Bordetella pertussis strain with increased yield of outer membrane vesicles

Abstract

The invention relates to the field of bioengineering, in particular to a bordetella pertussis strain with increased yield of outer membrane vesicles (OMV), and the BP2992 gene in the strain is partially or completely inactivated. The invention also relates to a method for producing said bordetella pertussis strain and to a method for producing OMV by said bordetella pertussis strain.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a Bordetella pertussis strain with increased production of outer membrane vesicles (OMVs), wherein the BP2992 gene in the strain is partially or completely inactivated. The present application also relates to a method for producing the Bordetella pertussis strain and a method for producing OMVs by the Bordetella pertussis strain. BACKGROUND

[0002] Bordetella pertussis causes human whooping cough, which is most severe in children under one year of age. The first vaccine against whooping cough was composed of inactivated whole cells and was introduced in the 1940s. The use of the vaccine led to a significant decrease in cases of whooping cough, but the side effects caused by the vaccine, such as neurological disorders and fever due to the presence of endotoxins in the vaccine, were of concern. In the 1980s, Japan developed the first acellular vaccine, and now acellular vaccines are widely used. Despite the high vaccination rate now, cases of whooping cough are on the rise again due to genetic changes in the prevalent Bordetella pertussis strains and differences in the immune protection induced after vaccination with whole-cell or acellular pertussis vaccines.

[0003] A new generation of vaccines can be based on outer membrane vesicles (OMVs). OMVs are blebs extruded from the outer membrane (OM) and range in size from 20 nm to 200 nm. OMVs are thought to form by a process of blebbing from the outer membrane and peptidoglycan layer, which is believed to be associated with a weakening of the association between the outer membrane and the peptidoglycan layer or accumulation of material such as proteins in the periplasmic space. OMVs play a role in long-distance delivery, biofilm formation, bacterial survival, and regulation of interactions between populations. On the one hand, OMVs are small in size and are easily taken up by antigen-presenting cells; on the other hand, OMVs have many things in common with bacteria and are closer to simulating natural infection; on the other hand, OMVs contain LPS and can act as a natural adjuvant. Therefore, Bordetella pertussis OMVs have enhanced immunogenicity compared to existing whole-cell pertussis vaccines and acellular pertussis vaccines.

[0004] The bottleneck of Bordetella pertussis OMVs secreted as a vaccine is that the production of Bordetella pertussis OMVs is extremely low, which makes the research and production of Bordetella pertussis OMV vaccine face great challenges. Increasing the production of OMVs has become a problem to be solved for the development of Bordetella OMV vaccine.

[0005] There are many ways to increase the production of bacterial OMVs, which can be divided into two categories: improving the yield from the fermentation process and improving the yield from the strain.

[0006] From the aspect of fermentation process, the secretion amount of OMV can be increased by induction, but there are deficiencies compared with naturally secreted OMV. For example, using a detergent to induce OMV secretion will cause the formed OMV to lose LPS with adjuvant effect and lipoprotein that may have immunogenicity; using ultrasonic to induce OMV secretion will cause the OMV product to be contaminated with inner membrane; using reagents such as EDTA to induce OMV secretion may reduce the stability of OMV membrane; using heating to induce OMV secretion may change the phospholipid composition of OMV membrane.

[0007] From the aspect of strains, the yield of OMV can be increased by editing the genes related to the secretion process of OMV. There are three common strategies. The first strategy is to weaken the combination of outer membrane and peptidoglycan layer, so that the outer membrane is loose and easy to form vesicle secretion. Outer membrane lipoprotein is the key bridge between outer membrane and peptidoglycan, and reducing the content of outer membrane lipoprotein is an important means to reduce the combination of outer membrane and peptidoglycan layer. For example, knocking out outer membrane lipoprotein genes lpp, ompA and their similar genes (such as rmpM), tol / pal, etc. can make the bacteria over-secrete OMV. The second strategy is to increase the content of phospholipid on the outer membrane to increase the curvature of the outer membrane. For example, knocking out the phospholipase gene pldA of the outer membrane to reduce the degradation of phospholipid, and destroying the transport system of outer membrane phospholipid to inner membrane—mla system can all achieve over-secretion of OMV. The third strategy is to increase the internal pressure of periplasmic space to promote the secretion of OMV. For example, the molecular chaperone gene degP of periplasmic space can be knocked out to increase the accumulation of misfolded proteins in periplasmic space, and destroy the recycling of peptidoglycan, etc.

[0008] Although knocking out the lpp gene in E. coli can increase the production of E. coli OMV by 160 times, there is no such gene and its homologous gene in B. pertussis. Eline F. de Jonge et al. found that the inactivation of rmpM, tolR and pal genes in B. pertussis failed, and it was speculated that the products of rmpM, tolR and pal (PG-associated lipoprotein) genes were essential in B. pertussis. Further, Eline F. de Jonge et al. constructed conditional pal gene mutants, which increased the production of B. pertussis OMV under pal-depleted conditions, but the composition of OMV released by the wild-type cells was different (Research in Microbiology, Volume 173, Issues 4-5, 2022, 103937). The ompA gene encodes an outer membrane protein of a B. pertussis strain. Studies have shown that knocking out ompA in E. coli can increase the production of OMV by 26 times. However, CN116438193A mentioned that OmpA and its homologues in B. pertussis appear to be essential proteins for the survival of B. pertussis, and the deletion of OmpA in B. pertussis is not conducive to its viability. Therefore, the method of increasing the production of OMV on other bacteria may not be applicable to B. pertussis.

[0009] Therefore, there is an urgent need to develop new methods to increase the production of B. pertussis OMV in order to develop new B. pertussis vaccines based on OMV. SUMMARY

[0010] The present application provides at least the following embodiments, but is not limited thereto: Embodiment 1. A B. pertussis strain, wherein a BP2992 gene of the strain is partially inactivated or completely inactivated, whereby the strain has increased production of bacterial outer membrane vesicles (OMV) compared to a control B. pertussis strain.

[0011] Embodiment 2. The B. pertussis strain of embodiment 1, wherein the BP2992 gene i) encodes a protein comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 18, or comprising the amino acid sequence set forth in SEQ ID NO: 18; or ii) comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 9 or comprises the nucleotide sequence set forth in SEQ ID NO: 9.

[0012] Embodiment 3. The B. pertussis strain of embodiment 1 or 2, wherein the pldA gene and / or the mltA gene of the strain is partially inactivated or completely inactivated.

[0013] Embodiment 4. The B. pertussis strain of embodiment 3, wherein the pldA gene i) encodes a protein comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 19 or comprises the amino acid sequence set forth in SEQ ID NO: 19; or ii) comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 12 or comprises the nucleotide sequence set forth in SEQ ID NO: 12, wherein the mltA gene i) encodes a protein comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 20 or comprises the amino acid sequence set forth in SEQ ID NO: 20; or ii) comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to SEQ ID NO: 15 or comprises the nucleotide sequence set forth in SEQ ID NO: 15.

[0014] Embodiment 5. The B. pertussis strain of embodiment 3 or 4, wherein the pldA gene and the mltA gene of the strain are partially inactivated or completely inactivated.

[0015] Embodiment 6. The B. pertussis strain of any one of embodiments 1-5, wherein the gene that is partially inactivated or completely inactivated in the strain comprises an introduced mutation that results in partial or complete inactivation of the gene.

[0016] Embodiment 7. The B. pertussis strain of embodiment 6, wherein the mutation is an addition, substitution or deletion of one or more nucleotides.

[0017] Embodiment 8. The B. pertussis strain of embodiment 7, wherein the mutation is introduced into an expression regulatory sequence of the gene, thereby resulting in reduced or no expression of the protein encoded thereby; or, the mutation is introduced into the coding sequence of the gene, resulting in an addition, substitution or deletion of one or more amino acids in the encoded protein, the addition, substitution or deletion of the one or more amino acids resulting in reduced or no activity of the encoded protein.

[0018] Embodiment 9. The B. pertussis strain of embodiment 6, wherein the mutation is a partial deletion or complete deletion of the gene, preferably a complete deletion.

[0019] Embodiment 10. The B. pertussis strain of any one of embodiments 6-9, wherein the mutation is introduced by homologous recombination or targeted mutagenesis mediated via CRISPR, TALEN or ZFN technology.

[0020] Embodiment 11. The B. pertussis strain of any one of embodiments 1-10, wherein the bacterial outer membrane vesicle (OMV) production of the B. pertussis strain is about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold or more than the OMV production of a control B. pertussis strain.

[0021] Embodiment 12. The B. pertussis strain of any one of embodiments 1-11, which is derived from the BAA-589 strain.

[0022] Embodiment 13. A method of producing a B. pertussis strain having increased bacterial outer membrane vesicle (OMV) production, the method comprising the steps of: a) partially inactivating or completely inactivating the BP2992 gene in a B. pertussis strain; and b) optionally, partially or completely inactivating the pldA gene and / or the mltA gene in the B. pertussis strain.

[0023] Embodiment 14. The method of embodiment 13, wherein in the optional step b) the pldA gene and the mltA gene in the B. pertussis strain are partially or completely inactivated.

[0024] Embodiment 15. The method of embodiment 14, wherein in the optional step b) the pldA gene in the B. pertussis strain is first partially or completely inactivated, and subsequently the mltA gene in the B. pertussis strain is partially or completely inactivated.

[0025] Embodiment 16. The method of any one of embodiments 13-15, wherein the optional step b) is performed prior to step a), or wherein the optional step b) is performed after step a).

[0026] Embodiment 17. The method of any one of embodiments 13-16, wherein the partial or complete inactivation of the gene is caused by introduction of a mutation.

[0027] Embodiment 18. The method of embodiment 17, wherein the mutation is an addition, substitution or deletion of one or more nucleotides.

[0028] Embodiment 19. The method of embodiment 18, wherein the mutation is introduced into an expression regulatory sequence of the gene, thereby causing reduced or no expression of the protein encoded thereby; or the mutation is introduced into the coding sequence of the gene, causing an addition, substitution or deletion of one or more amino acids in the encoded protein, which addition, substitution or deletion of one or more amino acids causes reduced or no activity of the encoded protein.

[0029] Embodiment 20. The method of embodiment 17, wherein the mutation is a partial or complete deletion of the gene, preferably a complete deletion.

[0030] Embodiment 21. The method of any one of embodiments 17-20, wherein the mutation is introduced by homologous recombination or targeted mutagenesis mediated via CRISPR, TALEN or ZFN technology.

[0031] Embodiment 22. The method of embodiment 21, wherein the method comprises the following steps: a) deleting the BP2992 gene in the B. pertussis strain by homologous recombination, thereby obtaining a modified B. pertussis strain with a deletion of the BP2992 gene; b) deleting the pldA gene in the B. pertussis strain obtained in step a) by homologous recombination, thereby obtaining a B. pertussis strain in which both the BP2992 gene and the pldA gene are deleted; and c) deleting the mltA gene in the B. pertussis strain obtained in step b) by homologous recombination, thereby obtaining a B. pertussis strain in which the BP2992 gene, the pldA gene and the mltA gene are deleted.

[0032] Embodiment 23. The method of embodiment 21 or 22, wherein the BP2992 gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 10 and 11; the pldA gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 13 and 14; and or, the mltA gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 16 and 17.

[0033] Embodiment 24. A B. pertussis strain produced by the method of any one of embodiments 13-23.

[0034] Embodiment 25. A method of producing a B. pertussis outer membrane vesicle (OMV), the method comprising the steps of: a) culturing the B. pertussis strain according to any one of embodiments 1-12 and 24 under conditions suitable for the production of outer membrane vesicles (OMVs); and b) recovering and optionally purifying the OMVs produced in step a).

[0035] Embodiment 26. A B. pertussis outer membrane vesicle (OMV) produced by the method of embodiment 25.

[0036] Embodiment 27. A composition comprising an effective amount of 1) a B. pertussis strain according to any one of embodiments 1-12 and 24; and / or 2) a B. pertussis outer membrane vesicle (OMV) according to embodiment 26.

[0037] Embodiment 28. The composition of embodiment 27, further comprising an adjuvant and / or a pharmaceutically acceptable carrier.

[0038] Embodiment 29. Use of a B. pertussis strain according to any one of embodiments 1-12 and 24, and / or a B. pertussis outer membrane vesicle according to embodiment 26, and / or a composition according to embodiment 27 or 28, for the manufacture of a medicament or vaccine for the prevention and / or treatment of a B. pertussis infection in a subject.

[0039] Embodiment 30. Use of a Bordetella pertussis strain according to any one of embodiments 1-12 in the production of Bordetella pertussis outer membrane vesicles (OMVs). BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 . Structure of the exogenous backbone plasmid used in the homologous recombination method.

[0041] Figure 2 . Sequencing alignment of the BAA-589 ΔpagP strain.

[0042] Figure 3 . Comparison of OMV particle concentration secreted by the BAA-589 ΔpagP strain and the wild-type strain BAA-589.

[0043] Figure 4 . Sequencing alignment of the BAA-589 ΔBP2992 strain.

[0044] Figure 5 . Comparison of growth curves of the BAA-589 ΔBP2992 strain and the wild-type strain BAA-589.

[0045] Figure 6 . Comparison of OMV particle size of the BAA-589 ΔBP2992 strain and the wild-type strain BAA-589.

[0046] Figure 7 . Sequencing alignment of the BAA-589 ΔBP2992 ΔpldA strain.

[0047] Figure 8 . Sequencing alignment of the BAA-589 ΔBP2992 ΔpldA ΔmltA strain.

[0048] Figure 9 . Comparison of OMV secretion of the BAA-589 ΔBP2992 strain, the BAA-589 ΔBP2992 ΔpldA strain, and the BAA-589 ΔBP2992 ΔpldA ΔmltA strain and the wild-type strain BAA-589. DETAILED DESCRIPTION

[0049] I. Definitions In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the terms and procedures employed in protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and molecular biology are those commonly used by those skilled in the corresponding fields and are generally described in the literature.

[0050] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0051] The terms “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” or “nucleic acid fragment” are used interchangeably and refer to single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanosine or deoxyguanosine, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “I” for inosine, and “N” for any nucleotide. Although nucleotide sequences may be represented as DNA sequences (containing T) herein, when referring to RNA, those skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0052] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0053] The term "gene" as used in this article can refer to the coding sequence of a protein, but it can also encompass expression regulatory elements / sequences, such as promoters and enhancers. "Gene" typically refers to the endogenous sequence of an organism, such as Bordetella pertussis.

[0054] When the term “comprising” is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid sequence, but still possess the activities described in this invention.

[0055] Sequence identity between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for assessing the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various known sequence analysis software. For example, sequence identity can be assessed using the online alignment tool EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm with default parameters. Sequence identity can be along the full length of a given sequence.

[0056] Endogenous genes or proteins encoded by bacteria such as Bordetella pertussis may have some natural sequence variation in their homologs in different strains. However, due to the high sequence similarity / homology between them (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even at least 99.5% sequence similarity), those skilled in the art will be able to expect them to have similar or identical functions.

[0057] The “BP2992 gene” mentioned in this article is an unknown functional gene in Bordetella pertussis that encodes the BP2992 protein, whose function is unknown.

[0058] The BP2992 protein in *Bordetella pertussis* may contain an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:18. In some embodiments, the BP2992 protein in *Bordetella pertussis* may contain the amino acid sequence shown in SEQ ID NO:18.

[0059] The BP2992 gene in *Bordetella pertussis* may contain a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:9. In some embodiments, the BP2992 gene in *Bordetella pertussis* may contain the nucleotide sequence shown in SEQ ID NO:9.

[0060] The "pldA gene" mentioned in this article encodes the PldA protein, which is a phospholipase. Its phospholipase activity can degrade phospholipids on the outer membrane and release fatty acids.

[0061] The PldA protein in *Bordetella pertussis* may contain an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:19. In some embodiments, the PldA protein in *Bordetella pertussis* may contain the amino acid sequence shown in SEQ ID NO:19.

[0062] The plcA gene in *Bordetella pertussis* may contain a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:12. In some embodiments, the plcA gene in *Bordetella pertussis* may contain the nucleotide sequence shown in SEQ ID NO:12.

[0063] The "mltA gene" described in this article encodes the MltA protein, a lipoprotein located on the outer membrane. This protein has peptidoglycan cleavage and transglycosylation activity, participates in peptidoglycan metabolism, and plays a role in cell separation and membrane construction.

[0064] The MltA protein in *Bordetella pertussis* may contain an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with SEQ ID NO:20. In some embodiments, the MltA protein in *Bordetella pertussis* may contain the amino acid sequence shown in SEQ ID NO:20.

[0065] The mltA gene in *Bordetella pertussis* may contain a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:15. In some embodiments, the MltA gene in *Bordetella pertussis* may contain the nucleotide sequence shown in SEQ ID NO:15.

[0066] As used herein, "effective amount" means an amount of a substance, compound, material, or composition containing a compound (such as the OMV of the present invention or the compositions of the present invention) that, when applied to a subject, is at least sufficient to produce a preventive or therapeutic effect. Therefore, an effective amount is the amount necessary to prevent, cure, improve, delay, or partially delay the symptoms of a disease or condition such as a bacterial infection.

[0067] The term "object" as used in this article preferably refers to a person.

[0068] II. Bordetella pertussis strains with increased OMV production In one aspect, the present invention provides a Bordetella pertussis strain in which the BP2992 gene is partially or completely inactivated, thereby the strain having an increased production of bacterial outer membrane vesicles (OMV) compared with a control Bordetella pertussis strain.

[0069] In some preferred embodiments, the plcA gene of the Bordetella pertussis strain is also partially or completely inactivated.

[0070] In some other preferred embodiments, the mltA gene of the Bordetella pertussis strain is also partially or completely inactivated.

[0071] In some preferred embodiments, the pldA and mltA genes of the Bordetella pertussis strain are also partially or completely inactivated.

[0072] In some embodiments, the genes of the Bordetella pertussis strain that are partially or completely inactivated are its endogenous genes.

[0073] As used herein, partial inactivation of a gene refers to a reduction in the expression and / or decreased activity of the product (e.g., protein) it encodes. For example, the expression of the product (e.g., protein) encoded by a partially inactivated gene is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more; or the expression activity of the partially inactivated gene is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more.

[0074] As used herein, complete gene inactivation means that the product it encodes (such as a protein) is not expressed and / or expresses an inactive product (such as a protein). For example, a completely inactivated gene does not express its encoded product (such as a protein), or a completely inactivated gene expresses an inactive product (such as a protein).

[0075] Partial or complete inactivation of a gene can be achieved through a variety of methods known in the art. For example, in some embodiments, partial or complete inactivation of the gene can be caused by introducing a mutation into the gene. The mutation can be the addition, substitution, or deletion of one or more nucleotides.

[0076] The mutation can be introduced into the expression regulatory sequence of the gene, thereby causing a reduction or absence of expression of its encoded product (such as a protein).

[0077] Alternatively, the mutation may be introduced into the coding sequence of the gene, resulting in the addition, substitution, or deletion of one or more amino acids in the protein it encodes, which leads to reduced or no activity of the encoded protein.

[0078] In some embodiments, the mutation is a partial or complete deletion of a gene. The gene may be completely deleted from the strain, such that the strain of the present invention does not express the product encoded by the gene (e.g., a protein). The gene may also be partially deleted, such that the strain of the present invention expresses only a truncated product with reduced or no activity (e.g., a truncated protein). In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the gene (e.g., its coding sequence) is deleted.

[0079] Introducing mutations into genes can be achieved through various means known in the art. In some embodiments, mutations are introduced into the genes of the *Bordetella pertussis* strain through genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, the mutation in the gene, such as gene deletion, can be achieved through homologous recombination. Methods of homologous recombination are described in patent document CN 117947054 B. In some embodiments, the mutation in the gene is performed through targeted mutagenesis, such as targeted mutagenesis mediated by CRISPR, TALEN, or ZFN technologies.

[0080] In some embodiments, the BP2992 gene is deleted via homologous recombination using the homologous arm sequences shown in SEQ ID NO: 10 and 11. In some embodiments, the pldA gene is deleted via homologous recombination using the homologous arm sequences shown in SEQ ID NO: 13 and 14. In some embodiments, the mltA gene is deleted via homologous recombination using the homologous arm sequences shown in SEQ ID NO: 16 and 17.

[0081] In some embodiments, compared with a corresponding control Bordetella pertussis strain (under similar or identical conditions), the Bordetella pertussis strain of the present invention exhibits an increase in bacterial outer membrane vesicle (OMV) yield of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or more. In some embodiments, under similar or identical conditions, the bacterial outer membrane vesicle (OMV) yield of the Bordetella pertussis strain of the present invention is approximately 1.1 times, approximately 1.2 times, approximately 1.3 times, approximately 1.4 times, approximately 1.5 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times or more of the OMV yield of the corresponding control Bordetella pertussis strain.

[0082] The “control Bordetella pertussis strain” mentioned herein can be a parent strain from which the Bordetella pertussis strain described in this invention is derived. For example, the BP2992 gene and optionally the mltA gene and / or pldA gene in the control Bordetella pertussis strain are not inactivated (partially or completely).

[0083] The Bordetella pertussis strains described herein can be derived from any known Bordetella pertussis strain. Exemplary originating Bordetella pertussis strains may be, for example, BAA-589, ATCC 9797, or CHLA-26. These originating Bordetella pertussis strains possess the plcA, mltA, or BP2992 genes. The Bordetella pertussis strains may also express one or more antigens, such as exogenous antigens; preferably, the antigens are antigens that can be displayed on the cell membrane and / or antigens that can be displayed on the OMV.

[0084] III. Methods of producing Bordetella pertussis strains with increased bacterial outer membrane vesicle (OMV) production In another aspect, the present invention provides a method for producing a Bordetella pertussis strain with increased production of bacterial outer membrane vesicles (OMV), the method comprising the following steps: a) To partially or completely inactivate the BP2992 gene in Bordetella pertussis strains; and b) Optionally, the pldA and / or mltA genes in the Bordetella pertussis strain are partially or completely inactivated.

[0085] In some embodiments, step b) optionally involves partially or completely inactivating the pldA and mltA genes in the Bordetella pertussis strain. In some preferred embodiments, step b) optionally involves first partially or completely inactivating the pldA gene in the Bordetella pertussis strain, followed by partially or completely inactivating the mltA gene in the Bordetella pertussis strain.

[0086] In some embodiments, optional step b) is performed before step a). In some embodiments, optional step b) is performed after step a).

[0087] In some embodiments, the genes of the Bordetella pertussis strain that are partially or completely inactivated are its endogenous genes.

[0088] Partial or complete inactivation of a gene can be achieved through a variety of methods known in the art. For example, in some embodiments, partial or complete inactivation of the gene can be caused by introducing a mutation into the gene. The mutation can be the addition, substitution, or deletion of one or more nucleotides.

[0089] The mutation can be introduced into the expression regulatory sequence of the gene, thereby causing a reduction or absence of expression of its encoded product (such as a protein).

[0090] Alternatively, the mutation may be introduced into the coding sequence of the gene, resulting in the addition, substitution, or deletion of one or more amino acids in the protein it encodes, which leads to reduced or no activity of the encoded protein.

[0091] In some embodiments, the mutation is a partial or complete deletion of a gene. The gene may be completely deleted from the strain, such that the strain of the present invention does not express the product encoded by the gene (e.g., a protein). The gene may also be partially deleted, such that the strain of the present invention expresses only a truncated product with reduced or no activity (e.g., a truncated protein). In some embodiments, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of the gene (e.g., its coding sequence) is deleted.

[0092] Introducing mutations into genes can be achieved through various means known in the art. In some embodiments, mutations are introduced into the genes of the *Bordetella pertussis* strain through genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, the mutation in the gene, such as gene deletion, can be achieved through homologous recombination. Methods of homologous recombination are described in patent document CN 117947054 B. In some embodiments, the mutation in the gene is performed through targeted mutagenesis, such as targeted mutagenesis mediated by CRISPR, TALEN, or ZFN technologies.

[0093] In some specific implementations, the method includes the following steps: a) By homologous recombination to delete the BP2992 gene in the Bordetella pertussis strain, a modified Bordetella pertussis strain with the BP2992 gene deleted was obtained. b) By deleting the plcA gene from the Bordetella pertussis strain obtained in step a), a Bordetella pertussis strain with both the BP2992 gene and the plcA gene deleted was obtained; and c) Obtain the mltA gene from the Bordetella pertussis strain obtained by homologous recombination deletion step b), thereby obtaining a Bordetella pertussis strain with deletions of the BP2992 gene, pldA gene, and mltA gene.

[0094] In some embodiments, the BP2992 gene is deleted via homologous recombination using the homologous arm sequences shown in SEQ ID NO: 10 and 11. In some embodiments, the pldA gene is deleted via homologous recombination using the homologous arm sequences shown in SEQ ID NO: 13 and 14. In some embodiments, the mltA gene is deleted via homologous recombination using the homologous arm sequences shown in SEQ ID NO: 16 and 17.

[0095] In another aspect, the present invention also provides a strain of Bordetella pertussis produced by the method of the present invention.

[0096] In some embodiments, the method of the present invention produces a bacterial outer membrane vesicle (OMV) yield of *Bordetella pertussis* strain that is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or more. In some embodiments, under similar or identical conditions, the bacterial outer membrane vesicle (OMV) yield of the Bordetella pertussis strain of the present invention is approximately 1.1 times, approximately 1.2 times, approximately 1.3 times, approximately 1.4 times, approximately 1.5 times, approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times or more of the OMV yield of the corresponding control Bordetella pertussis strain.

[0097] IV. Production and use of bacterial outer membrane vesicles (OMVs) In another aspect, the present invention provides the use of the Bordetella pertussis strain of the present invention in the production of Bordetella pertussis outer membrane vesicles (OMV).

[0098] In another aspect, the present invention provides a method for generating Bordetella pertussis outer membrane vesicles (OMV), the method comprising the following steps: a) The Bordetella pertussis strain of the present invention was cultured under conditions suitable for the production of outer membrane vesicles (OMV); and b) Recover and optionally purify the OMV generated in step a).

[0099] The process conditions known in the art for producing OMV by culturing / fermenting Bordetella pertussis strains can all be applied to this invention. For example, BG solid medium, SS medium, or Verwey medium can be used to culture / ferment Bordetella pertussis strains to produce OMV.

[0100] In another aspect, the present invention provides Bordetella pertussis outer membrane vesicles (OMV) generated by the method of the present invention.

[0101] In another aspect, the present invention provides a composition comprising an effective amount of 1) The Bordetella pertussis strain of the present invention; and / or 2) The Bordetella pertussis outer membrane vesicle (OMV) of the present invention.

[0102] In some embodiments, the composition is a vaccine. In some embodiments, the composition is used for the prevention and / or treatment of Bordetella pertussis infection in a subject.

[0103] The composition may also contain adjuvants. As used herein, "adjuvant" means an additional component in a vaccine that enhances the immune response, or an auxiliary molecule added to a vaccine, or an auxiliary molecule produced by the body after being induced by such additional components, such as, but not limited to, interferon, interleukin, or growth factors. "Adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions.

[0104] In some embodiments of the foregoing aspects, the composition may also comprise a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antifungal agents, isotonic agents, and absorption delay agents. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose), amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and colorants.

[0105] In some embodiments of the above aspects, the composition is formulated for intramuscular, intraperitoneal, subcutaneous, oral, or intranasal administration. In some embodiments, the composition is in lyophilized form and can be reconstituted prior to use.

[0106] In one aspect, the present invention provides the use of the Bordetella pertussis strain of the present invention, the Bordetella pertussis outer membrane vesicles of the present invention, and / or the compositions of the present invention in the preparation of a medicament or vaccine for the prevention and / or treatment of Bordetella pertussis infection in subjects.

[0107] In one aspect, the present invention provides a method for preventing and / or treating Bordetella pertussis infection in a subject, the method comprising administering to the subject an effective amount of the modified Bordetella pertussis strain of the present invention, the Bordetella pertussis outer membrane vesicle (OMV) of the present invention, and / or the composition of the present invention.

[0108] Example The present invention can be further understood by referring to the specific embodiments described herein. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Obviously, those skilled in the art will recognize that various modifications and variations can be made to the present invention without departing from its spirit; therefore, such modifications and variations also fall within the scope of the present invention. For illustrative purposes, the present invention uses the BAA-589 strain purchased from ATCC (American Type Culture Collection).

[0109] Example 1: Method for modifying Bordetella pertussis gene via homologous recombination 1.1 Construction of exogenous DNA donor plasmid The exogenous backbone plasmid contains ori-ColE1 / pMB1 / pBR322 / pUC, the transfer elements OriT and traJ, the gentamicin resistance gene (Gen), and the galk reverse screening gene. The sequence of the exogenous backbone plasmid is shown in SEQ ID NO:1, and the structure of the backbone plasmid is as follows. Figure 1 As shown in the diagram, the gentamicin resistance gene sequence is shown in SEQ ID NO:2, and the galk reverse screening gene sequence is shown in SEQ ID NO:3. Homologous arms of the target gene integration site are inserted between the upstream site-up (sequence shown in SEQ ID NO:4) and downstream site-down (sequence shown in SEQ ID NO:5) of the exogenous backbone plasmid to obtain the exogenous DNA donor plasmid.

[0110] 1.2 Electroporation to introduce donor plasmid Activated glycerol bacteria were cultured on Bordet-Gengo solid plates at 37°C until colonies appeared. The colonies were then inoculated into Stainer-Scholte liquid medium for further culture. Prepare electroporation competent cells of the strain by washing repeatedly with 10% glycerol buffer at approximately 4°C, and perform electroporation on an electroporator (BTX, ECM630). Electroporation parameters are set as follows: 1.6-2.2 kV, 200 Ω, 25 µF, and electroporation time not exceeding 5 ms. Immediately after electroporation, add SS liquid medium for resuscitation. Transfer the system to sterile EP tubes and incubate on a shaker at 35-37°C and 220-240 rpm for resuscitation. After resuscitation, centrifuge and discard the supernatant, add SS liquid medium to resuspend the cells, and plate them on BG solid plates containing 10 μg / mL gentamicin resistance (BG-G). Incubate the plates at 35-37°C. The emergence of single colonies indicates a possible primary recombinant strain (1HR).

[0111] 1.3 Validation of a single recombinant strain Randomly pick several single clones that have grown on BG-G selection plates using a sterile pipette tip and streak them onto a new BG-G plate. Dip the remaining bacterial cells on the pipette tip into 50µL of sterile water as a PCR verification template. Verify the clones by using upstream homologous arm integration-specific verification primers and / or downstream integration-specific verification primers, and select the clones that have successfully recombined.

[0112] 1.4 Reverse screening culture and verification of secondary recombinant strains Select clones that have successfully undergone recombination and streak them onto BG plates containing 2% DOG (2-Deoxy-D-galactose). Incubate the plates at 37°C. Single clones that grow are potential secondary recombination strains (2HR). Randomly pick several single clones that have grown on the BG-2% DOG selection plates using a sterile pipette tip and streak them onto a new BG plate. Dip the remaining cells on the pipette tip into 50 µL of sterile water as a PCR verification template. Use external genomic primers for verification and select clones that have successfully undergone secondary recombination.

[0113] Genome external primers refer to primers that use the upstream sequence of the upstream homologous arm as the forward primer (excluding the upstream homologous arm itself) and the downstream sequence of the downstream homologous arm as the reverse primer (excluding the downstream homologous arm itself).

[0114] Example 2: Screening for genes that increase OMV production in Bordetella pertussis Commonly used genes for increasing OMV production, such as rmpM, tolR, pal, and ompA, are either not present in pertussis or are essential for pertussis survival and therefore cannot achieve the effect of increasing OMV production in Bordetella pertussis. Therefore, the inventors further explored other previously unreported genes, such as pagP, ompP, and BP1721.

[0115] 2.1 No increase in OMV production was observed in strains after knocking out the outer membrane protein pagP gene.

[0116] The pagP gene (sequence shown in SEQ ID NO:6) encodes an outer membrane protein of a pertussis strain that degrades phospholipids and transfers myristoyl groups from phospholipids to lipopolysaccharide (LPS), thereby modifying the structure of LPS to adjust its toxicity. Based on its function, it is speculated that knockout of the pagP gene prevents phospholipid degradation, potentially increasing the phospholipid content of the outer membrane and ultimately increasing membrane curvature, thus promoting OMV secretion. However, there are currently no reports on OMV production in pertussis strains with pagP gene knockout. Following the method in Example 1, the inventors successfully constructed a pagP gene knockout strain BAA-589 (BAA-589ΔpagP, sequencing alignment results shown in Example 1) through homologous recombination. Figure 2(As shown), but tests revealed that the concentration of OMV particles secreted by the BAA-589ΔpagP strain was lower than that of the wild-type strain, and it had no effect on increasing yield. Figure 3 As shown.

[0117] 2.2 Strains from which ompP gene inactivation could not be obtained The ompP gene (sequence shown in SEQ ID NO:7) encodes an outer membrane protein of a pertussis strain, and its abundance is extremely high. However, using the homologous recombination method in Example 1, it was impossible to obtain a strain with an inactivated ompP gene. The inventors speculate that the ompP gene is essential for the survival of the pertussis BAA-589 strain, and that the strain cannot survive after the gene is knocked out.

[0118] 2.3 Strains from which BP1721 gene inactivation could not be obtained The protein encoded by the BP1721 gene (sequence shown in SEQ ID NO:8) has been predicted to be an outer membrane protein of the *Bacillus pertussis* BAA-589 strain, but this gene has not been studied in *Bacillus pertussis* strains. Therefore, the inventors attempted to investigate the effect of the BP1721 gene on *Bordetella pertussis* OMV. Using the homologous recombination method described in Example 1, the inventors were unable to obtain a strain with inactivated BP1721 gene. The inventors hypothesized that the BP1721 gene is essential for the survival of the *Bacillus pertussis* BAA-589 strain, and that knocking out this gene would prevent the strain from surviving.

[0119] Example 3: Knockout of the BP2992 gene in Bordetella pertussis leads to increased OMV production. Commonly reported OMV-boosting genes such as rmpM, tolR, pal, and ompA in existing technologies have failed to increase OMV production in Bordetella pertussis. The inventors further explored potential OMV-boosting genes such as pagP, ompP, and BP1721, all of which also failed. Unexpectedly, the inventors discovered that the previously unexplored BP2992 gene has a significant effect on increasing OMV production.

[0120] 3.1 Construction of strain BAA-589ΔBP2992 The inventors used the homologous recombination method described in Example 1 to knock out the BP2992 gene in the pertussis BAA-589 strain. Specifically, a plasmid carrying the upstream and downstream homologous arms of the BP2992 gene was electroporated into the pertussis BAA-589 strain. Through one and two homologous recombinations, sequencing confirmed that BP2992 had been successfully knocked out (sequencing results are shown in Figure 1). Figure 4 As shown in the figure, strain BAA-589ΔBP2992 was obtained.

[0121] The nucleotide sequence of BP2992 is shown in SEQ ID NO:9. The upstream homologous arm sequence used in the gene editing process using homologous recombination is shown in SEQ ID NO:10, and the downstream homologous arm sequence is shown in SEQ ID NO:11.

[0122] 3.2 Determination of growth curve and particle size of strain BAA-589ΔBP2992 To investigate whether knocking out the BP2992 gene affects the growth of the strain, the inventors inoculated BAA-589 and BAA-589ΔBP2992 strains activated on BG plates into shake flasks containing 10 ml of SS medium and cultured them overnight at 35°C and 220 rpm. The OD600 of the seed culture was measured, and a suitable amount of the seed culture was re-inoculated into shake flasks containing 1 L of SS medium to achieve an initial OD600 of 0.02. The shake flask culture system was cultured at 35°C and 220 rpm for approximately 24 hours, and the samples were collected. The OD600 of the fermentation broth was measured. Growth curves were plotted based on the measurement results. Figure 5 As shown in the curve, the growth of strain BAA-589ΔBP2992 is very close to that of the wild type (BAA-589), indicating that knocking out the BP2992 gene has no significant effect on the growth of the strain.

[0123] The particle size of OMV was measured using a nanoparticle size potentiometer (Malvern, Zetasizer Pro), and the results are as follows: Figure 6 As shown in the figure. The measurement results show that there was no significant difference in the particle size of OMV before and after the modification.

[0124] 3.3 Determination of OMV secretion of strain BAA-589ΔBP2992 BAA-589ΔBP2992, activated on BG plates, was inoculated into a test tube containing 3 ml of SS medium and cultured overnight at 35°C and 220 rpm on a shaker. The OD600 of the seed culture was measured, and an appropriate amount of the seed culture was re-inoculated into a test tube containing 3 ml of SS medium, maintaining an initial OD600 of 0.2. The test tube was then cultured at 35°C and 220 rpm for approximately 24 hours. The sample was then collected. 1 ml of the fermentation sample was centrifuged at 10,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter. The filtrate was collected. The OMV concentration was measured using a nanoparticle tracking analyzer (Malvern, NanoSight Pro). The results are as follows: Figure 9 As shown in the figure. The characterization results show that, under the above-mentioned in vitro culture conditions, BAA-589ΔBP2992 produced 2.8 times more than wild-type BAA-589.

[0125] Example 4: Construction of strains BAA-589ΔBP2992ΔpldA and BAA-589ΔBP2992ΔpldAΔmltA The inventors aimed to improve upon BAA-589ΔBP2992 to further increase OMV yield. They selected the pldA and mltA genes for combined knockout with the BP2992 gene.

[0126] The inventors used the homologous recombination method described in Example 1 to knock out the plcA gene in strain BAA-589ΔBP2992. Specifically, a plasmid carrying the upstream and downstream homologous arms of the plcA gene was electroporated into the pertussis strain BAA-589ΔBP2992. Through one and two homologous recombinations, sequencing confirmed that plcA had been successfully knocked out (sequencing results are shown below). Figure 7 As shown in the figure, strain BAA-589ΔBP2992ΔpldA was obtained.

[0127] The nucleotide sequence of the pldA gene is shown in SEQ ID NO:12. The upstream homologous arm sequence used in the gene editing process using homologous recombination is shown in SEQ ID NO:13, and the downstream homologous arm sequence is shown in SEQ ID NO:14.

[0128] The inventors used the homologous recombination method described in Example 1 to knock out the mltA gene in the pertussis strain BAA-589ΔBP2992ΔpldA. Specifically, a plasmid carrying the upstream and downstream homologous arms of the mltA gene was electroporated into the pertussis strain BAA-589ΔBP2992ΔpldA. Through one and two homologous recombinations, sequencing confirmed that mltA had been successfully knocked out (sequencing results are shown below). Figure 8 As shown in the figure, strain BAA-589ΔBP2992ΔpldAΔmltA was obtained.

[0129] The nucleotide sequence of the mltA gene is shown in SEQ ID NO:15. The upstream homologous arm sequence used in the gene editing process using homologous recombination is shown in SEQ ID NO:16, and the downstream homologous arm sequence is shown in SEQ ID NO:17.

[0130] The inventors also discovered that if the mltA gene is knocked out first, the success rate is very low, only 4.2%; while knocking out the pldA gene first can significantly improve the success rate of knocking out the mltA gene in the BAA-589 strain (up to 90%).

[0131] Example 6: Determination of OMV secretion by each strain BAA-589ΔBP2992, BAA-589ΔBP2992ΔpldA, and BAA-589ΔBP2992ΔpldAΔmltA strains activated on BG plates were inoculated into test tubes containing 3 ml of SS medium and cultured overnight at 35°C and 220 rpm on a shaker. The OD600 of the seed culture was measured, and an appropriate amount of seed culture was re-inoculated into test tubes containing 3 ml of SS medium, controlling the initial OD600 to 0.2. The test tubes were then cultured at 35°C and 220 rpm on a shaker for approximately 24 hours. The samples were then collected. 1 ml of the fermentation sample was centrifuged at 10000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was collected. The OMV concentration was measured using a nanoparticle tracking analyzer (Malvern, NanoSight Pro). The results are as follows: Figure 9 As shown in the figure. The characterization results show that, under the above-mentioned in vitro culture conditions, compared with the wild type BAA-589, BAA-589ΔBP2992 yielded 2.8 times more, BAA-589ΔBP2992ΔpldA yielded 3.4 times more, and BAA-589ΔBP2992ΔpldAΔmltA yielded 7.3 times more.

[0132] The sequence information involved in this application SEQ ID NO:1 Exogenous backbone plasmid for homologous recombination SEQ ID NO: 2 Nucleotide sequence of gentamicin resistance gene: ATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAA SEQ ID NO: 3 Nucleotide sequence of galk counter-selection gene: SEQ ID NO:4 Nucleotide sequence of the site-up upstream of the backbone plasmid insertion site: ACAACTGGCGGTATGGATGC SEQ ID NO:5 Nucleotide sequence of the site-down region downstream of the backbone plasmid insertion site: GGCGGGACCAGAGAAAAATC SEQ ID NO:6 pagP gene ATGACCCAGTATTTCCGGTCCCTGGCTTTCTTTCTTTTGCCGGTGCCGGCAACGGCCATGGCCTGTGACGGCTGGCCTTCCTGGGCGCGTGGCGCCTGCCAGCGCGTGGACCAGATATGGAATGAGGGCGGCAACGA CCTCTACCTGACCGGCTACTCGTGGCACAACCGGGCCATGTACAGCAGCGACAAGATCCGCAGCTTCAACGAGCTGGCCTGGGGCGGCGGACTGGGCAAGAGCATCTACGACGAGGACGGCGACTGGCAAGGGCTGT ACGCCATGGCGTTTCTCGATTCGCACAGCGACATCGAACCGATAGCCGGCTATGGTTTCCAGAAAATCGGCCGCATCGGCGCCGATACCCGCCTGGGCATCGGCTACACCGTCTTCCTGACTTCGCGCTCCGACATC ATGAGCCGGGTGCCGTTTCCCGGCATCCTGCCGCTGGTCTCTGCCGGCTACAGGGATGCCACCCTTCTACGCCACGTATATCCCGGGCGGCAAGGGCAACGGCAACGTGCTGTTCATGTTTGGCCGCTGGGAGTTCTAA SEQ ID NO:7 ompP gene SEQ ID NO:8 BP1721 gene ATGCTCAACGGGCAGTTGCAACTGACCGAATCCCAATCCGTGGCTGGCGCATCCGCCGCCGCCGCTTCCCGCCCCGTTCTCTGGGCGGGCGTGCTGGCCATCGCCTTGCTGGCCGGCTGCGCCTCGAAGGGCCCGCGCGCGCCTGTGGTCGACCTCACCGGACAGCCCGGCGCCTCCGGCCCGACCGACGGCAGCTATGTCGTCAAGCCTGGCGATACGCTCTACAAGATCGCGCGCGCCAACAATGTCGATATCGAAAACCTCAAGCGCTGGAACAACCTCACCGATCCCAACCAGATCAGTGTCGGCCAGGTGCTGCGCCTGTCGAGCTCGGGCGCCGGCGGCGCGCAGACCACGCCGGTGACCTCCTCCAAGCCGCAACCCAAGCCGCTGGACCAGGGAAGCGCCGAAACGCCGGCCGGCGGCATGGAAGCCGGCGCCGGCGGCGAAACCGGCGGGGCCACCACCCCGCCCGCGGCAACGGTGCCCGATCCCAAGCCCGCGCGCGCGGCCGATGCCGCCGTCATCAACTGGGGCTGGCCGGCCAATGGCGCCATCCTGCAGACCTTCAACAGCAACACCAAGGGCATCGACCTGGCCGGCTCGCTGGGCGATCCGGTGATCGCGGCGGCCGATGGCAAGGTGATGTACAGCGGCAATGGCGTGCGCGGCCTGGGCAACCTGATCATCATCAATCACCAGAACGGCTTCATCACGGCCTACGCGCACAACCGCGCGCTGCTGGTCAAGACCGGCCAGAACGTCAAGCGCGGGGCCAAGATCGCCGAGATCGGCGAGACCGACACCACGTCGCCGCGGCTGCATTTCGAGATCCGCCGCCAGGGCACGCCGGTCGACCCGATGCAGTATCTGCCGCCGCGATGA SEQ ID NO:9 BP2992 gene ATGAACTATATGCATTCCCCCTCTGTAGTTGCCGGGCGCGCCCGCCGCCTGCTGGCGGTAGCGGCGGTTGCCGGCTCGGTGGCCGTTCTGGCCGGCTGCGCCAATCCCAGCGCATCGAGTGGGGTGTACACGTACGGCCAGGCGCAGCGCGAGCAGATCGTGCGCACCGGCACGGTCACCGGCGTGCGTCCGATTACCATCCAGAACGACAAGTCCAGCGGCGTCGGCTTGGTGGCCGGTGGCGCGCTGGGCGGGGTAGCGGGCAATGCCGTCGGCGGCGGCACCGGCCGCACCATCGCCACGGTGGGCGGCGTCATCCTCGGCGCGCTGGCGGGCAACGCCATCGAGAACCGCGCGGGCAAGTCCTCCGGCTACGAAATCACGGTGCGCCTGGACAACGGCGAAACCCGGGTCGTGGCGCAGGAAGCCGACGTGCCCATCAGCGTGGGCCAGCGCGTGCAGGTCATCAGCGGCGCGGGCCCGACCCGCGTGACACCGTATTGA The upstream homologous arm of the BP2992 gene used in the homologous recombination method, SEQ ID NO:10 SEQ ID NO:11 Downstream homologous arm of the BP2992 gene used in the homologous recombination method SEQ ID NO:12 pldA gene SEQ ID NO:13 Upstream homologous arm of the plcA gene used in the homologous recombination method SEQ ID NO:14 Downstream homologous arm of the plcA gene used in the homologous recombination method SEQ ID NO:15 mltA gene SEQ ID NO:16 Upstream homologous arm of the mltA gene used in the homologous recombination method Downstream homologous arm of the mltA gene used in the homologous recombination method (SEQ ID NO:17) Amino acid sequence of SEQ ID NO:18 BP2992 protein MNYMHSPSVVAGRARRLLAVAAVAGSVAVLAGCANPSASSGVYTYGQAQREQIVRTGTVTGVRPITIQNDKSSGVGLVAGGALGGVAGNAVGGGTGRTIATVGGVILGALAGNAIENRAGKSSGYEITVRLDNGETRVVAQEADVPISVGQRVQVISGAGPTRVTPY* SEQ ID NO:19 Amino acid sequence of PldA protein MQVPDPRSPFIRPALAGALALALAGMSLPALAGISYRLDRPAAAPGETVRLEAVFFNDGSASAAWNAPQQLVLQWRGADDQTIRSLAQRPAGEAALNIPVNNFARMSWDAVVPVHARGLQAVSIEGESTMLALDATGREQDTLASTPADVPVTDARTGQPLPPAAVTAAGVSPDSGPAPAQVAVSQTFQPASAFDVFRSAISEYQPMYFDIGTREQT TARFQISAKYRLFSPKGDRPATFGENFYLGYTQTSLWDLEGDSKPFIDTTFNPSAFWLSDNIWSSASQNWRVGLNTGVEHQSNGKSGNDSRSLNDAYVQPALNYRFDS GSTLTFAPKVKTYFGVARENLDYADYAGYVDWNLRWAQDGGAVVSAMYRQGASSRRTTQLDFAWPLKRTWLDMNGYLHLQYFNGYGETLLGYNQRHDSQFRIGSLVP* SEQ ID NO:20 Amino acid sequence of MltA protein MKRLLCLSLLSVLLAACTTPSQIPPETAPGGVPPAAEGPLVVPPLSALSDTPPRALAGRYQRVAWTELPNWESDDLSRWWPLFLRNCKGLMRPTSGNLAAPARATPRAWQPVCAAAVDPSKAPAAGDSAAVRRFLQTWLQPWRIAGADGRPATNTVTGYYEPLVRGSRRQGGRYQWPLYAVPADLLVVDLGSVYPDLTGKRVRGRLDGRRVVPYDTRAAIEAGDRKPPAIVWVDDPVDNFFLQVQGSGRVQLTDGPDRGTTIRVAYADHNGQPYASIGRWLIDKGELRADQASMQNIRAWAQRNPSRVQEMLNANPAVVFFREEAVVDPEQGPKGAYGIPLAPQRSIAVDAGFVPLGTPVYLSTTLPASDRPLQRTVFAQDTGTAIRGAARADFYWGYGEEAGQQAGRMKQRGQMWLLWPKQAGEPSAR*。

Claims

1. A Bordetella pertussis strain, wherein the BP2992 gene of the strain is partially or completely inactivated, thereby the strain having an increased production of bacterial outer membrane vesicles (OMV) compared with a control Bordetella pertussis strain.

2. The Bordetella pertussis strain of claim 1, wherein the BP2992 gene... i) Encoding an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO: 18, or a protein containing the amino acid sequence shown in SEQ ID NO: 18; or ii) Contains a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:9, or contains the nucleotide sequence shown in SEQ ID NO:

9.

3. The Bordetella pertussis strain according to claim 1 or 2, wherein the pldA gene and / or mltA gene of the strain are partially or completely inactivated.

4. The Bordetella pertussis strain according to claim 3, wherein... The pldA gene i) Encoding an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO: 19, or a protein containing the amino acid sequence shown in SEQ ID NO: 19; or ii) Containing a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:12, or containing the nucleotide sequence shown in SEQ ID NO:

12. The mltA gene mentioned above i) Encoding an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:20, or a protein containing the amino acid sequence shown in SEQ ID NO:20; or ii) A nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with SEQ ID NO:15, or a nucleotide sequence containing the nucleotide sequence shown in SEQ ID NO:

15.

5. The Bordetella pertussis strain according to claim 3 or 4, wherein the pldA and mltA genes of the strain are partially or completely inactivated.

6. The Bordetella pertussis strain according to any one of claims 1-5, wherein the partially or completely inactivated gene in the strain contains an introduced mutation that results in the partial or complete inactivation of the gene.

7. The Bordetella pertussis strain of claim 6, wherein the mutation is the addition, substitution, or deletion of one or more nucleotides.

8. The Bordetella pertussis strain of claim 7, wherein the mutation is introduced into the expression regulatory sequence of the gene, thereby causing a reduction or absence of expression of the protein encoded therein; or, the mutation is introduced into the coding sequence of the gene, causing the addition, substitution or deletion of one or more amino acids in the protein encoded therein, the addition, substitution or deletion of one or more amino acids causing a reduction or absence of activity of the encoded protein.

9. The Bordetella pertussis strain of claim 6, wherein the mutation is a partial or complete deletion of a gene, preferably a complete deletion.

10. The Bordetella pertussis strain according to any one of claims 6-9, wherein the mutation is introduced by homologous recombination or by targeted mutagenesis mediated by CRISPR, TALEN or ZFN technology.

11. The Bordetella pertussis strain according to any one of claims 1-10, wherein the bacterial outer membrane vesicle (OMV) yield of the Bordetella pertussis strain is about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times or more of the OMV yield of the control Bordetella pertussis strain.

12. The Bordetella pertussis strain according to any one of claims 1-11, which is derived from strain BAA-589.

13. A method for producing a Bordetella pertussis strain with increased production of bacterial outer membrane vesicles (OMV), the method comprising the steps of: a) To partially or completely inactivate the BP2992 gene in Bordetella pertussis strains; and b) Optionally, the pldA and / or mltA genes in the Bordetella pertussis strain are partially or completely inactivated.

14. The method of claim 13, wherein, in optional step b), the pldA and mltA genes in the Bordetella pertussis strain are partially or completely inactivated.

15. The method of claim 14, wherein in optional step b), the pldA gene in the Bordetella pertussis strain is first partially or completely inactivated, and then the mltA gene in the Bordetella pertussis strain is partially or completely inactivated.

16. The method of any one of claims 13-15, wherein optional step b) is performed before step a), or wherein optional step b) is performed after step a).

17. The method of any one of claims 13-16, wherein the introduction of a mutation results in partial or complete inactivation of the gene.

18. The method of claim 17, wherein the mutation is the addition, substitution, or deletion of one or more nucleotides.

19. The method of claim 18, wherein the mutation is introduced into the expression regulatory sequence of the gene, thereby causing reduced or absent expression of the protein encoded therein; or, the mutation is introduced into the coding sequence of the gene, causing the addition, substitution, or deletion of one or more amino acids in the protein encoded therein, the addition, substitution, or deletion of the one or more amino acids causing reduced or absent activity of the encoded protein.

20. The method of claim 17, wherein the mutation is a partial or complete deletion of a gene, preferably a complete deletion.

21. The method of any one of claims 17-20, wherein the mutation is introduced by homologous recombination or by targeted mutagenesis mediated by CRISPR, TALEN, or ZFN technology.

22. The method of claim 21, wherein the method comprises the following steps: a) By homologous recombination to delete the BP2992 gene in the Bordetella pertussis strain, a modified Bordetella pertussis strain with the BP2992 gene deleted was obtained. b) By deleting the pldA gene from the Bordetella pertussis strain obtained in step a), a Bordetella pertussis strain with both the pldA gene and the BP2992 gene deleted is obtained. and c) Obtain the mltA gene from the Bordetella pertussis strain obtained by homologous recombination deletion step b), thereby obtaining a Bordetella pertussis strain with deletions of the BP2992 gene, pldA gene, and mltA gene.

23. The method of claim 21 or 22, wherein the BP2992 gene is deleted by homologous recombination using the homologous arm sequences shown in SEQ ID NO: 10 and 11; the pldA gene is deleted by homologous recombination using the homologous arm sequences shown in SEQ ID NO: 13 and 14; and / or, the mltA gene is deleted by homologous recombination using the homologous arm sequences shown in SEQ ID NO: 16 and 17.

24. A strain of Bordetella pertussis produced by the method of any one of claims 13-23.

25. A method for generating Bordetella pertussis outer membrane vesicles (OMV), the method comprising the steps of: a) Culture the Bordetella pertussis strain according to any one of claims 1-12 and 24 under conditions suitable for the production of outer membrane vesicles (OMV); and b) Recover and optionally purify the OMV generated in step a).

26. Bordetella pertussis outer membrane vesicles (OMV) produced by the method of claim 25.

27. A composition comprising an effective amount of 1) The Bordetella pertussis strain according to any one of claims 1-12 and 24; and / or 2) The Bordetella pertussis outer membrane vesicle (OMV) according to claim 26.

28. The composition of claim 27, further comprising an adjuvant and / or a pharmaceutically acceptable carrier.

29. The use of the Bordetella pertussis strain according to any one of claims 1-12 and 24, and / or the Bordetella pertussis outer membrane vesicle according to claim 26, and / or the composition according to claim 27 or 28 in the preparation of a medicament or vaccine for the prevention and / or treatment of Bordetella pertussis infection in a subject, preferably a human.

30. Use of the Bordetella pertussis strain according to any one of claims 1-12 in the production of Bordetella pertussis outer membrane vesicles (OMV).

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