Chaperone-based novel recombinant pertussis vaccine

Abstract

The present invention relates to an RNA-dependent chaperone-based cell-free pertussis antigen protein and a use thereof. A fusion protein according to the present invention not only exhibits improved water solubility and yield compared to existing cell-free pertussis (aP) vaccines, but can also induce a long-term and strong immune response mediated by the Th1 / Th17 pathway. Therefore, the fusion protein according to the present invention overcomes the limitations of Th2 immune response-based aP vaccines, and thus can be effectively used as a novel pertussis vaccine or therapeutic agent.

Description

Novel Chaperone-based Recombinant Pertussis Vaccine

[0001] The present invention relates to an RNA-dependent chaperone-based cell-free pertussis antigen protein and its use as a pertussis vaccine.

[0002]

[0003] Pertussis is a disease caused by infection with the bacterium Bordetella pertussis, and despite widespread vaccination efforts, it still poses a serious public health threat and is particularly fatal to vulnerable individuals, with cases of death reported (Nature Reviews Microbiology. 2014;12(4):274-88).

[0004] The whole-cell pertussis (wP) vaccine, developed in the 1940s, significantly reduced the incidence of pertussis, but various side effects such as high fever and neurological abnormalities were reported, leading to a transition to the acellular pertussis (aP) vaccine in the 1990s (Nature Reviews Microbiology. 2024). Generally, the acellular pertussis vaccine contains purified proteins derived from Bordetella pertussis, such as filamentous hemagglutinin (FHA), pertactin (PRN), and pertussis toxin (PTX), as antigens.

[0005] While aP vaccines have the advantage of exhibiting a good safety profile by significantly improving the side effects of wP vaccines, they present a problem in that they provide only short-term effects, referred to as the 'end-of-honeymoon' effect, and cannot provide long-term effects (Nature Immunology. 2023;24(11):1779-80). In particular, according to recent reports, unlike wP vaccines which induce potent Th1 / Th17 pathway immune responses, aP vaccines tend to induce primarily Th2 pathway immune responses, making them highly disadvantageous for long-term immunity and prevention of asymptomatic infections (The Journal of Clinical Investigation. 2018;128(9):3853-65). The mechanism of induction of the Th2 immune response by aP vaccines results in a rapid decline in immune effect and requires frequent booster shots (Frontiers in Immunology. 2019;10).

[0006] As such, although it has been reported that the Th1 / Th17 immune response is an important mechanism for providing effective and long-term immunity against Bordetella pertussis infection (Nature Reviews Microbiology. 2014;12(4):274-88; The Journal of Clinical Investigation. 2018;128(9):3853-65), the aP vaccines reported to date mainly induce a Th2-biased response, so the development of new aP vaccines capable of strongly stimulating the Th1 / Th17 immune response is required to provide a more long-term and effective immune effect.

[0007] Meanwhile, the inventors have developed and patented the Chaperna technology by confirming that fusing a fragment of aminoacyl tRNA synthase as an RNA binding domain promotes the proper folding and assembly of recombinant proteins based on the chaperone activity of RNA, and consequently improves the quality and production yield of the proteins (RNA Biology. 2021;18(1):16-23; Korean Registered Patent No. 10-1915740). It has also been confirmed that the Chaperna technology can be usefully applied to the production of virus-like particles or nanoparticles and can increase the solubility of proteins (RNA Biology. 2015;12(11):1198-208; Biomaterials. 2021;269:120650).

[0008]

[0009] Under the background technology described above, the inventors have made diligent efforts to develop a long-term and effective cell-free pertussis vaccine. As a result, they have newly confirmed that when a fragment of the RNA binding domain of aminoacyl-tRNA synthase is fused to and expressed on a cell-free pertussis antigen, it not only exhibits improved solubility and yield, but also induces a long-term immune response by inducing a Th1 / Th17 immune response when used as a vaccine, thereby overcoming the limitations of existing Th2 immune response-based aP vaccines, and thus have completed the present invention.

[0010]

[0011] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that forms prior art already known to those skilled in the art to which the present invention belongs.

[0012]

[0013] Summary of the Invention

[0014] The object of the present invention is to provide a fusion protein comprising an aminoacyl tRNA synthase or a fragment thereof; and an antigen protein derived from pertussis bacteria, and the use thereof.

[0015] Another objective of the present invention is to provide virus-like particles or nanoparticles comprising the fusion protein.

[0016] Another objective of the present invention is to provide a nucleic acid encoding the fusion protein.

[0017] Another objective of the present invention is to provide a recombinant vector comprising the nucleic acid.

[0018] Another objective of the present invention is to provide a host cell into which the nucleic acid or recombinant vector is introduced.

[0019] Another objective of the present invention is to provide a method for manufacturing the fusion protein.

[0020]

[0021] To achieve the above objective, the present invention provides a fusion protein comprising an aminoacyl tRNA synthase or a fragment thereof; and an antigen protein derived from pertussis bacteria.

[0022] The present invention also provides virus-like particles or nanoparticles comprising the fusion protein.

[0023] The present invention also provides a nucleic acid encoding the fusion protein.

[0024] The present invention also provides a vaccine composition comprising the fusion protein, nucleic acid, virus-like particles and / or nanoparticles.

[0025] The present invention also provides a use for the preparation of vaccine compositions of the fusion protein, nucleic acid, virus-like particles and / or nanoparticles.

[0026] The present invention also provides a method for vaccination against pertussis comprising the step of administering the fusion protein, nucleic acid, virus-like particles and / or nanoparticles to a target.

[0027] The present invention also provides a pharmaceutical composition for the prevention or treatment of pertussis comprising the fusion protein, nucleic acid, virus-like particles and / or nanoparticles as active ingredients.

[0028] The present invention also provides the use of the fusion protein, nucleic acid, virus-like particle and / or nanoparticle for the prevention or treatment of pertussis and for the preparation of a composition for the prevention or treatment of pertussis.

[0029] The present invention also provides a method for preventing or treating pertussis, comprising the step of administering the fusion protein, nucleic acid, virus-like particles and / or nanoparticles to a target.

[0030] The present invention also provides a recombinant vector comprising the nucleic acid.

[0031] The present invention also provides a host cell into which the nucleic acid or recombinant vector is introduced.

[0032] The present invention also provides a method for producing a fusion protein comprising the step of culturing the host cell.

[0033]

[0034] Figure 1. Expression levels and solubility of intact pertussis antigen (direct construct (left)) and ChaH2 fusion pertussis antigen (right) at various temperatures: (A) FHA, (B) PRN, and (C) PTX. Each lane contains a size marker (marker (M)), a lysate fraction (total lysate fraction (T)), a soluble fraction (soluble fraction (S)), and a pellet (P)).

[0035] Figure 2. Structure and characteristics of the novel recombinant pertussis vaccine (nrP). (A) Schematic representation of the nrP vaccine construct. It includes the following elements: ChaH2, a Chaperna fusion tag derived from the WHEP domain of human glutamyl-prolyl-tRNA synthase; 6xHis, six histidine tags for purification; TEV site, TEV cleavage recognition sequence; EK site, enterokinase cleavage recognition sequence; PTXDX, genetically detoxified pertussis toxin; FHA, filamentous hemagglutinin; PRN, pertactin. (B) Representative SDS-PAGE results of the purified novel recombinant pertussis vaccine (nrP). (C) Schematic representation of the prime-boost regimen via intramuscular injection of the nrP vaccine. (D) Antigen-specific serum IgG concentrations for each Pertussis pertussis antigen after immunization (FHA (left), PRN (center), PTX (right)). Data are expressed as mean ± SD and analyzed by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0036] Fig. 3. (A) A schematic representation of various PTX constructs (PTX1–4). (B) Representative SDS-PAGE analysis results of purified PTX constructs (PTX1–4). (C) PTX-specific serum IgG concentrations measured after two doses of immunization with each PTX antigen or the positive control, Pentaxim vaccine. Data are expressed as mean ± SD and analyzed by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0037] Fig. 4. This shows the gating strategy used in flow cytometry to determine the proportion of immune cells (A) or cytokine production (B). The gating strategy represents five independent experiments.

[0038] Fig. 5. (A) Gating results of Live / CD45+ cells in lung tissue of mice immunized with Vehicle or nrP. Visualized with UMAP and separated by Phenograph clustering. Each cluster is indicated as a corresponding immune cell cluster. (B) Frequencies of Treg, B cells, NK cells, neutrophils, and macrophages in lung tissue of mice immunized with Vehicle or nrP. (C, D) TBET expression (C) and GATA3 expression (D) in CD4+ T cells (top), CD8+ T cells (middle), and γδ T cells (bottom), along with frequencies for each group. Data are expressed as mean ± SD and analyzed using a two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0039] Figure 6. Results confirming that the nrP vaccine induces TH1 / TH17 responses in the lungs. (A) Results showing the gating of a subset of total TCRβ+ cells in the lungs of control and nrP-immunized mice. Visualized with UMAP and separated by Phenograph clustering. Each cluster is indicated as a corresponding immune cell cluster. (B) Representative flow cytometry plot (left) and frequency graph (right) regarding the proportion of CD45+TCRβ+ T cells. (C, D) Flow cytometry plots showing CD62L / CD44 expression (C) or RORγt expression (D) in CD4+ T cells (top), CD8+ T cells (middle), and γδ T cells (bottom), along with the frequencies of each T cell subset. (E) Cytokine production upon antigen stimulation via multiplex cytokine analysis. The heatmap indicates relative cytokine secretion compared to the Vehicle group. Data are expressed as mean ± SD and analyzed using a two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0040] Figure 7. This shows the preventive efficacy of the nrP vaccine against B. pertussis infection. (A) Schematic representation of vaccine administration to C57BL / 6. (B, C) CFU count (B) and mRNA expression of the is481 gene (C) in the lungs on day 3 after nasal infection with *Pertussis pertussis* in mice. (D) Representative H&E stained lung tissues of B. pertussis-infected and control mice. Bar scale = 50 μm. Histopathological analysis evaluated parenchymal inflammation, bronchial inflammation, and vascular inflammation, each rated on a scale of 0 to 5 (0 = none; 1 = weak; 2 = mild; 3 = moderate; 4 = severe; 5 = markedly severe). The total lung injury score was the sum of each inflammation score. Data were expressed as mean ± SD and analyzed using one-way ANOVA with Tukey's post-test. *p < 0.05; **p < 0.01; ***p < 0.005. (E, F) Representative plots showing the expression of (E) CD44 / CD62L and (F) RORγt with frequency in CD4+ T cells (top), CD8+ T cells (middle), and γδ T cells (bottom). Data are expressed as mean ± SD and analyzed by a two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0041] Fig. 8. (A) Gating results of Live / CD45+ cells in the lungs after B. pertussis infection in non-infected mice, and mice immunized with Vehicle or nrP. Visualized with UMAP and separated by Phenograph clustering. Each cluster is indicated as a corresponding immune cell cluster. (B) Frequency of B cells and neutrophils in lung tissue of mice immunized with Vehicle or nrP. (C, D) GATA3 expression (C) and TBET expression (D) in CD4+ T cells (top), CD8+ T cells (middle), and γδ T cells (bottom) in lung tissue, along with frequencies for each group. Data are expressed as mean ± SD and analyzed using a two-tailed Student's t-test. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0042] Figure 9. The results of comparing the immunogenicity of DTaP and nrP vaccines. (A) Antigen-specific serum IgG concentrations for FHA (left), PRN (center), and PTX (right) in mice primary and secondary immunized with Vehicle, IF, and nrP. (B) Results of measuring the levels of FHA (left), PRN (center), and PTX (right) specific IgG1, IgG2, and IgG3. (C) The IgG2 to IgG1 ratio and IgG3 to IgG1 ratio for mice immunized with Vehicle, IF, and nrP. Data are expressed as mean ± SD and analyzed by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0043] Figure 10. Results of the analysis of immune responses following immunization with DTaP and nrP in mucosal tissues. (A) Shows the gating results of a subset of whole Live / CD45+ cells in the lungs of Vehicle, IF, and nrP-immunized mice. Visualized with UMAP and separated by Phenograph clustering. Each cluster is indicated as a corresponding immune cell cluster. (B) Shows the frequencies of TCRβ+ (left), CD4+ T cells (center), and CD8+ T cells (right) in the lung tissues of Vehicle, IF, and nrP-immunized mice. (C, D) Representative flow cytometry plots showing CD62L / CD44 expression (C) or RORγt expression (D) in CD4+ T cells (top), CD8+ T cells (middle), and γδ T cells (bottom), and the frequencies of each population. (E) This shows the frequency of production of IL-4, IL-5, IL-17A, and IFN-γ in CD4+ T cells (top), CD8+ T cells (middle), and γδ T cells (bottom). Data are expressed as mean ± SD and analyzed by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0044] Fig. 11. (A) Frequencies of Treg, B cells, NK cells, neutrophils, macrophages, and CD11c+ MHCII+ populations in lung tissues of Vehicle-treated mice, IF-immunized mice, and nrP-immunized mice. (B, C) TBET expression (B) and GATA3 expression (C) in CD4+ T cells (top), CD8+ T cells (middle), and γδ T cells (bottom) in lung tissues, along with frequencies for each population. (D) Frequencies of IL-4, IL-5, IL-17A, and IFN-γ production in B cells (top), NK cells (middle), and macrophages (bottom). Data are expressed as mean ± SD and analyzed by two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0045] Figure 12. This shows the results of comparing the efficacy of the novel recombinant pertussis vaccine (nrP) and the Infanrix vaccine. (A, B) CFU count (A) and mRNA expression of the is481 gene (B) in lung tissues of non-infected mice, Vehicle-treated mice, and mice immunized with IF or nrP after infection with B. pertussis. (C) Representative H&E stained images of lung tissues of non-infected mice, Vehicle-treated mice, and mice immunized with IF or nrP after infection with B. pertussis. Bar scale = 50 μm. Histopathological analysis evaluated parenchymal inflammation, bronchial inflammation, and vascular inflammation, each rated on a scale of 0 to 5 (0 = none; 1 = weak; 2 = mild; 3 = moderate; 4 = severe; 5 = markedly severe). The total lung injury score is the sum of each inflammation score. (D) Flow cytometry plot showing the expression of RORγt in CD4+ T cells (top), CD8+ T cells (middle), and γδ T cells (bottom). (E) Cytokine production analysis results of lung immune cells measuring IL-2, IL-6, IL-17A, IL-17F, IL-22, and IFN-γ levels after stimulation with recombinant B. pertussis antigen (FHA, PRN, PTX). Data are expressed as mean ± SD and analyzed using one-way ANOVA with Tukey's post-test. *p < 0.05; **p < 0.01; ***p < 0.005.

[0046] Fig. 13. (A) Results of gating a subset of whole Live / CD45+ cells in the lungs of mice immunized with Vehicle, IF, and nrP after infection with B. pertussis. Visualized by UMAP and separated by Phenograph clustering. Each cluster is indicated as a corresponding immune cell cluster. (B, C) Frequency of Treg, NK cells, neutrophils, and macrophages (B) or flow cytometry plots and frequency of TCRβ+ T cells (C) in lung tissue of mice immunized with Vehicle, IF, and nrP after infection with B. pertussis. Data are expressed as mean ± SD and analyzed by two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.005; ns, not significant.

[0047]

[0048] Detailed Description of the Invention and Preferred Embodiments

[0049] The present invention will be described in detail below. However, the following detailed description is provided as an example of the present invention and is not intended to limit the present invention, and various modifications and applications are possible within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0050] Unless otherwise indicated, nucleic acids and amino acids are written from left to right with 5' to 3' and N-terminal to C-terminal orientations, respectively. Numerical ranges listed in the specification include a number defining the range and each integer or any non-integer fraction within the defined range.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Any method and material similar or equivalent to those described herein may be used in practice to test the invention, but preferred materials and methods are described herein.

[0052] Each fusion protein or domain included therein described in this specification may include not only the amino acid sequence described in connection therewith, but also a protein or polypeptide in which a part of the said amino acid sequence is substituted through conservative substitution.

[0053] In this specification, “conservative substitution” means a modification of a polypeptide comprising substituting one or more amino acids with amino acids having similar biochemical properties that do not cause a loss of the biological or biochemical function of the polypeptide.

[0054] “Conservative amino acid substitution” is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are defined in the art and are well known. These classes include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In general, conservative substitutions in the sequences of polypeptides and proteins described in this specification do not cause a loss of these functions.

[0055] With respect to all amino acid sequences described in this specification, an amino acid encoded by a start codon at the N-terminus, methionine, may be additionally included.

[0056]

[0057] Acellular pertussis vaccines (aP) have the advantage of reducing the side effects associated with whole-cell pertussis vaccines, but they induce a Th2 immune response. This results in a rapid recurrence period because they cannot provide long-term immunity, and they have the problem of being unable to prevent asymptomatic transmission. In particular, the Th2 response promoted by existing aP vaccines uses aluminum hydroxide as an adjuvant, so they cannot effectively induce the cellular immune response necessary to effectively eliminate bacteria such as B. pertussis (Trends in Microbiology. 2014;22(2):49-52).

[0058]

[0059] In one embodiment of the present invention, a novel recombinant cell-free pertussis antigen protein was prepared using Chaperna technology. In particular, in another embodiment of the present invention, it was confirmed that the fusion of an aminoacyl-tRNA synthase fragment not only induces solubility and proper folding of the B. pertussis-derived antigen, but also induces immune responses based on Th1 and Th17 pathways, thereby increasing the production of cytokines such as TNF-α, IFN-γ, and IL-17, and inducing cellular immune responses such as macrophage activation, neutrophil recruitment, and pluripotent T cell activation, which can exhibit a longer-lasting and more potent immune induction effect than existing aP vaccines.

[0060]

[0061] Accordingly, in one aspect, the present invention relates to a fusion protein comprising an aminoacyl-tRNA synthase or a fragment thereof; and an antigen protein derived from pertussis bacteria.

[0062] The term “aminoacyl-tRNA synthetase (aaRS or ARS)” in the present invention refers to an enzyme that binds an appropriate amino acid to each tRNA, and produces aminoacyl tRNA by attaching a specific homologous amino acid or precursor to the tRNA through an ester exchange reaction. The binding specificity of aminoacyl tRNA synthetase to amino acids and tRNAs serves as an important determinant in maintaining the accuracy of protein translation.

[0063] In the present invention, the aminoacyl-tRNA synthase may be derived from various organisms without limitation. Generally, there may be 20 aminoacyl-tRNA synthases corresponding to 20 natural amino acids, and depending on the organism, one or more aminoacyl-tRNA synthases may be included, or one aminoacyl-tRNA synthase may react with two or more amino acids.

[0064] The gene and amino acid sequences of various biologically derived aminoacyl-tRNA synthetases and their isoforms, as well as the sequences of each domain within the synthetase, are known in biological information databases such as UniProt, NCBI, and Gene Bank.

[0065] In the present invention, the aminoacyl-tRNA synthase may be derived from various organisms. For example, the aminoacyl-tRNA synthase may be an aminoacyl-tRNA synthase derived from prokaryotic or eukaryotic cells, specifically, bacteria, archaea, plants, fungi, plants, or animals, and may be characterized as preferably a mammalian, more preferably human-derived aminoacyl-tRNA synthase, but is not limited thereto.

[0066] In the present invention, where the aminoacyl-tRNA synthase is derived from a human, for example, among 19 types of human aminoacyl-tRNA synthases composed of glycyl-tRNA synthase, alanyl-tRNA synthase, valyl-tRNA synthase, leucyl-tRNA synthase, isoleucyl-tRNA synthase, methionyl-tRNA synthase, phenylalanyl-tRNA synthase, tryptophanyl-tRNA synthase, ceryl-tRNA synthase, threonyl-tRNA synthase, cysteinyl-tRNA synthase, tyrosyl-tRNA synthase, asparaginyl-tRNA synthase, aspartyl-tRNA synthase, glutaminyl-tRNA synthase, lysyl-tRNA synthase, arginyl-tRNA synthase, histidyl-tRNA synthase, and glutamyl-prolyl-tRNA synthase Can be selected.

[0067] In the present invention, the aminoacyl-tRNA synthase may more preferably be tryptophanyl-tRNA synthase, histidyl-tRNA synthase, glycyl-tRNA synthase, methionyl-tRNA synthase, lysyl-tRNA synthase, or glutamyl-prolyl-tRNA synthase, and most preferably glutamyl-prolyl-tRNA synthase.

[0068]

[0069] In the present invention, the fusion protein may be characterized by comprising one or more fragments of aminoacyl-tRNA synthase.

[0070] The term "fragment of aminoacyl-tRNA synthase" in the present invention refers to a protein fragment in which one or more amino acids are cleaved at the N' end, C' end, or both the N' end and C' end directions of the full-length amino acid sequence of aminoacyl-tRNA synthase.

[0071] In the present invention, the fragment of aminoacyl-tRNA synthase may preferably be characterized by comprising the RNA binding domain of aminoacyl-tRNA synthase.

[0072] The term “RNA binding domain” in the present invention refers to a domain that interacts with an anticodon in an aminoacyl-tRNA synthetase. In the present invention, the RNA binding domain may be a fragment comprising several to several hundred amino acid sequences that interact with the anticodon RNA in the sequence of the aminoacyl-tRNA synthetase. For example, the RNA binding domain may be located at the N' or C' end of the aminoacyl-tRNA synthetase, or, in some cases, at the junction between the synthetase domains, but is not limited thereto.

[0073] In the present invention, the fragment of the aminoacyl-tRNA synthase may be characterized by including a WHEP domain.

[0074] The term “WHEP domain” in the present invention is a domain having a sequence of about 40 to 50 amino acids in the form of a ‘helix-turn-helix’ present in an aminoacyl-tRNA synthase derived from eukaryotic cells, which may or may not be included depending on the type of aminoacyl-tRNA synthase, and one or more domains may be repeated continuously or spaced apart.

[0075] In the present invention, the location or repetition of the WHEP domain may differ depending on the origin. For example, it may exist as a single copy at the N'-terminus of tryptophanyl- (WRS), histidyl- (HRS), and glycyl- (GRS) tRNA synthases and at the C'-terminus of methionyl- (MRS) tRNA synthases, and three copies (TRS-1, TRS-2, and TRS-3) have been reported to exist in the central region of glutamyl-prolyl-tRNA synthase (EPRS), but is not limited thereto (Int J Mol Sci. 2021;22(9):4523; J Biol Chem. 2014;289(29):20359-20369; J. Biol. Chem. (2018) 293(23) 8843-8860 and PLOS ONE. 2014;9(6):e98493).

[0076] In the present invention, the fusion protein may be characterized by comprising a single copy or multiple copies of a WHEP domain.

[0077] In the present invention, preferably, the fusion protein may include a WHEP domain derived from glutamyl-prolyl-tRNA synthase, and more preferably, may include one or more of TRS-1, TRS-2, and TRS-3 derived from human glutamyl-prolyl-tRNA synthase.

[0078] In the present invention, the fragment of the aminoacyl-tRNA synthase may be, for example, part of the amino acid sequence represented by SEQ ID NO. 1, and preferably may include one or more amino acid sequences selected from the group consisting of SEQ ID NOs. 2 to 5, but is not limited thereto.

[0079]

[0080]

[0081] In the present invention, the fusion protein may include one or more antigen proteins derived from pertussis bacteria.

[0082] In the present invention, the term "pertussis bacteria" is used to include both Bordetella pertussis and closely related species of the genus Bordetella. In the present invention, the closely related species of the genus Bordetella include, but are not limited to, Bordetella parapertussis and Bordetella bronchiseptica.

[0083] The term “antigen protein derived from Bordetella pertussis” in this invention refers to any type of protein fragment or polypeptide derived from Bordetella pertussis that can be used as an antigen.

[0084] In the present invention, the antigen protein derived from the bacterium pertussis may be, for example, a protein selected from the group consisting of fibrous hemagglutinin (FHA), pertussis toxin (PTX), agglutinogen (AGG), adenylate cyclase (AC), pertactin (PRN), and fimbriae derived from the bacterium pertussis, a variant thereof, or a fragment derived therefrom, but is not limited thereto.

[0085] In the present invention, the fibrous hemagglutinin (FHA) may be a wild-type full-length FHA protein, a precursor or variant thereof (PNAS 2004, vol. 101, no. 16, 6194-6199). A person skilled in the art can easily obtain the sequences of mature FHA proteins or FHA precursor proteins derived from *Pertussis pertussis* from bioinformatics databases such as UniProt, NCBI, Gene Bank, etc. In the present invention, the antigen protein derived from the pertussis bacteria may comprise a fragment derived from the FHA protein known to possess immunodominance, and more specifically, may be characterized by comprising the Mal85 domain of the FHA protein (the 1655 to 2111 amino acid region of FHA), the GR7 domain of the FHA protein (the 862 to 2110 amino acid region of FHA), or the 1073 to 3590 amino acid region of the FHA protein, or some sequences included therein, but is not limited thereto (e.g., The Journal of Infectious Diseases, 175(6), 1423-1431).

[0086] In one embodiment of the present invention, a fusion protein was prepared using the FHA fragment of SEQ ID NO. 6 below as an antigen protein derived from the pertussis bacteria, but is not limited thereto.

[0087]

[0088] In the present invention, the pertussis toxin (PTX) may be wild-type full-length PTX, a variant thereof, or a fragment thereof. In the present invention, the fusion protein may include one or more subunits of the pertussis toxin or fragments thereof. For example, in the present invention, the antigen protein derived from *Pertussis* bacteria may include any one or more pertussis toxin subunits or fragments thereof selected from the group consisting of pertussis toxin subunit 1 (S1), pertussis toxin subunit 2 (S2), pertussis toxin subunit 3 (S3), pertussis toxin subunit 4 (S4), pertussis toxin subunit 5 (S5), and fragments thereof, but is not limited thereto.

[0089] In the present invention, the antigen protein derived from the pertussis bacteria may include sequences such as SEQ ID NOs. 7 to 9, but is not limited thereto.

[0090]

[0091]

[0092]

[0093] In the present invention, the antigen protein derived from *Pertussis pertussis* may be a variant that has been weakened or detoxified. Variants of weakened or detoxified pertussis toxin (PTX) or subunits are well known in the art, and a person skilled in the art may select and apply them to the present invention without limitation. For example, a variant PTX containing R9K and E129G mutations in the pertussis toxin subunit 1 sequence DXIt may include, but is not limited to, fragments from (Pizza M, et al., Science. 1989;246(4929):497-500).

[0094] In one embodiment of the present invention, the PTX of SEQ ID NO: 10 below DX A fusion protein was prepared using a fragment derived from a pertussis bacterium antigen protein, but is not limited thereto.

[0095]

[0096] In the present invention, the aglutinogen (AGG) may be a wild-type full-length AGG, a variant thereof, or a fragment thereof. In the present invention, the AGG may be classified according to serotype and may be selected from the group consisting of, for example, AGG1 to AGG6, but is not limited thereto.

[0097] In the present invention, the adenylate cyclase (AC, ACT, or CyaA) may be in the form of wild-type full-length AC (non-limiting example, SEQ ID NO. 11 (Uniprot No. P0DKX7)), variants thereof, fragments thereof, or precursors thereof (ProCyaA). In the present invention, for example, the AC may be divided into several domains according to functional characteristics, and such domains are well known in the art (Toxins, 10(7), 302, et al.).

[0098] In the present invention, the antigen protein derived from the pertussis bacteria may comprise a domain selected from the group comprising, for example, an adenylate cyclase catalytic domain (e.g., the region of amino acids 1 to 399 of SEQ ID NO. 11), or a hemolysin domain (e.g., the region of amino acids 400 to 1706 of SEQ ID NO. 11), but is not limited thereto.

[0099] In the present invention, the antigen protein derived from the pertussis bacteria may be selected from the group comprising, but not limited to, a translocation region of an adenylate cyclase (e.g., the 385 to 520 amino acid region of SEQ ID NO. 11), a hydrophobic region (e.g., the 520 to 720 amino acid region of SEQ ID NO. 11), an RTX domain (Repeat in Toxin, e.g., the 1000 to 1613 amino acid region of SEQ ID NO. 11), or an RD domain (e.g., the 1150 to 1300 amino acid region of SEQ ID NO. 11).

[0100] In the present invention, the antigen protein derived from the pertussis bacteria may include the sequence represented by SEQ ID NO. 12, but is not limited thereto.

[0101] Sequence No. 12: (GGXGXDXLX)n, where X is any amino acid and n is an integer greater than or equal to 1.

[0102] In the present invention, n of sequence number 12 may preferably be 5 to 50, more preferably 40 to 50.

[0103] In the present invention, L of sequence number 12 may be substituted with V, I, F, or Y.

[0104] In the present invention, the pertactin (PRN) may be a wild-type full-length PRN, a variant thereof, or a fragment thereof. In the present invention, the pertactin may be classified according to serotype, for example, Type 1 PRN or Type 2 PRN, but is not limited thereto.

[0105] In one embodiment of the present invention, a fusion protein was prepared using the PRN fragment of SEQ ID NO. 13 below as an antigen protein derived from the pertussis bacteria, but is not limited thereto.

[0106]

[0107] In the present invention, the fimbria may be wild-type full-length FIM, a variant thereof, or a fragment thereof. In the present invention, the fimbria may be classified according to serotype and may be, for example, FIM-2, FIM-3, or FIM-2 / 3, but is not limited thereto.

[0108] A person skilled in the art can easily obtain the protein sequence information of FHA, PTX, AGG, AC, PRN, and FIM derived from the aforementioned Bacillus pertussis, as well as their serotypes, subunits, and domain information, from biological information databases such as UniProt, NCBI, and Gene Bank, and use them without limitation as full-length proteins, fragments, or variants in the fusion protein of the present invention.

[0109] In the present invention, the antigen protein derived from the pertussis bacteria may preferably be one or more selected from FHA, PRN, and PTX.

[0110] In the present invention, the antigen protein derived from the pertussis bacteria may be a functional equivalent of the example of the antigen protein described above. A “functional equivalent” may have sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more of the wild-type sequence as a result of addition, substitution, or deletion of the amino acid sequence of the protein described above. Preferably, the functional equivalent may be characterized in that the antibody produced when administered as an antigen can neutralize the pertussis bacteria.

[0111]

[0112] In the present invention, the aminoacyl-tRNA synthase or a fragment thereof; and the antigen protein derived from pertussis bacteria may be directly connected through covalent bonds, or may be indirectly connected through linkers, proteins, polysaccharides, etc.

[0113] The term “linker” in the present invention means a molecule that links each domain of the fusion protein of the present invention, such as an aminoacyl-tRNA synthase or a fragment thereof and an antigen protein derived from pertussis bacteria.

[0114] In the present invention, the linker may be directly connected or connected through a linker, preferably connected by a flexible linker or a rigid linker, more preferably by a flexible linker. In the present invention, the linker may preferably be a glycine-serine linker (GS linker). The glycine-serine linker may be, for example, (GS)n or (G)nS (where n is an integer from 1 to 10), and more specifically, may be G2S, G3S, G4S, (GS)2, (GS)3, (GS)4, etc., but is not limited thereto.

[0115] In the present invention, the fusion protein may be characterized by including, for example, the following structural formula, but is not limited thereto:

[0116] Aminoacyl-tRNA synthase or a fragment thereof-(L1)n-one or more antigen proteins derived from Pertussis pertussis (I); or

[0117] One or more antigen proteins derived from Pertussis pertussis-(L1)n-aminoacyl-tRNA synthetase or fragments thereof (II),

[0118] Here, n is an integer from 1 to 10, and L1 represents the linker.

[0119] In the present invention, the structural formula (I) or (II) is intended to represent an aminoacyl-tRNA synthase or a fragment thereof; and an antigen protein derived from pertussis bacteria, and any amino acid sequence or functional amino acid sequence (e.g., restriction enzyme cleavage site, tag sequence, etc.) may be included between both ends or between each component by direct or indirect connection.

[0120]

[0121] In the present invention, the fusion protein may additionally include an aminoacyl-tRNA synthase or a fragment thereof; and, in addition to the antigen protein derived from *Pertussis pertussis*, additional domains such as a translation enhancement sequence for protein expression (e.g., IYIY sequence), a signal sequence, an Fc domain or HSA, an HSA binding domain for extending the in vivo half-life of the fusion protein.

[0122] In the present invention, the fusion protein may additionally include an aminoacyl-tRNA synthase or a fragment thereof; and a pertussis-derived antigen protein, as well as a restriction enzyme cleavage site, a tag sequence (e.g., 6xHis), etc.

[0123] In the present invention, the restriction enzyme cleavage site, Tag sequence (e.g., 6xHis) may be included between an aminoacyl-tRNA synthase or a fragment thereof; and an antigen protein derived from pertussis bacteria, but is not limited thereto.

[0124] In the present invention, most preferred example, it may be characterized by including the following structural formula, but is not limited thereto:

[0125] [aminoacyl-tRNA synthase or a fragment thereof]-[6x His]-[one or more antigen proteins derived from Pertussis pertussis]; or

[0126] [aminoacyl-tRNA synthase or a fragment thereof]-[6x His]-[one or more restriction enzyme cleavage sites]-[one or more antigen proteins derived from Pertussis pertussis],

[0127] Here, each domain can be connected sequentially or indirectly by a linker.

[0128]

[0129] In the present invention, the fusion protein may most preferably comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs 14 to 19, but is not limited thereto.

[0130] In the present invention, the fusion protein may be characterized by inducing a Th1 / Th17 immune response when administered to a subject.

[0131]

[0132] In another aspect, the present invention relates to a nucleic acid encoding a fusion protein of the present invention.

[0133] As used herein, nucleic acids may be present in cells, cell lysates, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially purified" when purified from other cellular components or other contaminants, e.g., nucleic acids or proteins of other cells, by standard techniques including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acids of the present invention may be, for example, DNA or RNA.

[0134] The term “nucleic acid” in the present invention refers to a polynucleotide of any length, which may include DNA, RNA, or a combination of DNA and RNA. In one embodiment of the present invention, the nucleic acid may be a deoxyribonucleotide, a ribonucleotide as well as a modified nucleotide or base, and / or an analogue thereof, or any substrate that may be incorporated by DNA or RNA polymerase. In one embodiment of the present invention, the nucleic acid may be RNA, DNA, TNA (Threose nucleic acids), GNA (glycol nucleic acids), PNA (peptide nucleic acids), LNA (locked nucleic acids, e.g., LNA having a β-D-ribo sequence, α-LNA having an α-ribo sequence (a diastereomer of LNA), 2'-amino-LNA or 2'-amino-α-LNA having 2'-amino functionalization), ENA (ethylene nucleic acid), CeNA (cyclohexenyl nucleic acids), or a hybrid or combination thereof, but is not limited thereto.

[0135] The term “coding nucleic acid” in this invention refers to a nucleic acid sequence encoding a specific protein or polypeptide. Where the sequence of a specific protein or polypeptide is disclosed in the art, methods for designing or deriving the nucleic acid encoding it are well known, and codons can be optimized according to the host cell to be expressed.

[0136] For example, the nucleic acid encoding the fusion protein of the present invention may include one or more nucleic acid sequences selected from the group consisting of SEQ ID NOs 20 to 25, but is not limited thereto.

[0137]

[0138] In another aspect, the present invention relates to virus-like particles (VLPs) comprising the fusion protein.

[0139] The term “virus-like particle” in this invention refers to a particle that presents a viral antigen and may be similar in shape and size to a virus, but does not possess genetic material. Virus-like particles can occur naturally or be synthesized through the recombination of viral structural proteins. VLPs can induce a stronger immune response than when used alone through the repetitive and high-density presentation of antigens, and generally, with a size ranging from tens of nanometers to several micrometers, they facilitate easy migration to lymph nodes.

[0140] In the field of this technology, the technology of constructing recombinant proteins based on the capsid proteins of various viruses and presenting antigens on their surfaces through in vivo assembly, in vitro assembly, etc., is well known in the art (Molecular Biotechnology. 53 (1): 92-107.). In addition, methods for producing recombinant VLPs using various culture systems such as bacteria, animal cell lines, insect cell lines, yeast, and plant cells are known in the art (Methods. 40 (1): 66-76; and npj Vaccines. 2 (1): 3).

[0141]

[0142] In another aspect, the present invention relates to nanoparticles comprising the nucleic acid or fusion protein.

[0143] The term “nano particle (NP)” in the present invention comprehensively refers to assembled particles having a size unit of several nanometers to several hundred nanometers, preferably 100 nm or less.

[0144] Techniques for loading nucleic acids or proteins onto the surface or interior of nanoparticles are well known in the art, and in particular, techniques for using nanoparticles as antigen carriers for vaccines are well known in the art. In the present invention, nanoparticles may be composed of, for example, carbon tubes, metals, polymers, lipids, proteins, etc., and are not limited to any type as long as they are capable of loading the fusion protein or nucleic acid of the present invention. Recently, various methods for manufacturing nanoparticles (NPs) with unique physicochemical characteristics are known, and they may be manufactured to have specific biological characteristics in terms of size, solubility, shape, hydrophilicity, and surface. Generally, antigens (e.g., the fusion protein of the present invention or the nucleic acid encoding it) may be loaded onto the exterior or interior of nanoparticles through covalent functionalization or encapsulation, and the antigens may be presented to a target at a desired location (Vaccines (Basel). 2022 Nov 17;10(11):1946).

[0145] In particular, in the technical field of the present invention, the use of lipid nanoparticles (LNPs) as carriers for RNA vaccines is well known, but is not limited thereto.

[0146]

[0147] In the embodiments of the present invention, it was confirmed that when the fusion protein of the present invention is used as an antigen for immunization, a surprisingly potent Th1 / Th17 immune response is induced, thereby inducing a long-term and potent immune response that prevents pertussis infection compared to existing cell-free pertussis vaccines that induce a Th2 immune response. In the case of existing cell-free pertussis vaccines, Alum-based substances were used as adjuvants, and Alum is known as a representative substance that enhances Th2 immunity. However, in the present invention, it was confirmed that a potent Th1 / Th17 immune response was induced despite the use of Alum as an adjuvant, thereby confirming that the fusion of aminoacyl-tRNA synthase in the present invention can induce a potent Th1 / Th17 immune response in addition to the previously reported effect of increasing protein solubility expression.

[0148]

[0149] Accordingly, in another aspect, the present invention relates to a vaccine composition comprising a fusion protein, nucleic acid, virus-like particle and / or nanoparticle of the present invention.

[0150] The term “vaccine composition” of the present invention refers to a composition comprising a substance capable of eliciting an immune response by acting as an antigen or immunogen in vivo or in vitro, and in the present invention, it may be used interchangeably with “vaccine” or “immunogenic composition” with the same meaning.

[0151] The term "immune response" in the present invention is a concept in the broadest sense that includes both innate immune responses and adaptive immune responses, e.g., complement-mediated immune responses, cell-mediated (T-cell) immune responses, and / or antibody (B-cell) responses.

[0152] The vaccine composition of the present invention can induce or increase an immune response against *Pertussis pertussis* in a subject administered with it, and more specifically, can induce and / or increase the formation of neutralizing antibodies against *Pertussis pertussis* or pertussis toxins, cellular immune responses, etc. More preferably, the vaccine composition of the present invention can induce a Th1 and / or Th17 immune response in a subject administered with it.

[0153] In the present invention, the vaccine composition may be characterized by additionally including an adjuvant.

[0154] The term “adjuvant” in this invention is a concept that originated from Alexander Glenny’s discovery that aluminum salts increase the immune response, and refers to an auxiliary component added to induce a stronger immune response in a subject receiving an immunogenic composition or vaccine. For example, the above adjuvant is an aluminum salt such as aluminum phosphate or aluminum hydroxide, squalene, MF59 or its analog (MF59 like), AS03 or its analog (AS03 like), AF03 or its analog (AF03 like), SE or its analog (SE like), a calcium salt, a dsRNA analog, a lipopolysaccharide, a Lipid A analog (MPL-A (e.g., Korean Patent Publications No. 2021-0027132 and No. 2022-0135935), GLA, etc.), flagellin, imidazoquinolines, CpG ODN, a mineral oil, an agonist of a Toll-like receptor (TLR), C-type Examples include lectin ligands, CD1d ligands (α-galactosylceramide, etc.), detergents, liposomes such as CoPoP (cobalt porphyrin-phospholipid nanoliposome, e.g., Korean Patent Publication No. 2022-0135935), saponins such as QS21, cytokines, peptides, etc., but are not limited thereto.

[0155] In the present invention, more preferably, the adjuvant may be characterized as being capable of stimulating a Th1 / Th17 pathway immune response, but is not limited thereto.

[0156] In the present invention, the adjuvant can be prepared without limitation in formulations known in the art, such as salts, emulsions, liposomes, aqueous solutions, etc.

[0157] In the present invention, the adjuvant may be prepared to be included in one formulation with the fusion protein of the present invention or prepared as a separate formulation.

[0158] The optimal dosage of the vaccine composition of the present invention can be determined by standard studies including the observation of a suitable immune response in subjects. After initial vaccination, subjects may be treated with one or several booster immunizations at appropriate intervals.

[0159] The vaccine composition of the present invention may be administered in a pharmaceutically effective amount. The term “pharmaceuticalally effective amount” in the present invention means an amount sufficient to induce or increase an immune response without causing side effects or severe or excessive immune responses, and the appropriate dosage may be determined by various factors such as the method of formulation, the mode of administration, the patient’s age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response responsiveness. Various general matters to be considered when determining the “pharmaceutically effective amount” are known to those skilled in the art, and are described, for example, in the literature [Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990] and [Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990].

[0160] The vaccine composition of the present invention may be administered simultaneously with a pertussis treatment or a symptomatic treatment, and may be administered together with other vaccines, antibiotics, treatments, adjuvants, symptom relievers, etc.

[0161] The vaccine composition of the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to methods that can be easily carried out by a person skilled in the art to which the invention pertains. The formulation may be used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. Suitable formulations known in the art may be those disclosed in the literature (Remington's Pharmaceutical Science, Mack Publishing Company, EastonPA).

[0162] The vaccine composition of the present invention may be administered orally or parenterally. The routes of administration of the composition according to the present invention are not limited to these, but may include, for example, intravenous, intravenous, subcutaneous, intramuscular, intra-arterial, intramedullary, intrathecal, intracardiac, transdermal, intraperitoneal, intestinal, sublingual, oral, or local administration. The dosage of the composition according to the present invention varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, or severity of the disease, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into a suitable dosage form using known techniques for clinical administration.

[0163] In another aspect, the present invention relates to the use of the fusion protein, nucleic acid, virus-like particle and / or nanoparticle of the present invention for the preparation of vaccine compositions.

[0164] In another aspect, the present invention relates to a method for vaccination against pertussis comprising the step of administering a fusion protein, nucleic acid, virus-like particle and / or nanoparticle of the present invention to a subject.

[0165]

[0166] The fusion protein of the present invention can be used for the prevention or treatment of pertussis because it induces a strong immune response against pertussis bacteria in the recipient.

[0167] In another aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of pertussis comprising the fusion protein, nucleic acid, virus-like particle, and / or nanoparticle of the present invention as an active ingredient.

[0168] As used in the present invention, the term "prevention" refers to any act of suppressing a target disease or delaying its onset by administering a pharmaceutical composition according to the present invention.

[0169] As used in the present invention, the term "treatment" refers to any act in which symptoms of a target disease are improved or beneficially altered by the administration of a pharmaceutical composition according to the present invention.

[0170] The above pharmaceutical composition may further include suitable carriers, excipients, and diluents commonly used in pharmaceutical compositions.

[0171] Carriers, excipients, and diluents that may be included in the above pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating the above composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0172] In the present invention, the pharmaceutical composition may be used in combination with other compositions or therapeutic regimens for treating whooping cough.

[0173] In another aspect, the present invention relates to the use of the fusion protein, nucleic acid, virus-like particle and / or nanoparticle of the present invention for the preparation of a pharmaceutical composition for the prevention or treatment of pertussis.

[0174] In another aspect, the present invention relates to a method for preventing or treating pertussis comprising the step of administering a fusion protein, nucleic acid, virus-like particle, and / or nanoparticle of the present invention to a target.

[0175] In another aspect, the present invention relates to the use of the fusion protein, nucleic acid, virus-like particle and / or nanoparticle of the present invention for the prevention or treatment of pertussis.

[0176]

[0177] In another aspect, the present invention relates to a recombinant vector containing nucleic acid of the present invention.

[0178] In the present invention, the recombinant vector may be used without limitation by a person skilled in the art, provided that it is a vector capable of inducing protein expression of the nucleic acid encoding the fusion protein. For example, when using E. coli as a host, a vector containing a T7 series (T7A1, T7A2, T7A3, etc.), lac, lacUV5, temperature-dependent (λphoA, phoB, rmB, tac, trc, trp, or 1PL) promoter may be used; when using yeast as a host, a vector containing an ADH1, AOX1, GAL1, GAL10, PGK, or TDH3 promoter may be used; and in the case of Bacillus, a vector containing a P2 promoter may be used. However, these are merely descriptions of some embodiments, and in addition to the vector containing the above promoter, a person skilled in the art may use various vectors known in the art without limitation, provided that they are suitable for the host as a vector containing a promoter for inducing the expression of the fusion protein according to the present invention.

[0179] In the present invention, the term "vector" refers to a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Upon transformation into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vector currently, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention. However, the present invention includes other forms of vectors having functions equivalent to those known or to be known in the art. Protein expression vectors used in E. coli may include the pET series from Novagen (USA); the pBAD series from Invitrogen (USA); pHCE or pCOLD from Takara (Japan); the pACE series from Genofocus (South Korea); and the like. In Bacillus subtilis, protein expression can be achieved by inserting a target gene into a specific part of the genome, or by using vectors such as the pHT series from MoBiTech (Germany). Protein expression is also possible in fungi and yeasts using genome insertion or self-replication vectors. Protein expression vectors for plants can be used by utilizing T-DNA systems such as those of Agrobacterium tumefaciens or Agrobacterium rhizogenes. Typical expression vectors for expression in mammalian cell cultures are based, for example, pRK5 (EP 307,247), pSV16B (WO 91 / 08291), and pVL1392 (Pharmingen).

[0180] The term "expression control sequence" refers to a DNA sequence essential for the expression of a coding sequence operably linked in a specific host organism. Such control sequences include a promoter for carrying out transcription, an optional operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. For example, a control sequence suitable for prokaryotes includes a promoter, optionally an operator sequence, and a ribosome binding site. For eukaryotes, it includes a promoter, a polyadenylation signal, and an enhancer. The factor that most influences the expression level of a gene in a plasmid is the promoter. For high expression, SRα promoters and cytomegalovirus-derived promoters are preferably used.

[0181] To express the nucleic acid sequence of the present invention, any of the very diverse expression regulatory sequences may be used in the vector. Examples of useful expression regulatory sequences include, in addition to the promoters described above, early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, T3 and T7 promoters, the major operator and promoter regions of phage lambda, the regulatory regions of fd code proteins, promoters for 3-phosphoglycerate kinase or other glycolases, promoters of said phosphatase, e.g., Pho5, promoters of the yeast alpha-mating system, and other sequences of configuration and induction known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. The T7 RNA polymerase promoter Φ can be usefully used to express proteins in E. coli.

[0182] Nucleic acids are "operably linked" when positioned in a functional relationship with other nucleic acid sequences. This may be a gene and regulatory sequence(s) linked in such a way that an appropriate molecule (e.g., a transcription-activating protein) enables gene expression when it binds to the regulatory sequence(s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a pre-sequence protein participating in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence when it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretion leader, they are in contact and exist within the reading frame. However, an enhancer does not need to be in contact. The linkage of these sequences is performed by ligation at a convenient restriction enzyme site. If such a site is not present, a synthetic oligonucleotide adapter or linker is used according to conventional methods.

[0183] As used in this specification, the term "expression vector" generally refers to a recombinant carrier into which a fragment of heterogeneous DNA is inserted, typically a fragment of double-stranded DNA. Here, heterogeneous DNA refers to DNA that is not naturally found in host cells. Once inside a host cell, the expression vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterogeneous) DNA may be produced.

[0184] As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression regulatory sequences that are functional within the selected expression host. Preferably, the expression regulatory sequences and the gene are contained within a single expression vector that includes both a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector may further include expression markers useful within the eukaryotic expression host.

[0185]

[0186] In another aspect, the present invention relates to a nucleic acid encoding a fusion protein of the present invention; and / or a recombinant host cell into which the recombinant vector is introduced.

[0187] In the present invention, the host cell refers to an expression cell into which a gene or recombinant vector, etc. has been introduced to produce a protein, etc. The host cell may be used without limitation as long as it is a cell capable of expressing the fusion protein of the present invention. For example, the host cell may be a prokaryotic cell including Escherichia coli, etc., or a eukaryotic cell including fungi, yeast, insect cells, or animal cells. More specifically, in the case of eukaryotic cells, CHO cell lines or HEK cell lines, which are mainly used for the expression of fusion proteins, may be used, but are not limited thereto. In the case of prokaryotic cells, lactic acid bacteria and Escherichia coli may be used, but are not limited thereto.

[0188] A wide variety of expression host / vector combinations may be used to express the fusion protein of the present invention. Expression vectors suitable for eukaryotic hosts include, for example, expression regulatory sequences derived from SV40, bovine papillomavirus, anenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors usable for bacterial hosts include bacterial plasmids that may be exemplified by those obtained from E. coli, such as pBluescript, pGEX2T, pUC vector, col E1, pCR1, pBR322, pMB9, and their derivatives; plasmids with a broader host range, such as RP4; phage DNA that may be exemplified by a wide variety of phage lambda derivatives, such as λ and λNM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells are 2μ plasmids and their derivatives. A useful vector for insect cells is pVL 941.

[0189] The above-mentioned recombinant vector may be introduced into a host cell by means such as transformation or transfection. As used herein, the term "transformation" means the introduction of DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through the completion of chromosomal integration. As used herein, the term "transfection" means the acceptance of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed.

[0190] Of course, it must be understood that not all vectors and expression regulatory sequences function equally in expressing the DNA sequence of the present invention. Likewise, not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate selections among various vectors, expression regulatory sequences, and hosts without departing from the scope of the present invention and without excessive experimental burden. For example, when selecting a vector, the host must be considered, as the vector must be replicated within it. The copy number of the vector, the ability to regulate the copy number, and the expression of other proteins encoded by said vector, such as antibiotic markers, must also be considered. When selecting an expression regulatory sequence, various factors must also be considered. For example, the relative strength of the sequence, its modulation capabilities, and compatibility with the DNA sequence of the present invention, particularly in relation to potential secondary structures, must be taken into account. A unicellular host must be selected by considering factors such as the selected vector, the toxicity and secretion characteristics of the product encoded by the DNA sequence of the present invention, the ability to accurately fold the protein, culture and fermentation requirements, and the ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within the range of these variables, a person skilled in the art may select various vector / expression control sequence / host combinations capable of expressing the DNA sequence of the present invention in fermentation or large-scale animal culture. Binding methods, panning methods, film emulsion methods, etc., may be applied as screening methods when attempting to clone cDNA by expression cloning.

[0191] The above-mentioned gene and recombinant vector can be introduced into a host cell through various conventionally known methods. The nucleic acid encoding the fusion protein of the present invention may be directly introduced into the genome of a host cell and exist as a chromosomal factor. It will be obvious to those skilled in the art to which the present invention pertains that inserting the gene into the genomic chromosome of a host cell will produce the same effect as introducing a recombinant vector into the host cell.

[0192]

[0193] In another aspect, the present invention relates to a method for producing a fusion protein comprising the step of culturing the host cell.

[0194] When a recombinant expression vector capable of expressing the above fusion protein is introduced into an E. coli host cell, the fusion protein can be produced by culturing the host cell for a period sufficient for it to be expressed in the host cell, or for a period sufficient for the fusion protein to be secreted into the culture medium in which the host cell is cultured.

[0195] In some cases, the expressed fusion protein may be isolated from the host cell and purified to a uniform degree. The isolation or purification of the fusion protein may be performed by separation and purification methods used for conventional proteins, such as chromatography. The chromatography may be, for example, one or more combinations selected from affinity chromatography, ion exchange chromatography, or hydrophobic chromatography, but is not limited thereto. In addition to the chromatography, filtration, ultrafiltration, salting out, dialysis, etc. may be used in combination.

[0196]

[0197] Although specific amino acid sequences and base sequences have been described in this invention, it will be obvious to those skilled in the art that amino acid sequences substantially identical to the enzyme intended for implementation in this invention and the base sequences encoding them fall within the scope of this invention. "Substantially identical" includes cases where the homology of amino acids or base sequences is very high, and also refers to proteins that share structural features or have the same function as those used in this invention, regardless of sequence homology. Proteins in which sequences other than the core sequence of this invention are partially deleted, or fragments of base sequences encoding them, may also be included in this invention; therefore, this invention includes all amino acid or base sequences having the same function as those used in this invention, regardless of the length of the fragment.

[0198]

[0199] Examples

[0200] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0201]

[0202] Example 1: Materials and Method

[0203] animal

[0204] Wild-type C57BL / 6(J) mice were purchased from Orient Bio (Korea) and reared under specific pathogen-free (SPF) conditions at the Avison Biomedical Research Center (ABMRC) of Yonsei University College of Medicine. All experiments were performed in accordance with guidelines approved by the Association for the Evaluation and Accreditation of Laboratory Animal Care.

[0205]

[0206] Preparation of novel recombinant aP vaccine antigen

[0207] To manufacture the aP vaccine, an expression vector of the recombinant protein was constructed. The pGE-ChaH2 vector was prepared by replacing the PCR-amplified WHEP domain of human glutamyl-prolyl-tRNA synthase (hEPRS) (also known as ChaH2) in the pGE-LysRS vector using NdeI and BamHI restriction enzymes. EPRS is a dual-function tRNA synthase composed of glutamyl-tRNA synthase (ERS) and prolyl-tRNA synthase (PRS), and the WHEP domain is located in a flexible linker region connecting enzyme domains that attach glutamate and proline to their respective tRNAs (Ray PS, PLOS ONE. 2014;9(6):e98493.). The pGE-ChaH2 expression vector was prepared by linking the ChaH2 PCR product, which includes a 6x-His tag at the C-terminus and TEV and enterokinase cleavage sites, to the cleaved vector. To express pertussis antigens, filamentous hemagglutinin (FHA, NCBI gene ID 69601663; amino acids 1655-2111), pertussis toxin A subunit (PTX, NCBI gene ID 69599992; amino acids 35-269), and pertactin (PRN, NCBI gene ID 69600977; amino acids 35-631) were cloned into the pGE-ChaH2 vector using BamHI and HindIII to construct pGE-ChaH2-PTX, pGE-ChaH2-FHA, and pGE-ChaH2-PRN constructs. For the PTX segment, it was further modified by site-directed mutagenesis to introduce R9K and E129G mutations for genetic detoxification (Pizza M, et al., Science. 1989;246(4929):497-500.). Genetically detoxified PTX PTX DXIt is said that. The FHA protein segment was selected based on known immunogenic properties (Leininger E, et al., J Infect Dis. 1997;175(6):1423-31). ChaH2-PTX DX The expression vectors were transformed into E. coli SHuffle® T7 competent cells, and the ChaH2-FHA and ChaH2-PRN vectors were transformed into E. coli BL21 Star™ (DE3) pLysS competent cells. Cultures were started in 50 mL of medium containing appropriate antibiotics and then increased to 1 L in fresh medium. Protein expression was induced in SHuffle® T7 cells using 1 mM IPTG at 30°C, and in BL21 Star™ (DE3) pLysS cells using 1 mM IPTG at 37°C. After induction, cells were harvested by centrifugation, suspended in lysis buffer containing lysozyme and PMSF, and sonicated. The resulting lysate was centrifuged to obtain the supernatant, which was filtered through a 0.45 μm polyethersulfone syringe filter for protein purification. Proteins were purified by immobilized metal affinity chromatography (IMAC) on a nickel column pre-equilibrated with buffer A. After loading the filtered supernatant, the column was washed with buffer C to remove endotoxins, and ChaH2-PTX DX Proteins were eluted with buffer B containing 1 M imidazole for ChaH2-FHA and 1.5 M imidazole for ChaH2-PRN. The eluted fractions were analyzed by SDS-PAGE, and the fractions containing the target proteins were collected and dialized into a storage buffer.

[0208]

[0209] The amino acid and nucleic acid sequences of the prepared recombinant aP antigen are as shown in Tables 2 and 3 below.

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221] Endotoxin removal through phase separation

[0222] Endotoxins were removed from the purified protein using phase separation with Triton X-114. The protein was mixed with 1% Triton X-114 and reacted in a rotary mixer at 4°C for 1 hour. Subsequently, the mixture was reacted at 37°C for 20 minutes and centrifuged at 20,000 RCF at 25°C for 10 minutes. The upper aqueous phase was collected and concentrated using an Amicon® Ultra centrifugal filter. Endotoxin levels were measured using the Pierce™ Chromogenic Endotoxin Quant Kit, and the maximum value was set to 100 EU / mL in this study.

[0223]

[0224] Bacterial culture and respiratory infection

[0225] Bordetella pertussis was cultured on Bordet-Gengou agar (BGA) plates supplemented with 10% glycerol, 15% defibrinated horse blood, and 1% Bordetella supplement at 37°C. After an initial 7-day culture, the bacteria were subcultured onto new BGA plates for another 7 days. Bacterial concentration was estimated by calculating colony-forming units (CFU) based on optical density (OD600) measured at 600 nm. For in vivo respiratory infection, 6-week-old C57BL / 6(J) mice were anesthetized with ketamine (50 mg / kg) and xylarzine (5 mg / kg), and B. pertussis at a concentration of 8×10^8 CFU / 40 μL was administered intranasally to each mouse.

[0226]

[0227] Histological analysis

[0228] The lungs were washed with PBS and fixed in 4% paraformaldehyde (PFA) at 4°C for 24 hours. The tissues were washed, embedded in paraffin, and sectioned to a thickness of 4 μm. Sections were stained with H&E for histological analysis. Histopathological analysis evaluated parenchymal inflammation, bronchitis, and vascular inflammation, each rated on a scale of 0 to 5 (0 = none, 1 = mild, 2 = minor, 3 = moderate, 4 = severe, 5 = very severe). The sum of these parameters represents a lung injury score ranging from 0 to 15. Histological scores were calculated using a double-blind method to ensure accuracy and minimize bias.

[0229]

[0230] CFU (Colony-Forming Unit) Count Test

[0231] Lungs were sonicated in PBS, and serial dilutions were plated onto BGA plates. Plates were incubated at 37°C for 7 days, and bacterial counts were expressed as log10 CFU per 1g of lung tissue.

[0232]

[0233] Quantitative Real-Time PCR (qRT-PCR)

[0234] Genomic DNA (gDNA) from total lungs was extracted using a gDNA extraction kit (Qiagen) according to the manufacturer's instructions. After phenol-chloroform extraction, the samples were washed with 70% ethanol and centrifuged. The extracted gDNA was eluted in RNase-free water. qRT-PCR was performed using the QuantStudio3 Real-Time PCR System with SYBR™ Green PCR Master Mix (Thermo-Fisher, 4309155). The mouse gene-specific primers used are as follows.

[0235] gapdhsense 5′-GAGAAACCTGCCAAGTATGATGAC-3′(Sequence No.: 26)

[0236] antisense 5′-ATCGAAGGTGGAAGAGTGGG-3′(Sequence No.: 27);

[0237] is481sense 5′-GCCGGATGAACACCCATAAG-3′(Sequence No.: 28)

[0238] antisense 5′-GCGATCAATTGCTGGACCAT-3′(Sequence No.: 29).

[0239]

[0240] Mouse Immunization

[0241] Infanrix-IPV (GlaxoSmithKline) or Pentaxim (Sanofi) were used as commercial DTaP vaccines. Recombinant proteins ChaH2-FHA (0.25 mg / kg), ChaH2-PRN (0.08 mg / kg), and ChaH2-PTX were administered for immunization. DX A dose of 0.25 mg / kg was prepared to match the protein amounts of the two DTaP vaccines mentioned above. The recombinant antigen was mixed with PBS and allium hydroxide (Invivogen, 21645-51-2), and a final dose of 100 μl was administered by intramuscular injection. For lower doses, the protein was diluted 1 / 10 in sterile PBS. Control mice were administered PBS containing allium hydroxide. Mice were immunized at Week 0 and booster-vaccinated at Week 2. Serum samples were collected at Week 4 to measure antibody titers.

[0242]

[0243] Measurement of antigen-specific IgG

[0244] Antigen-specific IgG was quantified using an indirect ELISA kit (Aloha Diagnostic International, FHA: 960-300-FMG, PTX: 960-130-PMG, and RPN: 960-230-PGG). For isotype-specific Igs, serum samples were diluted at a ratio of 1:250 and incubated with an antigen-specific ELISA kit at room temperature (RT) for 1 hour. Subsequently, the plates were further incubated with isotype-specific Ig-HRP conjugates including IgG1-HRP (Southern Biotech: 1073-05), IgG2-HRP (Southern Biotech: 1093-05), and IgG3-HRP (Southern Biotech: 1103-05). After incubating at room temperature for 30 minutes, the plates were washed and developed using TMB substrate reagent. The enzyme reaction was terminated by adding 2 N sulfuric acid, and the absorbance was measured at dual wavelengths of 450 nm and 630 nm.

[0245]

[0246] Mouse lymphocyte isolation and flow cytometry analysis

[0247] Mice were euthanized at 2 weeks after secondary immunization and perfused with 20 mL of PBS. Immune cells were obtained from tissues by a known method (Journal of Allergy and Clinical Immunology. 2021;147(4):1517-21.). Specifically, spleen and lungs were harvested and RPMI 1640 supplemented with 2% FBS was added to 14 mL of digestion buffer composed of collagenase type IV (3 mg / mL) and DNase I (5 mg / mL), and the mixture was incubated at 37°C for 30 minutes. A cell suspension was obtained from the digested tissues using a 40 μm cell filter. This suspension was washed with MACS buffer (PBS containing 0.5% EDTA and 0.5% FBS), and red blood cells were lysed using RBC lysis buffer (Invitrogen, 00-4300-54).

[0248] Single-cell suspensions of spleen or lung tissue were stained with LIVE / DEAD™ Fixable Aqua (Invitrogen, L34965) and the immunomarkers listed in Table 4 below were stained. Cells were fixed at room temperature for 1 hour using the Foxp3 / Transcription Factor Fixation / Permeabilization buffer set (Invitrogen, 00-5521-00), and permeability was enhanced using permeation buffer (Invitrogen, 00-5523-00). Intracellular staining was performed using an antibody mixture for 30 minutes at room temperature. Intracellular cytokines were detected after treatment with a protein transport inhibitor (Invitrogen, 00-4970-03) at 37°C for 4 hours. Flow cytometry data were collected using a SONY ID7000 Spectrum Cell Analyzer (Sony) or Symphony A5 SE (BD Biosciences) and analyzed using FlowJo software v10.10.0 (BD Biosciences).

[0249]

[0250]

[0251]

[0252] Ex Vivo restimulation

[0253] Immune cells were isolated and deposited at 5×10⁶ per RPMI 1640 medium supplemented with 10% FBS and penicillin / streptomycin. 6The cells were resuspended at a concentration of cell / mL. Cells were stimulated at 37°C for 18 hours using 5 μg / mL of recombinant FHA (MyBioSource), PRN (MyBioSource), and PTX (Merck: P2980). Brefeldin A (BD Biosciences, 555029) was added to the culture medium during the last 5 hours of stimulation. 1 μg / mL of anti-mouse CD3ε (BioXcell, BE0001-1) was used for positive control stimulation, and 5 μg / mL of ovalbumin (Sigma-Aldrich, A5503) was used as a negative control. In some experiments, the culture supernatant was collected 72 hours after protein stimulation, and cytokine analysis was performed using the LEGENDplex™ MU Th Cytokine Panel (12-plex) (BioLegend, 741044).

[0254]

[0255] Statistical analysis

[0256] Comparisons between groups of data were performed using t-tests, ordinary one-way ANOVA, and two-way ANOVA. Statistical significance was determined as *P < 0.5, **P < 0.01, and ***P < 0.001.

[0257]

[0258] Example 2: Construction and Characterization of a Novel Recombinant Pertussis (nrP) Vaccine

[0259] In the field of production technology for recombinant antigens for cell-free pertussis (aP) vaccines, the formation of misfolded, insoluble aggregates in E. coli is a major technical limitation. This problem is particularly concerning pure FHA, PRN, and PTX DXThis is prominently displayed during the initial generation of the antigen (Fig. 1A). To address this technical issue, ChaH2 was adopted as a fusion partner for each aP protein antigen. This approach enables the successful production of soluble immunogenic antigens (Fig. 2A).

[0260] As a result of evaluating various PTX constructs, PTX4 was selected as the PTX antigen based on its excellent solubility and immunogenicity characteristics (Fig. 3). ChaH2-FHA, ChaH2-PRN, and ChaH2-PTX DX The solubility of (hereinafter referred to as FHA, PRN, and PTX) was significantly improved compared to the pure protein antigen, and was particularly pronounced when incubated at 30°C or below (Fig. 1B). The presence of purified soluble proteins with expected molecular weights was confirmed by SDS-PAGE analysis: PTX (52 kDa), FHA (75 kDa), and PRN (85 kDa) (Fig. 2B). Protein yields were determined by densitometric scanning of band intensity and were FHA (5.64 mg / L), PRN (2.58 mg / L), and PTX (3.5 mg / L), respectively, and the final endotoxin levels were maintained below 50 EU / mL (Fig. 1C).

[0261] To evaluate the immunogenicity of the novel nrP antigen, mice were immunized with FHA, PRN, and PTX at concentrations identical to those found in commercially available DTAP (1X) or reduced relative to them (0.1X). ​​Antigen-specific IgG production was analyzed 2 weeks after the first and second immunizations (Figs. 2C and 3D). As a result, FHA, PRN, and PTX-specific antibodies were potently produced when using the recombinant aP vaccine of the present invention, and a significant boost was observed during the second immunization. These results indicate that the vaccine containing the nrP antigen of the present invention exhibits potent immunogenicity (Fig. 2D).

[0262]

[0263] Example 3: Confirmation of TH1 / TH17 Response Induction in the Lungs by Novel Recombinant Pertussis (nrP) Vaccine

[0264] It is well known that whole-cell pertussis (wP) vaccines induce a potent TH1 / TH17 immune response, whereas acellular pertussis (aP) vaccines primarily induce a TH2 response, which is ineffective for long-term immunity and prevention of asymptomatic infections (The Journal of Clinical Investigation. 2018;128(9):3853-65.). To address this issue, we evaluated whether a novel recombinant vaccine (nrP) could induce a more balanced immune response by incorporating the TH1 and TH17 pathways, which are crucial for effective mucosal immunity. The evaluation involved analyzing immune cell populations and their functions within lung tissue using multiparameter flow cytometry (Fig. 4). The overall impact of the nrP vaccine on mucosal immunity was assessed using the PhenoGraph clustering algorithm. As a result, it was confirmed that there were no significant immunological changes in the spleen (data not shown) or lung tissue, except for a decrease in CD19+ B cells (Fig. 5). However, significant changes were observed in the T cell population after nrP vaccination (Figs. 6A, 6B), in particular, CD4+ T cells and γδ T cells were preferentially activated, while no significant activation of CD8+ T cells was observed (Fig. 6C). Importantly, lung T cells in nrP-vaccinated mice showed distinct polarization toward type 17 T cells expressing RORγt, which indicates the effect of the present invention in inducing a type 17 response essential for mucosal immunity against Bordetella pertussis (Figs. 6D, 7C, 7D). This is consistent with results observed in other studies highlighting the role of specific immune pathways in enhancing the efficacy of recombinant vaccines against various pathogens (MedComm. 2021;2(3):430-41.). To further investigate the specific T cell responses induced by the nrP vaccine, lung immune cells from Vehicle-treated mice and nrP-vaccinated mice were stimulated with each recombinant antigen.The production of various cytokines was analyzed using a multiplex assay. As expected, TH2-type cytokines such as IL-5, IL-9, and IL-13 were produced due to antigen-specific stimulation, but IL-4 was not detected. This is attributed to the use of Alum as an adjuvant (Fig. 6E). Furthermore, the nrP vaccine significantly increased the production of TH1 cytokines (IL-2, IFN-γ, TNF-α) and TH17 cytokines (IL-17A, IL-22), and these responses were more pronounced compared to TH2 cytokines (Fig. 6E). These results suggest that the nrP vaccine effectively induces both TH1 and TH17 immune responses in the respiratory system and can provide superior protective effects compared to the existing aP vaccine.

[0265]

[0266] Example 4: B. Preventive efficacy of nrP vaccine against pertussis infection

[0267] To evaluate the preventive efficacy of a new recombinant pertussis (nrP) vaccine against Bordetella pertussis infection, prime-boost immunization was performed in mice.

[0268] As shown in Fig. 7, the nrP vaccine was administered at a full dose (1x) or a reduced dose (0.1x) equivalent to the amount of aP antigen used in the currently administered DTaP vaccine. After immunization on days 0 and 14, mice were infected with B. pertussis on day 28. Three days after infection, the bacterial burden in the lungs was evaluated by colony-forming unit (CFU) counting and qRT-PCR analysis of the is481 gene, a specific marker for B. pertussis. The results indicate that the bacterial burden in the lungs of the immunized group using the nrP vaccine was significantly reduced compared to the vehicle treatment group (Fig. 7B). Notably, mice immunized with the full dose of nrP showed almost no bacterial burden, and the CFU count was similar to the levels observed in the uninfected control group. This reduction was confirmed by qRT-PCR analysis, where is481 expression was significantly lower in the nrP-treated group, and transcription levels were similar to those of non-infected mice, particularly in the total dose group (Fig. 7C).

[0269] Histopathological examination of lung tissue further supports the protective effect of the nrP vaccine. Mice in the vehicle treatment group exhibited distinct lung pathology characterized by severe parenchymal inflammation, bronchitis, and vascular inflammation typical of B. pertussis infection (Fig. 7D). In contrast, the lungs of nrP-immunized mice, particularly those administered the full dose, showed a significant reduction in inflammation and tissue damage. Quantitative analysis of histopathological data indicates that parenchymal and vascular inflammation, bronchitis, and overall lung damage were significantly reduced in the nrP vaccination group compared to the vehicle administration group (Fig. 7D). These findings suggest that the nrP vaccine provides potent protection against B. pertussis infection, effectively reduces bacterial counts, alleviates lung pathology, and that the full-dose regimen provides nearly perfect protection.

[0270] To investigate the immune response associated with these protective effects in more detail, lung tissues from mice treated with the vehicle and mice vaccinated with the nrP vaccine were analyzed after B. pertussis infection. Consistent with previous reports, B. pertussis infection reduced CD19+ B cells in the airways while significantly increasing neutrophil recruitment, an effect that was completely suppressed by nrP vaccination (Figs. 8A, 8B). Additionally, the nrP vaccine significantly increased the expansion of effector CD4+ T cells, γδ T cells, and RORγt+ TH17 populations in mucosal tissues (Figs. 7E, 7F, Figs. 8C, 8D). Furthermore, nrP vaccination significantly increased IL-17A and / or TNF-α production in T cells without a corresponding increase in IFN-γ production. IL-17A was produced in both CD4+ and γδ T cells, while TNF-α production was observed in CD4+, CD8+, and γδ T cells. In particular, a population of pluripotent T cells expressing both IL-17A and TNF-α was also observed (Figure 3G). Collectively, these results indicate that the nrP vaccine effectively activates Th17-type immunity, which plays an important role in suppressing B. pertussis infection.

[0271]

[0272] Example 5: Comparison of Immunogenicity of DTaP and nrP Vaccines

[0273] Next, we compared the immunogenicity of the new nrP vaccine with a commercially available DTaP vaccine (Infanrix, referred to as IF). Humoral immune responses were evaluated by measuring antigen-specific antibody levels in serum samples collected at 2 and 4 weeks post-vaccination. While the IF vaccine induced higher levels of PTX-specific binding antibodies, the nrP vaccine exhibited significantly higher PRN-specific IgG titers after both the first and second vaccinations. The nrP vaccine also induced levels of FHA-specific antibodies similar to those induced by IF, exhibiting different immunogenicity profiles for each antigen (Fig. 9A). Given the significant role of antibody levels and isotype conversion in vaccine efficacy against B. pertussis (Pathogens and Disease. 2015;73(7); and JCI Insight. 2021;6(7).), specific Ig isotypes induced by the two vaccines were further analyzed. Consistent with previous findings, the IF vaccine induced IgG1 antibodies, which are generally associated with TH2-biased immune responses to FHA, PRN, and PTX. In contrast, the nrP vaccine not only induced IgG1 but also significantly higher levels of IgG2 and IgG3 isoforms, which indicate TH1 and TH17 immune responses (Fig. 9B) (PLOS Pathogens. 2013;9(4):e1003264. and BMC Microbiology. 2018;18(1):45.). The higher IgG2 / IgG1 and IgG3 / IgG1 ratios observed in the nrP vaccination group imply a stronger induction of TH1 / TH17 responses compared to the TH2-biased response induced by the IF vaccine (Fig. 9D).Considering the importance of TH1 / TH17 responses in providing protection against pertussis infection, these study results indicate that while DTaP vaccines primarily promote TH2 responses, nrP vaccines induce TH1 / TH17 responses more effectively, which means that the nrP vaccine of the present invention can enhance protection against B. pertussis infection compared to the existing DTaP vaccine.

[0274]

[0275] Example 6: Analysis of Immunological Response in Mucosal Tissues Following DTaP and nrP Vaccination

[0276] Multiparameter flow cytometry was performed to further investigate the immune responses induced by IF and nrP vaccination in lung tissue. Uniform Manifold Approximation and Projection (UMAP) analysis revealed no significant differences in the total immune cell populations among the vehicle, IF, and nrP groups (Figs. 10A and 11A, 11B). However, detailed analysis confirmed distinct immune responses between the two vaccines. The nrP group showed a significant increase in MHCII expression within the CD11c+ population, indicating enhanced antigen presentation ability—a change not observed in the IF group (Fig. 11A). Consistent with this, nrP significantly increased the proportion of TCRβ+CD4+ helper T cells in mucosal tissue compared to the vehicle-treated group, a response not observed in the IF group (Fig. 10B). Furthermore, nrP increased the CD4+CD62L- effector population and exhibited higher expression levels of RORγt and / or TBET in CD4+ T cells and γδ T cells, whereas not in CD8+ T cells, implying that the nrP vaccine of the present invention induces a potent type 1 / 17 immune response (Figs. 10C, 10D and Figs. 11C, 11D). In contrast, IF did not induce these specific immune changes but instead elicited a mixed response involving significant production of IL-4 and IL-5 along with IFN-γ (Fig. 10E), which implies a potent TH2 response as previously reported (The Journal of Clinical Investigation. 2018;128(9):3853-65.). Conversely, nrP preferentially induced IFN-γ and IL-17A without significantly increasing IL-4 and IL-5 in T cells (Figs. 10E and 11E), which indicates a unique and potent 1 / 17 type profile induced by nrP compared to the TH2-biased response induced by IF.

[0277]

[0278] Example 7: Evaluation of the efficacy of the novel recombinant pertussis (nrP) vaccine

[0279] The therapeutic efficacy of the recombinant pertussis vaccine (nrP) of the present invention was evaluated by comparing it with the commercially available Infanrix vaccine, focusing on its ability to suppress Bordetella pertussis infection. CFU analysis showed that both vaccines achieved effective bacterial clearance, which was evidenced by undetectable levels of B. pertussis bacteria in the lung tissues of both vaccine groups (Fig. 12A). However, a more detailed analysis quantifying B. pertussis is481 gene expression in lung tissues using qRT-PCR revealed a significant reduction in bacterial load in the nrP treatment group. Bacterial DNA levels in these samples approached those observed in the uninfected control group and were significantly lower than the detectable levels remaining in the Infanrix treatment group (Fig. 12B). Consistent with this, histopathological analysis of lung tissues indicated that nrP vaccination significantly alleviated infection-induced lung pathologies, including parenchymal inflammation, bronchitis, vascular inflammation, and overall lung damage. On the other hand, Infanrix reduced inflammation but did not completely prevent these pathological changes, and the severity of tissue damage was more pronounced compared to the nrP group (Fig. 12C).

[0280] Next, we examined the immune responses in lung tissue induced by IF and nrP vaccination following B. pertussis infection. UMAP analysis indicated no significant changes in the total immune cell population across the various treatment groups (Figs. 13A, 13B). However, both IF and nrP vaccines effectively suppressed neutrophil infiltration in lung tissue, which is generally associated with B. pertussis infection (Fig. 13B). Additionally, the proportion of TCRβ+ T cells in lung mucosal tissue significantly increased in the vaccinated groups (Fig. 13C). To confirm the functional changes in T cells induced by vaccination, we analyzed the expression of major T cell lineage markers, including TBET (indicating a type 1 response), GATA3 (indicating a type 2 response), and RORγt (indicating a type 17 response). While no significant differences in TBET and GATA3 expression were found between the groups (data not shown), RORγt expression was significantly upregulated in CD4+ T cells of the nrP-vaccinated group compared to the vehicle and IF groups (Fig. 12D). This implies that the nrP vaccine specifically induces a type 17 cellular immune response. To evaluate antigen-specific immune responses, immune cells were isolated from the lungs of mice in the vehicle, IF, and nrP groups after B. pertussis infection and stimulated with recombinant FHA, PRN, and PTX proteins. In response to all three antigens, cytokine production was minimal in the IF group. In contrast, the nrP-vaccinated group showed significant induction of TH1 / TH17 cytokines (Fig. 12E). These results imply that while the IF vaccine does not effectively induce a T-cell-mediated immune response, the nrP vaccine potently promotes this response, specifically type 1 / 17 cellular immunity. This enhanced cellular immunity is consistent with the B. It is believed to contribute to a strong inhibitory effect on pertussis infection.

[0281]

[0282] Foregoing, specific parts of the content of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0283]

[0284] The fusion protein of the present invention not only exhibits improved solubility and yield compared to existing cell-free pertussis (aP) vaccines, but can also induce a long-term and potent immune response based on the Th1 / Th17 pathway. Therefore, the fusion protein of the present invention overcomes the limitations of Th2 immune response-based aP vaccines and can be usefully employed as a novel pertussis vaccine or therapeutic agent.

[0285]

[0286] I have attached the electronic file.

Claims

Aminoacyl-tRNA synthase or a fragment thereof; and a fusion protein comprising an antigen protein derived from *Pertussis pertussis*. A fusion protein according to claim 1, wherein the aminoacyl-tRNA synthase is selected from the group consisting of glycyl-tRNA synthase, alanyl-tRNA synthase, valyl-tRNA synthase, leucyl-tRNA synthase, isoleucyl-tRNA synthase, methionyl-tRNA synthase, phenylalanyl-tRNA synthase, tryptophanyl-tRNA synthase, ceryl-tRNA synthase, threonyl-tRNA synthase, cysteinyl-tRNA synthase, tyrosyl-tRNA synthase, asparaginyl-tRNA synthase, aspartyl-tRNA synthase, glutaminyl-tRNA synthase, lysyl-tRNA synthase, arginyl-tRNA synthase, histidyl-tRNA synthase, and glutamyl-prolyl-tRNA synthase. A fusion protein according to claim 1, characterized in that the fragment of the aminoacyl-tRNA synthase comprises a WHEP domain. A fusion protein according to claim 3, characterized in that the aminoacyl-tRNA synthase fragment comprises the WHEP domain of human-derived tryptophanyl-tRNA synthase, histidyl-tRNA synthase, glycyl-tRNA synthase, methionyl-tRNA synthase, or glutamyl-prolyl-tRNA synthase. A fusion protein according to claim 4, characterized in that the WHEP domain of the human glutamyl-prolyl-tRNA synthase comprises one or more selected from the group consisting of WHEP-TRS1, WHEP-TRS2, and WHEP-TRS3. A fusion protein according to claim 3, characterized in that the fragment of the aminoacyl-tRNA synthase comprises a sequence selected from the group consisting of SEQ ID NOs 2 to 5. A fusion protein according to claim 1, characterized in that the antigen protein derived from Bordetella pertussis is derived from Bordetella pertussis. A fusion protein according to claim 1, characterized in that the antigen protein derived from the bacterium pertussis is one or more selected from the group consisting of fibrous hemagglutinin (FHA), pertussis toxin (PTX), aglutinogen (AGG), adenylate cyclase (AC), pertactin (PRN), and fimbriae (FIM). A fusion protein according to claim 1, characterized in that the antigen protein derived from pertussis bacteria comprises one or more sequences selected from the group consisting of SEQ ID NOs 6 to 13. A fusion protein according to claim 1, characterized by comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs 14 to 19. Nucleic acid encoding the fusion protein of claim 1. A virus-like particle (VLP) comprising the fusion protein of claim 1. Nanoparticles comprising the fusion protein of claim 1 or the nucleic acid encoding it. A vaccine composition comprising one or more selected from the group consisting of the fusion protein of claim 1, the nucleic acid of claim 11, the virus-like particle of claim 12, and the nanoparticle of claim 13. A vaccine composition characterized by additionally including adjuvants in claim 14. A pharmaceutical composition for the prevention or treatment of pertussis comprising any one or more selected from the group consisting of the fusion protein of claim 1, the nucleic acid of claim 11, the virus-like particle of claim 12, and the nanoparticle of claim 13. A recombinant vector containing the nucleic acid of claim 11. A recombinant host cell into which the nucleic acid of claim 11 or the recombinant vector of claim 17 has been introduced. A method for producing a fusion protein comprising the step of culturing the host cells of claim 18. A method for the prevention or treatment of pertussis comprising the step of administering the vaccine composition of claim 14 or the pharmaceutical composition of claim 16 to a subject.

Images