Modified Bordetella pertussis strains

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a modified Bordetella pertussis strain, wherein the mltA gene and the pldA gene are mutated, thereby having an increased bacterial outer membrane vesicle (OMV) yield. The present invention also relates to a method for producing the modified Bordetella pertussis strain and a method for producing OMVs by the modified Bordetella pertussis strain. Background Art

[0002] Bordetella pertussis Bordetella pertussis ) causes whooping cough in humans, which is most severe in children under one year of age. The first pertussis vaccine consisted of inactivated whole cells and was introduced around the 1940s. Use of the vaccine has led to a significant decline in pertussis cases, but vaccine-induced side effects, such as neurological disorders and fever due to the presence of endotoxins in the vaccine, are a cause of concern. In the 1980s, the first acellular vaccine was developed in Japan, and acellular vaccines are now widely used. Despite high vaccination rates today, pertussis cases are resurging due to genetic changes in circulating strains of B. pertussis and differences in immunity induced after vaccination with whole cell or acellular pertussis vaccines.

[0003] The new generation of vaccines can be based on outer membrane vesicles (OMVs). OMVs are vesicles extruded from the outer membrane (OM) and range in size from 20 nm to 200 nm. OMVs are believed to be formed by Gram-negative bacteria through a budding process, and budding is believed to be associated with a weakening of the connection 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 the regulation of long-distance delivery, biofilm formation, bacterial survival, and bacterial interactions. On the one hand, OMVs are small in size and can be easily absorbed by antigen-presenting cells; on the other hand, OMVs have a lot in common with bacteria and are closer to simulating natural infections; on the other hand, OMVs contain LPS, which can act as a natural adjuvant. Therefore, compared with existing whole-cell pertussis vaccines and acellular pertussis vaccines, OMVs of Bordetella pertussis have enhanced immunogenicity.

[0004] It is worth noting that the production of OMVs varies by species, strain, and growth period. The bottleneck of using OMVs secreted by Bordetella pertussis as a vaccine is that the production of OMVs is extremely low, which makes the research and development and production of Bordetella pertussis OMV vaccines face huge challenges. Increasing OMV production has become an urgent problem to be solved in the development of pertussis OMV vaccines.

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

[0006] From the perspective of fermentation technology, the secretion of OMVs can be increased by induction, but there are some shortcomings compared with the OMVs obtained by natural secretion. For example, the use of detergents to induce OMV secretion will cause the formed OMVs to lose LPS with adjuvant effect and lipoproteins that may be immunogenic; the use of ultrasound to induce OMV secretion will cause the OMV products to be contaminated with the inner membrane; the use of reagents such as EDTA to induce OMV secretion may reduce the stability of the OMV membrane; the use of heating to induce OMV secretion may change the phospholipid composition of the OMV membrane.

[0007] From the strain aspect, the OMV production can be increased by editing genes related to the OMV secretion process. There are three commonly used strategies. The first strategy is to weaken the binding of the outer membrane to the peptidoglycan layer, making the outer membrane loose and easy to form vesicle secretion. Outer membrane lipoprotein is the key bridge for the binding of the outer membrane and peptidoglycan. Reducing the content of outer membrane lipoprotein is an important means to reduce the binding of the outer membrane and the peptidoglycan layer. For example, knocking out the outer membrane lipoprotein genes lpp, ompA and similar genes (such as rmpM), tol / pal, etc. can enable bacteria to achieve excessive secretion of OMV. The second strategy is to increase the phospholipid content 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 transport system of outer membrane phospholipids to the inner membrane-mla system can achieve excessive secretion of OMV. The third strategy is to increase the internal pressure of the periplasmic space to promote OMV secretion. For example, the molecular chaperone protein gene degP in the periplasmic space can be knocked out to increase the accumulation of misfolded proteins in the periplasmic space and destroy the recycling of peptidoglycan.

[0008] Although knocking out the lpp gene in E. coli can increase the production of E. coli OMVs by 160 times, this gene and its homologous genes are not present in Bordetella pertussis. Eline F. de Jonge et al. found that the inactivation of rmpM, tolR and pal genes in Bordetella pertussis failed, and speculated that the reason may be that the products of rmpM, tolR and pal (PG-associated lipoprotein) genes are essential in Bordetella pertussis. Furthermore, Eline F. de Jonge et al. constructed conditional pal gene mutants, which increased the production of Bordetella pertussis OMVs under pal depletion conditions, but the composition of OMVs released by wild-type cells was different (Research in Microbiology, Volume 173, Issues 4-5,2022, 103937). The ompA gene encodes an outer membrane protein of Bordetella pertussis strains. Studies have shown that knocking out ompA in E. coli can increase OMV production by 26 times. However, CN116438193A mentioned that OmpA and its homologues in Bordetella pertussis seem to be proteins necessary for the survival of Bordetella pertussis, and the absence of OmpA in Bordetella pertussis is not conducive to its survival. It can be seen that the method that can increase the production of OMVs in other bacteria may not be applicable to Bordetella pertussis.

[0009] Therefore, it is urgent to develop new methods to increase the production of OMVs of B. pertussis in order to develop new pertussis vaccines based on OMVs. Summary of the invention

[0010] In one aspect, the present invention provides a modified Bordetella pertussis strain comprising a mutation in the mltA gene compared to a corresponding control Bordetella pertussis strain, and thereby having increased bacterial outer membrane vesicle (OMV) production. In some embodiments, the modified Bordetella pertussis strain further comprises a mutation in the pldA gene.

[0011] In another aspect, the present invention provides a method for producing a modified Bordetella pertussis strain, the method comprising the steps of:

[0012] 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

[0013] 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.

[0014] In another aspect, the present invention provides a method for producing Bordetella pertussis outer membrane vesicles (OMVs), the method comprising the following steps:

[0015] a) culturing the modified Bordetella pertussis strain of the present invention under conditions suitable for producing outer membrane vesicles (OMVs); and

[0016] b) recovering and optionally purifying the OMVs produced in step a).

[0017] In another aspect, the invention provides an OMV produced by the method of the invention.

[0018] In another aspect, the present invention also provides a composition comprising

[0019] 1) a modified Bordetella pertussis strain of the present invention; and / or

[0020] 2) The Bordetella pertussis outer membrane vesicles (OMV) of the present invention.

[0021] In one aspect, the present invention provides use of the modified Bordetella pertussis strain of the present invention and / or the Bordetella pertussis outer membrane vesicles (OMV) of the present invention in the preparation of a composition for preventing or treating Bordetella pertussis infection.

[0022] In one aspect, the present invention provides a method for preventing and / or treating a Bordetella pertussis infection in a subject, the method comprising administering to the subject an effective amount of a modified Bordetella pertussis strain of the present invention, a Bordetella pertussis outer membrane vesicle (OMV) of the present invention and / or a composition of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 . Homologous recombination backbone plasmid map.

[0024] Figure 2 . PCR verification of the secondary recombinant strain in which mltA gene was knocked out in BAA-589 strain using homologous recombination method.

[0025] Figure 3 . Comparison of sequencing results of BAA-589ΔmltA strain.

[0026] Figure 4 . Comparison of strain OD concentrations between BAA-589ΔmltA strain and wild-type strain BAA-589 after 24 hours of culture.

[0027] Figure 5 . Comparison of OMV particle concentrations secreted by the BAA-589ΔmltA strain and the wild-type strain BAA-589.

[0028] Figure 6. PCR verification of the secondary recombinant strain in which the pldA gene was knocked out in the BAA-589 strain using the homologous recombination method.

[0029] Figure 7 . Comparison of sequencing results of BAA-589ΔpldA strain.

[0030] Figure 8 . Comparison of strain OD concentrations between BAA-589ΔpldA strain and wild-type strain BAA-589 after 24 hours of culture.

[0031] Fig. 9 . Comparison of OMV particle concentrations secreted by the BAA-589ΔpldA strain and the wild-type strain BAA-589.

[0032] Fig.10 . PCR verification of the secondary recombinant strain in which mltA gene was knocked out in BAA-589ΔpldA strain using homologous recombination method.

[0033] Fig.11 . Comparison of sequencing results of BAA-589ΔpldAΔmltA strain.

[0034] Fig.12 . Comparison of OMV particle concentrations secreted by the BAA-589ΔpldAΔmltA strain and the wild-type strain BAA-589.

[0035] Fig.13 . Comparison of strain OD concentrations between BAA-589ΔpldAΔmltA strain and wild-type strain BAA-589 after 24 hours of culture. DETAILED DESCRIPTION

[0036] I. Definition

[0037] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are the terms and routine procedures widely used in the corresponding fields.

[0038] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0039] "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 names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide. Although nucleotide sequences herein may be represented as DNA sequences (including T), when RNA is referred to, a person skilled in the art can easily determine the corresponding RNA sequence (i.e., replacing T with U).

[0040] "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 residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0041] The term "gene" as used herein may refer to a coding sequence for a protein, but may also encompass expression regulatory elements / sequences, such as promoters, enhancers, etc. "Gene" typically refers to an endogenous sequence of an organism such as Bordetella pertussis.

[0042] When the term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid sequence, but still have the activity described in the present invention.

[0043] "Sequence identity" between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for evaluating the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be evaluated using various known sequence analysis software. For example, sequence identity can be evaluated by the online alignment tool of EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ). The sequence identity between two sequences can be evaluated using the Needleman-Wunsch algorithm using default parameters. The sequence identity can be the sequence identity along the entire length of a given sequence.

[0044] The "mltA gene" described in this article encodes the MltA protein, which is a lipoprotein located on the outer membrane. The protein has peptidoglycan cleavage exotransglycosylase activity, participates in the metabolism of peptidoglycan, and plays a role in the separation of cells and the construction of membranes.

[0045] The MItA protein in Bordetella pertussis may 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 MItA protein in Bordetella pertussis may comprise the amino acid sequence shown in SEQ ID NO: 6.

[0046] The mltA gene in Bordetella pertussis may 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 may comprise the nucleotide sequence shown in SEQ ID NO: 7.

[0047] The "pldA gene" described herein encodes the PldA protein, which is a phospholipase that can degrade phospholipids on the outer membrane and release fatty acids.

[0048] The PldA protein in Bordetella pertussis may 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 may comprise the amino acid sequence shown in SEQ ID NO: 10.

[0049] The pldA gene in Bordetella pertussis may 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 may comprise the nucleotide sequence set forth in SEQ ID NO: 11.

[0050] As used herein, "effective amount" refers to an amount of a substance, compound, material, or composition containing a compound (such as an OMV of the present invention or a composition of the present invention) that is at least sufficient to produce a preventive or therapeutic effect after administration to a subject. Therefore, an effective amount is an amount necessary to prevent, cure, improve, delay, or partially delay the symptoms of a disease or condition such as a bacterial infection.

[0051] The "subject" mentioned herein preferably refers to a human.

[0052] 2. Modified Bordetella pertussis strains

[0053] In one aspect, the present invention provides a modified Bordetella pertussis strain comprising a mutation in the mltA gene and thereby having increased bacterial outer membrane vesicle (OMV) production compared to a corresponding control Bordetella pertussis strain.

[0054] In some embodiments, the mutation in the mltA gene results in reduced expression of the MItA protein. In some embodiments, the mutation in the mltA gene results in reduced expression of the MItA protein 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, preferably, the mutation in the mltA gene results in the strain not expressing the MItA protein.

[0055] In some embodiments, the mutation in the mltA gene results in the expression of a mutant MItA protein with reduced activity. In some embodiments, the activity of the mutant MItA 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 MItA protein, preferably, the mutant MItA protein is an inactivated MItA protein. The activity is, for example, peptidoglycan cleavage exotransglycosylase activity.

[0056] 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 includes 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 so that the mltA gene is not present in the strain of the present invention, thereby the MltA protein is not present. The mltA gene can also be partially deleted so that only a truncated MltA protein with reduced activity or no activity is present in the strain of the present invention.

[0057] In some embodiments, the modified Bordetella pertussis strain further comprises a mutation in the pldA gene compared to a corresponding control Bordetella pertussis strain.

[0058] In some embodiments, the mutation in the pldA gene results in reduced expression of the PldA protein. In some embodiments, the mutation in the pldA gene results in a reduction in expression of the PldA protein 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, preferably, the mutation in the pldA gene results in the strain not expressing the PldA protein.

[0059] In some embodiments, the mutation in the pldA gene results in the expression of 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 the wild-type PldA protein, preferably, the mutant PldA protein is an inactivated PldA protein. The activity is, for example, phospholipase activity.

[0060] 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 includes 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 so that there is no pldA gene in the strain of the present invention, thereby there is no PldA protein. The pldA gene can also be partially deleted so that only a truncated PldA protein with reduced activity or inactivity is present in the strain of the present invention.

[0061] The mutation in the gene can be achieved by various means known in the art. In some embodiments, the mutation is introduced into the Bordetella 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 the deletion of the gene, can be carried out by homologous recombination. The method of homologous recombination can be found in patent document CN 117947054 B. In some embodiments, the mutation in the gene is carried out by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

[0062] In some embodiments, the mltA gene is deleted by homologous recombination using the homology arm sequences shown in SEQ ID NOs: 8 and 9. In some embodiments, the pldA gene is deleted by homologous recombination using the homology arm sequences shown in SEQ ID NOs: 12 and 13.

[0063] In some embodiments, the bacterial outer membrane vesicle (OMV) production of the modified Bordetella pertussis strain of the present invention 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 compared to the corresponding control Bordetella pertussis strain. In some embodiments, the bacterial outer membrane vesicle (OMV) production of the modified Bordetella pertussis strain of the present invention is about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times or more times the OMV production of the corresponding control Bordetella pertussis strain.

[0064] The "control Bordetella pertussis strain" described herein may be a parent strain from which the modified Bordetella pertussis strain is derived. For example, the control Bordetella pertussis strain does not comprise a mutation in the mltA gene and / or a mutation in the pldA gene.

[0065] The modified Bordetella pertussis strains described herein may be derived from any known Bordetella pertussis strain, such as strain BAA-589.

[0066] 3. Methods for Producing Modified Bordetella Pertussis Strains

[0067] The present inventors surprisingly found that first deleting the pldA gene in Bordetella pertussis can significantly increase the success rate of subsequently deleting the mltA gene by homologous recombination.

[0068] Therefore, in another aspect, the present invention provides a method for producing a modified Bordetella pertussis strain, the method comprising the steps of:

[0069] 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

[0070] 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.

[0071] In some embodiments, the mutation introduced into the pldA gene results in reduced expression of the PldA protein in the Bordetella pertussis strain. In some embodiments, the mutation introduced into the pldA gene results in a reduction in expression of the PldA protein 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, preferably, the mutation in the pldA gene results in the strain not expressing the PldA protein.

[0072] In some embodiments, the mutation introduced into the pldA gene causes the Bordetella pertussis strain to express 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 the wild-type PldA protein, preferably, the mutant PldA protein is an inactivated PldA protein. The activity is, for example, phospholipase activity.

[0073] 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 includes 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 so that the pldA gene is not present in the modified strain, thereby the PldA protein is not present. The pldA gene can also be partially deleted so that only a truncated PldA protein with reduced activity or no activity is present in the modified strain.

[0074] In some embodiments, the mutation introduced into the mltA gene results in reduced expression of the MItA protein. In some embodiments, the mutation introduced into the mltA gene results in reduced expression of the MItA protein 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, preferably, the mutation introduced into the mltA gene results in the strain not expressing the MItA protein.

[0075] In some embodiments, the mutation introduced into the mltA gene results in the expression of a mutant MItA protein with reduced activity. In some embodiments, the activity of the mutant MItA 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 MItA protein, preferably, the mutant MItA protein is an inactivated MItA protein. The activity is, for example, peptidoglycan cleavage exotransglycosylase activity.

[0076] The mutation introduced into the mltA gene can be an addition, substitution or deletion of one or more nucleotides. In some embodiments, the mutation introduced into the mltA gene includes 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, so that the mltA gene is not present in the modified strain, and thus the MltA protein is not present. The mltA gene can also be partially deleted, so that only a truncated MltA protein with reduced activity or no activity is present in the modified strain.

[0077] In some embodiments, the modified B. pertussis strain obtained comprises mutations in both the mltA gene and the pldA gene.

[0078] In some embodiments, mutations are introduced into the genes of the Bordetella pertussis strain by genetic engineering. For example, the mutations in a gene, such as deletions of a gene, can be introduced by homologous recombination. The method of homologous recombination can be found in patent document CN 117947054 B. In some embodiments, the mutations in a gene are introduced by targeted mutagenesis, such as via CRISPR, TALEN or ZFN technology.

[0079] In some specific embodiments, the method comprises the following steps:

[0080] a) deleting the pldA gene in the Bordetella pertussis strain by homologous recombination, thereby obtaining a modified Bordetella pertussis strain in which the pldA gene is deleted; and

[0081] 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 deleted in both the pldA gene and the mltA gene.

[0082] In another aspect, the present invention also provides a modified Bordetella pertussis strain produced by the method of the present invention.

[0083] In some embodiments, the bacterial outer membrane vesicle (OMV) production of the modified Bordetella pertussis strain produced by the method of the present invention 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 compared to the corresponding control Bordetella pertussis strain. In some embodiments, the bacterial outer membrane vesicle (OMV) production of the modified Bordetella pertussis strain of the present invention is about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times or more times the OMV production of the corresponding control Bordetella pertussis strain.

[0084] 4. Production and Use of Bacterial Outer Membrane Vesicles (OMVs)

[0085] In another aspect, the present invention provides a method for producing Bordetella pertussis outer membrane vesicles (OMVs), the method comprising the following steps:

[0086] a) culturing the modified Bordetella pertussis strain of the present invention under conditions suitable for producing outer membrane vesicles (OMVs); and

[0087] b) recovering and optionally purifying the OMVs produced in step a).

[0088] In another aspect, the present invention provides Bordetella pertussis outer membrane vesicles (OMVs) produced by the methods of the present invention.

[0089] In another aspect, the present invention provides a composition comprising an effective amount of

[0090] 1) a modified Bordetella pertussis strain of the present invention; and / or

[0091] 2) The Bordetella pertussis outer membrane vesicles (OMV) of the present invention.

[0092] In some embodiments, the composition is a vaccine. In some embodiments, the composition is used to prevent and / or treat Bordetella pertussis infection in a subject.

[0093] The composition may also include an adjuvant. As used herein, "adjuvant" refers to an additional component in a vaccine that enhances the immune response, or an auxiliary molecule added to a vaccine, or an auxiliary molecule produced by the body after being induced by such additional components, such as but not limited to interferons, interleukins, or growth factors. "Adjuvants" used herein may include aluminum hydroxide and aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions.

[0094] In some embodiments of the above aspects, the composition may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, saline, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose), amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, and colorants.

[0095] In some embodiments of the above aspects, the composition is formulated 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 before use.

[0096] In one aspect, the present invention provides use of the modified Bordetella pertussis strain of the present invention and / or the Bordetella pertussis outer membrane vesicles (OMV) of the present invention in the preparation of a composition for preventing or treating Bordetella pertussis infection.

[0097] In one aspect, the present invention provides a method for preventing and / or treating a Bordetella pertussis infection in a subject, the method comprising administering to the subject an effective amount of a modified Bordetella pertussis strain of the present invention, a Bordetella pertussis outer membrane vesicle (OMV) of the present invention and / or a composition of the present invention.

[0098] Example

[0099] The present invention can be further understood by reference to the specific embodiments described herein, which are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Obviously, those skilled in the art know that various modifications and changes can be made to the present invention without departing from the spirit of the present invention, so such modifications and changes also belong to the scope of the present invention. In order to illustrate the technical scheme and technical effects of the present invention, the present invention uses the BAA-589 strain purchased from ATCC (American type culture collection).

[0100] Example 1: Homologous recombination method

[0101] 1.1 Construction of exogenous DNA donor plasmid

[0102] The exogenous backbone plasmid contains ori-ColE1 / pMB1 / pBR322 / pUC, the transfer elements OriT and traJ, the gentamicin resistance gene (Gen) and the galk anti-screening 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 Figure 1 As shown, the gentamicin resistance gene sequence is shown in SEQ ID NO: 2, and the galk anti-screening gene sequence is shown in SEQ ID NO: 3. The upstream and downstream homology arms of the target gene integration site are inserted between the upstream site-up (sequence shown in SEQ ID NO: 4) and the downstream site-down (sequence shown in SEQ ID NO: 5) of the exogenous backbone plasmid to obtain the exogenous DNA donor plasmid.

[0103] 1.2 Electroporation of donor plasmid

[0104] Activate the glycerol bacteria on a BG plate (Bordet-Gengo solid plate) and culture at 37°C until colonies grow. Inoculate the colonies in a SS liquid culture (Stainer-Scholte liquid culture medium) to expand the culture.

[0105] Use 10% glycerol buffer at about 4 degrees Celsius to wash several times, prepare the electroporation competence of the strain, and perform electroporation on an electroporator (BTX, ECM630). The electroporation parameters are set as follows: 1.6-2.2kV, 200Ω, 25uF, and the electroporation time does not exceed 5ms. After electroporation, quickly add SS liquid culture medium to recover. Transfer the system to a sterile EP tube and culture it on a shaker at 35-37℃ and 220-240rpm for recovery. After recovery, centrifuge and discard the supernatant, add SS liquid culture medium, resuspend the bacteria, and spread it on a BG solid plate containing 10ug / mL gentamicin resistance (BG-G). The plate is placed in an environment of 35-37℃ and cultured. A single clone is grown, which is a possible primary recombinant strain (1HR).

[0106] 1.3 Verification of primary recombinant strain

[0107] Use a sterile pipette tip to randomly pick single clones grown on multiple BG-G screening plates and streak them on new BG-G plates. Dip the remaining bacteria on the pipette tip into 50uL sterile water as a PCR verification template. Use the upstream homology arm integration-specific verification primers and / or downstream integration-specific verification primers for verification, and select the clones with the correct recombination.

[0108] 1.4 Counter-screening culture and verification of secondary recombinant strains

[0109] Select the clones with the correct primary recombination and streak them on a BG plate containing 2% DOG (2-Deoxy-D-galactose). The plate is placed in a 37°C environment and the single clone that grows is the possible secondary recombination strain (2HR). Use a sterile pipette tip to randomly pick a number of single clones grown on the BG-2% DOG screening plate and streak them on a new BG plate. Dip the remaining bacteria on the tip of the pipette into 50uL sterile water as a PCR verification template, and use the genome external primers for verification to select the clones with the correct secondary recombination.

[0110] The genomic external primer refers to a primer using the upstream sequence of the upstream homology arm as a forward primer (excluding the upstream homology arm itself) and a primer using the downstream sequence of the downstream homology arm as a reverse primer (excluding the downstream homology arm itself).

[0111] Example 2: Screening for genes that increase OMV production in Bordetella pertussis

[0112] The common bacterial OMV production-increasing genes rmpM, tolR, pal, and ompA reported in the prior art cannot achieve the effect of increasing OMV production in Bordetella pertussis because they do not exist in pertussis or are essential genes for the survival of pertussis. Therefore, the inventors further tried other potential production-increasing genes pagP and ompP that have not been reported.

[0113] 2.1 No increase in OMV production after knocking out the outer membrane protein pagP gene

[0114] The pagP gene encodes an outer membrane protein of a pertussis strain, which can degrade phospholipids and transfer the myristoyl group on the phospholipids to the lipopolysaccharide LPS, thereby adjusting its toxicity by modifying the structure of LPS. The inventor speculated based on its function that after the pagP gene was knocked out, phospholipids could not be degraded, which may increase the phospholipid content of the outer membrane, and ultimately increase the curvature of the membrane and promote the secretion of OMVs. However, in pertussis strains, there is currently no report on the OMV yield of strains characterized by knocking out the pagP gene. The inventor successfully constructed a BAA-589 strain (BAA-589ΔpagP) with a pagP gene knockout by homologous recombination according to the method of Example 1, but after testing, it was found that the OMV particle number concentration secreted by the BAA-589ΔpagP strain was slightly lower than that of the wild-type strain, and there was no yield-increasing effect.

[0115] 2.2 Unable to obtain strains with inactivated ompP gene

[0116] The ompP gene encodes an outer membrane protein of a pertussis strain, which is extremely abundant. However, the homologous recombination method of Example 1 cannot obtain a strain with an inactivated ompP gene. The inventors speculate that the ompP gene is essential for the survival of the pertussis BAA-589 strain, and after inactivating or knocking out the gene, the strain cannot survive.

[0117] Example 3: Knockout of mltA gene increased OMV production of Bordetella pertussis by 3.1 times, but the gene editing success rate was too low

[0118] After the screening failure in Example 2, the inventors noticed that the mltA gene encodes an outer membrane lipoprotein of a pertussis strain. There has been no report on the successful knockout of mltA in pertussis strains. Therefore, the inventors tried to explore the effect of the mltA gene on the OMV of Bordetella pertussis.

[0119] The inventors used the homologous recombination method in Example 1 to knock out the mltA gene in the BAA-589 strain of pertussis, specifically, the exogenous donor DNA plasmid carrying the upstream homologous arm and the downstream homologous arm of the mltA gene was electrotransduced into the BAA-589 strain of pertussis. The primary recombinant strain was screened in BG-G medium, and the secondary recombinant strain was screened in galk medium. The nucleotide sequence of mltA is shown in SEQ ID NO: 7, the upstream homologous arm sequence used in the gene editing process using the homologous recombination method 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-specific verification primers for the primary homologous recombination are PTTU344+BpeLYF004, and the downstream integration-specific verification primers are (BpeLYF003+PTTU361). The genomic external primers for the secondary homologous recombination are PTTU344 and PTTU361. The sequencing primers are PTTU344, PTTU361, PTTU183 and PTTU229. Primer information is shown in Table 1.

[0120] Table 1: Primer information for mltA knockout

[0121]

[0122] The inventors found that the editing success rate was very low during the process of knocking out the mltA gene. The success rate of the first recombination was 100% (all 8 clones were positive), but in the second recombination stage, the knockout success rate was only 1 / 24. Specifically, according to the method of Example 1.4, 24 clones were selected for PCR verification, and the results were as follows: Figure 2 As shown in the figure, secondary recombination occurred in all 24 clones, but only clone 2 had mltA gene knockout, and the other 23 clones returned to the wild type, so the gene editing success rate was only 1 / 24 (4.2%). Figure 3As shown, clone 2 was further sequenced to confirm that mltA had been successfully knocked out, obtaining strain BAA-589ΔmltA. The applicant speculates that the reason why there is no report on the knockout of mltA gene in Bordetella pertussis in the prior art is that the mltA gene is difficult to be successfully knocked out.

[0123] After the strain was successfully constructed, the inventors further evaluated the growth status of the BAA-589ΔmltA strain and the yield of secreted OMVs. The BAA-589 and BAA-589ΔmltA strains activated on the BG plate were inoculated in test tubes containing 3 ml of SS culture medium and cultured overnight on a shaker at 35°C and 220 rpm. Measure the OD600 of the seed liquid, take an appropriate amount of the seed liquid and re-inoculate it into a test tube containing 3 ml of SS culture medium, control the starting OD600 = 0.2, place the test tube on a shaker at 35°C and 220 rpm and culture for about 24 hours, and collect the sample. Measure the OD600 of the fermentation liquid, and the results are as follows: Figure 4 As shown, it can be seen that the absence of mltA in the BAA-589 strain does not affect its survival and growth. Take 1 ml of the fermentation sample, centrifuge at 10,000 rpm for 5 minutes, filter the supernatant using a 0.22 um filter membrane, and collect the filtrate. The OMV concentration is measured using a nanoparticle tracking analyzer (Malvern, NanoSight Pro). The results are shown in Figure 5 As shown in the characterization results, under the above test tube culture conditions, the OMV particle number concentration secreted by the BAA-589ΔmltA strain was 3.1 times that of the wild-type strain, and the yield-increasing effect was significant.

[0124] Example 4: Knocking out the pldA gene first can improve the success rate of knocking out the mltA gene in the BAA-589 strain

[0125] The inventors accidentally discovered that by knocking out the mltA gene in the BAA-589 strain in which the pldA gene had been knocked out, the gene knockout success rate reached 90%. The knockout success rate was significantly improved from the previous 4.2% to 90%, and the gene editing success rate was increased by nearly 22 times.

[0126] pldA is the phospholipase gene of Bordetella pertussis, which can degrade phospholipids on the outer membrane and release fatty acids. Eline F. de Jonge et al. found that when a large amount of phospholipids accumulate in the outer membrane of the cell, pldA functions to degrade phospholipids and produce a large amount of fatty acids, leading to cytotoxicity, which seriously affects the growth of the strain ((Research in Microbiology, Volume 173, Issues 4–5, 2022, 103937)). However, the inventors did not find a significant effect of the deletion of the pldA gene on the survival and growth of the BAA-589ΔmltA strain (such as Figure 8 shown).

[0127] The pldA gene was knocked out in the BAA-589 strain according to the homologous recombination method in Example 1 to obtain the BAA-589ΔpldA strain.

[0128] The nucleotide sequence of pldA is shown in SEQ ID NO: 11, the upstream homology arm sequence used in the gene editing process using the homologous recombination method is shown in SEQ ID NO: 12, and the downstream homology arm sequence is shown in SEQ ID NO: 13. The upstream homology arm integration-specific verification primers for the first homologous recombination are PTTU135+PTTU222, and the downstream integration-specific verification primers (PTTU221+PTTU136). The genomic external primers for the second homologous recombination are PTTU354+PTTU380. The sequencing primers are PTTU76, PTTU354 and PTTU380. The primer information is shown in Table 2.

[0129] Table 2: Primer information for pldA knockout

[0130]

[0131] The success rate of the first recombination was 100% (all 8 clones were positive), and the knockout success rate of the second recombination was 3 / 8 ( Figure 6 ).like Figure 7 As shown, clone 2 was further sequenced to confirm that pldA had been successfully knocked out, obtaining strain BAA-589ΔpldA.

[0132] After the strain was successfully constructed, the applicant further evaluated the growth status and secreted OMV production of the BAA-589ΔpldA strain. According to the culture and detection methods in Example 3, the growth of the BAA-589ΔpldA strain was not significantly different from that of the wild type ( Figure 8 ), and the concentration of OMV particles secreted by the BAA-589ΔpldA strain was not significantly different from that of the wild type ( Fig. 9 ).

[0133] Then, the mltA gene was knocked out in the BAA-589ΔpldA strain according to the homologous recombination method and experimental materials of Example 1.

[0134] During the process of knocking out the mltA gene in the BAA-589ΔpldA strain, the secondary recombinant strain PCR verification stage, e.g. Fig.10 As shown in the figure, secondary recombination occurred in all 20 clones. Among them, clones 9# and 16# returned to wild type (WT), and the remaining 18 clones all achieved mltA knockout, with a gene editing success rate of 90%. The sequencing verification results of clone 2 are shown in the figure. Fig.11 shown.

[0135] Example 5: OMV production increased 4.7-fold in BAA-589ΔpldAΔmltA strain

[0136] The inventors found that knocking out mltA on BAA-589, where the pldA gene had been knocked out, not only significantly improved the gene editing success rate, but also the OMV production of the BAA-589ΔpldAΔmltA strain was more significantly increased (4.7 times, as compared to the BAA-589ΔmltA strain (3.1 times). Fig.12 ), while the growth and survival of BAA-589ΔpldAΔmltA were not affected (as shown in Fig.13 shown).

[0137] Sequence information involved in this application

[0138] SEQ ID NO: 1 Nucleotide sequence of homologous recombination backbone plasmid

[0139] SEQ ID NO: 2 Nucleotide sequence of gentamicin resistance gene

[0140] SEQ ID NO: 3 Nucleotide sequence of galk anti-screening gene

[0141] SEQ ID NO: 4 Site-up nucleotide sequence of the backbone plasmid insertion site

[0142] SEQ ID NO: 5 Nucleotide sequence of site-downstream of backbone plasmid insertion site

[0143] SEQ ID NO: 6 Amino acid sequence of mltA from B. pertussis strain BAA-589

[0144] SEQ ID NO: 7 Nucleotide sequence of mltA from B. pertussis strain BAA-589

[0145] SEQ ID NO: 8 Nucleotide sequence of the upstream homology arm used for mltA knockout of B. pertussis BAA-589 strain

[0146] SEQ ID NO: 9 Nucleotide sequence of the downstream homology arm used for mltA knockout of B. pertussis BAA-589 strain

[0147] SEQ ID NO: 10 Amino acid sequence of pldA of B. pertussis strain BAA-589

[0148] SEQ ID NO: 11 Nucleotide sequence of pldA of B. pertussis strain BAA-589

[0149] SEQ ID NO: 12 Nucleotide sequence of the upstream homology arm used for pldA knockout of B. pertussis BAA-589 strain

[0150] SEQ ID NO: 13 Nucleotide sequence of the downstream homology arm used for the pldA knockout of B. pertussis BAA-589 strain.

Claims

1. A modified Bordetella pertussis strain comprising a mutation in the mltA gene and thereby having increased bacterial outer membrane vesicle (OMV) production compared to a corresponding control Bordetella pertussis strain, wherein the mutation in the mltA gene causes the strain to not express the MItA protein or to express an inactivated MItA protein, and The modified Bordetella pertussis strain is derived from strain BAA-589.

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

3. The modified Bordetella pertussis strain according to claim 1, wherein the mltA gene encodes the amino acid sequence shown in SEQ ID NO: 6 or the nucleotide sequence thereof is shown in SEQ ID NO:

7.

4. The modified Bordetella pertussis strain according to any one of claims 1 to 3, further comprising a mutation in the pldA gene, wherein the mutation in the pldA gene causes the strain to not express the PldA protein or to express an inactive PldA protein.

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

6. The modified Bordetella pertussis strain according to claim 4, wherein the pldA gene encodes the amino acid sequence shown in SEQ ID NO: 10 or the nucleotide sequence thereof is shown in SEQ ID NO:

11.

7. A method for 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, wherein the mutation introduced into the pldA gene causes the strain to not express PldA protein or to express an inactivated PldA protein; the mutation introduced into the mltA gene causes the strain to not express MltA protein or to express an inactivated MltA protein, The modified Bordetella pertussis strain is derived from strain BAA-589.

8. The method according to claim 7, wherein the mutation introduced into the pldA gene comprises a deletion of the pldA gene, and the mutation introduced into the mltA gene comprises a deletion of the mltA gene.

9. The method according to claim 7, wherein the pldA gene encodes the amino acid sequence shown in SEQ ID NO: 10 or its nucleotide sequence is shown in SEQ ID NO: 11; the mltA gene encodes the amino acid sequence shown in SEQ ID NO: 6 or its nucleotide sequence is shown in SEQ ID NO:

7.

10. The method according to any one of claims 7 to 9, wherein the mutation is introduced by homologous recombination.

11. A method for producing Bordetella pertussis outer membrane vesicles (OMV), the method comprising the following steps: a) culturing a modified Bordetella pertussis strain according to any one of claims 1 to 6 or a modified Bordetella pertussis strain produced by the method of any one of claims 7 to 10 under conditions suitable for producing outer membrane vesicles (OMVs); and b) recovering and optionally purifying the OMVs produced in step a).

12. A composition comprising an effective amount of the modified Bordetella pertussis strain according to any one of claims 1 to 6 or a modified Bordetella pertussis strain produced by the method of any one of claims 7 to 10.

13. Use of a modified Bordetella pertussis strain according to any one of claims 1 to 6, and / or a Bordetella pertussis strain produced by the method of any one of claims 7 to 10, and / or a composition according to claim 12 in the preparation of a medicament or vaccine for preventing and / or treating Bordetella pertussis infection in a subject.

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