A recombinant cell wall antigen display technology of bacillus calmette-guerin (bcg) and its application in vaccine preparation

Abstract

The present application provides a kind of recombinant BCG cell wall antigen display technology and its application in vaccine preparation.The present application provides a kind of recombinant protein carrying antigen protein or its fragment, 19-KD antigen signal peptide, corresponding polynucleotide, vector, host cell and the like, and its application in the preparation of pharmaceutical composition, vaccine composition.The 19-KD antigen signal peptide of the present application can effectively express and enrich antigen-specific genes on the surface (especially cell wall) of recombinant BCG, so that the immune strategy of the recombinant BCG of the present application and the immune strategy combined with antigen protein vaccine can induce long-term neutralizing antibody production, and strong antigen-specific memory T cell response is generated, which provides a new idea for improving humoral immune response and vaccine combination immunization.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a cell wall antigen display technology for recombinant BCG and its application in vaccine preparation. Background Technology

[0002] Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is widely used in tuberculosis vaccines due to its high safety profile. It is also used in post-treatment therapy for bladder cancer and as an immune adjuvant. Due to its intracellular immunogenic properties, BCG has the potential to serve as a multifunctional vaccine vector against human pathogens and cancers, such as human immunodeficiency virus (HIV), human respiratory syncytial virus (RSV), and human parainfluenza virus (hMPV). Studies have shown that these recombinant BCG (rBCG) strains have demonstrated the potential to activate T cells in mouse models, for example, inducing hMPV antigen Th1 responses and HCV antigen CD8+ T cell responses, demonstrating that rBCG can induce immune protective responses against viral infections. However, it still cannot elicit high levels of antigen-specific humoral immune responses, especially the production of neutralizing antibodies, which remains a problem to be solved in current rBCG vaccine development. Although the induced T cells can assist B cells in antibody production, these T cells are usually insufficient to induce high levels of antigen-specific antibodies.

[0003] Therefore, there is an urgent need in this field to develop rBCG that can induce humoral immune responses, thereby improving the strength of antigen-specific immune responses elicited by rBCG vaccines. Summary of the Invention

[0004] The purpose of this invention is to provide a cell wall antigen display technology for recombinant BCG and its application in vaccine preparation.

[0005] In a first aspect of the invention, a recombinant protein carrying an antigen protein or a fragment thereof is provided, said recombinant protein comprising a structure as shown in Formula I:

[0006] Z0-Z1-Z2(I)

[0007] In the formula, Z0 is the 19-KD antigen or a fragment thereof;

[0008] Z1 is either a non-peptide or a flexible peptide;

[0009] Z2 is an antigen protein or a fragment thereof;

[0010] The "-" indicates a linking peptide or peptide bond.

[0011] In another preferred embodiment, the recombinant protein consists of a structure as shown in Formula I.

[0012] In another preferred embodiment, Z0 is a 19-KD antigen signal peptide sequence.

[0013] In another preferred embodiment, Z0 is the full-length sequence of the 19-KD antigen, having the amino acid sequence shown in SEQ ID NO:10.

[0014] In another preferred embodiment, the 19-KD antigen signal peptide sequence is selected from the group consisting of:

[0015] (a) An amino acid sequence as shown in SEQ ID NO:13 or 14;

[0016] (b) A sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 13 or 14;

[0017] (c) A sequence obtained by adding, deleting, modifying and / or substituting at least one (e.g., 1-6) amino acids based on the amino acid sequence shown in SEQ ID NO:13 or 14.

[0018] In another preferred embodiment, the 19-KD antigen signal peptide sequence is as shown in SEQ ID NO:13 or 14.

[0019] In another preferred embodiment, the amino acid sequence of Z0 is selected from the group consisting of:

[0020] (a) An amino acid sequence as shown in SEQ ID NO:10, 13 or 14;

[0021] (b) A sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, 13, or 14;

[0022] (c) A sequence obtained by adding, deleting, modifying and / or substituting at least one (e.g., 1-6) amino acids based on the amino acid sequence shown in SEQ ID NO: 10, 13 or 14.

[0023] In another preferred embodiment, the encoding sequence of Z0 is as shown in SEQ ID NO:2 or 9.

[0024] In another preferred embodiment, Z1 has a structure of (GGGGS)n, where n is a positive integer from 1 to 5, i.e., n is 1, 2, 3, 4 or 5.

[0025] In another preferred embodiment, the amino acid sequence of Z1 is GGGGSGGGGSGGGGS (SEQ ID NO:15) or GGGGSGGGGSGGGGSQA (SEQ ID NO:16).

[0026] In another preferred embodiment, the encoding sequence of Z1 is as shown in SEQ ID NO:3.

[0027] In another preferred embodiment, the antigen protein or a fragment thereof includes an antigenic epitope.

[0028] In another preferred embodiment, the antigen protein or a fragment thereof is a pathogen protein.

[0029] In another preferred embodiment, Z2 is selected from the group consisting of: viral antigen proteins, bacterial antigen proteins, parasitic antigen proteins, chlamydia antigen proteins, mycoplasma antigen proteins, autoantigen proteins, tumor antigen proteins, allergen proteins, toxin proteins, or fragments thereof, or combinations thereof.

[0030] In another preferred embodiment, the viral antigen protein includes viral surface proteins (such as glycoproteins, hemagglutinin proteins, etc.), viral transmembrane proteins, and viral spike proteins.

[0031] In another preferred embodiment, the viral antigen protein is selected from the group consisting of: influenza virus hemagglutinin (HA) or neuraminidase (NA), HIV virus gp120 or gp41, HBV surface antigen (HBsAg), coronavirus spike protein, Zika virus envelope (E) protein, yellow fever virus viral envelope protein, or combinations thereof.

[0032] In another preferred embodiment, the bacterial antigen protein is selected from the group consisting of: ESAT-6 or CFP-10 of Mycobacterium tuberculosis, capsular polysaccharide of Streptococcus pneumoniae, CTA1 or CTB of Vibrio cholerae, protein A of Staphylococcus aureus, alpha-toxin of Staphylococcus aureus, alginate lyase of Pseudomonas aeruginosa, exotoxin S of Pseudomonas aeruginosa, or combinations thereof.

[0033] In another preferred embodiment, the parasite antigen protein is selected from the group consisting of: CSP of Plasmodium, LACK of Leishmania, Sj23 of Schistosoma, VSG of Trypanosoma brucellosis (African trypanosomiasis), cathepsin L protease of Fasciola hepatica (liver fluke), or combinations thereof.

[0034] In another preferred embodiment, the chlamydia antigen protein includes Chlamydia trachomatis Pgp3.

[0035] In another preferred embodiment, the mycoplasma antigen proteins include: LppA, LppB, LppC, LppQ of Mycoplasma filamentosa, GAPDH of Mycoplasma bovis, GroEL, EF-Tu, greA, PDHC, DnaK, P67 (pMGA), P52, etc. of Mycoplasma gallisepticum.

[0036] In another preferred embodiment, the autoantigen protein is selected from the group consisting of: rheumatoid factor (RF), myelin basic protein (MBP), insulin, proteinase 3 (PR3), tyrosinase, or combinations thereof.

[0037] In another preferred embodiment, the tumor antigen protein is selected from the group consisting of: alpha-fetoprotein (AFP), HER2, CD19, CD20, PD-1, PD-L1, and Claudin. 18.2, BCMA, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD52, CD56, CD70, CD96, CD97, CD99, CD123, EGFR, HER2, HER3, CD117, C-Met, EGFR, EGFRvIII, ERBB3, ERBB4, VEGFR1, VEGFR2, ROR1, P THR2, B7-H1(PD-L1), B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, Trop-2, GPC-3, EPCAM, DLL- 3. Nectin-4, Claudin6, Muc-1, PSMA, GD3, FAP, CEA, EphA2, CTLA-4, LAG-3, TIGIT, MSLN, or combinations thereof.

[0038] In another preferred embodiment, the allergenic protein includes pollen and dust mites.

[0039] In another preferred embodiment, the toxin protein includes botulinum toxin.

[0040] In another preferred embodiment, Z2 is the SARS-CoV-2 RBD protein or the SARS-CoV-2 Spike protein; more preferably, Z2 is the SARS-CoV-2 RBD protein.

[0041] In another preferred embodiment, the coding sequence of the SARS-CoV-2RBD protein is shown in SEQ ID NO:4.

[0042] In another preferred embodiment, the amino acid sequence of the SARS-CoV-2RBD protein is shown in SEQ ID NO:17:

[0043] QPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNY NYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDP QTLERS (SEQ ID NO:17).

[0044] In another preferred embodiment, the amino acid sequence of Z0 is as shown in SEQ ID NO:10, 13 or 14, and the amino acid sequence of Z2 is as shown in SEQ ID NO:17.

[0045] In another preferred embodiment, the sequence of the recombinant protein is selected from the group consisting of:

[0046] (a) An amino acid sequence as shown in SEQ ID NO:11 or 12;

[0047] (b) A sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 11 or 12;

[0048] (c) A sequence obtained by adding, deleting, modifying and / or substituting at least one (e.g., 1-6) amino acids based on the amino acid sequence shown in SEQ ID NO:11 or 12.

[0049] In another preferred embodiment, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO:11 or 12; preferably, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO:11.

[0050] In a second aspect of the invention, a 19-KD antigen signal peptide is provided, the sequence of which is selected from the group consisting of:

[0051] (a) The amino acid sequence as shown in SEQ ID NO:13;

[0052] (b) A sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:13;

[0053] (c) A sequence obtained by adding, deleting, modifying and / or substituting at least one (e.g., 1-6) amino acids based on the amino acid sequence shown in SEQ ID NO:13.

[0054] In another preferred embodiment, the 19-KD antigen signal peptide sequence is shown in SEQ ID NO:13.

[0055] In a third aspect of the invention, a polynucleotide is provided that encodes a recombinant protein as described in the first aspect of the invention or a 19-KD antigen signal peptide as described in the second aspect of the invention.

[0056] In another preferred embodiment, the polynucleotide is selected from RNA (such as mRNA) and DNA (such as cDNA).

[0057] In another preferred embodiment, the polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:2 or 9.

[0058] In a fourth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the third aspect of the invention.

[0059] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0060] In another preferred embodiment, the vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0061] In another preferred embodiment, the vector is a plasmid, a retrovirus, or a lentiviral vector.

[0062] In another preferred embodiment, the vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, pMV261, etc.

[0063] In another preferred embodiment, the vector further includes elements selected from the group consisting of: promoters (e.g., the Rv3763 promoter of Mycobacterium tuberculosis H37Rv strain), transcriptional enhancement elements WPRE, long terminal repeat sequences (LTR), etc.

[0064] In another preferred embodiment, the vector comprises: an Rv3763 promoter sequence, the full-length Rv3763 gene or an Rv3763 signal peptide nucleotide sequence, a linker nucleotide sequence, and a SARS-CoV-2 RBD nucleotide sequence.

[0065] In another preferred embodiment, the Rv3763 promoter sequence is shown in SEQ ID NO:1.

[0066] In another preferred embodiment, the full length of the Rv3763 gene is as shown in SEQ ID NO:9.

[0067] In another preferred embodiment, the Rv3763 signal peptide nucleotide sequence is shown in SEQ ID NO:2.

[0068] In another preferred embodiment, the Linker nucleotide sequence is shown in SEQ ID NO:3.

[0069] In another preferred embodiment, the SARS-CoV-2RBD nucleotide sequence is shown in SEQ ID NO:4.

[0070] In a fifth aspect of the invention, a host cell is provided, the host cell containing a vector as described in the fourth aspect of the invention, or having a genome integrated with polynucleotides as described in the third aspect of the invention.

[0071] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0072] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0073] In another preferred embodiment, the host cell is Mycobacterium bovis, preferably BCG.

[0074] In a sixth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0075] (i) the recombinant protein as described in the first aspect of the invention, the polynucleotide as described in the third aspect of the invention, the vector as described in the fourth aspect of the invention, the host cell as described in the fifth aspect of the invention, or a combination thereof; and

[0076] (ii) Pharmaceutically acceptable carriers and / or excipients.

[0077] In another preferred embodiment, the pharmaceutical composition is a vaccine.

[0078] In another preferred embodiment, the pharmaceutical composition further includes the RBD-His recombinant protein.

[0079] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections and lyophilized preparations.

[0080] In a seventh aspect of the invention, a vaccine composition is provided, the vaccine composition comprising:

[0081] (i) the recombinant protein as described in the first aspect of the invention, the polynucleotide as described in the third aspect of the invention, the vector as described in the fourth aspect of the invention, the host cell as described in the fifth aspect of the invention, or a combination thereof; and

[0082] (ii) Immunologically acceptable carriers and / or excipients.

[0083] In another preferred embodiment, the vaccine composition further includes the RBD-His recombinant protein.

[0084] In another preferred embodiment, the vaccine composition further includes an adjuvant.

[0085] In another preferred embodiment, the adjuvant includes: alumina, saponins, quil A, muramyl dipeptide, mineral oil or vegetable oil, vesicle-based adjuvants, nonionic block copolymers or DEAE dextran, and cytokines (including IL-1, IL-2, IFN-γ, GM-CSF, IL-6, IL-12, CpG, etc.).

[0086] In another preferred embodiment, the adjuvant is AS01 adjuvant.

[0087] In another preferred embodiment, the vaccine composition is a nucleic acid vaccine composition, which contains a polynucleotide as described in the third aspect of the invention or a carrier as described in the fourth aspect of the invention.

[0088] In an eighth aspect of the invention, a use of the recombinant protein as described in the first aspect of the invention is provided for:

[0089] (a) Preparing antibodies against the antigen protein or fragments thereof; and / or

[0090] (b) To prepare a medicine for treating and / or preventing diseases associated with the said antigen protein or fragments thereof.

[0091] In another preferred embodiment, the disease includes, but is not limited to: viral infection, diseases or symptoms caused by viral infection (such as chronic COVID-19), autoimmune diseases, tumors, cardiovascular diseases, etc.

[0092] In another preferred embodiment, the drug comprises a pharmaceutical composition as described in the sixth aspect of the invention and a vaccine composition as described in the seventh aspect of the invention.

[0093] In a ninth aspect of the invention, a method for treatment and / or prevention is provided, the method comprising the steps of administering to a desired object a recombinant protein as described in the first aspect of the invention, a polynucleotide as described in the third aspect of the invention, a vector as described in the fourth aspect of the invention, a host cell as described in the fifth aspect of the invention, a pharmaceutical composition as described in the sixth aspect of the invention, or a vaccine composition as described in the seventh aspect of the invention.

[0094] In a tenth aspect of the invention, the use of the 19-KD antigen signal peptide as described in the second aspect of the invention is provided for:

[0095] (a) preparing the recombinant protein as described in the first aspect of the invention; and / or

[0096] (b) Prepare a vector for expressing and enriching the target protein in the cell wall of the host cell.

[0097] In another preferred embodiment, the target protein includes an antigen protein or a fragment thereof (the antigen protein or a fragment thereof is as described in the first aspect of the invention).

[0098] In another preferred embodiment, the host cell is Mycobacterium bovis, preferably BCG.

[0099] In another preferred embodiment, the carrier comprises the carrier as described in the fourth aspect of the invention.

[0100] In another preferred embodiment, the vector is a plasmid.

[0101] In an eleventh aspect of the present invention, a cell wall display technique for recombinant BCG (rBCG) expressing antigen protein or fragments thereof is provided, the technique comprising the steps of:

[0102] (i) providing or constructing a carrier as described in the fourth aspect of the invention; and

[0103] (ii) The vector from step (i) is transferred into wild-type BCG to obtain recombinant BCG (rBCG) expressing the antigen protein or fragment thereof carried by the vector, wherein the antigen protein or fragment thereof is enriched on the cell wall of the recombinant BCG (rBCG).

[0104] In another preferred embodiment, the vector is a plasmid.

[0105] In another preferred embodiment, the vector comprises the promoter of Mycobacterium tuberculosis H37Rv strain Rv3763, preferably having a nucleotide sequence as shown in SEQ ID NO:1.

[0106] In another preferred embodiment, the antigen protein or a fragment thereof is as described in the first aspect of the invention.

[0107] In another preferred embodiment, the antigen protein or a fragment thereof is the SARS-CoV-2RBD protein.

[0108] In another preferred embodiment, the method of transfer in step (ii) is electric shock transfer.

[0109] In another preferred embodiment, the wild-type BCG is a Danish strain.

[0110] In another preferred embodiment, the primers used in step (i) include: sequences as shown in SEQ ID NO:5 or 6; and / or sequences as shown in SEQ ID NO:7 or 8.

[0111] In another preferred embodiment, the technique further includes the step of:

[0112] (iii) Expanding the culture of the recombinant BCG (rBCG); and / or

[0113] (iv) Screening and / or purification of the recombinant BCG (rBCG); and / or

[0114] (v) Identify the expression of the antigen protein or a fragment thereof.

[0115] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0116] Figure 1 A schematic diagram of the pMV261 recombinant plasmid expressing the RBD antigen is shown. VKRGLTVAVAGAAILVAGLSGCSSNKSTTG (SEQ ID NO:13) is the 19-KD antigen signal peptide sequence (i.e., the amino acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO:2), where LSG represents the amino acid sequence at the signal peptidase cleavage site, and CSSNKSTTG (SEQ ID NO:14) is the mature signal peptide sequence. The gray dashed box represents the CDS region, which is the 867 bp gene sequence encoding the rBCG::RBD protein, including the mature 19-KD antigen signal peptide gene sequence, the linker sequence, and the RBD protein gene sequence.

[0117] Figure 2 This study demonstrates the expression and quantitative analysis of RBD antigen protein in the recombinant BCG cell wall, which displays the RBD antigen, using Western blotting. Figure 2A: Western blotting was used to verify the subcellular localization of the RBD antigen in the CW-rBCG::RBD vector and the expression level of the RBD antigen in the subcellular components. Using the pMV261 empty vector BCG as a control, the presence of the RBD antigen in the rBCG::RBD subcellular components was verified, with the target RBD protein band located at 35 kDa-40 kDa. Lanes 1-7 represent the components of CW-rBCG::RBD, lane 8 represents the protein marker, and lanes 9-15 represent the components of the pMV261 empty vector BCG. Figure 2 B: According to Figure 2 C and Figure 2 The quantitative results of the RBD content of cell wall and cell membrane components in D were calculated, and the result was obtained per 1×10 7 The content of RBD antigen in the cell wall, cell membrane and secretory components of CFU cells; Figure 2 C-2D: Western blotting was used to quantify antigen expression levels in the cell wall and cell membrane, and ImageJ software was used to analyze the band gray values ​​and the fitting standard curve with the standard. Figure 2 E: Western blotting was used to verify the expression of CW-rBCG::RBD secretory RBD protein. Lane 2 contained MPT64 protein, which served as an internal control for detecting secretory proteins in mycobacterial culture supernatant. The RBD secretory protein was located between 35-40 kDa. Compared with the control group, no target band of RBD secretory protein was detected in the CW-rBCG::RBD culture supernatant.

[0118] Figure 3 A schematic diagram of the Triton X-114 subcellular component separation process is shown. Figure 3 A), and a schematic diagram of the enrichment of subcellular components in Triton X-114 (A). Figure 3 B).

[0119] Figure 4 This study demonstrated that immunization of BALB / c mice with CW-rBCG::RBD induced RBD-specific IgG antibodies and neutralizing antibodies (Nab) in the early stages. Figure 4 A is an overview of the immunization procedures for the experimental and control groups; Figure 4 B-4C is used in ELISA experiments to detect immunized mice in week 4. Figure 4 B) and week 8 ( Figure 4 C) Serum RBD-specific IgG titer, where "A" represents the CW-rBCG::RBD group and A6 represents the standard dose group (10 μL / 100 μL). 6 cfu); Figure 4 D represents the neutralizing activity detected in the serum of immunized mice. NT was measured at week 8 using a SARS-CoV-2 RBD pseudovirus (PsV) neutralization assay.50 Values. Serum samples were obtained by pooling equal amounts of serum from 6 samples in each group; data are presented as geometric mean ± standard error of three independent replicates. Student's t-test was used to determine significant differences between the two groups, *p<0.05, ****p<0.0001.

[0120] Figure 5 The results showed that immunization of mice with recombinant BCG induced antigen-specific T cell responses. This was compared with parental BCG strains carrying the pMV261 empty vector plasmid and RBD. AS01 Compared with the PBS group, CW-rBCG::RBD induced an RBD-specific cellular immune response. At 12 weeks of immunization, the spleen, inguinal lymph nodes, and lungs of mice (6 mice per group) were combined and processed into single-cell suspensions. The cells were stimulated in vitro with a 2.5 μg / mL SARS-CoV-2 RBD peptide library for 44 h, and the proportions of Tfh and Tcm cells were then detected by flow cytometry. Figure 5 A, Figure 5 C: In the reinforced group ( Figure 5 A) and the primary immunization group ( Figure 5 C) Detection of Tfh(CD4) + CD44 + CD62L - GL7 + CXCR5 + PD-1 + )cell; Figure 5 B. Figure 5 D: In the reinforced group ( Figure 5 B) and the primary immunization group ( Figure 5 D) Detection of CD4+Tcm(CD4+) + CD44 + CD62L + )cell; Figure 5 E: Using the ELISPOT experiment, for every 10 5 The number of IFN-γ-secreting cells in each lung cell was quantified. Data are expressed as mean ± standard error, and flow cytometry data were from four independent experiments. Student's t-test was used to determine significant differences between the two groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0121] Figure 6 The combined immunization with recombinant BCG and RBD protein subunit vaccines induced long-acting neutralizing antibodies and antigen-specific memory T cell responses. CW-rBCG::RBD and RBD AS01 The RBD-specific memory T cells and neutralizing antibodies are combined via a subcutaneous route (sc.). Figure 6A: Schematic diagram of the immunization protocol. BALB / c mice (6 mice per group) were subcutaneously inoculated with a standard dose (10...) via the paw pads. 6 cfu)BCG and RBD AS01 Combinations (denoted as BCGscRBD) AS01 ), standard dose CW-rBCG::RBD and RBD AS01 The combination (denoted as A6scRBD) AS01 (or RBD alone) AS01 (referred to as RBD) AS01 All groups received booster immunizations against RBD after 2 weeks. AS01 ; Figure 6 B. Figure 6 E: At 16 weeks ( Figure 6 B) and 31 weeks ( Figure 6 E) The neutralizing antibody titer (NT) in serum samples (4 animals per group) was detected by a SARS-CoV-2RBD PsV (pseudovirus) neutralization assay. 50 (Through three independent repeated experiments); Figure 6 C Figure 6 D: Memory T cell responses were detected by flow cytometry at week 12 of immunization. Cell proportion analysis of the spleen was performed, including follicular helper T cells (Tfh). Figure 6 C, CD4 + CD44 + CD62L - GL7 + CXCR5 + PD-1 + ); Cell proportion analysis of the lungs, central memory T cells (Tcm) ( Figure 6 D, CD4 + CD44 + CD62L + The cells were stimulated in vitro with a 2.5 μg / mL SARS-CoV-2 RBD peptide library. Data were analyzed using the Student-t test to determine statistical significance between the two groups: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0122] Figure 7 A schematic diagram of the recombinant pMV261 plasmid vector in Example 6 is shown. Figure 7 A) and Western Blot experiments were used to verify the expression of the fusion protein 19-KD-RBD in each component of recombinant BCG. Figure 7 B). Figure 7In lane B, M: protein marker; lane 1: control group - BCG empty vector group - cell wall (TritonX-114 insoluble); lane 2: control group - BCG empty vector group - cell membrane connected to the cell wall; lane 3: control group - BCG empty vector group - supernatant after centrifugation (PBS lysate); lane 4: control group - BCG empty vector group - cytoplasm; lane 5: control group - BCG empty vector group - cell membrane; lane 6: rBCG-19-KD-RBD cell wall (TritonX-114 insoluble); lane 7: cell membrane connected to the rBCG-19-KD-RBD cell wall; lane 8: rBCG- Supernatant after centrifugation of 19-KD-RBD (PBS-cracked bacteria); Lane 9: rBCG-19-KD-RBD cytoplasm; Lane 10: rBCG-19-KD-RBD cell membrane; Loading volumes of each component: Lane 1: 30 μL; Lane 2: 21.6 μL (0.1 μg); Lane 3: 20.2 μL (20 μg); Lane 4: 18.4 μL (10 μg); Lane 5: 18.4 μL (2 μg); Lane 6: 30 μL; Lane 7: 28 μL (0.1 μg); Lane 8: 21.6 μL (20 μg); Lane 9: 30.4 μL (10 μg); Lane 10: 32 μL (2 μg).

[0123] Figure 8 A schematic diagram of the recombinant pMV261 plasmid vector in Example 7 is shown. Figure 8 A) and Western Blot experiments were used to verify the expression of the fusion protein 19-KD-RBD in each component of recombinant BCG. Figure 8 B). Figure 8In lane B, M: protein marker; lane 1: control group - BCG empty vector group - cell wall (TritonX-114 insoluble); lane 2: control group - BCG empty vector group - cell wall; lane 3: control group - empty vector group - supernatant after centrifugation (PBS-cracked bacteria); lane 4: control group - BCG empty vector group - cytoplasm; lane 5: control group - BCG empty vector group - cell membrane; lane 6: rBCG cell wall (TritonX-114 insoluble); lane 7: cell membrane connected to the rBCG cell wall; lane 8: supernatant after centrifugation (PBS-cracked bacteria); lane 9: rBCG cytoplasm. Lane 10: rBCG cell membrane (isolated from the supernatant of cell lysis); Lane 11: rBCG secretory protein; Loading volumes of each component: Lane 1: 30 μL (15 μg); Lane 2: 21.6 μL (0.1 μg); Lane 3: 20.2 μL (20 μg); Lane 4: 18.4 μL (10 μg); Lane 5: 18.4 μL (2 μg); Lane 6: 20 μL; Lane 7: 9.6 μL (0.1 μg); Lane 8: 19.2 μL (20 μg); Lane 9: 16.8 μL (10 μg); Lane 10: 22.4 μL (2 μg); Lane 11: 20 μL.

[0124] Figure 9 A schematic diagram of the recombinant pMV261 plasmid vector in Example 8 is shown. Figure 9 A), and Western Blot experiments were used to verify the expression of the fusion protein 19-KD-S in each component of recombinant BCG. Figure 9 B). Figure 9 In section B, lane 1: supernatant obtained by centrifugation of whole-cell lysate (PBS buffer); lane 2: secreted protein; lane 3: rBCG-19-KD-S cytoplasm; lane 4: cell wall of rBCG-19-KD-S fusion expression vector; lane 5: rBCG-19-KD-S cell membrane; lane 6: marker; lane 7: other rBCG vectors as control group for WB experiment; loading volumes of each component: lane 1: 15 μL (15 μg); lane 2: 32 μL (0.04 μg); lane 3: 32 μL (15 μg); lane 4: 32 μL (0.4 μg); lane 5: 32 μL (0.4 μg); lane 6: 2 μL. Detailed Implementation

[0125] Through extensive and in-depth research and numerous screenings, the inventors have, for the first time, creatively discovered an rBCG antigen surface display technology to improve the efficacy of rBCG vaccines. Specifically, through innovative genetic engineering methods, antigens are presented on the surface of rBCG (especially the cell wall), and simultaneous immunization of mice to verify antigen-specific responses has confirmed that both immunization with rBCG alone to induce an immune response, and combined immunization with rBCG and antigen proteins (such as SARS-CoV-2 RBD subunit protein) vaccines, can promote and enhance the production of recombinant BCG-induced antibodies, providing new insights into improving humoral immune responses and combined vaccine immunization. This invention was completed based on these findings.

[0126] the term

[0127] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0128] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0129] The terms “administration” or “giving” refer to the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0130] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also known as "sequence consistency") refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Generally, sequence alignment is performed and gaps (if any) are introduced before calculating the percentage of similarity between two amino acid or nucleotide sequences. If, at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be identical or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, sequence consistency is obtained by dividing the number of matching positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and / or gap length are also taken into account. For the purposes of this invention, the publicly available alignment software BLAST (available at ncbi.nlm.nih.gov) can be used to obtain the optimal sequence alignment and calculate the sequence consistency between two amino acid or nucleotide sequences using default settings.

[0131] When referring to pharmaceutical compositions, the term "pharmaceuticalally acceptable carrier" refers to substances such as solid or liquid diluents, fillers, antioxidants, and stabilizers that can be safely administered to humans and / or animals without excessive adverse side effects, and which are suitable for maintaining the activity of the drug or active agent contained therein.

[0132] 19-KD antigen

[0133] The 19-KD antigen of Mycobacterium tuberculosis is a protein encoded by the Rv3763 gene, which is expressed on the cell membrane surface of Mycobacterium tuberculosis. This antigen exhibits strong immunogenicity during the interaction between Mycobacterium tuberculosis and host immune cells. Furthermore, it can serve as a biomarker for detecting serum samples from patients with active tuberculosis and has significant potential value in research on candidate vaccines.

[0134] Composition and method of application

[0135] The present invention also provides a composition comprising: (i) the recombinant protein or polynucleotide or carrier or host cell of the present invention, or a combination thereof, and (ii) a pharmaceutically or immunologically acceptable excipient or adjuvant.

[0136] In this invention, the term "containing" indicates that various ingredients may be used together or present in the composition of this invention. Therefore, the terms "consistent with" and "composed of" are included in the term "containing".

[0137] The compositions of the present invention include pharmaceutical compositions and vaccine compositions.

[0138] The compositions of the present invention can be monovalent (containing only one recombinant protein, polynucleotide, carrier, or host cell) or polyvalent (containing multiple recombinant proteins, polynucleotides, carriers, or host cells).

[0139] The pharmaceutical or vaccine compositions of the present invention can be prepared into various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc.

[0140] (i) Pharmaceutical Composition

[0141] The pharmaceutical compositions of the present invention comprise (or contain) a therapeutically effective amount of the recombinant protein or polynucleotide or carrier or host cell of the present invention.

[0142] As used herein, the term "therapeutic effective dose" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or the amount that exhibits a detectable therapeutic or preventative effect. This effect can be detected, for example, by antigen levels. Therapeutic effects also include a reduction in physiological symptoms. The precise effective dose for a given subject depends on that subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. Therefore, it is not useful to pre-specify an exact effective dose. However, for a given condition, the effective dose can be determined using routine laboratory testing.

[0143] For the purposes of this invention, an effective dose is approximately 0.001 mg / kg to 1000 mg / kg, preferably approximately 0.01 mg / kg to 100 mg / kg body weight, of recombinant protein administered to an individual. An effective dose may also be approximately 10 mg / kg per 100 μL. 3 CFU-10 12 CFU, preferably about 10 μL per 100 μL 5 CFU-10 10 CFU, preferably about 10 μL per 100 μL 6 CFU-10 7 CFU in host cells (such as recombinant BCG).

[0144] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent (such as the recombinant protein or polynucleotide of the present invention, or a carrier or host cell). This term refers to pharmaceutical carriers that do not induce antibodies harmful to the individual receiving the composition and do not cause excessive toxicity after administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, etc. These carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0145] Pharmaceutically acceptable carriers in a composition may include liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc. Typically, the composition can be formulated as an injectable preparation, such as a liquid solution or suspension; it can also be formulated as a solid form suitable for reconstitution into solutions or suspensions, or liquid excipients, prior to injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

[0146] (ii) Vaccine composition

[0147] The vaccine (composition) of the present invention can be preventive (i.e., prevent disease) or therapeutic (i.e., treat disease after infection).

[0148] These vaccines contain immune antigens (including, for example, the recombinant proteins of this invention) and are typically combined with pharmaceutically acceptable carriers, including any carrier that does not itself induce antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those skilled in the art. Additionally, these carriers can act as immunostimulants (“adjuvants”). Furthermore, the antigens can also be conjugated to bacterial toxoids (such as toxoids of pathogens such as diphtheria, tetanus, cholera, and Helicobacter pylori).

[0149] Preferred adjuvants for enhancing the efficacy of immune compositions include, but are not limited to: (1) aluminum salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, such as (a) MF59 (see WO 90 / 14837), (b) SAF, and (c) Ribi. TM (2) Adjuvant systems (RAS) (Ribi Immunochem, Hamilton, MT); (3) Saponin adjuvants; (4) Freund complete adjuvants (CFA) and Freund incomplete adjuvants (IFA); (5) Cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon), macrophage colony-stimulating factor (M-CFS), tumor necrosis factor (TNF), etc.; (6) Detoxified variants of bacterial ADP-ribosylated toxins (e.g., Escherichia coli heat unstable toxin LT); and (7) Other substances that enhance the effect of the composition as immunostimulants.

[0150] Vaccine compositions, including immunogenic compositions (e.g., may include antigens, pharmaceutically acceptable carriers, and adjuvants), typically contain diluents such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances such as wetting agents or emulsifiers, pH buffers, etc., may be present in these carriers.

[0151] More specifically, vaccines, including immunogenic compositions, contain an immunologically effective amount of an immunogenic peptide, as well as the other required components mentioned above. An "immunologically effective amount" refers to the amount administered to an individual as a single or partial dose that is effective for treatment or prevention. This dosage can be determined based on the individual's health and physiological condition, the individual's class (e.g., human), the individual's immune system's ability to synthesize antibodies, the required level of protection, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. This dosage is expected to be within a relatively wide range and can be determined through routine laboratory testing.

[0152] Typically, vaccine compositions or immunogenic compositions can be formulated as injectable preparations, such as liquid solutions or suspensions; they can also be formulated as solid forms suitable for reconstitution into solutions or suspensions or liquid excipients prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance adjuvant effects.

[0153] Furthermore, the vaccine composition of the present invention can be a monovalent or multivalent vaccine.

[0154] (iii) Route of administration and dosage

[0155] Once formulated into the composition of the present invention, it can be administered directly to the subject. The subject to be treated can be a mammal, especially a human.

[0156] When used as a vaccine, the recombinant proteins or polynucleotides or vectors or host cells of the present invention can be directly administered to an individual using known methods. These vaccines are typically administered via the same route of administration as conventional vaccines and / or by mimicking the pathogen infection pathway.

[0157] The routes of administration for the pharmaceutical or vaccine compositions of the present invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral, or other parenteral routes. Routes of administration may be combined if necessary, or adjusted according to the disease condition. Vaccine compositions may be administered in single or multiple doses, and may include booster doses to induce and / or maintain immunity.

[0158] Recombinant protein vaccines should be administered in an "effective amount," meaning that the amount of recombinant protein is sufficient to elicit an immune response and effectively protect the host against the associated disease via the chosen route of administration.

[0159] Representative diseases include (but are not limited to): autoimmune diseases, tumors, etc.

[0160] The amount of recombinant protein selected in each vaccine dose is determined based on the amount that can elicit a protective immune response without significant side effects. Typically, after infecting host cells, each dose of vaccine is sufficient to contain approximately 1 μg-1000 mg, preferably 1 μg-100 mg, and more preferably 10 μg-50 mg of protein. The optimal dosage of a specific vaccine can be determined using standard research methods, including antibody titers and other responses in the subjects. Whether a booster dose is needed can be determined by monitoring the level of immunity provided by the vaccine. After assessing the antibody titer in the serum, a booster dose immunization may be necessary. Administration of adjuvants and / or immunostimulants can enhance the immune response to the proteins of this invention.

[0161] The preferred method is to administer the immunogenic composition by injection via a parenteral (e.g., subcutaneous) or intravenous route.

[0162] Furthermore, the vaccine of the present invention can be administered in combination with other immunomodulators or with other therapeutic agents.

[0163] Main advantages of the invention

[0164] 1. The recombinant BCG of this invention can significantly induce the production of neutralizing antibodies: This invention proposes a novel design strategy in the design and construction of recombinant BCG vaccines, which effectively presents antigens on the surface (especially the cell wall) of recombinant BCG through genetic engineering, achieving the production of neutralizing antibodies per 10 7 The amount of antigen per colony-forming unit is approximately 850 ng. The recombinant BCG carrier and immunogenicity are used to deliver the antigen RBD to induce a Tfh cell response, promote the production of neutralizing antibodies, and simultaneously generate a strong antigen-specific memory T cell response.

[0165] 2. The recombinant BCG of the present invention can also be used in combination with RBD subunit vaccines for immunization, inducing the production of long-acting neutralizing antibodies, enhancing humoral immune responses, and providing a new approach for combined vaccine immunization.

[0166] 3. This invention is the first to discover that the 19-KD antigen signal peptide can be used to express and enrich the target protein on the cell wall of the host cell, and thus can be used to prepare corresponding recombinant proteins and vectors (such as plasmids) to guide, display and enrich the target protein on the cell surface (especially the cell wall).

[0167] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0168] Example 1: Construction of recombinant BCG with RBD antigen displayed on its surface

[0169] 1. Construct a recombinant pMV261 plasmid vector expressing SARS-CoV-2 RBD antigen:

[0170] In this embodiment, after extensive screening of suitable signal peptides and other elements, a shuttle-type pMV261 recombinant plasmid for Escherichia coli and Mycobacterium was constructed.

[0171] The basic method is as follows:

[0172] First, the heat shock protein Hsp60 promoter of the pMV261 plasmid (starting plasmid) is removed and replaced with the Rv3763 promoter encoding the 19-KD antigen of Mycobacterium tuberculosis H37Rv strain. The Rv3763 signal peptide, the gene linker sequence (such as a flexible peptide), and the antigen-specific gene (in this embodiment, an exemplary example is the novel coronavirus SARS-CoV-2RBD) are inserted at the multiple cloning site. The expression cassette of the pMV261 recombinant plasmid is constructed in this order to obtain the antigen fusion plasmid (in this embodiment, an exemplary example is the SARS-CoV-2RBD antigen fusion plasmid).

[0173] The nucleotide sequence of the promoter of Mycobacterium tuberculosis H37Rv strain Rv3763 is shown in SEQ ID NO:1, specifically:

[0174] ACCTCGGGCCCTGCTAACGCGCATACTGCCGAAGCGGTCCTCAATGC CGATGGACCGCTACGACAGGCAAAGGAGCACAGG;

[0175] The nucleotide sequence of the Rv3763 signal peptide is shown in SEQ ID NO:2, specifically:

[0176] GTGAAGCGTGGACTGACGGTCGCGGTAGCCGGAGCCGCCATTCTGGT CGCAGGTCTTTCCGGATGTTCAAGCAACAAGTCGACTACAGGA;

[0177] The gene linker nucleotide sequence is shown in SEQ ID NO:3, specifically:

[0178] GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGC AAGCT;

[0179] The nucleotide sequence of the novel coronavirus SARS-CoV-2RBD is shown in SEQ ID NO:4, specifically:

[0180] .

[0181] A schematic diagram of the pMV261 recombinant plasmid is shown below. Figure 1 As shown.

[0182] The specific steps are as follows:

[0183] a) Using the SARS-CoV-2S antigen plasmid as a template, the SARS-CoV-2 RBD antigen sequence containing the linker sequence was amplified using a high-fidelity PCR enzyme. The primers used for amplifying the RBD sequence were:

[0184] F1 primer:

[0185] GCAACAAGTCGACTACAGGAGGTGGAGGCGGTTCAGGCG(SEQ ID NO:5)

[0186] R1 primer:

[0187] CTTCGAATTCTGCAGCTGGATCTTAACTACGCTCAAGTGTCTGTGGAT CACG(SEQ ID NO:6)

[0188] Using the M.tb H37RV genome as a template, the 19KD gene promoter and the 19KD gene signal peptide sequence were amplified. The primers used for amplifying the 19KD promoter and signal peptide sequence were:

[0189] F2 primer:

[0190] GCTCTAGAGGCAGTGGGAGGTTTGTGTTCCATCG(SEQ ID NO:7)

[0191] R2 primer:

[0192] CGGGATCCTTATCCTGTAGTCGACTTGTTGCTTGAACATC(SEQ ID NO:8)

[0193] b) PCR amplification program: 98℃ pre-denaturation for 2 min, melting temperature 95℃ for 10 s, annealing temperature 63℃ for 1 min, 35 cycles, cooling temperature 10℃.

[0194] c) Digest the pMV261 plasmid and the amplified 19KD sequence described above using enzymes, with restriction sites XbaI and BamHI. The digested 19KD fragment is then ligated to the vector using T4 DNA ligase. The promoter sequence for constructing the pMV261 plasmid expressing the RBD antigen is the 19KD gene promoter from the M.tb H37RV genome, and the signal peptide sequence is the 19KD signal peptide.

[0195] d) The pMV261 plasmid was digested at the bamHI and SalI sites, and the amplified RBD sequence was ligated by homologous recombination with the plasmid digested with bamHI and SalI.

[0196] e) Transform 20 μL of the homologous recombinant product into 100 μL of DH5αE.coli competent cells, heat shock at 42℃ for 90 s, place on ice for 30 s, add LB medium, and incubate at 37℃ for 45 min. Spread 20 μL onto LB solid medium containing 50 μg / mL kanamycin and incubate at 37℃ for 24 h.

[0197] f) Select clones, extract plasmids, and perform gene sequencing verification.

[0198] 2. Construct a recombinant BCG vector expressing SARS-CoV-2 RBD antigen:

[0199] The basic method is as follows:

[0200] The SARS-CoV-2 RBD antigen fusion plasmid obtained in step 1 was electroporated into BCG (Danish strain) to obtain rBCG. rBCG was then cultured in kanamycin-resistant medium to amplify the bacterial suspension. The bacterial suspension was washed with PBS buffer to adjust the bacterial concentration to 10. 7 One colony-forming unit per milliliter, stored at 4°C, is the recombinant BCG displaying the RBD antigen for immunizing mice, named CW-rBCG::RBD.

[0201] The specific steps are as follows:

[0202] a) Preparation of BCG (Danish strain) competent cells: Take 10 mL OD 600 The BCG bacterial culture was prepared at 1.0 g / mL, centrifuged at 3000 g for 10 min at room temperature, washed three times with 10% glycerol, centrifuged to a volume of 1.5 mL, aliquoted, and stored at -80°C. The competent cells contained 10% glycerol.

[0203] b) Electroporate the pMV261-19KD-19KD Signal peptide-RBD plasmid into BCG (Danish strain): Add 1 μg of plasmid to 400 μL of competent cells and perform electroporation at 2.5 kV, 25 μF, and 1000 Ω. Add 4.5 mL of 7H9 and 0.5 mL of OADC, incubate at 37°C in a shaker for 24 h, centrifuge, plate onto 25 μg / mL kanamycin-resistant 7H10 solid medium, and incubate statically for 3-4 weeks.

[0204] c) Select single clones and incubate them in 7H9 medium containing 0.05% Tween80 at 100 rpm and 37°C for 2 weeks.

[0205] d) The bacterial culture was subsequently ground and broken up for protein level verification of RBD antigen expression.

[0206] Example 2: Quantitative analysis and subcellular localization analysis of RBD antigen expressed by recombinant BCG

[0207] 1. The basic method for Western Blot validation and quantification of RBD protein antigen expressed by CW-rBCG::RBD is as follows:

[0208] The same concentration and volume of bacterial suspension containing the parental BCG strain (Danish) carrying the pMV261 empty vector plasmid (as a control) and CW-rBCG::RBD were centrifuged to collect bacterial cells for separating the components. Equal volumes of protein electrophoresis samples were taken and electrophoresed on the same gel, then imaged on the same PVDF membrane. Heat shock protein 65 (Hsp65) is a mycobacterial internal reference protein with a molecular weight of approximately 65 kDa. The theoretical molecular weight of the BCG-expressed RBD fusion protein with a 19 kDa signal peptide is approximately 35 kDa. During electrophoresis, the band size was between 35 kDa and 40 kDa. Figure 2 As shown in Figure A, lanes 1-7 represent the components of CW-rBCG::RBD, lane 8 represents the protein marker, and lanes 9-15 represent the components of the pMV261 empty vector BCG.

[0209] like Figure 2 Figures B, 2C, and 2D show the quantitative analysis of Western Blot 10. 7 RBD expression in cfu CW-rBCG::RBD was analyzed by comparing the RBD protein content in the cell wall, cell membrane, and secretory components of rBCG. The results showed that RBD expression was primarily enriched in the cell wall, with approximately 850 ng of recombinant RBD protein detected per 10^10 cells in the cell wall component. 7 In each colony-forming unit, relatively little RBD recombinant protein expression was detected in the cell membrane component, and even less RBD recombinant protein was present in the cytoplasm, while virtually no RBD recombinant protein expression was detected in the supernatant (secretory component).

[0210] like Figure 2 As shown in Figure E, Western blotting validates the expression of CW-rBCG::RBD secretory RBD protein. Lane 2 contains MPT64 protein, which serves as an internal control for detecting secretory proteins in the mycobacterial culture supernatant. The RBD secretory protein is located between 35-40 kDa. Compared to the control group, no target band of RBD secretory protein was detected in the CW-rBCG::RBD culture supernatant. This Western blotting validation further confirms that the expression of recombinant RBD protein was essentially undetectable in the CW-rBCG::RBD supernatant (secretory fraction).

[0211] 2. The specific steps for subcellular localization analysis of SARS-CoV-2 RBD antigen expressed by recombinant BCG vector are as follows:

[0212] 1) Bacterial culture:

[0213] Inoculate 10 mL of 7H9 medium containing 10% OADC with glycerol culture culture at -80℃. Add Kanamycin and 0.05% Tween 80 to a final concentration of 25 μg / mL. Incubate at 37℃ with shaking at 100 rpm until OD500 is reached. 600 The value is 0.8. Take 5 mL of OD. 600 The bacterial culture was prepared at 0.8 μg / mL in 7H9 medium containing 10% OADC, 25 μg / mL Kanamycin, and 0.05% Tween 80. The culture was incubated at 37°C with shaking at 100 rpm until OD500 was reached. 600 1.0.

[0214] 2) Collect bacterial cells:

[0215] Cultivate to OD 600 1.0 The bacterial culture was placed on ice and transferred to a sterile centrifuge tube. The cells were collected by centrifugation at 3000g, 4°C for 7 min. The cells were then resuspended in 30 mL of pre-chilled sterile PBS (pH 7.4) and collected by centrifugation. The process was repeated three times. During the final wash, the PBS was completely discarded, and any remaining PBS buffer was aspirated with a pipette tip. Meanwhile, the culture supernatant was stored at 4°C (using Soton medium, suitable for ultrafiltration-concentrated media for secreted proteins).

[0216] 3) Weigh the collected bacterial cells:

[0217] Weigh and record the wet weight and volume of the bacterial cells (for bacterial cells not used immediately, divide them into 3 tubes and store them at -80℃ without repeated freeze-thaw cycles).

[0218] 4) Cell lysis:

[0219] Resuspend 300 μL of collected bacterial cells in 600 μL of sterile PBS (pH 7.4), then add quartz sand and grind the bacterial cells by shaking. Repeat 30 seconds each time, 6 times, with a 5-minute interval between each time, and place the lysis buffer on ice.

[0220] 5) Cell component separation:

[0221] a) Preparation of whole-cell component samples:

[0222] Take 100 μL of the homogenized bacterial lysis buffer and add 100 μL of 5× loading buffer. Incubate at 98 °C for 15 min.

[0223] b) Method for separating the supernatant from whole-cell lysate:

[0224] The lysate was centrifuged at 21000g at 4℃ for 15 min. Unlyzed cells were visible. The unlyzed cells were removed, and the remaining cells were centrifuged at 21000g at 4℃ for 1 h. Clear stratification was observed. The upper layer consisted of bacterial proteins dissolved in PBS buffer (enriched with cytoplasmic and cell membrane proteins), while the lower layer consisted of bacterial fragments (enriched with cell wall components).

[0225] Note: The Bradford protein concentration assay (595nm) is used to detect protein concentration. If the protein concentration in the soluble fraction reaches 3-5 mg / mL, subsequent separation operations can be performed.

[0226] c) Methods for separating cell wall components:

[0227] The precipitate from the lower layer of bacterial cell lysis obtained by centrifugation was resuspended in 1 mL of sterile PBS (pH 7.4) by pipetting, centrifuged at 21000g at 4°C for 30 min, and the process was repeated three times with PBS. The same volume of PBS (pH 7.4) as the supernatant was added, followed by 1 mL of 2% Triton X-114. The mixture was inverted and mixed at 4°C for 16 h. After incubating at 37°C for 10 min and centrifuging at 23°C at 21000g for 1 h to separate the two phases, the Triton X-114-insoluble precipitate in the bacterial cell lysis buffer (rich in cell wall components) was at the bottom of the centrifuge tube, the cell wall-soluble proteins extracted by Triton X-114 were in the lower layer, and the upper layer was the aqueous phase. 300 μL of 2% Triton X-114 was added to each of the two phases, and the mixture was inverted and mixed at 4°C for 1-2 h. The mixture was then incubated at 37°C for 10 min and centrifuged at 21000g for 1 h at room temperature. The lower phase was collected as cell wall components, and the precipitate insoluble in Triton X-114 also contained cell wall components.

[0228] d) Methods for separating cytoplasmic components:

[0229] Take 600 μL of the whole-cell lysate, centrifuge the supernatant, add 5% Triton X-114 to a final concentration of 2% Triton X-114, mix thoroughly at 4°C for 16 h, incubate at 37°C for 10 min, and centrifuge at 21000g for 1 h at room temperature. Aspirate the upper aqueous phase (excluding the interphase) and transfer it to a new 1.5 mL EP tube. Add 300 μL of 2% Triton X-114, mix thoroughly at 4°C for 1-2 h, and centrifuge at 21000g for 1 h at room temperature. Collect the upper aqueous phase (containing cytoplasmic proteins) and the lower and interphase phases (containing cell membrane proteins). Collect the upper phase from each centrifugation into one tube, and the lower and interphase phases into another tube. All operations were performed on ice.

[0230] e) Methods for separating cell membrane components:

[0231] After centrifugation, the cytoplasmic components were separated and transferred, with the lower and middle layers being the cell membrane components. 5% Triton X-114 was added to prepare a final concentration of 2%, and the mixture was inverted and mixed at 4°C for 16 h. The mixture was then placed in a 37°C water bath for 10 min and centrifuged at 21000g for 1 h at room temperature. The upper aqueous phase, excluding the middle layer, was aspirated and transferred to a new 1.5 mL EP tube. 300 μL of 2% Triton X-114 was added, and the mixture was inverted and mixed at 4°C for 1-2 h. The tube was then centrifuged at 21000g for 1 h at room temperature. The upper phase from each centrifugation was collected in one tube, and the lower and middle layers were collected in another tube. All operations were performed on ice.

[0232] f) Precipitated proteins:

[0233] Add 5 mL of acetone to each isolated fraction and incubate at -20°C for 16 h to precipitate the protein. Centrifuge at 21000 g for 30 min, pour the acetone into a hazardous liquid collection bottle, and remove any residual acetone with a pipette tip. Immediately add an appropriate volume of 75 mM Tris (pH 7.5) buffer.

[0234] g) Method for extracting secreted proteins from bacterial culture supernatant:

[0235] The bacterial cells were cultured according to the above method, centrifuged at 3000g for 7 min, and 60 mL of OD was collected. 600 The bacterial culture was at pH 1.0. The cells were resuspended in preheated Suton's medium (pH 7.0) at 37°C, centrifuged, washed three times, and the supernatant was discarded. The cells were then transferred to 200 mL of Suton's medium (pH 7.0), inoculated with 25 μg / mL kanamycin, 0.05% Tween 80, and cultured at 37°C with shaking at 100 rpm until OD500 was reached. 600 Centrifuge at 3000g for 7 min at 4℃ for 1.0-1.5 μm, collect bacterial cells and supernatant. Sterilize the supernatant by filtration through a 0.22 μm filter membrane, store on ice or at 4℃, add 40% ammonium sulfate to a final concentration, and stir at a constant speed for 16 h at 4℃ to precipitate the protein (4℃, 52g ammonium sulfate added to make a 40% ammonium sulfate solution). Centrifuge the supernatant at 27000g for 1 h, transfer the precipitate to a dialysis bag, mix by pipetting, and heat to boiling for 15 min. Place the dialysis bag in 2L of pre-chilled sterile PBS (pH 7.4) buffer at 4℃ and dialyze for 8 h. Replace the sterile PBS (pH 7.4) buffer and dialyze again for 8 h. The protein in the dialysis bag should be clear and transparent.

[0236] h) Add 70% ammonium sulfate to the supernatant after centrifugation and stir at 4°C for 16 hours to precipitate the protein. Centrifuge again at 27000g and 4°C for 1 hour. Dialyze the precipitate according to the above method.

[0237] i) Transfer the protein obtained from the two dialysis cycles to an ultrafiltration tube of appropriate molecular weight, and concentrate the volume by ultrafiltration at 4000 rpm and 4°C.

[0238] 6) Quantitative analysis of RBD antigen:

[0239] Integrated density analysis of the electrophoretic bands was performed using ImageJ software. A standard curve was constructed based on the integrated intensity of the RBD-His protein band (range 1.1 ng to 35.2 ng). Protein mass was quantified using the standard curve by measuring the integrated density values ​​of the electrophoretic bands obtained from Western blot analysis, and the RBD antigen content in the subcellular components was calculated. The antigen content in the subcellular components was further calculated by calculating the volume ratio of the subcellular components to the whole-cell lysate determined during separation. The RBD antigen content corresponding to 10⁷ colony-forming units (cfu) was calculated by multiplying the component volume ratio by the optical density (OD) of the bacteria used for separation at 600 nm.

[0240] like Figure 3 The diagram shown illustrates the process of separating subcellular components using Triton X-114. Figure 3 A), and a schematic diagram of the enrichment of subcellular components in Triton X-114 (A). Figure 3 B). The results also showed that in CW-rBCG::RBD, the recombinant RBD protein could be effectively expressed and enriched on the surface of rBCG (especially the cell wall).

[0241] The above results indicate that the Rv3763 signal peptide and Rv3763 promoter in the pMV261 recombinant plasmid effectively guide and promote the expression of the RBD recombinant protein in the insoluble part of Triton X-114, indicating that it can effectively express and enrich antigen-specific genes on the surface of rBCG (especially the cell wall) with almost no secretion. Therefore, it can efficiently and with almost no loss display the antigen on the surface of rBCG.

[0242] Example 3: Evaluation of humoral immune response in mice immunized with recombinant BCG

[0243] To assess whether recombinant BCG (rBCG) strains can induce antibody responses after immunization, such as... Figure 4 As shown in Figure A, in the experiment with immunized BALB / c mice (supplementary mice purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., SPF grade BALB / c female mice, 7 weeks old), the experimental groups were given a single intravenous injection of 10 6 cfu rBCG::RBD (tail vein injection), and at 4 weeks 10 6 The cfu rBCG::RBD immunization group received a booster immunization (two-dose immunization strategy); the control group received PBS via tail vein injection (this group was the blank control group), 106 CFU parental BCG tail vein injection, or RBD protein subunit vaccine (i.e., RBD) AS01 The vaccine was administered via intramuscular injection. It contained 10 μg of RBD-His recombinant protein and 5 μg of AS01 adjuvant (a liposome-based adjuvant designed to rapidly induce a humoral immune response). A booster immunization was also administered to the control group (a two-dose immunization strategy).

[0244] The vaccine preparation methods are as follows:

[0245] BCG and rBCG::RBD were aseptically cultured in a biosafety level 2 laboratory. CW-rBCG::RBD was inoculated into 7H9 medium containing 25 μg / mL kanamycin resistance, 0.05% Tween 80, and 10% OADC, and cultured in 50 mL on a shaker at 100 rpm and 37°C with constant temperature and ventilation until the bacterial culture reached OD500. 600 Passaged from 0.8 to 1.0 cells and amplified in 300 mL of culture, 50 mL was then used for target protein expression validation. Wild-type BCG was used as a control, and the OD values ​​of the same bacterial culture were controlled. 600 The concentration should be 0.8-1.0. Take 3000g of the same volume of bacterial culture, centrifuge at room temperature for 10 min to remove the culture medium, add PBS buffer to resuspend, centrifuge and wash 4 times to completely remove the culture medium, and sonicate for 30 s to disperse the bacteria. Adjust the bacterial concentration (OD) to 0.8-1.0. 600 The value is 0.8-1.0, at which point the colony count is approximately 10. 7 CFU / mL. The bacterial suspension used for immunization also needs to be serially diluted 10-fold, plated on 7H10 solid medium, and incubated at 37°C for about 20 days before counting to predict the immunization dose.

[0246] The method for collecting whole blood from the orbit of mice is as follows:

[0247] Hold the mouse by the neck with your left hand to expose its eyeballs, and insert a sterile blood collection needle into the eye socket with your right hand to collect venous blood from the mouse's eye socket. After the collected blood is left to stand at 37°C for 20 minutes, it is centrifuged at 3000 rpm and 4°C for 15 minutes to obtain the serum of the immunized mouse, which is used to detect the titers of RBD-specific IgG antibodies and neutralizing antibodies.

[0248] like Figure 4 As shown in B and Table 1, the titer of specific IgG antibodies against RBD was measured 4 weeks post-immunization. A single intravenous injection of 10... 6 The cfu CW-rBCG::RBD assay showed an IgG titer of 1600, while the same dose of parental BCG, PBS, or RBD protein subunit vaccine RBD... AS01 Then, significant IgG antibodies were not produced.

[0249] Table 1 Figure 4 B RBD-specific IgG antibody titer data

[0250] Immune strategy RBD-specific IgG antibody titer <![CDATA[10 6 cfu CW-rBCG::RBD(vein)]]> 1600 <![CDATA[10 6 cfu BCG (intravenous) Below the detection limit <![CDATA[Protein vaccine RBD AS01 (Intramuscular injection)]]> Below the detection limit PBS Below the detection limit

[0251] like Figure 4 As shown in C, a two-dose immunization strategy was adopted, with 10 mg of iodine administered intravenously at 8 weeks. 6 The IgG titer detected by cfu CW-rBCG::RBD further increased to 3200, which was significantly better than that of parental BCG or PBS.

[0252] like Figure 4 As shown in D and Table 2, using a two-dose immunization strategy, the RBD Nab titer in CW-rBCG::RBD mice immunized at 8 weeks reached approximately 4565.67, significantly higher than that obtained with parental BCG, PBS, or RBD. AS01 Nab titers induced in immunized mice.

[0253] Table 2 Figure 4 RBD neutralizing antibody titer data for D

[0254]

[0255] These findings suggest that CW-rBCG::RBD can effectively induce a strong humoral immune response, including specific IgG and Nab against RBD, and elicits higher antibody levels than other immunization strategies.

[0256] Example 4: Evaluation of antigen-specific cellular immune response in mice immunized with recombinant BCG

[0257] The same dose of CW-rBCG::RBD immunization group and the parental BCG immunization group carrying pMV261 empty vector plasmid, RBD AS01 The immunized group or the PBS group (serving as controls to measure RBD-specific immune responses) were compared. All vaccines were boosted in week 2. At the detection time point, mice were euthanized, and the spleen, lungs, and inguinal lymph nodes were isolated. Single-cell samples from the mouse organs were prepared, and T cells in the lungs, spleen, and lymphocytes were detected by flow cytometry after stimulation with the SARS-CoV-2 RBD peptide library.

[0258] At the 12-week endpoint of the experiment, compared with the parental BCG immunization group and RBD group... AS01 Compared to the PBS group, such as Figure 5 As shown in Figure A, the spleen of CW-rBCG::RBD-immunized mice showed a significantly increased proportion of activated Tfh cells. These Tfh cells were detected by flow cytometry and labeled with CD4+. + CD44 + CD62L -Tfh testing identifiers CXCR5 and PD-1, and hair regrowth center testing identifier GL7.

[0259] Even after a single immunization, such as Figure 5 As shown in Figure C, compared with the parental BCG and PBS groups, CW-rBCG::RBD induced a stronger and more durable Tfh response in lymph nodes.

[0260] This indicates that CW-rBCG::RBD induced sustained Tfh cell activation and antigen-specific Tfh cell proliferation. In contrast, compared to the PBS control, RBD... AS01 The vaccine failed to induce a significant Tfh response.

[0261] These results indicate that CW-rBCG::RBD induces Tfh cell responses.

[0262] In addition to Tfh cells, such as Figure 5 As shown in B, with BCG and RBD AS01 Compared with the PBS group, at week 12, CW-rBCG::RBD stimulation of mice significantly increased central memory T (Tcm) cells, which are crucial for T cell immune memory. Furthermore, as... Figure 5 As shown in D, when it was initially exempt, RBD AS01 The group could not elicit a response from Tcm cells (therefore no experimental data were presented).

[0263] like Figure 5 As shown in Figure E, the ELISPOT assay further demonstrated that the number of cells secreting IFN-γ was significantly increased in the CW-rBCG::RBD group, and IFN-γ is a marker of memory T cell response.

[0264] These results indicate that CW-rBCG::RBD induces a strong memory T cell response.

[0265] Example 5: Immunization of mice with a combined recombinant BCG and RBD protein subunit vaccine induces the production of long-acting neutralizing antibodies.

[0266] like Figure 6 As shown in A, the same dose of CW-rBCG::RBD and RBD protein subunit vaccines RBD AS01 Combined immunization via subcutaneous route (denoted as A6scRBD) AS01 ), and the parental BCG and RBD carrying the pMV261 empty plasmid. AS01 Combined immunization group (i.e., BCG combined immunization group, denoted as BCGscRBD) AS01 ), standalone RBD AS01 Group (denoted as RBD) AS01 The results were compared with either the PBS group (which served as controls to measure RBD-specific immune responses) or the PBS group (which served as controls).

[0267] like Figure 6 As shown in B, A6scRBD AS01 The group showed a significant improvement in humoral immune response and maintained high levels of Nab antibodies. At week 16, the group receiving the combined CW-rBCG::RBD treatment (i.e., A6scRBD) AS01 The Nabs titer of the group was 5,635, and the BCG combined application group (i.e., BCGscRBD) had a Nabs titer of 5,635. AS01 The Nabs titer of group (1,909) was 1,909 when RBD was used alone. AS01 The Nabs titer in the immunized group was 1,778. The BCG combined immunization group did not significantly enhance the production of RBD-specific neutralizing antibodies at week 16, while the CW-rBCG::RBD combined immunization significantly increased the production of neutralizing antibodies.

[0268] like Figure 6 As shown in Figure E, at week 31 of immunization, the endpoint neutralizing antibody titer in the CW-rBCG::RBD combined immunization group was 1,795, and the endpoint neutralizing antibody titer in the BCG combined application group was 414.5, while that in the RBD monotherapy group was [missing data]. AS01 The endpoint neutralizing antibody titer in the immunization group was 0.7. The antibody titer in the CW-rBCG::RBD group was significantly higher than that in the BCG combined application group (p<0.01) and the RBD monotherapy group. AS01 The immune group (p<0.001) indicates that it can maintain RBD-specific neutralizing antibodies for a longer period of time.

[0269] like Figure 6 As shown in Figure C, spleen samples from mice immunized for 12 weeks were analyzed by flow cytometry. The CW-rBCG::RBD combined immunization group showed increased CD4 count. + The proportion of Tfh cells was significantly higher in the BCG combined immunization group and the RBD alone group. AS01 Immunization group and PBS group.

[0270] like Figure 6 As shown in Figure D, in the lungs, the proportion of Tcm cells was significantly increased in the group receiving combined CW-rBCG::RBD immunization, while the proportion of Tcm cells was significantly increased in the group receiving combined BCG immunization and RBD immunization. AS01 The Tcm cell frequency in the immunization group alone was almost zero, similar to that in the PBS group.

[0271] These results indicate that CW-rBCG::RBD co-immunization induced stronger Tfh and Tcm cell responses, especially under in vitro RBD peptide library stimulation, demonstrating enhanced RBD-specific memory T cell responses, significantly superior to BCG co-immunization which primarily induces non-specific T cell responses. Furthermore, this suggests that the subcutaneous immunization route may be a better pathway for inducing long-acting antibody-mediated immunity.

[0272] Example 6: The recombinant BCG constructed after changing the antigen protein still enabled the antigen protein to accumulate on the cell wall.

[0273] In this embodiment, a construction method similar to that in Example 1 is used, employing the Rv3763 promoter (same as in Example 1) and the Rv3763 gene signal peptide (same as in Example 1) to express the replaced protein: a fusion protein of 19-KD antigen, linker, and RBD. The full-length sequence of the Rv3763 gene in the coding sequence of the recombinant plasmid is shown in SEQ ID NO:9, and the corresponding amino acid sequence is the full-length amino acid sequence of the 19-KD antigen, as shown in SEQ ID NO:10. A schematic diagram of the recombinant pMV261 plasmid vector is shown below. Figure 7 A. Recombinant BCG was constructed using the same method as in Example 1.

[0274] The fusion protein consists of a 19-KD antigen, a linker, and an RBD protein, with a theoretical protein mass of 48 KD, higher than that of the SARS-CoV-2 RBD antigen expressed by recombinant BCG in Example 1. The amino acid sequence of the expressed fusion protein is shown in SEQ ID NO:11, where positions 1-138 are the 19-KD antigen sequence (i.e., SEQ ID NO:10), positions 139-155 are the linker sequence (i.e., SEQ ID NO:16), and positions 156-420 are the RBD protein sequence (i.e., SEQ ID NO:17, which is also the amino acid sequence encoded by SEQ ID NO:4).

[0275] Full-length sequence of the Rv3763 gene:

[0276] TGTTCAAGCAACAAGTCGACTACAGGAAGCGGTGAGACCACGACCGCGGCAGGCACGACGGCAAGCCCCGGCGCCGCCTCCGGGCCGAAGGTCGTCATCGACGGTAAGGACCAGAACGTCACCGGCTCCGTGGTGTGCACAACCGCGGCCGGCAATGTCAACATCGCGATCGGCGGGGCGGCGACCGGCATTGCCGCCGTGCTCACCGACGGCAACCCTCCGGAGGTGAAGTCCGTTGGGCTCGGTAACGTCAACGGCGTCACGCTGGGATACACGTCGGGCACCGGACAGGGTAACGCCTCGGCAACCAAGGACGGCAGCCACTACAAGATCACTGGGACCGCTACCGGGGTCGACATGGCCAACCCGATGTCACCGGTGAACAAGTCGT TCGAAATCGAGGTGACCTGTTCC(SEQ ID NO:9)

[0277] Amino acid sequence of the expressed 19-KD antigen:

[0278] CSSNKSTTGSGETTTAAGTTASPGAASGPKVVIDGKDQNVTGSVVCTTA AGNVNIAIGGAATGIAAVLTDGNPPEVKSVGLGNVNGVTLGYTSGTGQGNA SATKDGSHYKITGTATGVDMANPMSPVNKSFEIEVTCS(SEQID NO:10)

[0279] Amino acid sequence of the expressed fusion protein:

[0280] CSSNKSTTGSGETTTAAGTTASPGAASGPKVVIDGKDQNVTGSVVCTTAAGNVNIAIGGAATGIAAVLTDGNPPEVKSVGLGNVNGVTLGYTSGTGQGNASATKDGSHYKITGTATGVDMANPMSPVNKSFEIEVTCSGGGGSGGGGSGGGGSQAQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTF KCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGV EGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLERS(SEQ ID NO:11)

[0281] like Figure 7 As shown in Figure B, Western blotting experiments confirmed that the RBD / 19-KD fusion antigen was mainly expressed on the cell wall, with a small amount present in the cell membrane connected to the cell wall, and was not detected in the supernatant. This indicates that the Rv3763 signal peptide, especially the mature signal peptide sequence, used in Example 1 as a signal peptide, along with the Rv3763 promoter, can not only be used to construct recombinant plasmids of recombinant BCG expressing SARS-CoV-2 RBD protein, but also to construct recombinant plasmids of recombinant BCG expressing other proteins (such as the RBD / 19-KD fusion antigen), so as to express and enrich the carried antigen proteins on the surface of rBCG (especially the cell wall).

[0282] Example 7: In the recombinant BCG constructed after changing the expression promoter (Hsp60), the antigen protein was only enriched in small amounts in the cells. cell wall

[0283] In this embodiment, a construction method similar to that in Example 1 was used, but the Hsp60 (heat shock protein 60) promoter and Rv3763 gene signal peptide (same as in Example 1) were replaced to express the replaced protein: a fusion protein of 19-KD antigen, linker, and SARS-CoV-2RBD. The recombinant plasmid encoding the Rv3763 gene and the corresponding amino acid sequence are the same as in Example 6 (SEQ ID NO:9 and SEQ ID NO:10). A schematic diagram of the recombinant pMV261 plasmid vector is shown below. Figure 8 A. Recombinant BCG was constructed using the same method as in Example 1.

[0284] The fusion protein consists of 19-KD antigen, linker, and SARS-CoV-2 RBD protein, with a theoretical protein mass of 48KD, which is higher than that of the SARS-CoV-2 RBD antigen expressed by recombinant BCG in Example 1. The amino acid sequence of the expressed fusion protein is shown in SEQ ID NO:11.

[0285] like Figure 8 As shown in Figure B, Western blotting experiments confirmed that only a small amount of the SARS-CoV-2 RBD protein and 19-KD fusion antigen were expressed on the cell wall, and it was not detected in the cell membrane or supernatant connected to the cell wall. This indicates that the Rv3763 promoter and Rv3763 signal peptide used in Example 1 can better promote the enrichment of expressed antigen on the cell wall. When the Hsp60 promoter and Rv3763 signal peptide are combined, although they can be expressed on the cell wall, the expression level is low, indicating that the ability to enrich antigen on the cell wall is weakened.

[0286] Example 8: The main antigen protein in recombinant BCG constructed by replacing the expression promoter (Hsp60) and the expressed S antigen. To accumulate in the cytoplasm

[0287] In this embodiment, a construction method similar to that in Example 1 was used, but the Hsp60 (heat shock protein 60) promoter and Rv3763 gene signal peptide (same as in Example 1) were replaced to express the replaced proteins: the fusion protein of 19-KD antigen, linker, and SARS-CoV-2 Spike. The recombinant plasmid encoding the Rv3763 gene and the corresponding amino acid sequence are the same as in Example 6 (SEQ ID NO: 9 and SEQ ID NO: 10). A schematic diagram of the recombinant pMV261 plasmid vector is shown below. Figure 9 A. Recombinant BCG was constructed using the same method as in Example 1.

[0288] The fusion protein consists of 19-KD antigen, linker, and SARS-CoV-2 Spike protein, with a theoretical protein mass of 156 KD, higher than that of the SARS-CoV-2 RBD antigen expressed by recombinant BCG in Example 1. The amino acid sequence of the expressed fusion protein is shown in SEQ ID NO:12.

[0289] The amino acid sequence of the expressed fusion protein:

[0290] CSSNKSTTGSGETTTAAGTTASPGAASGPKVVIDGKDQNVTGSVVCTTAAGNVNIAIGGAATGIAAVLTDGNPPEVKSVGLGNVNGVTLGYTSGTGQGNASATKDGSHYKITGTATGVDMANPMSPVNKSFEIEVTCSGGGGSGGGGSGGGGGSGFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCLADPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATR FASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGV SVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTTPWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIATMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVV NQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGV VFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGD ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:12)

[0291] like Figure 9 As shown in Figure B, Western blotting experiments confirmed that the SARS-CoV-2 Spike protein fusion antigen with 19-KD was mainly expressed in the cytoplasm and no longer enriched in the cell wall (not detected by WB). Furthermore, it was not detected in the cell membrane or supernatant. This indicates that the Rv3763 promoter and Rv3763 signal peptide used in Example 1 are more effective in promoting antigen enrichment in the cell wall. However, when the Hsp60 promoter is combined with the Rv3763 signal peptide, the ability to enrich the antigen in the cell wall becomes very weak, even undetectable by WB. Of course, the localization and expression level of the antigen are also influenced by some of the antigen's own characteristics.

[0292] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A recombinant protein carrying an antigen protein or a fragment thereof, characterized in that, The recombinant protein comprises a structure as shown in Formula I: Z0-Z1-Z2(I) In the formula, Z0 is the 19-KD antigen or a fragment thereof; Z1 is either a non-peptide or a flexible peptide; Z2 is an antigen protein or a fragment thereof; The "-" indicates a linking peptide or peptide bond.

2. The recombinant protein as described in claim 1, characterized in that, Z0 is a 19-KD antigen signal peptide sequence or a full-length 19-KD antigen sequence; preferably, the amino acid sequence of Z0 is selected from the following group: (a) An amino acid sequence as shown in SEQ ID NO:10, 13 or 14; (b) A sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 10, 13, or 14; (c) A sequence obtained by adding, deleting, modifying and / or substituting at least one (e.g., 1-6) amino acids based on the amino acid sequence shown in SEQ ID NO: 10, 13 or 14.

3. The recombinant protein as described in claim 1, characterized in that, Z2 is either the SARS-CoV-2 RBD protein or the SARS-CoV-2 Spike protein; preferably, Z2 is the SARS-CoV-2 RBD protein.

4. A 19-KD antigen signal peptide, characterized in that, The sequence of the 19-KD antigen signal peptide is selected from the following group: (a) The amino acid sequence as shown in SEQ ID NO:13; (b) A sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:13; (c) A sequence obtained by adding, deleting, modifying and / or substituting at least one (e.g., 1-6) amino acids based on the amino acid sequence shown in SEQ ID NO:

13.

5. A polynucleotide, characterized in that, The polynucleotide encodes the recombinant protein as described in any one of claims 1-3 or the 19-KD antigen signal peptide as described in claim 4.

6. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the vector as described in claim 6, or its genome is integrated with the polynucleotide as described in claim 5.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the recombinant protein as described in any one of claims 1-3, the polynucleotide as described in claim 5, the vector as described in claim 6, the host cell as described in claim 7, or a combination thereof; and (ii) Pharmaceutically acceptable carriers and / or excipients.

9. A vaccine composition, characterized in that, The vaccine composition contains: (i) the recombinant protein as described in any one of claims 1-3, the polynucleotide as described in claim 5, the vector as described in claim 6, the host cell as described in claim 7, or a combination thereof; and (ii) Immunologically acceptable carriers and / or excipients.

10. A cell wall display technology for recombinant BCG (rBCG) antigen protein or fragments thereof, characterized in that, The technology includes the following steps: (i) providing or constructing the carrier as described in claim 6; and (ii) The vector from step (i) is transferred into wild-type BCG to obtain recombinant BCG (rBCG) expressing the antigen protein or fragment thereof carried by the vector, wherein the antigen protein or fragment thereof is enriched on the cell wall of the recombinant BCG (rBCG).

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