Modified bordetella pertussis strain

Abstract

The present invention relates to the field of bioengineering, and specifically relates to a modified Bordetella pertussis strain in which the mltA gene and pldA gene are mutated, thereby having an increased bacterial outer membrane vesicle (OMV) yield. The present invention further relates to a method for producing the modified Bordetella pertussis strain and a method for producing OMVs by means of the modified Bordetella pertussis strain.

Description

Modified bordetella pertussis strain TECHNICAL FIELD

[0001] The present invention relates to the field of bioengineering, in particular to a modified Bordetella pertussis strain, the mltA gene and the pldA gene of which are mutated, thereby having an increased production of bacterial outer membrane vesicles (OMVs). The present invention also relates to a method for producing said modified Bordetella pertussis strain and a method for producing OMVs by said modified 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 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, which is now widely used. Despite the high vaccination rate nowadays, cases of whooping cough are on the rise again due to genetic changes in the prevalent Bordetella pertussis strains and differences in the immunity induced after vaccination with whole-cell or acellular whooping cough 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 have a size between 20 nm and 200 nm. OMVs are thought to be formed by the process of blebbing in Gram-negative bacteria, which is believed to be related to the weakening of the association between the outer membrane and the peptidoglycan layer or the accumulation of substances such as proteins in the periplasmic space. OMVs play a role in long-distance delivery, biofilm formation, bacterial survival, and the regulation of interactions between populations. On the one hand, OMVs are small in volume and are easily taken up by antigen-presenting cells; on the other hand, OMVs have many things in common with bacteria, being closer to simulating a natural infection; on the other hand, OMVs contain LPS, which can act as a natural adjuvant. Therefore, Bordetella pertussis OMVs have enhanced immunogenicity compared to existing whole-cell whooping cough vaccines and acellular whooping cough vaccines.

[0005] It is worth noting that the production of OMVs varies depending on the species, strain, and growth phase. The bottleneck of Bordetella pertussis OMVs secreted as a vaccine is that the production of Bordetella pertussis OMVs is extremely low, which makes the development and production of Bordetella pertussis OMV vaccines a great challenge. Increasing the production of OMVs has become a problem to be solved for the development of whooping cough OMV vaccines.

[0006] There are many ways to increase the yield of bacterial OMVs, which can be divided into two categories: improving yield from fermentation process and improving yield from strain.

[0007] From the fermentation process, the secretion of OMVs can be improved by induction. However, compared with naturally secreted OMVs, there are some deficiencies. For example, using detergents to induce OMV secretion will cause the formed OMVs to lose LPS with adjuvant effect and lipoprotein with possible immunogenicity; using ultrasound 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; and using heating to induce OMV secretion may change the phospholipid composition of OMV membrane.

[0008] From the strain, the yield of OMVs can be improved by editing the genes related to the secretion process of OMVs. 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 OMVs. The second strategy is to increase the content of phospholipids 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 phospholipids and destroying the mla system, the transport system of outer membrane phospholipids to inner membrane, can all achieve over-secretion of OMVs. The third strategy is to increase the internal pressure of the periplasmic space to promote the secretion of OMVs. For example, knocking out the chaperone protein gene degP of the periplasmic space to increase the accumulation of misfolded proteins in the periplasmic space, destroying the recycling of peptidoglycan, etc.

[0009] Although knocking out the lpp gene in E. coli results in a 160-fold increase in E. coli OMV production, there is no such gene and its homologues in B. pertussis. Eline F. de Jonge et al. found that 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 achieve a 26-fold increase in OMV production. 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 OMV production 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] In one aspect, the present application provides a modified B. pertussis strain comprising a mutation in the mltA gene and thereby having an increased production of bacterial outer membrane vesicles (OMV) as compared to a corresponding control B. pertussis strain. In some embodiments, the modified B. pertussis strain further comprises a mutation in the pldA gene.

[0013] In another aspect, the present application provides a method of producing a modified B. pertussis strain, the method comprising the steps of:

[0014] a) introducing a mutation into the pldA gene in a B. pertussis strain, thereby obtaining a modified B. pertussis strain comprising a mutation in the pldA gene; and

[0015] b) introducing a mutation into the mltA gene in the modified B. pertussis strain obtained in step a), thereby obtaining a modified B. pertussis strain comprising a mutation in the mltA gene.

[0016] In another aspect, the present application provides a method of producing a Bordetella pertussis outer membrane vesicle (OMV), said method comprising the steps of:

[0017] a) culturing a modified Bordetella pertussis strain of the present application under conditions suitable for the production of outer membrane vesicles (OMVs); and

[0018] b) recovering and optionally purifying the OMVs produced in step a).

[0019] In another aspect, the present application provides an OMV produced by the method of the present application.

[0020] In another aspect, the present application also provides a composition comprising

[0021] 1) a modified Bordetella pertussis strain of the present application; and / or

[0022] 2) a Bordetella pertussis outer membrane vesicle (OMV) of the present application.

[0023] In one aspect, the present application provides the use of a modified Bordetella pertussis strain of the present application and / or a Bordetella pertussis outer membrane vesicle (OMV) of the present application for the preparation of a composition for the prevention or treatment of a Bordetella pertussis infection.

[0024] In one aspect, the present application provides a method of preventing and / or treating a Bordetella pertussis infection in a subject, the method comprising administering to said subject an effective amount of a modified Bordetella pertussis strain of the present application, a Bordetella pertussis outer membrane vesicle (OMV) of the present application and / or a composition of the present application.

[0025] BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1. Homologous recombination backbone plasmid map.

[0027] Figure 2. Secondary recombination strain PCR verification of the homologous recombination method knocking out the mltA gene on the BAA-589 strain.

[0028] Figure 3. Sequencing results alignment of the BAA-589 AmltA strain.

[0029] Figure 4. Comparison of the OD concentration of the BAA-589 AmltA strain and the wild type strain BAA-589 after 24 hours of culture.

[0030] Figure 5. Comparison of the OMV particle concentration secreted by the BAA-589 AmltA strain and the wild type strain BAA-589.

[0031] Figure 6. Secondary recombination strain PCR verification of the homologous recombination method knocking out the pldA gene on the BAA-589 strain.

[0032] Figure 7. Sequencing results alignment of BAA-589 ApldA strain.

[0033] Figure 8. Comparison of the OD concentration of BAA-589 ApldA strain and wild type BAA-589 strain after 24 hours of culture.

[0034] Figure 9. Comparison of the OMV particle concentration secreted by BAA-589 ApldA strain and wild type BAA-589 strain.

[0035] Figure 10. PCR verification of secondary recombination strain of mltA gene knockout in BAA-589 ApldA strain using homologous recombination method.

[0036] Figure 11. Sequencing results alignment of BAA-589 ApldA AmltA strain.

[0037] Figure 12. Comparison of the OMV particle concentration secreted by BAA-589 ApldA AmltA strain and wild type BAA-589 strain.

[0038] Figure 13. Comparison of the OD concentration of BAA-589 ApldA AmltA strain and wild type BAA-589 strain after 24 hours of culture.

[0039] DETAILED DESCRIPTION

[0040] I. DEFINITIONS

[0041] 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 related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunology, and laboratory operations procedures used herein are terms and procedures commonly used in the respective fields.

[0042] As used herein, the term "and / or" encompasses all combinations of the items linked by the term. It will be recognized by those of ordinary skill that each combination is individually contemplated herein, even though the term "and / or" is used. For example, "A and / or B" encompasses "A," "B," "A and B," "A and C," "B and C," and "A and B and C." As used herein, the term "comprises" means "includes, but is not limited to."

[0043] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. A polynucleotide can be either sense or anti-sense, and can be of synthetic, non-natural, or altered composition. 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 (including T), the corresponding RNA sequence (i.e., with U in place of T) can be readily determined by one of skill in the art when RNA is referred to.

[0044] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers 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.

[0045] 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. "Gene" generally refers to an endogenous sequence of an organism, such as B. pertussis.

[0046] The word "comprising" is used herein to mean that a protein or nucleic acid sequence can consist of the recited elements, or that additional amino acids or nucleotides can be present at either or both ends of the protein or nucleic acid sequence, but that the recited activity is still present.

[0047] "Sequence identity" between two polypeptide sequences or between 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 over the full length of a given sequence.

[0048] The "mltA gene" described herein encodes a MltA protein, which is a lipoprotein located in the outer membrane, the protein has a peptidoglycan cleaving exosaccharide glycosylase activity, is involved in the metabolism of peptidoglycan, and plays a function in the process of cell separation and membrane construction.

[0049] The MltA 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: 6. In some embodiments, the MltA protein in Bordetella pertussis can comprise the amino acid sequence set forth in SEQ ID NO: 6.

[0050] The mltA gene in Bordetella 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: 7. In some embodiments, the mltA gene in Bordetella pertussis can comprise the nucleotide sequence set forth in SEQ ID NO: 7.

[0051] The "pldA gene" described herein encodes a PldA protein, which is a phospholipase that can degrade phospholipids on the outer membrane and release fatty acids.

[0052] The PldA 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: 10. In some embodiments, the PldA protein in Bordetella pertussis can comprise the amino acid sequence set forth in SEQ ID NO: 10.

[0053] The pldA gene in Bordetella 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: 11. In some embodiments, the pldA gene in Bordetella pertussis can comprise the nucleotide sequence set forth in SEQ ID NO: 11.

[0054] 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, that is sufficient, at least, to produce a prophylactic or therapeutic effect after administration to a subject. 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.

[0055] "Subject" as described herein preferably means a human.

[0056] II. Modified Bordetella pertussis strains

[0057] In one aspect, the present application provides a modified Bordetella pertussis strain comprising a mutation in the mltA gene and thereby having an increased production of bacterial outer membrane vesicles (OMVs) compared to a corresponding control Bordetella pertussis strain.

[0058] In some embodiments, the mutation in the mltA gene results in reduced expression of MltA protein. In some embodiments, the mutation in the mltA gene results in 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 expression of MltA protein, preferably the mutation in the mltA gene results in no expression of MltA protein by the strain.

[0059] In some embodiments, the mutation in the mltA gene results in a mutant MltA protein having reduced activity. In some embodiments, the activity of the mutant MltA protein 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 relative to wild-type MltA protein, preferably the mutant MltA protein is an inactive MltA protein. The activity is, for example, peptidoglycan-lytic exoglycosylase activity.

[0060] The mutation in the mltA gene can be an addition, substitution or deletion of one or more nucleotides. In some embodiments, the mutation in the mltA gene comprises a deletion of the mltA gene, for example, a complete deletion or a partial deletion of the mltA gene. The mltA gene can be completely deleted from the strain, such that the mltA gene is not present in the strain of the application, and thereby no MltA protein is present. The mltA gene can also be partially deleted, such that only a truncated MltA protein having reduced activity or no activity is present in the strain of the application.

[0061] In some embodiments, the modified Bordetella pertussis strain further comprises a mutation in the pldA gene compared to a corresponding control Bordetella pertussis strain.

[0062] In some embodiments, the mutation in the pldA gene results in reduced PldA protein expression. In some embodiments, the mutation in the pldA gene results in 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 reduction in PldA protein expression, preferably, the mutation in the pldA gene results in the strain not expressing PldA protein.

[0063] In some embodiments, the mutation in the pldA gene results in a mutant PldA protein with reduced activity. In some embodiments, the activity of the mutant PldA protein 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 relative to wild-type PldA protein, preferably, the mutant PldA protein is an inactive PldA protein. The activity is, for example, phospholipase activity.

[0064] The mutation in the pldA gene can be an addition, substitution, or deletion of one or more nucleotides. In some embodiments, the mutation in the pldA gene comprises a deletion of the pldA gene, for example, a complete deletion or a partial deletion of the pldA gene. The pldA gene can be completely deleted from the strain, such that the pldA gene is not present in the strain of the application, and thus no PldA protein is present. The pldA gene can also be partially deleted, such that only a truncated PldA protein with reduced activity or no activity is present in the strain of the application.

[0065] The mutation in the gene can be achieved by various means known in the art. In some embodiments, the mutation is introduced into 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 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 by targeted mutagenesis, such as via CRISPR, TALEN, or ZFN technology.

[0066] In some embodiments, the mltA gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 8 and 9. In some embodiments, the pldA gene is deleted by homologous recombination using the homology arm sequences set forth in SEQ ID NOs: 12 and 13.

[0067] In some embodiments, the yield of bacterial outer membrane vesicles (OMVs) of the modified 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% or more, as compared to the yield of OMVs of a corresponding control B. pertussis strain. In some embodiments, the yield of bacterial outer membrane vesicles (OMVs) of the modified 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 or more, as compared to the yield of OMVs of a corresponding control B. pertussis strain.

[0068] A "control B. pertussis strain" as described herein can be a parental strain from which the modified B. pertussis strain is derived. For example, the control B. pertussis strain does not comprise a mutation in the mltA gene and / or a mutation in the pldA gene.

[0069] The modified B. pertussis strain as described herein can be derived from any known B. pertussis strain, for example the BAA-589 strain.

[0070] III. Methods of producing a modified B. pertussis strain

[0071] The present inventors surprisingly found that a prior deletion of the pldA gene in B. pertussis can significantly increase the success rate of a subsequent deletion of the mltA gene by homologous recombination.

[0072] Accordingly, in another aspect, the present application provides a method of producing a modified B. pertussis strain, the method comprising the steps of:

[0073] a) introducing a mutation into the pldA gene in a B. pertussis strain, thereby obtaining a modified B. pertussis strain comprising a mutation in the pldA gene; and

[0074] b) introducing a mutation into the mltA gene in the modified B. pertussis strain obtained in step a), thereby obtaining a modified B. pertussis strain comprising a mutation in the mltA gene.

[0075] In some embodiments, the mutation introduced into the pldA gene results in reduced expression of PldA protein in the B. pertussis strain. In some embodiments, the mutation introduced into the pldA gene results in 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 reduction in PldA protein expression, preferably the mutation in the pldA gene results in the strain not expressing PldA protein.

[0076] In some embodiments, the mutation introduced into the pldA gene results in the B. pertussis strain expressing a mutant PldA protein with reduced activity. In some embodiments, the activity of the mutant PldA protein 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 relative to wild-type PldA protein, preferably the mutant PldA protein is an inactive PldA protein. The activity is, for example, phospholipase activity.

[0077] The mutation introduced into the pldA gene can be an addition, substitution, or deletion of one or more nucleotides. In some embodiments, the mutation introduced into the pldA gene comprises a deletion of the pldA gene, for example, a complete deletion or a partial deletion of the pldA gene. The pldA gene can be completely deleted from the strain such that the pldA gene is not present in the modified strain, and thus no PldA protein is present. The pldA gene can also be partially deleted such that only a truncated PldA protein with reduced or no activity is present in the modified strain.

[0078] In some embodiments, the mutation introduced into the mltA gene results in reduced expression of MltA protein. In some embodiments, the mutation introduced into the mltA gene results in 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 reduction in MltA protein expression, preferably the mutation introduced into the mltA gene results in the strain not expressing MltA protein.

[0079] In some embodiments, the mutation introduced into the mltA gene results in a mutant MltA protein with reduced activity. In some embodiments, the activity of the mutant MltA protein 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 relative to the wild-type MltA protein. Preferably, the mutant MltA protein is an inactive MltA protein. The activity is, for example, the peptidoglycan-lytic ectoglycosylase activity.

[0080] The mutation introduced into the mltA gene can be an addition, a substitution, or a deletion of one or more nucleotides. In some embodiments, the mutation introduced into the mltA gene comprises a deletion of the mltA gene, for example, a complete deletion or a partial deletion of the mltA gene. The mltA gene can be completely deleted from the strain, such that the mltA gene is absent in the modified strain, and thus the MltA protein is absent. The mltA gene can also be partially deleted, such that only a truncated MltA protein with reduced or no activity is present in the modified strain.

[0081] In some embodiments, the obtained modified Bordetella pertussis strain comprises a mutation in both the mltA gene and the pldA gene.

[0082] In some embodiments, the mutations are introduced into the genes of the Bordetella pertussis strain by genetic engineering. For example, the mutation in the gene, such as a deletion of the gene, can be introduced 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 introduced by targeted mutagenesis, such as via CRISPR, TALEN, or ZFN technology.

[0083] In some specific embodiments, the method comprises the following steps:

[0084] a) deleting the pldA gene in a Bordetella pertussis strain by homologous recombination, thereby obtaining a modified Bordetella pertussis strain with a deletion of the pldA gene; and

[0085] b) deleting the mltA gene in the modified Bordetella pertussis strain obtained in step a) by homologous recombination, thereby obtaining a modified Bordetella pertussis strain with a deletion in both the pldA gene and the mltA gene.

[0086] In another aspect, the present application also provides a modified Bordetella pertussis strain produced by the method of the present application.

[0087] In some embodiments, the yield of bacterial outer membrane vesicles (OMVs) from the modified B. pertussis strain produced by the methods 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%, or more, as compared to the yield of OMVs from a corresponding control B. pertussis strain. In some embodiments, the yield of bacterial outer membrane vesicles (OMVs) from the modified 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, or more, as compared to the yield of OMVs from a corresponding control B. pertussis strain.

[0088] IV. Production and use of bacterial outer membrane vesicles (OMVs)

[0089] In another aspect, the application provides a method of producing B. pertussis outer membrane vesicles (OMVs), the method comprising the steps of:

[0090] a) culturing a modified B. pertussis strain of the application under conditions suitable for production of outer membrane vesicles (OMVs); and

[0091] b) recovering and optionally purifying the OMVs produced in step a).

[0092] In another aspect, the application provides B. pertussis outer membrane vesicles (OMVs) produced by the methods of the application.

[0093] In another aspect, the application provides a composition comprising an effective amount of

[0094] 1) a modified B. pertussis strain of the application; and / or

[0095] 2) a B. pertussis outer membrane vesicle (OMV) of the application.

[0096] 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.

[0097] The composition can further 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 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.

[0098] 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.

[0099] 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 lyophilized form, which can be reconstituted prior to use.

[0100] In one aspect, the present application provides the use of the modified Bordetella pertussis strain of the present application and / or the Bordetella pertussis outer membrane vesicle (OMV) of the present application in the manufacture of a composition for preventing or treating a Bordetella pertussis infection.

[0101] In one aspect, the present application provides a method of preventing and / or treating a 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 application, the Bordetella pertussis outer membrane vesicle (OMV) of the present application, and / or the composition of the present application. EXAMPLE

[0102] The present application can be further understood by reference to the specific examples described herein, which are intended to be purely exemplary of the application and are not intended to limit the scope of the application. Obviously, many modifications and variations of the present application are possible in light of the above teachings, the scope of the application is not limited to the examples described herein. For the purpose of illustrating the technical solutions and technical effects of the present application, the present application selects the BAA-589 strain purchased from ATCC (American type culture collection).

[0103] Example 1, method of homologous recombination

[0104] 1.1 Construction of exogenous DNA donor plasmid

[0105] The exogenous backbone plasmid comprises ori-ColEl / pMB1 / pBR322 / pUC, OriT with mobilization element and traJ, a gentamicin resistance gene (Gen) and a 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. 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.

[0106] 1.2 Electroporation of the donor plasmid

[0107] The 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,

[0108] The electroporation of the strain was prepared by multiple washes with 10% glycerol buffer at about 4°C, and the electroporation was performed on an electroporator (BTX, ECM630). The electroporation parameters were set as follows: 1.6-2.2 kV, 200Ω, 25 uF, and the electroporation time was not more than 5 ms. After the electric shock, SS liquid medium was quickly added for recovery. The system was transferred to a sterile EP tube and cultured at 35-37°C, 220-240 rpm on a shaker for recovery. After recovery, the supernatant was discarded by centrifugation, SS liquid medium was added, the bacterial cells were resuspended, and were plated on BG solid plates containing 10 ug / mL gentamicin resistance (BG-G). The plates were incubated at 35-37°C, and the single colonies that grew were the possible recombinant strains (1HR).

[0109] 1.3 Verification of the first recombinant strain

[0110] The single colonies that grew on the BG-G screening plates were randomly picked with a sterile gun tip and streaked on new BG-G plates, and the remaining bacterial cells on the gun tip were dipped in 50 uL sterile water as a PCR verification template. Verification was performed using the upstream homologous arm integration-specific verification primer and / or the downstream integration-specific verification primer, and the correct 1HR clones were selected.

[0111] 1.4 Counter-selection culture and verification of the second recombinant strain

[0112] The correct recombinant clone is selected by streaking on BG plates containing 2% DOG (2-Deoxy-D-galactose), and the plates are incubated at 37°C. The single colonies that grow are possible secondary recombinant strains (2HR). A sterile gun tip is used to randomly pick several single colonies that grow on the BG-2% DOG screening plates and streak them on new BG plates. The remaining bacteria on the gun tip are dipped in 50 uL sterile water as a PCR verification template. The correct secondary recombinant clone is selected by using external genome primers for verification.

[0113] The external genome primers refer to the upstream sequence of the upstream homology arm as the forward primer (not including the upstream homology arm itself) and the downstream sequence of the downstream homology arm as the reverse primer (not including the downstream homology arm itself).

[0114] Example 2, screening of genes for increased production of OMVs in Bordetella pertussis

[0115] The common bacterial OMV production genes rmpM, tolR, pal, and ompA reported in the prior art cannot achieve the effect of increasing the production of OMVs in Bordetella pertussis because they do not exist in Bordetella pertussis or are essential for the survival of Bordetella pertussis. Therefore, the inventors further tried other potential production genes pagP and ompP that have not been reported.

[0116] 2.1 The strain without the outer membrane protein pagP gene does not increase the production of OMVs.

[0117] The pagP gene encodes an outer membrane protein of a Bordetella strain that 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, the inventors speculate that after the pagP gene is knocked out, 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 OMVs. However, there is currently no report on the characterization of OMV production in a strain with a pagP gene knocked out in a Bordetella strain. The inventors successfully constructed the BAA-589 strain with the pagP gene knocked out (BAA-589ΔpagP) by the method of homologous recombination according to the method of Example 1, but found that the OMV particle concentration secreted by the BAA-589ΔpagP strain was slightly lower than that of the wild-type strain, and there was no effect of increasing production.

[0118] 2.2 Unable to obtain a strain with an inactivated ompP gene

[0119] The ompP gene encodes an outer membrane protein of a pertussis strain, which is extremely abundant. However, using the method of homologous recombination of Example 1, the strain in which the ompP gene is inactivated cannot be obtained, and the inventors speculate that the ompP gene is essential for the survival of the pertussis BAA-589 strain, and after inactivating or knocking out the gene, the strain cannot survive.

[0120] Example 3, Knocking out the mltA gene increases the yield of pertussis Bordetella OMV by 3.1 times, but the success rate of gene editing is too low

[0121] After the screening failure of Example 2, the inventors noticed that the mltA gene encodes an outer membrane lipoprotein of a pertussis strain. In the pertussis strain, there is no report of successfully knocking out mltA. Therefore, the inventors tried to explore the effect of the mltA gene on the pertussis Bordetella OMV.

[0122] The inventors knocked out the mltA gene in the pertussis BAA-589 strain using the method of homologous recombination in Example 1, specifically by electroporating the exogenous donor DNA plasmid carrying the upstream and downstream homologous arms of the mltA gene into the pertussis BAA-589 strain. BG-G medium was used to screen the recombinant strain once, and galk medium was used to screen the recombinant strain twice. The nucleotide sequence of mltA is shown in SEQ ID NO: 7, the upstream homologous arm sequence used in the process of gene editing using homologous recombination is shown in SEQ ID NO: 8, and the downstream homologous arm sequence is shown in SEQ ID NO: 9. The upstream homologous arm integration specificity verification primer for the first homologous recombination is PTTU344+BpeLYF004, and the downstream integration specificity verification primer (BpeLYF003+PTTU361). The external primer for the second homologous recombination is PTTU344 and PTTU361. The sequencing primer is PTTU344, PTTU361, PTTU183 and PTTU229. The primer information is shown in Table 1.

[0123] Table 1: Primer information for mltA knockout

[0124] The inventors found that the success rate of editing was low in the process of knocking out the mltA gene. The success rate of one recombination was 100% (8 clones were all positive), but in the second recombination stage, the success rate of knocking out was only 1 / 24. Specifically, according to the method of Example 1.4, 24 clones were picked for PCR verification, and the results are shown in Figure 2. Secondary recombination occurred in all 24 clones, but only clone No. 2 had mltA gene knockout, and the remaining 23 clones all returned to the wild type, so the success rate of gene editing was only 1 / 24 (4.2%). As shown in Figure 3, clone No. 2 was further sequenced to confirm that mltA had been successfully knocked out, and strain BAA-589ΔmltA was obtained. The applicant speculates that the reason why there is no report of knocking out the mltA gene of Bordetella pertussis in the prior art should be that it is difficult to successfully knock out the mltA gene.

[0125] After the strain was successfully constructed, the inventors further evaluated the growth status and OMV secretion yield of the BAA-589ΔmltA strain. The BAA-589 and BAA-589ΔmltA 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, with the initial OD600 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. The fermentation liquid OD600 was measured, and the results are shown in Figure 4. It can be seen that the absence of mltA does not affect the survival and growth of the BAA-589 strain. 1 ml of 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 5. According to the characterization results, under the above test tube culture conditions, the OMV particle concentration secreted by the BAA-589ΔmltA strain was 3.1 times that of the wild type strain, and the yield was significantly increased.

[0126] Example 4, knocking out the pldA gene first can improve the success rate of knocking out the mltA gene on the BAA-589 strain.

[0127] The inventors accidentally found that knocking out the mltA gene on the BAA-589 strain with knocked out pldA gene had a gene knockout success rate of 90%, which was significantly improved from the previous 4.2% to 90%, and the gene editing success rate was improved by nearly 22 times.

[0128] pldA is the phospholipase gene of B. pertussis, which can degrade the phospholipids on the outer membrane and release fatty acids. Eline F. de Jonge et al. found that when a large amount of phospholipids accumulated on the outer membrane of the cell, pldA played a function to degrade phospholipids and produce a large amount of fatty acids, which caused cell toxicity, seriously affecting the growth of the strain ((Research in Microbiology, Volume 173, Issues 4-5, 2022, 103937)). However, the inventors did not find that the deletion of pldA gene had a significant effect on the survival and growth of BAA-589ΔmltA strain (as shown in Figure 8).

[0129] The pldA gene was knocked out on the BAA-589 strain according to the method of homologous recombination in Example 1 to obtain the BAA-589ΔpldA strain.

[0130] The nucleotide sequence of pldA is shown as SEQ ID NO: 11, the upstream homologous arm sequence used in the process of gene editing by homologous recombination is shown as SEQ ID NO: 12, and the downstream homologous arm sequence is shown as SEQ ID NO: 13. The upstream homologous arm integration specificity verification primer for the first homologous recombination is PTTU135+PTTU222, and the downstream integration specificity verification primer (PTTU221+PTTU136). The external primer for the second homologous recombination is PTTU354+PTTU380. The sequencing primers are PTTU76, PTTU354 and PTTU380. The primer information is shown in Table 2.

[0131] Table 2: Primer information for pldA knockout

[0132] The success rate of the first recombination was 100% (8 clones were all positive), and the knockout success rate of the second recombination was 3 / 8 (Figure 6). As shown in Figure 7, No. 2 clone was further sequenced to confirm that pldA had been successfully knocked out, and the strain BAA-589ΔpldA was obtained.

[0133] After the construction of the strain, the applicant further evaluated the growth condition and the yield of OMV secreted by the BAA-589ΔpldA strain. According to the culture and detection method in Example 3, the growth of BAA-589ΔpldA strain had no significant difference compared with the wild type (Figure 8), and the concentration of OMV particles secreted by BAA-589ΔpldA strain also had no significant difference compared with the wild type (Figure 9).

[0134] Then the mltA gene was knocked out on the BAA-589ΔpldA strain according to the method of homologous recombination in Example 1 and the experimental materials.

[0135] In the process of knocking out the mltA gene on the BAA-589ΔpldA strain, secondary recombination strain PCR verification stage, as shown in Figure 10, 20 clones all occurred secondary recombination. Among them, 9# and 16# clones returned to wild type (WT), and the remaining 18 clones all achieved mltA knockout, and the gene editing success rate reached 90%. The sequencing verification result of No. 2 clone is shown in Figure 11.

[0136] Example 5, BAA-589ΔpldAΔmltA strain OMV increased by 4.7 times.

[0137] The inventors found that on BAA-589 with pldA gene knocked out, knocking out mltA not only significantly improved the gene editing success rate, but also compared with BAA-589ΔmltA strain (3.1 times), BAA-589ΔpldAΔmltA strain OMV increased more significantly (4.7 times, as shown in Figure 12), and the growth and survival of BAA-589ΔpldAΔmltA were not affected (as shown in Figure 13).

[0138] Sequence information related to the present application

[0139] SEQ ID NO: 1 nucleotide sequence of homologous recombination backbone plasmid

[0140] SEQ ID NO: 2 nucleotide sequence of gentamicin resistance gene

[0141] SEQ ID NO: 3 nucleotide sequence of galk counter-selection gene

[0142] SEQ ID NO: 4 nucleotide sequence of site-up upstream of the insertion site of the backbone plasmid

[0143] SEQ ID NO: 5 nucleotide sequence of site-down downstream of the insertion site of the backbone plasmid

[0144] SEQ ID NO: 6 amino acid sequence of mltA of pertussis BAA-589 strain

[0145] SEQ ID NO: 7 nucleotide sequence of mltA of pertussis BAA-589 strain

[0146] SEQ ID NO: 8 nucleotide sequence of the upstream homologous arm used for knocking out mltA of pertussis BAA-589 strain

[0147] SEQ ID NO: 9 nucleotide sequence of the downstream homologous arm used for knocking out mltA of pertussis BAA-589 strain

[0148] SEQ ID NO: 10 Amino acid sequence of pldA of Bordetella pertussis BAA-589 strain

[0149] SEQ ID NO: 11 Nucleotide sequence of pldA of Bordetella pertussis BAA-589 strain

[0150] SEQ ID NO: 12 Nucleotide sequence of the upstream homology arm used for pldA knockout of Bordetella pertussis BAA-589 strain

[0151] SEQ ID NO: 13 Nucleotide sequence of the downstream homology arm used for pldA knockout of Bordetella pertussis BAA-589 strain.

Claims

1. A modified Bordetella pertussis strain comprising a mutation in the mltA gene and thereby having increased production of bacterial outer membrane vesicles (OMVs) as compared to a corresponding control Bordetella pertussis strain.

2. The modified Bordetella pertussis strain of claim 1, wherein the mutation in the mltA gene results in reduced expression of MltA protein or a mutant MltA protein having reduced activity.

3. The modified Bordetella pertussis strain of claim 1, wherein the mutation in the mltA gene results in no expression of MltA protein or expression of an inactive MltA protein by the strain.

4. The modified Bordetella pertussis strain of claim 1, wherein the mutation in the mltA gene comprises a deletion of the mltA gene.

5. The modified Bordetella pertussis strain of claim 1, wherein the mltA gene encodes the amino acid sequence set forth in SEQ ID NO: 6 or comprises the nucleotide sequence set forth in SEQ ID NO:

7.

6. The modified Bordetella pertussis strain of any one of claims 1-5, further comprising a mutation in the pldA gene.

7. The modified Bordetella pertussis strain of claim 6, wherein the mutation in the pldA gene results in reduced expression of PldA protein or a mutant PldA protein having reduced activity.

8. The modified Bordetella pertussis strain of claim 6, wherein the mutation in the pldA gene results in no expression of PldA protein or expression of an inactive PldA protein by the strain.

9. The modified Bordetella pertussis strain of claim 6, wherein the mutation in the pldA gene comprises a deletion of the pldA gene.

10. The modified Bordetella pertussis strain of claim 6, wherein the pldA gene encodes the amino acid sequence set forth in SEQ ID NO: 10 or comprises the nucleotide sequence set forth in SEQ ID NO:

11.

11. The modified Bordetella pertussis strain of claim 1, which is derived from the BAA-589 strain.

12. A method of producing a modified Bordetella pertussis strain, the method comprising the steps of: a) introducing a mutation into the pldA gene in a Bordetella pertussis strain, thereby obtaining a modified Bordetella pertussis strain comprising a mutation in the pldA gene; and b) introducing a mutation into the mltA gene in the modified Bordetella pertussis strain obtained in step a), thereby obtaining a modified Bordetella pertussis strain comprising a mutation in the mltA gene.

13. The method of claim 12, wherein the mutation introduced into the pldA gene results in reduced expression of PldA protein or a mutant PldA protein having reduced activity; and the mutation introduced into the mltA gene results in reduced expression of MltA protein or a mutant MltA protein having reduced activity.

14. The method of claim 12, wherein the mutation introduced in the pldA gene results in the strain not expressing a PldA protein or expressing an inactivated PldA protein; and the mutation introduced in the mltA gene results in the strain not expressing a MltA protein or expressing an inactivated MltA protein.

15. The method of claim 12, wherein the mutation introduced in the pldA gene comprises a deletion of the pldA gene and the mutation introduced in the mltA gene comprises a deletion of the mltA gene.

16. The method of claim 12, wherein the pldA gene encodes the amino acid sequence set forth in SEQ ID NO: 10 or comprises the nucleotide sequence set forth in SEQ ID NO: 11; and the mltA gene encodes the amino acid sequence set forth in SEQ ID NO: 6 or comprises the nucleotide sequence set forth in SEQ ID NO:

7.

17. The method of claim 12, wherein the modified Bordetella pertussis strain is derived from the BAA-589 strain.

18. The method of any one of claims 12-17, wherein the mutation is introduced by homologous recombination or targeted mutagenesis.

19. A method of producing a Bordetella pertussis outer membrane vesicle (OMV), the method comprising the steps of: a) culturing the modified Bordetella pertussis strain of any one of claims 1-11 or the modified Bordetella pertussis strain produced by the method of any one of claims 12-18 under conditions suitable for production of outer membrane vesicles (OMVs); and b) recovering and optionally purifying the OMVs produced in step a).

20. A Bordetella pertussis outer membrane vesicle (OMV) produced by the method of claim 19.

21. A composition comprising an effective amount of 1) the modified Bordetella pertussis strain of any one of claims 1-11 or the modified Bordetella pertussis strain produced by the method of any one of claims 12-18; and / or 2) the Bordetella pertussis outer membrane vesicle (OMV) of claim 20.

22. Use of the modified Bordetella pertussis strain of any one of claims 1-11 and / or the modified Bordetella pertussis strain produced by the method of any one of claims 12-18 and / or the Bordetella pertussis outer membrane vesicle of claim 20 and / or the composition of claim 21 in the manufacture of a medicament or vaccine for the prevention and / or treatment of a Bordetella pertussis infection in a subject.

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