Bordetella pertussis strain with increased outer membrane vesicle yield

Abstract

The present invention relates to the field of bioengineering, and specifically relates to a Bordetella pertussis strain with an increased outer membrane vesicle (OMV) yield, wherein a BP2992 gene in the strain is partially or completely inactivated. The present invention further relates to a method for producing the Bordetella pertussis strain and a method for producing OMVs by means of the Bordetella pertussis strain.

Description

A bordetella pertussis strain with increased outer membrane vesicle production TECHNICAL FIELD

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

[0002] BACKGROUND

[0003] 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 whooping cough cases, 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, whooping cough cases are re-emerging 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.

[0004] 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 weakening of the contact 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 natural adjuvants. Therefore, Bordetella pertussis OMVs have enhanced immunogenicity compared to existing whole-cell pertussis vaccines and acellular pertussis vaccines.

[0005] The bottleneck of Bordetella pertussis OMVs secreted as vaccines is that the production of Bordetella pertussis OMVs is extremely low, which makes the research and production of Bordetella pertussis OMV vaccines face great challenges. Increasing the production of OMVs has become a problem to be solved for the development of Bordetella OMV vaccines.

[0006] 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.

[0007] From the aspect of fermentation process, the secretion amount of OMV can be improved 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.

[0008] From the aspect of strains, the OMV yield can be improved by editing the genes related to OMV secretion process. 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, destroying the mla system which is the transport system of outer membrane phospholipid to inner membrane, can all achieve over-secretion of OMV. The third strategy is to increase the internal pressure of periplasmic space to promote OMV secretion. For example, the molecular chaperone protein gene degP in periplasmic space can be knocked out to increase the accumulation of misfolded proteins in periplasmic space, destroy the recycling of peptidoglycan, etc.

[0009] 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 mentions 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, methods that can increase the production of OMV on other bacteria may not be suitable for B. pertussis.

[0010] 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.

[0011] SUMMARY

[0012] The present application provides at least the following embodiments, but is not limited thereto:

[0013] 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.

[0014] Embodiment 2. The B. pertussis strain of embodiment 1, wherein the BP2992 gene

[0015] 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

[0016] 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.

[0017] 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.

[0018] Embodiment 4. The B. pertussis strain of embodiment 3, wherein

[0019] the pldA gene

[0020] 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

[0021] 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,

[0022] wherein the mltA gene

[0023] 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

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Embodiment 7. The B. pertussis strain of embodiment 6, wherein the mutation is an addition, substitution or deletion of one or more nucleotides.

[0028] Embodiment 8. The B. pertussis strain of embodiment 7, wherein the mutation is introduced into an expression regulatory sequence of the gene, whereby it results in reduced or no expression of the protein encoded by the gene; 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 protein encoded by the gene, which addition, substitution or deletion of one or more amino acids results in reduced or no activity of the encoded protein.

[0029] Embodiment 9. The B. pertussis strain of embodiment 6, wherein the mutation is a partial deletion or a complete deletion of the gene, preferably a complete deletion.

[0030] 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.

[0031] 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.

[0032] Embodiment 12. The B. pertussis strain of any one of embodiments 1-11, which is derived from the BAA-589 strain.

[0033] Embodiment 13. A method of producing a B. pertussis strain having increased production of bacterial outer membrane vesicles (OMVs), the method comprising the steps of:

[0034] a) partially or completely inactivating the BP2992 gene in the B. pertussis strain; and

[0035] b) optionally, partially or completely inactivating the pldA gene and / or the mltA gene in the B. pertussis strain.

[0036] Embodiment 14. The method of embodiment 13, wherein the pldA gene and the mltA gene in the B. pertussis strain are partially or completely inactivated in the optional step b).

[0037] Embodiment 15. The method of embodiment 14, wherein in the optional step b), the pldA gene in the B. pertussis strain is partially or completely inactivated first, followed by the mltA gene in the B. pertussis strain being partially or completely inactivated.

[0038] 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).

[0039] Embodiment 17. The method of any one of embodiments 13-16, wherein the partial or complete inactivation of the gene is caused by introducing a mutation.

[0040] Embodiment 18. The method of embodiment 17, wherein the mutation is an addition, substitution or deletion of one or more nucleotides.

[0041] Embodiment 19. The method of embodiment 18, wherein the mutation is introduced into the 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.

[0042] Embodiment 20. The method of embodiment 17, wherein the mutation is a partial or complete deletion of the gene, preferably a complete deletion.

[0043] 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.

[0044] Embodiment 22. The method of embodiment 21, wherein the method comprises the following steps:

[0045] a) deleting the BP2992 gene in a B. pertussis strain by homologous recombination, thereby obtaining a modified B. pertussis strain with a BP2992 gene deletion;

[0046] b) deleting the pldA gene in the B. pertussis strain obtained in step a) by homologous recombination, thereby obtaining a B. pertussis strain with both a BP2992 gene and a pldA gene deletion; and

[0047] c) deleting the mltA gene in the B. pertussis strain obtained in step b) by homologous recombination, thereby obtaining a B. pertussis strain with deletions of the BP2992 gene, the pldA gene and the mltA gene.

[0048] 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.

[0049] Embodiment 24. A B. pertussis strain produced by the method of any one of embodiments 13-23.

[0050] Embodiment 25. A method of producing B. pertussis outer membrane vesicles (OMVs), the method comprising the steps of:

[0051] a) culturing a 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

[0052] b) recovering and optionally purifying the OMVs produced in step a).

[0053] Embodiment 26. B. pertussis outer membrane vesicles (OMVs) produced by the method of embodiment 25.

[0054] Embodiment 27. A composition comprising an effective amount of

[0055] 1) a Bordetella pertussis strain according to any one of embodiments 1-12 and 24; and / or

[0056] 2) a Bordetella pertussis outer membrane vesicle (OMV) according to embodiment 26.

[0057] Embodiment 28. The composition of embodiment 27, further comprising an adjuvant and / or a pharmaceutically acceptable carrier.

[0058] Embodiment 29. Use of a Bordetella pertussis strain according to any one of embodiments 1-12 and 24, and / or a Bordetella 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 Bordetella pertussis infection in a subject.

[0059] Embodiment 30. Use of a Bordetella pertussis strain according to any one of embodiments 1-12, for the production of a Bordetella pertussis outer membrane vesicle (OMV).

[0060] BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1. Structure of the exogenous backbone plasmid used in the homologous recombination method.

[0062] Figure 2. Sequencing alignment of the BAA-589 ApagP strain.

[0063] Figure 3. Comparison of the concentration of OMV particles secreted by the BAA-589 ApagP strain and the wild-type strain BAA-589.

[0064] Figure 4. Sequencing alignment of the BAA-589 ABP2992 strain.

[0065] Figure 5. Comparison of the growth curves of the BAA-589 ABP2992 strain and the wild-type strain BAA-589.

[0066] Figure 6. Comparison of the OMV particle size of the BAA-589 ABP2992 strain and the wild-type strain BAA-589.

[0067] Figure 7. Sequencing alignment of the BAA-589 ABP2992 ApldA strain.

[0068] Figure 8. Sequencing alignment of the BAA-589 ABP2992 ApldA AmItA strain.

[0069] Figure 9. Comparison of the amount of OMV secreted by the BAA-589 ABP2992 strain, the BAA-589 ABP2992 ApldA strain and the BAA-589 ABP2992 ApldA AmItA strain and the wild-type strain BAA-589.

[0070] DETAILED DESCRIPTION

[0071] I. DEFINITIONS

[0072] 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 phrases used in this document, and variations thereof, unless otherwise indicated, are to be construed to have the meaning most often used in the corresponding field of art. The terms "protein" and "nucleic acid chemistry," "molecular biology," "cell and tissue culture," "microbiology," "immunology," and related terms and laboratory procedures steps used herein are terms and procedures that are well known in the corresponding fields.

[0073] As used herein, the term "and / or" encompasses all combinations of the items connected by the term. For example, "A and / or B" covers A, B, and "A 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."

[0074] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single letter designation: "A" for adenosine or deoxyadenosine (RNA or DNA, respectively), "C" for cytosine or deoxycytosine, "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 or C or T, "I" for inosine, and "N" for any nucleotide. While nucleotide sequences herein can be represented in DNA sequence format (including T), the corresponding RNA sequences are readily determined by one of skill in the art (i.e., substituting U for T).

[0075] "Polypeptide," "peptide," and "protein" are used interchangeably herein in reference to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" can also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0076] The term "gene" as used herein can refer to a coding sequence for a protein, but can also encompass expression regulatory elements / sequences, such as promoters, enhancers, etc. A "gene" generally refers to an endogenous sequence of an organism, such as B. pertussis.

[0077] The word "comprising" is used herein to describe a protein or nucleic acid sequence which can consist of the sequence, or which can have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid sequence, but still have the activity described in the present application.

[0078] "Sequence identity" between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods to assess the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various sequence analysis software known. For example, sequence identity can be assessed by the online alignment tool of EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm, using default parameters. Sequence identity can be sequence identity over the full length of a given sequence.

[0079] Endogenous genes in bacteria such as Bordetella pertussis or the proteins they encode, homologues of which in different strains can exhibit some natural variation in sequence, will nevertheless have a sufficiently high sequence identity / 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%, even at least 99.5% sequence identity) to enable the skilled person to expect that they have similar or identical function.

[0080] A "BP2992 gene" as described herein is an unknown function gene in Bordetella pertussis, encoding an unknown function BP2992 protein.

[0081] A BP2992 protein in Bordetella pertussis can comprise 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. In some embodiments, a BP2992 protein in Bordetella pertussis can comprise the amino acid sequence set forth in SEQ ID NO: 18.

[0082] The BP2992 gene in B. pertussis can comprise 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. In some embodiments, the BP2992 gene in B. pertussis can comprise the nucleotide sequence set forth in SEQ ID NO: 9.

[0083] The "pldA gene" described herein encodes a PldA protein, which is a phospholipase that degrades phospholipids on the outer membrane and releases fatty acids by its phospholipase activity.

[0084] The PldA protein in B. pertussis can comprise 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. In some embodiments, the PldA protein in B. pertussis can comprise the amino acid sequence set forth in SEQ ID NO: 19.

[0085] The pldA gene in B. pertussis can comprise 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. In some embodiments, the pldA gene in B. pertussis can comprise the nucleotide sequence set forth in SEQ ID NO: 12.

[0086] The "mltA gene" described herein encodes a MltA protein, which is a lipoprotein located on the outer membrane, the protein has a peptidoglycan cleaving exo-transglycosylase activity, is involved in peptidoglycan metabolism, and plays a function in cell separation and membrane construction.

[0087] The MltA protein in B. pertussis can comprise 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. In some embodiments, the MltA protein in B. pertussis can comprise the amino acid sequence set forth in SEQ ID NO: 20.

[0088] The mltA gene in the B. pertussis can comprise 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. In some embodiments, the MltA gene in the B. pertussis can comprise the nucleotide sequence set forth in SEQ ID NO: 15.

[0089] As used herein, "effective amount" means an amount of a substance, compound, material, or composition containing a compound, such as an OMV of the application or a composition of the application, which following administration to a subject, is at least sufficient to produce a prophylactic or therapeutic effect. Thus, an effective amount is that amount necessary to prevent, cure, ameliorate, delay or partially delay the symptoms of a disease or disorder, such as a bacterial infection.

[0090] "Subject" as described herein preferably means a human.

[0091] II. B. pertussis strains with increased OMV production

[0092] In one aspect, the present application provides a B. pertussis strain, wherein the BP2992 gene is partially or completely inactivated, whereby said strain has increased production of bacterial outer membrane vesicles (OMVs) compared to a control B. pertussis strain.

[0093] In some preferred embodiments, the pldA gene of said B. pertussis strain is also partially or completely inactivated.

[0094] In other preferred embodiments, the mltA gene of said B. pertussis strain is also partially or completely inactivated.

[0095] In some preferred embodiments, the pldA gene and the mltA gene of said B. pertussis strain are also partially or completely inactivated.

[0096] In some embodiments, the gene of said B. pertussis strain that is partially or completely inactivated is an endogenous gene thereof.

[0097] As used herein, partial inactivation of a gene refers to reduced expression of its encoded product (e.g., protein) and / or a product (e.g., protein) with reduced activity. For example, the encoded product (e.g., protein) of a gene that is partially inactivated is expressed 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 less; or a gene that is partially inactivated expresses a product (e.g., protein) with 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 reduced activity.

[0098] As used herein, complete inactivation of a gene refers to no expression of its encoded product (e.g., protein) and / or expression of a product (e.g., protein) with no activity. For example, a gene that is completely inactivated does not express its encoded product (e.g., protein), or a gene that is completely inactivated expresses a product (e.g., protein) with no activity.

[0099] Partial or complete inactivation of a gene can be achieved by a variety of methods known in the art. For example, in some embodiments, a mutation can be introduced into a gene, thereby resulting in partial or complete inactivation of the gene. The mutation can be an addition, substitution, or deletion of one or more nucleotides.

[0100] The mutation can be introduced into an expression regulatory sequence of the gene, thereby resulting in reduced expression or no expression of its encoded product (e.g., protein).

[0101] Alternatively, the mutation can be 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 that results in reduced activity or no activity of the encoded protein.

[0102] In some embodiments, the mutation is a partial deletion or complete deletion of the gene. The gene can be completely deleted from the strain, such that no expression of the product (e.g., protein) encoded by the gene occurs in the strain of the application. The gene can also be partially deleted, such that only a truncated product (e.g., truncated protein) with reduced activity or no activity is expressed in the strain of the application. 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.

[0103] Introducing a mutation into a gene can be achieved by various means known in the art. In some embodiments, the mutation is introduced into the gene of the B. pertussis strain by genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, the mutation in the gene, such as a deletion of the gene, can be achieved by homologous recombination. Methods of homologous recombination can be found in patent document CN 117947054 B. In some embodiments, the mutation in the gene is made by targeted mutagenesis, such as targeted mutagenesis mediated via CRISPR, TALEN or ZFN technology.

[0104] In some embodiments, the BP2992 gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 10 and 11. In some embodiments, the pldA gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 13 and 14. In some embodiments, the mltA gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 16 and 17.

[0105] In some embodiments, the yield of bacterial outer membrane vesicles (OMVs) of the B. pertussis strain of the application is increased 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%, 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, compared to the yield of OMVs of a corresponding control B. pertussis strain (under similar or identical conditions). In some embodiments, the yield of bacterial outer membrane vesicles (OMVs) of the B. pertussis strain of the application 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, compared to the yield of OMVs of a corresponding control B. pertussis strain (under similar or identical conditions).

[0106] A “control B. pertussis strain” as described herein can be a parental strain from which the B. pertussis strain of the application is derived. For example, the BP2992 gene, and optionally the mltA gene and / or the pldA gene, in the control B. pertussis strain is not (partially or completely) inactivated.

[0107] The B. pertussis strain described herein can be derived from any known B. pertussis strain. Exemplary starting B. pertussis strains can be, for example, the BAA-589 strain, the ATCC 9797 strain, the CHLA-26 strain. These starting B. pertussis strains have a pldA, mltA or BP2992 gene. The B. pertussis strain can also express one or more antigens, such as a foreign antigen, preferably the antigen is an antigen that can be displayed on the cell membrane and / or an antigen that can be displayed on OMVs.

[0108] III. Methods of producing a B. pertussis strain having increased bacterial outer membrane vesicle (OMV) production

[0109] In another aspect, the present application provides a method of producing a B. pertussis strain having increased bacterial outer membrane vesicle (OMV) production, the method comprising the steps of:

[0110] a) partially or completely inactivating the BP2992 gene in a B. pertussis strain; and

[0111] b) optionally, partially or completely inactivating the pldA gene and / or the mltA gene in the B. pertussis strain.

[0112] In some embodiments, the pldA gene and the mltA gene in the B. pertussis strain are partially or completely inactivated in the optional step b). In some preferred embodiments, in the optional step b), the pldA gene in the B. pertussis strain is first partially or completely inactivated, followed by the mltA gene in the B. pertussis strain.

[0113] In some embodiments, the optional step b) is performed before step a). In some embodiments, the optional step b) is performed after step a).

[0114] In some embodiments, the gene in the B. pertussis strain that is partially or completely inactivated is an endogenous gene thereof.

[0115] The partial or complete inactivation of a gene can be achieved by various methods known in the art. For example, in some embodiments, the partial or complete inactivation of a gene can be achieved by introducing a mutation into the gene. The mutation can be an addition, a substitution or a deletion of one or more nucleotides.

[0116] The mutation can be introduced into the expression regulatory sequence of the gene, thereby resulting in reduced or no expression of the product (such as a protein) encoded thereby.

[0117] Alternatively, the mutation can be introduced into the coding sequence of the gene, resulting in the addition, substitution, or deletion of one or more amino acids in the encoded protein, which results in reduced activity or no activity of the encoded protein.

[0118] In some embodiments, the mutation is a partial or complete deletion of the gene. The gene can be completely deleted from the strain, such that the product (e.g., protein) encoded by the gene is not expressed in the strain of the application. The gene can also be partially deleted, such that only a truncated product (e.g., truncated protein) with reduced activity or no activity is expressed in the strain of the application. 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.

[0119] Introducing a mutation into a gene can be achieved by various means known in the art. In some embodiments, the mutation is introduced into the gene of the B. pertussis strain by genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, the mutation in the gene, e.g., deletion of the gene, can be achieved by homologous recombination. Methods of homologous recombination can be found in patent document CN 117947054 B. In some embodiments, the mutation in the gene is performed by targeted mutagenesis, such as targeted mutagenesis mediated via CRISPR, TALEN, or ZFN technology.

[0120] In some specific embodiments, the method comprises the following steps:

[0121] a) deleting the BP2992 gene in a B. pertussis strain by homologous recombination, thereby obtaining a modified B. pertussis strain with a deletion of the BP2992 gene;

[0122] b) deleting the pldA gene in the B. pertussis strain obtained in step a) by homologous recombination, thereby obtaining a B. pertussis strain with a deletion of both the BP2992 gene and the pldA gene; and

[0123] c) deleting the mltA gene in the B. pertussis strain obtained in step b) by homologous recombination, thereby obtaining a B. pertussis strain with a deletion of the BP2992 gene, the pldA gene, and the mltA gene.

[0124] In some embodiments, the BP2992 gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 10 and 11. In some embodiments, the pldA gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 13 and 14. In some embodiments, the mltA gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 16 and 17.

[0125] In another aspect, the present application also provides a Bordetella pertussis strain produced by the method of the present application.

[0126] In some embodiments, the bacterial outer membrane vesicle (OMV) yield of a Bordetella pertussis strain produced by the method of the present application 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, compared to a corresponding control Bordetella pertussis strain (under similar or identical conditions). In some embodiments, the bacterial outer membrane vesicle (OMV) yield of a Bordetella pertussis strain of the present application 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, compared to the OMV yield of a corresponding control Bordetella pertussis strain (under similar or identical conditions).

[0127] IV. Production and use of bacterial outer membrane vesicles (OMVs)

[0128] In another aspect, the present application provides the use of a Bordetella pertussis strain of the present application for the production of Bordetella pertussis outer membrane vesicles (OMVs).

[0129] In another aspect, the present application provides a method for producing Bordetella pertussis outer membrane vesicles (OMVs), the method comprising the steps of:

[0130] a) culturing a Bordetella pertussis strain of the present application under conditions suitable for the production of outer membrane vesicles (OMVs); and

[0131] b) recovering and optionally purifying the OMVs produced in step a).

[0132] Process conditions for producing OMVs by culturing / fermenting B. pertussis strains are known in the art, all of which can be applied to the present application. For example, B. pertussis strains can be cultured / fermented using BG solid medium, SS medium medium, or Verwey medium medium, among others, to produce OMVs.

[0133] In another aspect, the present application provides a B. pertussis outer membrane vesicle (OMV) produced by the method of the present application.

[0134] In another aspect, the present application provides a composition comprising an effective amount of

[0135] 1) a B. pertussis strain of the present application; and / or

[0136] 2) a B. pertussis outer membrane vesicle (OMV) of the present application.

[0137] In some embodiments, the composition is a vaccine. In some embodiments, the composition is used to prevent and / or treat a B. pertussis infection in a subject.

[0138] The composition can also comprise an adjuvant. As used herein, “adjuvant” refers to an additional component in a vaccine that enhances the immune response, or a helper molecule that is added to the vaccine, or a helper molecule that is produced by the body after induction by such additional component, respectively, such as, but not limited to, interferons, interleukins, or growth factors. “Adjuvant” as used herein can include aluminum hydroxide and aluminum phosphate, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.

[0139] In some embodiments of each of the above aspects, the composition can further comprise a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, normal saline, lactated Ringer’s, standard sucrose, standard dextrose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer’s solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and coloring agents.

[0140] In some embodiments of each of the above aspects, the composition is formulated in a form for intramuscular administration, intraperitoneal administration, subcutaneous administration, oral administration, or intranasal administration. In some embodiments, the composition is in a lyophilized form, which can be reconstituted prior to use.

[0141] In one aspect, the present application provides the use of the B. pertussis strain of the present application, the B. pertussis outer membrane vesicle of the present application and / or the composition of the present application in the manufacture of a medicament or vaccine for preventing and / or treating B. pertussis infection in a subject.

[0142] In one aspect, the present application provides a method for preventing and / or treating B. pertussis infection in a subject, comprising administering to the subject an effective amount of the modified B. pertussis strain of the present application, the B. pertussis outer membrane vesicle (OMV) of the present application and / or the composition of the present application. Examples

[0143] The present application can be further understood by reference to the specific examples described herein, which are intended for purposes of illustration only and are not intended to limit the scope of the present application. Obviously, many modifications and variations of this application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the present application there is a full equivalency of all such modifications and variations as would be suggested by the above teachings. For the purpose of illustration, the present application selects BAA-589 strain purchased from ATCC (American type culture collection).

[0144] Example 1, a method for modifying B. pertussis gene by homologous recombination

[0145] 1.1 Construction of exogenous DNA donor plasmid

[0146] The exogenous backbone plasmid contains ori-ColE1 / pMB1 / pBR322 / pUC, OriT and traJ with mobilization element, gentamicin resistance gene (Gen) and galk counter-selection gene. The sequence of the exogenous backbone plasmid is shown in SEQ ID NO: 1, and the structure of the backbone plasmid is shown in FIG. 1, wherein the sequence of the gentamicin resistance gene is shown in SEQ ID NO: 2, and the sequence of the galk counter-selection gene is shown in SEQ ID NO: 3. The upstream and downstream homologous arms of the target gene integration site are inserted between the upstream site-up (the sequence is shown in SEQ ID NO: 4) and the downstream site-down (the sequence is shown in SEQ ID NO: 5) of the exogenous backbone plasmid, thereby obtaining the exogenous DNA donor plasmid.

[0147] 1.2 Electroporation of donor plasmid

[0148] Glycerol bacteria were activated on BG plates (Bordet-Gengo solid plates) and cultured at 37°C until colonies grew. The colonies were inoculated in SS liquid medium (Stainer-Scholte liquid medium) for expansion culture,

[0149] The strain is prepared for electroporation by washing in 10% glycerol buffer at about 4 degrees Celsius several times. The electroporation is performed on an electroporator (BTX, ECM630). The electroporation parameters are set as follows: 1.6-2.2 kV, 200 Ω, 25 μF, and the electroporation time is not more than 5 ms. After the electric shock, the SS liquid medium is added quickly for recovery. The system is transferred to a sterile EP tube and cultured at 35-37 degrees Celsius on a shaker at 220-240 rpm for recovery. After recovery, the supernatant is discarded by centrifugation, and the SS liquid medium is added to resuspend the bacteria, which are then spread on BG solid plates containing 10 μg / mL gentamicin resistance (BG-G). The plates are incubated at 35-37 degrees Celsius, and the single colonies that grow are the possible recombinant strains (1HR).

[0150] 1.3 Verification of the first recombinant strain

[0151] The single colonies that grow on the BG-G screening plates are randomly picked with a sterile gun tip and streaked on new BG-G plates. The remaining bacteria on the gun tip are dipped in 50 μL sterile water as a PCR verification template. The verification is performed using the upstream homology arm integration-specific verification primer and / or the downstream integration-specific verification primer. The correct clones of the first recombination are selected.

[0152] 1.4 Counter-screening culture and verification of the second recombinant strain

[0153] The correct clones of the first recombination are selected and streaked on BG plates containing 2% DOG (2-Deoxy-D-galactose). The plates are incubated at 37 degrees Celsius, and the single colonies that grow are the possible second recombinant strains (2HR). The single colonies that grow on the BG-2% DOG screening plates are randomly picked with a sterile gun tip and streaked on new BG plates. The remaining bacteria on the gun tip are dipped in 50 μL sterile water as a PCR verification template. The verification is performed using the external genome primer. The correct clones of the second recombination are selected.

[0154] The external genome primer refers to the upstream sequence of the upstream homology arm as a forward primer (not containing the upstream homology arm itself) and the downstream sequence of the downstream homology arm as a reverse primer (not containing the downstream homology arm itself).

[0155] Example 2, screening of genes for increased production of OMV in Bordetella pertussis

[0156] The common genes for increased production, rmpM, tolR, pal, and ompA, in the prior art cannot achieve the effect of increased production of OMV in Bordetella pertussis because they do not exist in pertussis or are essential for the survival of pertussis. Therefore, the inventors further attempt other genes, pagP, ompP, and BP1721, which have not been reported.

[0157] 2.1 Knocking out the outer membrane protein pagP gene does not increase the production of OMV by the strain.

[0158] The pagP gene (sequence shown as SEQ ID NO: 6) encodes an outer membrane protein of a Bordetella pertussis strain, which can degrade phospholipids and transfer the myristoyl group on the phospholipids to the lipopolysaccharide LPS, thereby adjusting the toxicity of LPS by modifying the structure of LPS. According to its function, it is speculated that after knocking out the pagP gene, the phospholipids cannot be degraded, which may increase the phospholipid content of the outer membrane, ultimately leading to an increase in the curvature of the membrane and promoting the secretion of OMV. However, in Bordetella pertussis strains, there is currently no report on knocking out the pagP gene to characterize the OMV production of the strain. The inventors successfully constructed the pagP gene knockout strain BAA-589 (BAA-589ΔpagP, the sequencing alignment result is shown in Figure 2) by the method of homologous recombination according to the method of Example 1, but it was found through testing that the OMV particle concentration secreted by the BAA-589ΔpagP strain was lower than that of the wild-type strain, and there was no yield-increasing effect, as shown in Figure 3.

[0159] 2.2 Unable to obtain a strain with an inactivated ompP gene

[0160] The ompP gene (sequence shown as SEQ ID NO: 7) encodes an outer membrane protein of a Bordetella pertussis strain, which is extremely abundant. However, using the method of homologous recombination of Example 1, the inventors were unable to obtain a strain with an inactivated ompP gene, and the inventors speculated that the ompP gene is essential for the survival of Bordetella pertussis BAA-589 strain, and after knocking out the gene, the strain cannot survive.

[0161] 2.3 Unable to obtain a strain with an inactivated BP1721 gene

[0162] The BP1721 gene (sequence shown as SEQ ID NO: 8) encodes a protein predicted to be an outer membrane protein of Bordetella pertussis BAA-589 strain, but this gene has not been studied in Bordetella pertussis strains. Therefore, the inventors attempted to explore the effect of the BP1721 gene on the OMV of Bordetella pertussis. The inventors were unable to obtain a strain with an inactivated BP1721 gene using the method of homologous recombination of Example 1, and the inventors speculated that the BP1721 gene is essential for the survival of Bordetella pertussis BAA-589 strain, and after knocking out the gene, the strain cannot survive.

[0163] Example 3, Knocking out the BP2992 gene in Bordetella pertussis leads to an increase in OMV production

[0164] The common yield-increasing genes rmpM, tolR, pal, and ompA reported in the prior art cannot achieve the effect of increasing the yield of OMV on Bordetella pertussis. The inventors further attempted the potential yield-increasing genes pagP, ompP, and BP1721, all of which failed. The inventors unexpectedly found that the BP2992 gene, which has not been studied so far, has a good effect on OMV yield increase.

[0165] 3.1 Construction of strain BAA-589ΔBP2992

[0166] The inventors knocked out the BP2992 gene in the Bordetella pertussis BAA-589 strain using the homologous recombination method of Example 1, specifically, the plasmid carrying the upstream and downstream homologous arms of the BP2992 gene was electroporated into the Bordetella pertussis BAA-589 strain, and through primary and secondary homologous recombination, sequencing confirmed that BP2992 had been successfully knocked out (the sequencing results are shown in FIG. 4), and the strain BAA-589ΔBP2992 was obtained.

[0167] The nucleotide sequence of BP2992 is shown as SEQ ID NO: 9, the upstream homologous arm sequence used in the gene editing process using the homologous recombination method is shown as SEQ ID NO: 10, and the downstream homologous arm sequence is shown as SEQ ID NO: 11.

[0168] 3.2 Determination of the growth curve and particle size of strain BAA-589ΔBP2992

[0169] In order to study whether knocking out the BP2992 gene affects the growth of the strain, the inventors inoculated the BAA-589 and BAA-589ΔBP2992 strains activated on BG plates into shake flasks containing 10 ml of SS medium, and cultured overnight at 35°C on a shaker at 220 rpm. The seed liquid OD600 was measured, and an appropriate amount of seed liquid was inoculated into shake flasks containing 1 L of SS medium, so that the initial OD600 was 0.02. The shake flask culture system was cultured at 35°C on a shaker at 220 rpm for about 24 h, and the sample was collected. The fermentation liquid OD600 was measured. According to the measurement results, the growth curve is shown in FIG. 5. It can be seen from the curve that the growth of the BAA-589ΔBP2992 strain is very close to that of the wild type (BAA-589), indicating that knocking out the BP2992 gene has no obvious effect on the growth of the strain.

[0170] The particle size of OMV was measured using a nanoparticle size potential analyzer (Malvern, Zetasizer Pro), and the results are shown in FIG. 6. It can be seen from the measurement results that there is no significant difference in the particle size of OMV before and after modification.

[0171] 3.3 Determination of the OMV secretion amount of strain BAA-589ΔBP2992

[0172] The BG plate activated BAA-589ABP2992 was inoculated in a test tube containing 3 ml of SS medium and cultured overnight at 35°C in a shaker at 220 rpm. The OD600 of the seed liquid was measured, and an appropriate amount of seed liquid was inoculated into a test tube containing 3 ml of SS medium, and the initial OD600 was controlled at 0.2. The test tube was placed in a shaker at 35°C and 220 rpm for about 24 h, and the sample was collected. 1 ml of the fermentation sample was centrifuged at 10,000 rpm for 5 min, and the supernatant was filtered using a 0.22 um filter. The OMV concentration was measured using a nanoparticle tracking analyzer (Malvern, NanoSight Pro), and the results are shown in Figure 9. According to the characterization results, the BAA-589ABP2992 produced 2.8 times more than the wild type BAA-589 under the above test tube culture conditions.

[0173] Example 4, Construction of Strains BAA-589ABP2992ApldA and BAA-589ABP2992ApldAAmItA

[0174] The inventors wanted to improve BAA-589ABP2992 and further improve OMV production. The pldA gene and the mltA gene were selected for combined knockout with the BP2992 gene.

[0175] The inventors used the homologous recombination method of Example 1 to knockout the pldA gene in the BAA-589ABP2992 strain. Specifically, the plasmid carrying the upstream and downstream homologous arms of the pldA gene was electroporated into the B. pertussis BAA-589ABP2992 strain, and through primary and secondary homologous recombination, sequencing confirmed that the pldA gene had been successfully knocked out (the sequencing results are shown in Figure 7), and the strain BAA-589ABP2992ApldA was obtained.

[0176] 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.

[0177] The inventors used the homologous recombination method of Example 1 to knock out the mltA gene on the strain BAA-589ΔBP2992ΔpldA, specifically, the plasmid carrying the upstream and downstream homologous arms of the mltA gene was electroporated into the Bordetella pertussis BAA-589ΔBP2992ΔpldA strain, and through primary and secondary homologous recombination, sequencing confirmed that the mltA gene had been successfully knocked out (the sequencing results are shown in Figure 8), and the strain BAA-589ΔBP2992ΔpldAΔmltA was obtained.

[0178] The nucleotide sequence of the mltA gene is shown as SEQ ID NO: 15, the upstream homologous arm sequence used in the gene editing process using the homologous recombination method is shown as SEQ ID NO: 16, and the downstream homologous arm sequence is shown as SEQ ID NO: 17.

[0179] The inventors also found 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 on the BAA-589 strain (as high as 90%).

[0180] Example 6, determination of OMV secretion amount of each strain

[0181] The BAA-589ΔBP2992, BAA-589ΔBP2992ΔpldA, and BAA-589ΔBP2992ΔpldAΔmltA strains activated on BG plates were inoculated in test tubes containing 3 ml of SS medium and cultured overnight at 35°C in a shaker at 220 rpm. The seed liquid OD600 was measured, and an appropriate amount of seed liquid was inoculated into test tubes containing 3 ml of SS medium, and the initial OD600 was controlled at 0.2. The test tubes were placed in a shaker at 35°C and 220 rpm for about 24 h, and the sample was collected. 1 ml of the fermentation sample was centrifuged at 10,000 rpm for 5 min, and the supernatant was filtered using a 0.22 um filter. The OMV concentration was measured using a nanoparticle tracking analyzer (Malvern, NanoSight Pro), and the results are shown in Figure 9. According to the characterization results, under the above test tube culture conditions, compared with the wild type BAA-589, the yield of BAA-589ΔBP2992 was increased by 2.8 times, the yield of BAA-589ΔBP2992ΔpldA was increased by 3.4 times, and the yield of BAA-589ΔBP2992ΔpldAΔmltA was increased by 7.3 times.

[0182] Sequence information related to the present application

[0183] SEQ ID NO: 1 exogenous backbone plasmid for homologous recombination SEQ ID NO: 1 exogenous backbone plasmid for homologous recombination

[0184] SEQ ID NO: 2 nucleotide sequence of gentamicin resistance gene:

[0185] SEQ ID NO: 3 nucleotide sequence of galk counter-selectable gene:

[0186] SEQ ID NO: 4 nucleotide sequence of site-up of backbone plasmid insertion site:

[0187] SEQ ID NO: 5 nucleotide sequence of site-down of backbone plasmid insertion site:

[0188] SEQ ID NO: 6 pagP gene

[0189] SEQ ID NO: 7 ompP gene

[0190] SEQ ID NO: 8 BP1721 gene

[0191] SEQ ID NO: 9 BP2992 gene

[0192] SEQ ID NO: 10 upstream homology arm of BP2992 gene used in homologous recombination method

[0193] SEQ ID NO: 11 downstream homology arm of BP2992 gene used in homologous recombination method

[0194] SEQ ID NO: 12 pldA gene

[0195] SEQ ID NO: 13 upstream homology arm of pldA gene used in homologous recombination method

[0196] SEQ ID NO: 14 downstream homology arm of pldA gene used in homologous recombination method

[0197] SEQ ID NO: 15 mltA gene

[0198] SEQ ID NO: 16 Upstream homology arm of mltA gene used in homologous recombination method

[0199] SEQ ID NO: 17 Downstream homology arm of mltA gene used in homologous recombination method

[0200] SEQ ID NO: 18 Amino acid sequence of BP2992 protein

[0201] SEQ ID NO: 19 Amino acid sequence of PldA protein

[0202] SEQ ID NO: 20 Amino acid sequence of MltA protein

Claims

1. A Bordetella pertussis strain, wherein the BP2992 gene of the strain is partially or completely inactivated, whereby the strain has increased production of bacterial outer membrane vesicles (OMVs) compared to a control Bordetella pertussis strain.

2. The Bordetella pertussis strain of claim 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 comprising the nucleotide sequence set forth in SEQ ID NO:

9.

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

4. The Bordetella pertussis strain of claim 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 comprising 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 comprising the nucleotide sequence set forth in SEQ ID NO: 12, and 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 comprising 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: 13 or comprising the nucleotide sequence set forth in SEQ ID NO:

13. ​ ​ ​ ​ ​ ​ ​ 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.

5. The B. pertussis strain of claim 3 or 4, wherein the pldA gene and the mltA gene of the strain are partially inactivated or completely inactivated.

6. The B. pertussis strain of any one of claims 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.

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

8. The B. pertussis strain of claim 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 by the gene; 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 protein encoded by the gene, the addition, substitution or deletion of the one or more amino acids resulting in reduced activity or no activity of the encoded protein.

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

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

11. The B. pertussis strain of any one of claims 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.

12. The B. pertussis strain of any one of claims 1-11, which is derived from the BAA-589 strain.

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 inactivating or completely inactivating the pldA gene and / or the mltA gene in the B. pertussis strain.

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

15. The method of claim 14, wherein in optional step b) the pldA gene in the B. pertussis strain is first partially or completely inactivated, followed by the mltA gene in the B. pertussis strain being partially or completely inactivated.

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

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

18. The method of claim 17, wherein the mutation is an 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 no expression of the encoded protein; 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.

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

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

22. The method of claim 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 in 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 with a deletion in both the BP2992 gene and the pldA gene; and c) deleting the mltA gene in the B. pertussis strain obtained in step b) by homologous recombination, thereby obtaining a B. pertussis strain with a deletion in the BP2992 gene, the pldA gene and the mltA gene.

23. The method of claim 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.

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

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

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

27. A composition comprising an effective amount of 1) a Bordetella pertussis strain according to any one of claims 1-12 and 24; and / or 2) a 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. Use of a Bordetella pertussis strain according to any one of claims 1-12 and 24, and / or a Bordetella pertussis outer membrane vesicle according to claim 26, and / or a composition according to claim 27 or 28, for the manufacture of a medicament or vaccine for the prevention and / or treatment of a Bordetella pertussis infection in a subject, preferably a human.

30. Use of a Bordetella pertussis strain according to any one of claims 1-12, for the production of a Bordetella pertussis outer membrane vesicle (OMV).

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