III, C57 mice are immunized, 5 mice per group in the vaccine inoculation group, and 10 μg is inoculated intramuscularly. The control group is empty vector, and 4 mice per group. Ten days after the booster, the mice are sacrificed, and the spleen cells are taken. The spleen cells are stimulated with the prototype strain RBD and CoV-T peptide library of the new coronavirus, respectively, and the T cell immune response level is detected by ELISpot.

[0243] Results show that: BA.2 trimer-CoV-T mRNA vaccine can produce high levels of T cell responses in mice, and the T cell responses induced by RBD and CoV-T peptide library are strong, and the GMT of positive cells per million spleen cells is 7457 and 15765, respectively, while in the control group, it is at a low level (Figure 4A). Subsequently, the T cell response to each peptide library was analyzed, and the results showed that 13 peptides R1-R13 in the RBD peptide library can induce effective responses after vaccination (Figure 4B), and the response to the CoV-T immunogen is mainly concentrated in the ORF1ab region (Figure 4C), further suggesting that the use of neutralizing antibodies and focusing on conserved T cell immunogens can more effectively respond to infection of different mutant strains.

[0244] Example 5. Evaluation of RBD-trimer-CoV-T mRNA-LNP vaccine in K18-hACE2 transgenic mice for protection against challenge

[0245] In view of the BA.2 trimer-CoV-T mRNA vaccine in Example 3 and Example 4 can induce better antibody and T cell responses in C57 mice, this example further explores the protective effect of BA.2 trimer-CoV-T mRNA vaccine in k18-hACE2 transgenic mice against different SARS-CoV-2 prototype strains and mutant strains.

[0246] According to the third group of immunization experiments described in the animal immunization scheme in Experimental Method III, 6-8 week old k18-hACE2 transgenic mice were used as challenge models, and the immunization strategy is shown in Figure 5A. Three weeks apart, three doses were administered, and blood was collected two weeks after the third dose. Subsequently, intranasal challenge was performed three weeks after the third dose, with each virus dose being 1E4 PFU, including SARS-CoV-2 virus prototype and different mutant strains including BA.2, BF.7, XBB.1, with a volume of 20 μL, and the protective effect of AdC68-panCoV / Flu vaccine against other coronaviruses was detected by monitoring body weight changes and mortality within 14 days after challenge.

[0247] Results show that, as shown in Figures 5B and 5C, the vaccine can induce high-titer specific binding antibodies and pseudovirus neutralizing antibodies against different mutants of COVID-19 in k18-hACE2 transgenic mice. The neutralizing antibody titers against Omicron variants such as XBB.1, XBB.1.5 can reach 220 and 461, respectively, two weeks after the second booster dose; and against BA.2, BA.5, BF.7, BQ.1, it can reach about 1300-3000.

[0248] Subsequently, we continuously observed the weight changes and survival of mice in different groups, and found that the body weight of the control group mice continued to decrease, and the empty vector control group of the prototype strain infection group all died at 4 days, and the empty vector control groups of other mutant strains BA.2, BF.7, XBB.1 all died within 6-7 days, while the BA.2 trimer-CoV-T mRNA vaccination group did not change in body weight and all survived within the 2-week observation period, which could effectively resist the attack of lethal SARS-CoV-2 virus prototype strain and other BA.2, BF.7, XBB.1 mutant strains (Figures 5D and 5E). In addition, the geometric mean titer of neutralizing antibodies of the RBD-trimer-CoV-T mRNA vaccine of the present application to the prototype strain WT was only 38.7, and it could provide complete protection against lethal SARS-CoV-2 prototype strain virus, which further illustrated that virus-specific T cells played a key role in the clearance of virus, and these data provided preliminary evidence for the RBD-trimer-CoV-T mRNA-LNP vaccine as a universal coronavirus vaccine.

[0249] As can be seen from the above embodiments, the anti-COVID-19 immunogenic composition provided in the present application, which combines the multimeric chimeric RBD immunogen and the T cell immunogen, can not only induce a broad-spectrum neutralizing antibody response, but also activate an effective cellular immune response to better inhibit viral replication; based on the unpredictability of the current epidemic and future variant coronavirus strains, the anti-COVID-19 immunogenic composition provided in the present application has broad application prospects for preventing changes in epidemic strains or co-epidemic of new multiple strains.

[0250] All documents mentioned in the present disclosure are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various modifications or changes can be made to the present disclosure by those skilled in the art upon reading the above teachings of the present disclosure, and such equivalent forms are also within the scope of the appended claims of the present application.

[0251] Appendix: Sequence Information

[0252] SEQ ID NO: 1: Full-length amino acid sequence of S protein RBD derived from Omicron variant BA.2 strain

[0253] SEQ ID NO: 2: Full-length amino acid sequence of S protein RBD derived from Omicron variant BA.2.75 strain

[0254] SEQ ID NO: 3: Full-length amino acid sequence of S protein RBD derived from Omicron variant XBB.1.5 strain

[0255] SEQ ID NO: 4: Full-length amino acid sequence of S protein RBD derived from Omicron variant BA.4 / 5 strain

[0256] SEQ ID NO: 5: Amino acid sequence of linker 1

[0257] SEQ ID NO: 6: Amino acid sequence of linker 2

[0258] SEQ ID NO: 7: Amino acid sequence of linker 3

[0259] SEQ ID NO: 8: Amino acid sequence of Foldon domain of T4 phage fibritin protein

[0260] SEQ ID NO: 9: Amino acid sequence of multimerized chimeric antigen BA.2-BA.2.75-BA.4 / 5-foldon construct

[0261] SEQ ID NO: 10: Amino acid sequence of multimerized chimeric antigen BA.2-XBB.1.5-BA.4 / 5-foldon construct

[0262] SEQ ID NO: 11: Amino acid sequence of T cell immunogen CoV-T

[0263] SEQ ID NO: 12: Amino acid sequence of T2A self-cleaving peptide

[0264] SEQ ID NO: 13: Amino acid sequence of BA.2-BA.2.75-BA.4 / 5-foldon-CoV-T construct (multimerized chimeric antigen-T2A self-cleaving peptide-T cell immunogen)

[0265] SEQ ID NO: 14: Amino acid sequence of BA.2-XBB.1.5-BA.4 / 5-foldon-CoV-T construct (multimerized chimeric antigen-T2A self-cleaving peptide-T cell immunogen)

[0266] SEQ ID NO: 15: Codon-optimized BA.2-BA.2.75-BA.4 / 5-foldon construct polynucleotide sequence

[0267] SEQ ID NO: 16: Codon-optimized BA.2-XBB.1.5-BA.4 / 5-foldon construct nucleic acid sequence

[0268] SEQ ID NO: 17: Codon-optimized T cell antigen CoV-T polynucleotide sequence

[0269] SEQ ID NO: 18: Codon-optimized BA.2-BA.2.75-BA.4 / 5-foldon-CoV-T construct polynucleotide sequence

[0270] SEQ ID NO: 19: Codon-optimized BA.2-XBB.1.5-BA.4 / 5-foldon-CoV-T construct polynucleotide sequence

[0271] SEQ ID NO: 20: Multimerization chimeric antigen N-terminal signal peptide amino acid sequence

[0272] SEQ ID NO: 21: CoV-T immunogen C-terminal fusion flag tag amino acid sequence

[0273] SEQ ID NO: 22: DNA template for BA.2-BA.2.75-BA.4 / 5-foldon mRNA

[0274] SEQ ID NO: 23: DNA template for BA.2-BA.2.75-BA.4 / 5-foldon-CoV-T

[0275] SEQ ID NO: 24: DNA template for BA.2-XBB.1.5-BA.4 / 5-foldon-CoV-T

[0276] SEQ ID NO: 25: aa319-aa591 amino acid sequence of wild-type RBD

Claims

1. An immunogen composition comprising RBD recombinant chimeric antigens, wherein the chimeric antigens comprise S protein RBD domains or functional fragments thereof from two or more SARS-CoV-2 subtypes.

2. The immunogen composition of claim 1, further comprising one or more domains selected from the group consisting of: multimerization domains; T cell immunogens.

3. The immunogenic composition of claim 1, wherein, the S protein RBD domains or functional fragments thereof are from a SARS-CoV-2 prototype strain and / or two or more SARS-CoV-2 variant strains selected from the group consisting of: variant strains selected from SARS-CoV-2 Omicron, such as Omicron subtypes BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, BA.4, BA.5, XBB, XBB.1.5, XBB.1.6, BQ.1, BQ.1.1, BF.7, EG.5, BA.2.86; and / or the S protein RBD domains or functional fragments thereof are from two or more SARS-CoV-2 variant strains selected from the group consisting of: BA.2, BA.2.75, XBB.1.2, and BA.4 / BA.5; and / or the S protein RBD domains or functional fragments thereof are from SARS-CoV-2 variant strains in the group consisting of: BA.2, BA.2.75, and BA.4 / BA.5, or BA.2, XBB.1.5, and BA.4 / BA.5; and / or the S protein RBD domains or functional fragments thereof from two or more SARS-CoV-2 variant strains are linked directly or via a linker peptide.

4. The immunogenic peptide of claim 1, wherein, the S protein RBD domains or functional fragments thereof have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to, and have the same or substantially the same immunogenicity as, the S protein RBD domains or functional fragments thereof of native SARS-CoV-2 variant strains; and / or wherein the S protein RBD domains or functional fragments thereof comprise a terminal cysteine modification to form a sRBD region; and / or wherein the S protein RBD domains or functional fragments thereof have an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

5. The immunogenic composition of claim 2, wherein, the multimerization domains are foldon domains of T4 bacteriophage fibritin protein, For example, a foldon domain having an amino acid sequence as set forth in SEQ ID NO: 8, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto and having a multimerization domain that promotes multimerization.

6. The immunogenic composition of claim 2, wherein, The T cell immunogen is ORF1ab or fragments thereof from SARS-CoV-2, ORF1ab derived sequences in combination with S, M, E, N, etc. derived sequences, or conserved and shared sequences from different coronaviruses; and / or, wherein the T cell immunogen has an amino acid sequence as set forth in SEQ ID NO: 11 or 21, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto and has T cell immunogenicity.

7. The immunogenic composition of any one of claims 1-6, further comprising one or more elements selected from the group consisting of: a signal peptide, for example, a signal peptide having an amino acid sequence as set forth in SEQ ID NO: 20; a linker peptide, e.g., one or more linker peptides selected from the group consisting of: (G4S) n (n = 1-8, e.g., (G4S)3, G4S, GSAGSAAGSGEF, (Gly)6, IRES, P2A, T2A (e.g., T2A having the sequence set forth in SEQ ID NO: 12), EFPKPSTPPGSSGGAP, KESGSVSSEQLAQFRSLD, (Gly)8, EGKSSGSGSESKST; and / or a molecular tag, for example, one or more tags selected from the group consisting of: FLAG tag, His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Sof-tagl, Sof-tag3, Strep-tag, TC tag, V5 tag, T7 tag, VSV tag, Xpress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag, and PNE tag; other proteins of viral or host origin, for example, other proteins for broadening the antiviral spectrum, increasing the ability to induce an immune response, such as transferrin (Fn), HIV p24, a stem of a capsid virus, such as influenza HA2, gp41 of HIV, Fc fragment of antibody, GM-CSF, IL-21, CD40L, or CD40 antibody.

8. The immunogenic composition of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 13, or 14, or having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or, the immunogenic composition is capable of inducing broad-spectrum anti-COVID-19 neutralizing antibodies and is capable of inducing cross-protective T cells against different coronaviruses.

9. A polynucleotide molecule encoding the immunogenic composition of any one of claims 1-8.

10. The polynucleotide molecule of claim 9, wherein, the nucleotide molecule is codon-optimized; and / or wherein the polynucleotide molecule comprises a sequence selected from the group consisting of SEQ ID NO: 15, 16, 17, 18, 19.

11. A vector comprising the polynucleotide molecule of claim 9 or 10.

12. A host cell comprising the vector of claim 11 and capable of expressing the immunogenic composition of any one of claims 1-8.

13. A vaccine against the novel coronavirus SARS-CoV-2 comprising the immunogenic composition of any one of claims 1-8, the polynucleotide molecule of claim 9 or 10, the vector of claim 11 and / or the host cell of claim 12.

14. Use of the immunogenic composition of any one of claims 1-8, the polynucleotide molecule of claim 9 or 10, the vector of claim 11 and / or the host cell of claim 12 for the manufacture of a vaccine for the prevention or treatment of the novel coronavirus SARS-CoV-2.

15. A method of manufacturing a vaccine against the novel coronavirus SARS-CoV-2, the method comprising: (a) providing the immunogenic composition of any one of claims 1-8, the polynucleotide molecule of claim 9 or 10, the vector of claim 11 and / or the host cell of claim 12; (b) combining the active substance provided in (a) with an immunologically or pharmaceutically acceptable carrier.

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