CN117940554A - Live bacterial strains of the genus Pseudomonas - Google Patents
Live bacterial strains of the genus Pseudomonas Download PDFInfo
- Publication number
- CN117940554A CN117940554A CN202380013541.5A CN202380013541A CN117940554A CN 117940554 A CN117940554 A CN 117940554A CN 202380013541 A CN202380013541 A CN 202380013541A CN 117940554 A CN117940554 A CN 117940554A
- Authority
- CN
- China
- Prior art keywords
- bacterial strain
- live bacterial
- oprf
- protein
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 174
- 241000589516 Pseudomonas Species 0.000 title claims description 23
- 238000000034 method Methods 0.000 claims abstract description 55
- 230000002829 reductive effect Effects 0.000 claims abstract description 45
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 39
- 241000894007 species Species 0.000 claims abstract description 36
- 230000000694 effects Effects 0.000 claims abstract description 29
- 229960005486 vaccine Drugs 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 99
- 101150071603 oprF gene Proteins 0.000 claims description 83
- 102000004169 proteins and genes Human genes 0.000 claims description 78
- 230000035772 mutation Effects 0.000 claims description 51
- 230000014509 gene expression Effects 0.000 claims description 47
- 239000000203 mixture Substances 0.000 claims description 27
- 238000012217 deletion Methods 0.000 claims description 23
- 230000037430 deletion Effects 0.000 claims description 23
- 208000035143 Bacterial infection Diseases 0.000 claims description 22
- 208000022362 bacterial infectious disease Diseases 0.000 claims description 22
- 108091026890 Coding region Proteins 0.000 claims description 20
- 230000001018 virulence Effects 0.000 claims description 17
- 230000005847 immunogenicity Effects 0.000 claims description 16
- 239000000427 antigen Substances 0.000 claims description 15
- 108091007433 antigens Proteins 0.000 claims description 15
- 102000036639 antigens Human genes 0.000 claims description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 14
- 208000015181 infectious disease Diseases 0.000 claims description 13
- 210000004027 cell Anatomy 0.000 claims description 12
- 238000002744 homologous recombination Methods 0.000 claims description 10
- 230000006801 homologous recombination Effects 0.000 claims description 10
- 101150026476 PAO1 gene Proteins 0.000 claims description 8
- 108091033409 CRISPR Proteins 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 238000010354 CRISPR gene editing Methods 0.000 claims description 6
- 101100244394 Cereibacter sphaeroides pmtA gene Proteins 0.000 claims description 6
- 101100295833 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) oprI gene Proteins 0.000 claims description 6
- 241000191967 Staphylococcus aureus Species 0.000 claims description 6
- 241000193998 Streptococcus pneumoniae Species 0.000 claims description 6
- 238000010459 TALEN Methods 0.000 claims description 6
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims description 6
- 101150016690 esxA gene Proteins 0.000 claims description 6
- 101150079015 esxB gene Proteins 0.000 claims description 6
- 238000002703 mutagenesis Methods 0.000 claims description 6
- 231100000350 mutagenesis Toxicity 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 230000036961 partial effect Effects 0.000 claims description 6
- 229940031000 streptococcus pneumoniae Drugs 0.000 claims description 6
- 239000002671 adjuvant Substances 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 5
- 230000002265 prevention Effects 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 2
- 230000003248 secreting effect Effects 0.000 claims description 2
- 208000032536 Pseudomonas Infections Diseases 0.000 abstract description 5
- 241000589774 Pseudomonas sp. Species 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 description 9
- 230000000890 antigenic effect Effects 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 210000002540 macrophage Anatomy 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000011725 BALB/c mouse Methods 0.000 description 4
- -1 coatings Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241001240958 Pseudomonas aeruginosa PAO1 Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 230000032770 biofilm formation Effects 0.000 description 2
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 2
- 229960003669 carbenicillin Drugs 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000017555 immunoglobulin mediated immune response Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 108020005065 3' Flanking Region Proteins 0.000 description 1
- 108020005029 5' Flanking Region Proteins 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 238000011814 C57BL/6N mouse Methods 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 229920002444 Exopolysaccharide Polymers 0.000 description 1
- 101710082714 Exotoxin A Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 101710105759 Major outer membrane porin Proteins 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 108010025955 Pyocins Proteins 0.000 description 1
- 208000035415 Reinfection Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 108091027544 Subgenomic mRNA Proteins 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 108010069584 Type III Secretion Systems Proteins 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 238000011203 antimicrobial therapy Methods 0.000 description 1
- 229960001212 bacterial vaccine Drugs 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000007110 pathogen host interaction Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 210000000680 phagosome Anatomy 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000018612 quorum sensing Effects 0.000 description 1
- 231100000272 reduced body weight Toxicity 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
- C12R2001/385—Pseudomonas aeruginosa
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to the biomedical field. In particular, the invention relates to a live bacterial strain from a species of Pseudomonas sp, such as Pseudomonas aeruginosa Pseudomonas aeruginosa, and uses thereof. More particularly, the present invention relates to a live bacterial strain of pseudomonas aeruginosa with reduced OprF activity, a vaccine against pseudomonas aeruginosa infection comprising said live bacterial strain, and a method for preventing and/or treating pseudomonas aeruginosa infection in a subject by administering said live bacterial strain.
Description
Technical Field
The present invention relates to the biomedical field. In particular, the invention relates to a live bacterial strain from a species of Pseudomonas sp, such as Pseudomonas aeruginosa Pseudomonas aeruginosa, and uses thereof. More particularly, the present invention relates to a live bacterial strain of pseudomonas aeruginosa with reduced OprF activity, a vaccine against pseudomonas aeruginosa infection comprising said live bacterial strain, and a method for preventing and/or treating pseudomonas aeruginosa infection in a subject by administering said live bacterial strain.
Background
Pseudomonas aeruginosa is a ubiquitous gram-negative bacterium that can survive in a broad range of natural environments. It is also an opportunistic human pathogen associated with hospital acquired infections such as sepsis in immunocompromised patients, intestinal and pulmonary infections, and is also a major cause of morbidity and mortality in individuals with cystic fibrosis. Since pseudomonas aeruginosa is resistant to many of the currently available antibiotics, treatment of this bacteria has become a significant challenge. Pseudomonas aeruginosa strains are known to resist most antibiotics by their high level of intrinsic and acquired resistance mechanisms. Furthermore, the adaptive antibiotic resistance of pseudomonas aeruginosa is a newly disclosed mechanism that involves biofilm-mediated drug resistance and the formation of multi-drug resistant durable cells and can lead to persistent and recurrent infections 1. The adaptability of this opportunistic pathogen hampers the development of antimicrobial therapy and therefore it remains a major threat to public health.
Pseudomonas aeruginosa was originally classified as an extracellular pathogen. However, many reports underscores their ability to enter host cells, resulting in an intracellular residence phase, which may be an important infection beyond traditional extracellular infections. Recently, pseudomonas aeruginosa has been shown to be located within cultured macrophages. Fate studies of this bacterium within macrophages have found that vacuole escape of pseudomonas aeruginosa and macrophage death driven by intracellular bacteria is likely to be related to cytoplasmic location of the bacterium 2. Bacterial factors involved in this macrophage inner step were also studied. Among these, oprF has been found to be one of the key factors involved in the survival of P.aeruginosa in macrophages 3.
OprF is a major outer membrane porin that is involved in maintenance of cellular structure, outer membrane permeability, environmental sensing, adhesion, biofilm formation and virulence. It allows non-specific diffusion of ionic species and small polar nutrients, including polysaccharides 4 with molecular weights up to 1.5 kDa. OprF anchors OM to the peptidoglycan layer and participates in host-pathogen interactions. The absence of OprF leads to increased biofilm formation and production of Pel exopolysaccharides and has been shown to be necessary for expression of complete virulence 5,6. Furthermore, recent studies indicate that OprF regulates transcription of type III secretion system (T3 SS) genes. T3SS and its ExoS effectors play a major role in the survival of P.aeruginosa macrophages, allowing internalized bacteria to escape the phagosome and promote macrophage lysis. Consistent with the effect of OprF on T3SS gene transcription, oprF regulates production of T3SS PCRV CAP protein and secretion of ExoT and ExoS toxins 3. Furthermore, oprF also regulates production 5 of quorum sensing-dependent virulence factors, pyocin, elastase, lectin PA-1L, and exotoxin A, indicating that OprF acts as a sensor of the host immune system and plays a role in the immune escape of P.aeruginosa infected hosts.
Vaccines represent an alternative strategy to combat pathogens due to their antimicrobial resistance, and despite more than 50 years of research into anti-pseudomonas vaccines, no vaccine has been licensed 7. There remains a need for other methods to produce effective vaccines.
Disclosure of Invention
With the rapid development of genome modification technology, the use of synthetic bacterial vectors in vaccines has become one of the promising strategies. The aim of this study was to construct a bacterial vaccine vector that could be modulated using efficient antigen display and elicit immunogenicity in various models of pseudomonas aeruginosa infection. Aiming at immune escape of the targeting pseudomonas aeruginosa, the low toxicity is designed by knocking out the oprF gene in the pseudomonas aeruginosa. By doing so, the novel attenuated strain further exhibits high immunogenicity in a mouse model.
In this regard, the invention provides at least the following embodiments:
Embodiment 1. A live bacterial strain from a species of the genus pseudomonas, wherein the live bacterial strain lacks or has reduced OprF activity and/or the expression of the OprF gene in the live bacterial strain is reduced and/or the live bacterial strain of the invention contains a mutation of the OprF gene, e.g. compared to a corresponding control strain.
Embodiment 2. The live bacterial strain according to embodiment 1, wherein said species from the genus Pseudomonas is Pseudomonas aeruginosa.
Embodiment 3. The live bacterial strain of embodiment 1 or 2, wherein the activity of OprF in the live bacterial strain 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 compared to a corresponding control strain, preferably the live bacterial strain lacks OprF activity.
Embodiment 4. The live bacterial strain of any one of embodiments 1 to 3, wherein the expression of the oprF gene in the live bacterial strain is reduced, e.g., 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, as compared to a corresponding control strain, preferably the live bacterial strain lacks oprF gene expression.
Embodiment 5. The live bacterial strain of any of embodiments 1 to 4, wherein the oprF gene encodes an oprF protein having an amino acid sequence with 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 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID No. 1 or an amino acid sequence with SEQ ID No. 1.
Embodiment 6. The live bacterial strain of any one of embodiments 1 to 5, wherein the live bacterial strain contains a mutation of the OprF gene that results in a reduction or lack of OprF activity.
Embodiment 7. The live bacterial strain of embodiment 6, wherein mutation of the oprF gene results in reduced expression of the oprF protein or in reduced expression of a mutated oprF protein, preferably the mutation of the oprF gene results in non-expression of the oprF protein or in expression of an inactive mutated oprF protein.
Embodiment 8. The live bacterial strain according to embodiment 6 or 7, wherein the mutation comprises a deletion of the oprF gene, e.g. a complete deletion or a partial deletion of the oprF gene.
Embodiment 9. The live bacterial strain according to any of embodiments 6 to 8, the mutation is achieved by homologous recombination or by targeted mutagenesis, such as via CRISPR, TALEN or ZFN techniques.
Embodiment 10. The live bacterial strain of any of embodiments 1 to 9, wherein the live bacterial strain has a reduced virulence, e.g., the virulence of the live bacterial strain 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, as compared to a corresponding control strain.
Embodiment 11. The live bacterial strain of any of embodiments 1 to 10, wherein the live bacterial strain has increased immunogenicity as compared to a corresponding control strain, e.g., the immunogenicity of the live bacterial strain 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% or more.
Embodiment 12. The live bacterial strain of any of embodiments 1 to 11, wherein the live bacterial strain is derived from a parent strain as a clinical isolate.
Embodiment 13. The live bacterial strain of any of embodiments 1 to 11, wherein the live bacterial strain is derived from a parent strain that already has low virulence.
Embodiment 14. The live bacterial strain according to any of embodiments 1 to 11, wherein the live bacterial strain is derived from a pseudomonas aeruginosa strain PAO1 or PA14.
Embodiment 15. The live bacterial strain according to any one of embodiments 1 to 14, which is used as a live expression vector for expressing a protein of interest.
Embodiment 16. The live bacterial strain according to any of embodiments 1 to 15, further comprising a coding sequence for a protein of interest, and thereby being capable of expressing said protein of interest.
Embodiment 17. The live bacterial strain of embodiment 16, wherein the coding sequence for the protein of interest is introduced into the live bacterial strain, for example by a nucleic acid expression construct.
Embodiment 18. The live bacterial strain of embodiment 17, wherein the introduced coding sequence for the protein of interest is integrated into the genome of the live bacterial strain.
Embodiment 19 the live bacterial strain of any one of embodiments 16 to 18, wherein the protein of interest is expressed and displayed on the cell surface of the live bacterial strain; or cells of the living bacterial strain expressing and secreting the protein of interest.
Embodiment 20. The live bacterial strain according to any of embodiments 16 to 19, wherein the protein of interest is selected from antibodies or antigens, preferably the protein of interest is an antigen,
For example, the antigen is selected from: pcrV, oprI or oprJNM from pseudomonas aeruginosa; adsA, esxA, esxB, pmtA or PmtC from staphylococcus aureus (s.aureus); or PspA from Streptococcus pneumoniae (S.pneumoniae).
Embodiment 21. Use of the live bacterial strain according to any of embodiments 1 to 20 in the preparation of a composition, such as a vaccine, for the prevention or treatment of a bacterial infection.
Embodiment 22. The use according to embodiment 21, wherein the bacterial infection is an infection caused by a species from the genus Pseudomonas, such as Pseudomonas aeruginosa.
Embodiment 23. A composition, such as a vaccine, for preventing or treating a bacterial infection, comprising the live bacterial strain according to any one of embodiments 1 to 20.
Embodiment 24. The composition of embodiment 23, wherein the composition further comprises an adjuvant and/or a pharmaceutically acceptable carrier.
Embodiment 25. The use according to embodiment 23 or 24, wherein the bacterial infection is an infection caused by a species from the genus Pseudomonas, such as Pseudomonas aeruginosa.
Embodiment 26 a method for preventing and/or treating a bacterial infection in a subject, the method comprising administering to the subject an effective amount of a live bacterial strain according to any one of embodiments 1 to 20, or a composition according to embodiments 24 or 25.
Embodiment 27. The method of embodiment 26, wherein the bacterial infection is an infection caused by a species from the genus Pseudomonas, such as Pseudomonas aeruginosa.
Embodiment 28. A method for producing a live bacterial strain having reduced virulence and/or increased immunogenicity from a species of the genus pseudomonas, the method comprising reducing the OprF activity in the live bacterial strain and/or reducing expression of an OprF gene in the live bacterial strain and/or introducing a mutation into the OprF gene of the live bacterial strain.
Embodiment 29. The method of embodiment 28, wherein the method comprises reducing expression of an oprF gene of the live bacterial strain.
Embodiment 30. The method according to embodiment 28 or 29, wherein the method comprises introducing a mutation into the oprF gene of the live bacterial strain.
Embodiment 31. The method of embodiment 30, wherein said mutation comprises a complete deletion or a partial deletion of said oprF gene.
Embodiment 32. The method of embodiment 30 or 31, wherein said mutation results in reduced or no expression of said OprF protein.
Embodiment 33. The method of embodiment 30 or 31, wherein the mutation results in a mutated OprF protein with reduced or no activity.
Embodiment 34. The method of any one of embodiments 28 to 33, wherein the species from the genus pseudomonas is pseudomonas aeruginosa.
Embodiment 35 the method according to any one of embodiments 29 to 33, wherein said oprF gene encodes an oprF protein having an amino acid sequence with 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 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO. 1 or having the amino acid sequence shown in SEQ ID NO. 1.
Embodiment 36. The method of any one of embodiments 28 to 35, wherein the mutation is achieved by homologous recombination, e.g., double homologous recombination, or by targeted mutagenesis, such as via CRISPR, TALEN, or ZFN techniques.
Embodiment 37 the method of any one of embodiments 28 to 36, wherein the method further comprises introducing a coding sequence for a protein of interest into the live bacterial strain, thereby enabling the live bacterial strain to express the protein of interest.
Embodiment 38. The method of embodiment 37, wherein the coding sequence for the protein of interest is introduced into the live bacterial strain by a nucleic acid expression construct.
Embodiment 39. The method of embodiment 37 or 38, wherein the introduced coding sequence for the protein of interest is integrated into the genome of the live bacterial strain.
Embodiment 40. The method according to any of embodiments 37 to 39, wherein the protein of interest is selected from the group consisting of antibodies and antigens, preferably antigens,
For example, the antigen is selected from: pcrV, oprI or oprJNM from pseudomonas aeruginosa; adsA, esxA, esxB, pmtA or PmtC from staphylococcus aureus (s.aureus); or PspA from Streptococcus pneumoniae (S.pneumoniae).
Drawings
FIG. 1 knockdown of the oprF gene in Pseudomonas aeruginosa.
FIG. 2. Delta. OprF strain shows an extended lag phase and lower growth rate compared to the isogenic wt strain.
Figure 3.Δoprf strain shows lower virulence. BALB/c mice administered with Δoprf strain (2×10 7 CFU) showed 100% survival, whereas mice injected with the same dose of wt strain all died on day 2.
Fig. 4. Pao1Δoprf strain showed a significant increase in antibody titer on day 14 compared to the control group.
FIG. 5 survival studies show improved protection of ΔoprF strain compared to wt strain.
Figure 6 bacterial loads from different tissues.
Detailed Description
Before describing aspects of the present invention, it must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The term "and/or" is intended to include any combination of items connected by that term, equivalent to listing all combinations individually. 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". Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In one aspect, the invention provides a live bacterial strain from a species of pseudomonas, wherein the live bacterial strain lacks or has reduced OprF activity, and/or wherein expression of an OprF gene in the live bacterial strain is reduced, and/or wherein the live bacterial strain of the invention contains a mutation of the OprF gene, e.g. compared to a corresponding control strain.
In some embodiments, the species from the genus pseudomonas is pseudomonas aeruginosa.
In some embodiments, the OprF activity in a live bacterial strain of the invention 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 as compared to a corresponding control strain.
In some preferred embodiments, the viable bacterial strain lacks OprF activity, e.g., no detectable OprF activity.
In some embodiments, expression of the oprF gene is reduced in a live bacterial strain. In some embodiments, expression of the oprF gene in the live bacterial strain 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 as compared to a corresponding control strain.
In some preferred embodiments, the oprF gene is not expressed in the viable bacterial strain of the invention.
As used herein, an "oprF gene" may refer to the coding sequence of an oprF protein in the genome of the bacterium. However, the oprF gene can also cover expression regulatory elements/sequences, such as promoters, enhancers, etc.
In some embodiments, exemplary OprF proteins have an amino acid sequence that has 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 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO. 1. In some embodiments, the OprF protein has the amino acid sequence set forth in SEQ ID NO. 1.
In some embodiments, exemplary oprF genes have 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 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO. 2. In some embodiments, the oprF gene has the amino acid sequence shown in SEQ ID NO. 2.
In some embodiments, the viable bacterial strains of the invention contain mutations in the oprF gene, e.g., mutations that result in reduced or absent oprF activity. Such mutations may be additions, substitutions or deletions of one or more nucleotides.
In some embodiments, mutation of the oprF gene results in reduced expression of the oprF protein, or expression of a mutated oprF protein that results in reduced activity. In some embodiments, mutation of the oprF gene results in non-expression of the oprF protein or results in expression of an inactive mutant oprF protein. In some embodiments, the mutation is a frame shift mutation that results in mistranslation of the oprF gene.
In some embodiments, the mutation comprises a deletion of the oprF gene, e.g., a complete deletion or a partial deletion of the oprF gene. The oprF gene can be deleted completely from the strain so that the oprF gene is not present in the live strain of the invention. The oprF gene can also be partially deleted, so that only the oprF protein with reduced or no activity is present in the live strain of the invention.
Mutation of the oprF gene can be achieved by various means known in the art. In some embodiments, the mutation is introduced into the live bacterial strain by genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, mutations such as deletions may be achieved by homologous recombination, e.g., double homologous recombination. In some embodiments, the mutation is performed by targeted mutagenesis, such as via CRISPR, TALEN, or ZFN techniques.
In some embodiments, reduced or absent activity of an OprF protein, reduced or absent expression of an OprF gene, and/or reduced virulence caused by mutation of an OprF gene in a live bacterial strain of the invention.
For example, the virulence of a live bacterial strain of the invention can be 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 as compared to a corresponding control strain.
In some embodiments, reduced or absent activity of the OprF protein, reduced or absent expression of the OprF gene, and/or mutation of the OprF gene in the viable bacterial strain of the invention results in increased immunogenicity. Immunogenicity may refer to the ability to elicit an immune response (e.g., an antibody-mediated immune response) in a host.
For example, the immunogenicity of a live bacterial strain of the invention can be 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% or more as compared to a corresponding control strain.
In some embodiments, a "control strain" may be a parent strain derived from a live bacterial strain of the invention. In some embodiments, a "control strain" may refer to a strain of the same species whose oprF gene or oprF activity is not altered. In some embodiments, a "control strain" may also refer to a strain of the same species that does not comprise an oprF gene mutation as described above.
The live bacterial strain of the invention may be derived from a parent strain, which is a wild-type strain of the same species. In some embodiments, the wild-type strain may be a strain that is not genetically engineered. In some embodiments, the wild-type strain may be a strain whose oprF gene or oprF activity is not genetically engineered. In some embodiments, the wild-type strain may be clinically isolated.
The live bacterial strain of the present invention may be derived from a parent strain that already has low virulence. For example, the parent strain is an attenuated strain. The parent strain may contain other mutations (not within the oprF gene) that may lead to attenuation.
Exemplary pseudomonas aeruginosa strains that can be used as parent strains for the live bacterial strains of the present invention include, but are not limited to, PAO1, PA14, and the like.
In some embodiments, the viable bacterial strains of the present invention are also used as viable expression vectors for expressing a protein of interest. The protein of interest may confer certain properties on the viable bacterial strains of the invention.
In some embodiments, a live bacterial strain of the invention may comprise a coding sequence for a protein of interest, and thereby be capable of expressing the protein of interest.
In some embodiments of the aspects, the protein of interest may be an endogenous protein, i.e., a protein derived from a bacterial species of a live bacterial strain. In some embodiments, the protein of interest may be an exogenous protein, i.e., a protein of a different species than the bacterial species derived from the live bacterial strain.
In some embodiments of the aspects, the coding sequence for the protein of interest is introduced into a live bacterial strain of the invention, for example by a nucleic acid expression construct. In some embodiments, the introduced coding sequence for the protein of interest is integrated into the genome of a live bacterial strain of the invention.
As used herein, an "expression construct" refers to a vector, such as a recombinant vector suitable for expressing a nucleotide sequence of interest in a host cell. "expression" means the production of a functional product. For example, expression of a nucleotide sequence may refer to transcription of the nucleotide sequence, and/or translation of RNA into a precursor or mature protein. The "expression construct" of the invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or in some embodiments, may be an RNA (such as mRNA) capable of translation.
In some embodiments of the aspects, the protein of interest may be expressed and displayed on the cell surface of a live bacterial strain of the invention. In some embodiments, the protein of interest may be expressed and secreted by cells of a live bacterial strain of the invention.
Proteins of interest include, but are not limited to, antibodies, antigens, and the like.
In some preferred embodiments of the aspects, the protein of interest is an antigenic protein. Expression or display of the antigenic protein may further increase the immunogenicity of the live bacterial strain of the invention.
In some embodiments of the aspects, the protein of interest is an antigenic protein of a species different from a bacterial species derived from the live bacterial strain. Expression or display of antigenic proteins of other species may confer immunogenicity to the live bacterial strain against the other species.
Exemplary antigenic proteins include, but are not limited to, pcrV, oprI or oprJNM from pseudomonas aeruginosa; adsA, esxA, esxB, pmtA or PmtC from staphylococcus aureus; or PspA from Streptococcus pneumoniae.
In some embodiments, the viable bacterial strains of the present invention are used for preventing and/or treating bacterial infections. In some embodiments, the bacterial infection is an infection caused by a species from the genus pseudomonas, such as pseudomonas aeruginosa. In some particular embodiments, the bacterial infection is an infection caused by a pseudomonas aeruginosa PAO1 strain.
As used herein, preventing and/or treating a bacterial infection also encompasses preventing and/or treating a disease or clinical sign or symptom caused by a bacterial infection.
In one aspect, the invention provides the use of a live bacterial strain of the invention in the preparation of a composition for the prevention or treatment of a bacterial infection. In some embodiments, the composition is a vaccine.
In one aspect, the invention provides a composition for preventing or treating a bacterial infection, the composition comprising a viable bacterial strain of the invention. In some embodiments, the composition comprises an effective amount of a live bacterial strain of the invention. In some embodiments, the composition is a vaccine.
In one aspect, the invention provides a method of preventing and/or treating a bacterial infection in a subject, the method comprising administering to the subject an effective amount of a live bacterial strain of the invention or a composition of the invention.
In some embodiments of the above aspects, the bacterial infection is an infection caused by a species from the genus pseudomonas, such as pseudomonas aeruginosa.
In some embodiments of the above aspects, the composition may further comprise an adjuvant. As used herein, "adjuvant" refers to an additional component in a vaccine that enhances an immune response, or an auxiliary molecule added to a vaccine, or an auxiliary molecule produced by the body after separate induction by such additional component, such as, but not limited to, an interferon, interleukin, or growth factor. "adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions.
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 and absorption delaying agents, and the like that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers include water, naCl, physiological saline, lactated ringer's solution, standard sucrose, standard dextrose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, saline solutions (such as ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose), amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and coloring agents.
In some embodiments of the above aspects, the composition is formulated for intramuscular administration, intraperitoneal administration, subcutaneous administration, oral administration, or intranasal administration. In one embodiment, the composition is not for intravenous administration. In some embodiments, the composition is in a lyophilized form, which can be reconstituted prior to use.
As used herein, an "effective amount" refers to an amount of a substance, compound, material, or composition containing a compound (such as a modified live bacterial strain of the invention or a composition of the invention) that is at least sufficient to produce a prophylactic or therapeutic effect upon administration to a subject. Thus, an effective amount is that amount necessary to prevent, cure, ameliorate, delay or partially delay symptoms of a disease or disorder, such as a bacterial infection.
The actual dose of the live strain or composition of the invention to be administered to a subject may be determined according to the following physical and physiological factors: body weight, sex, severity of symptoms, type of disease to be treated, previous or current therapeutic intervention, disease of unknown etiology in the patient, time of administration, route of administration, etc. In any event, the amount of viable strains in the composition and the appropriate dosage for the individual subject will be determined by the medical personnel responsible for administration.
In one aspect, the invention provides a method of attenuating and/or increasing the immunogenicity of a live bacterial strain from a species of the genus pseudomonas, or a method for producing a live bacterial strain with reduced virulence and/or increased immunogenicity from a species of the genus pseudomonas, the method comprising reducing the activity of OprF in the live bacterial strain, and/or reducing the expression of an OprF gene in the live bacterial strain, and/or introducing a mutation into the OprF gene of the live bacterial strain.
In some embodiments, the method comprises reducing expression of an oprF gene of the live bacterial strain.
In some embodiments, the method comprises introducing a mutation into the oprF gene of the live bacterial strain. Such mutations may be additions, substitutions or deletions of one or more nucleotides.
In some embodiments, the mutation comprises a complete deletion or a partial deletion of the oprF gene. In some embodiments, the mutation results in reduced expression of the OprF protein. In some embodiments, the mutation results in the OprF protein not being expressed. In some embodiments, the mutation results in a mutated OprF protein with reduced activity. In some embodiments, the mutation results in an inactive mutant OprF protein.
In some embodiments, the species from the genus pseudomonas is pseudomonas aeruginosa.
In some embodiments, the oprF gene encodes an oprF protein having an amino acid sequence with 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 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO. 1. In some embodiments, the OprF protein has the amino acid sequence set forth in SEQ ID NO. 1.
In some embodiments, exemplary oprF genes have 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 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO. 2. In some embodiments, the oprF gene has the amino acid sequence shown in SEQ ID NO. 2.
In some embodiments, mutations such as deletions may be achieved by homologous recombination, e.g., double homologous recombination. In some embodiments, the mutation is performed by targeted mutagenesis, such as via CRISPR, TALEN, or ZFN techniques.
In some embodiments, the method further comprises introducing a coding sequence for the protein of interest into the live bacterial strain, thereby enabling the live bacterial strain to express the protein of interest. In some embodiments, the protein of interest may be an endogenous protein or an exogenous protein.
In some embodiments, the coding sequence for the protein of interest is introduced into a live bacterial strain by a nucleic acid expression construct. In some embodiments, the introduced coding sequence for the protein of interest is integrated into the genome of the live bacterial strain.
Proteins of interest include, but are not limited to, antibodies, antigens, and the like.
In some preferred embodiments of the aspects, the protein of interest is an antigenic protein. In some embodiments of the aspects, the protein of interest is an antigenic protein of a species different from a bacterial species derived from the live bacterial strain.
Exemplary antigenic proteins include, but are not limited to, pcrV, oprI or oprJNM from pseudomonas aeruginosa; adsA, esxA, esxB, pmtA or PmtC from staphylococcus aureus; or PspA from Streptococcus pneumoniae.
Examples
A further understanding of the present invention may be obtained by reference to the specific examples illustrated herein which are intended to illustrate the invention and are not intended to limit the scope of the invention. It will be apparent that various modifications and variations can be made to the present invention without departing from the spirit of the invention, and such modifications and variations are also within the scope of the invention.
Methods and materials
Construction of strains with knockdown of oprF
The previously developed pCasPA/pACRISPR system was used to construct the marker-free deletion 8 of oprF in PAO 1. Briefly, one-way guide RNA (sgRNA) was designed for a highly efficient gRNA target sequence, followed by protospacer adjacent motif nucleotide sequence NGG (20 nt: ATCTACCTTACCGTCGGTACCCC). The linearized pACRISPR plasmid was ligated with annealed spacer oligomers oprF-spacer-F (GTGGATCTACCACTTCGGTACCCC) and oprF-spacer-R (AAACGGGGTACCGAAGTGGTAGAT) to produce the pACRISPR-sgRNA plasmid. Primers oprF-upstream-F (TGTCCATACCCATGGTCTAGAATGAAGAATTGATGCGGCGT), oprF-upstream-R (CTTGGCTTCAGTTTCATCCGTTAAATCCCC), oprF-downstream-F (CGGATGAAACTGAAGCCAAGTAATCGGCTGAGC) and oprF-downstream-R (GGGAGTATGAAAAGTCTCGAGTTCATCCAGCGCCTGATGC) were used to PCR amplify the 5 '-and 3' -flanking regions of oprF from the chromosomal DNA of the P.aeruginosa PAO1 strain. The individual PCR products were then mixed to generate the deletion pattern of the oprF (repair template) and subcloned into the pACRISPR-sgRNA plasmid to generate plasmid pACRISPR-sgRNA-oprF. Plasmid pACRISPR-sgRNA-oprF was introduced into DH5a and screened on carbenicillin plates. The pCasPA plasmid was transferred into PAO1 electrocompetent cells (PAO 1-pCasPA) and expression of the Cas9 nuclease and lambda-Red system was induced by adding L-arabinose to a final concentration of 2 mg/mL. The pACRISPR-sgRNA-oprF plasmid, equipped with spacer and repair template, was further electroporated into PAO1-pCasPA electrocompetent cells. Cells were allowed to recover in LB at 37℃for 1 to 2 hours and plated onto LB agar plates containing 100. Mu.g/mL tetracycline and 150. Mu.g/mL carbenicillin. The correct deletion of the defective mutant (PAO1ΔoprF) was verified by PCR and sequencing using primers chr-oprF-F (ATCTCACTTGAATAAGCCTCACCC) and chr-oprF-R (AACTGTTGACCCTGAAGGCAG). The plasmids were cured by streaking the culture mutants on LB plates supplemented with 5% (w/v) sucrose.
Animal infection experiment
Each BALB/c mouse (7 week old, male) was immunized intraperitoneally with a single dose of 200 μl of lyophilized bacterial suspension (2×10 7 CFU). Animals were monitored daily for weight loss for 14 days, and serum was sampled on days 14 and 35. On day 35, mice were challenged by intraperitoneal route with 200 μl of lyophilized PAO1 wt strain (1×10 7 CFU) and survival was monitored for 14 days. Serum samples were tested for antibody titration by whole bacterial ELISA (coated PAO wt,1 x 10 7 CFU/well).
Bacterial load detection was performed in a separate experiment. C57BL/6N mice (females) were immunized intraperitoneally on day 0 and day 15, 2X 10 7 CFU per mouse. On day 35, mice were challenged with a sublethal dose (control group n=5, vaccinated group n=5) by intraperitoneal route, 2×10 7 CFU per mouse of PAO1 wt. Tissues were collected 24 hours after challenge and homogenized in sterile PBS. Bacterial load in each organ was determined by serial dilution and plating onto LB agar plates.
EXAMPLE 1 construction and characterization of the ΔoprF Pseudomonas aeruginosa Strain
The oprF gene was targeted, no marker deletion was performed, and successful removal of the gene from the PAO 1wt strain was confirmed by PCR (FIG. 1). Growth curves in LB broth showed that the ΔoprF strain showed an extended lag phase and lower growth rate compared to the isogenic wt strain. (FIG. 2).
EXAMPLE 2 characterization of the ΔoprF Pseudomonas aeruginosa Strain
1. The ΔoprF strain showed lower virulence
In the BALB/c mouse model, animals immunized with the Δoprf strain had slightly reduced body weight, but were rapidly stable after day 2. Mice immunized with Δoprf strain (2×10 7 CFU) showed 100% survival, whereas mice injected with the same dose of wt strain all died on day 2, indicating that Δoprf strain has lower virulence (fig. 3).
2. The delta oprF strain shows higher immunogenicity
To measure the antibody-mediated immune response, BALB/c mice were immunized with the Δoprf strain and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA). The pao1Δoprf strain showed a significantly increased antibody titer on day 14 compared to the control group (fig. 4).
3. The delta oprF strain shows higher protective effect
Survival studies also showed protection against PAO1 wt. All mice immunized with the Δoprf strain and subsequently challenged with PAO1 wt survived, whereas only 10% survived in the control group (fig. 5). Furthermore, in pao1 Δoprf vaccine vaccinated mice, bacterial loads from different tissues were significantly reduced (fig. 6), indicating protection from the vaccine.
Sequence information
SEQ ID NO.1 oprF_amino_sequence
MKLKNTLGVVIGSLVAASAMNAFAQGQNSVEIEAFGKRYFTDSVRNMKNADLYGGSIGY
FLTDDVELALSYGEYHDVRGTYETGNKKVHGNLTSLDAIYHFGTPGVGLRPYVSAGLAHQ
NITNINSDSQGRQQMTMANIGAGLKYYFTENFFAKASLDGQYGLEKRDNGHQGEWMAGL
GVGFNFGGSKAAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSKVKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEGRAINRRVEAEVEAEAK*
SEQ ID NO. 2 OprF coding sequence
ATGAAACTGAAGAACACCTTAGGCGTTGTCATCGGCTCGCTGGTTGCCGCTTCGGCAATGAACGCCTTTGCCCAGGGCCAGAACTCGGTAGAGATCGAAGCCTTCGGCAAGCGCTACTTCACCGACAGCGTTCGCAACATGAAGAACGCGGACCTGTACGGCGGCTCGATCGGTTACTTCCTGACCGACGACGTCGAGCTGGCGCTGTCCTACGGTGAGTACCATGACGTTCGTGGCACCTACGAAACCGGCAACAAGAAGGTCCACGGCAACCTGACCTCCCTGGACGCCATCTACCACTTCGGTACCCCGGGCGTAGGTCTGCGTCCGTACGTGTCGGCTGGTCTGGCTCACCAGAACATCACCAACATCAACAGCGACAGCCAAGGCCGTCAGCAGATGACCATGGCCAACATCGGCGCTGGTCTGAAGTACTACTTCACCGAGAACTTCTTCGCCAAGGCCAGCCTCGACGGCCAGTACGGTCTGGAGAAGCGTGACAACGGTCACCAGGGCGAGTGGATGGCTGGCCTGGGCGTCGGCTTCAACTTCGGTGGTTCGAAAGCCGCTCCGGCTCCGGAACCGGTTGCCGACGTTTGCTCCGACTCCGACAACGACGGCGTTTGCGACAACGTCGACAAGTGCCCGGATACCCCGGCCAACGTCACCGTTGACGCCAACGGCTGCCCGGCTGTCGCCGAAGTCGTACGCGTACAGCTGGACGTGAAGTTCGACTTCGACAAGTCCAAGGTCAAAGAGAACAGCTACGCTGACATCAAGAACCTGGCTGACTTCATGAAGCAGTACCCGTCCACTTCCACCACCGTTGAAGGTCACACCGACTCCGTCGGCACCGACGCTTACAACCAGAAGCTGTCCGAGCGTCGTGCCAACGCCGTTCGTGACGTACTGGTCAACGAGTACGGTGTAGAAGGTGGTCGCGTGAACGCTGTTGGTTACGGCGAGTCCCGCCCGGTTGCCGACAACGCCACCGCTGAAGGCCGCGCTATCAACCGTCGCGTTGAAGCCGAAGTAGAAGCTGAAGCCAAGTAA
Reference to the literature
1.Pang,Z.,Raudonis,R.,Glick,B.R.,Lin,T.-J.&Cheng,Z.Antibiotic resistance in Pseudomonas aeruginosa:mechanisms and alternative therapeutic strategies.Biotechnol Adv 37,177–192(2018).
2.Moussouni,M.,Berry,L.,Sipka,T.,Nguyen-Chi,M.&Blanc-Potard,A.-B.Pseudomonas aeruginosa OprF plays a role in resistance to macrophage clearance during acute infection.Sci Rep-uk 11,359(2021).
3.Garai,P.,Berry,L.,Moussouni,M.,Bleves,S.&Blanc-Potard,A.-B.Killing from the inside:Intracellular role of T3SS in the fate of Pseudomonas aeruginosa within macrophages revealed by mgtC and oprF mutants.Plos Pathog 15,e1007812(2019).
4.Chevalier,S.et al.Structure,function and regulation of Pseudomonas aeruginosa porins.Fems Microbiol Rev 41,698–722(2017).
5.Fito-Boncompte,L.et al.Full virulence of Pseudomonas aeruginosa requires OprF.Infect Immun79,1176–86(2010).
6.Bouffartigues,E.et al.The absence of the Pseudomonas aeruginosa OprF protein leads to increased biofilm formation through variation in c-di-GMP level.Front Microbiol 6,630(2015).
7.Sainz-Mejías,M.,Jurado-Martín,I.&McClean,S.Understanding Pseudomonas aeruginosa–Host Interactions:The Ongoing Quest for an Efficacious Vaccine.Cells 9,2617(2020).
8.Chen,W.et al.CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species.Iscience 6,222–231(2018).
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the relevant art (including the contents of the documents cited and incorporated by reference herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the relevant art.
While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example, and not limitation. Various changes in form and detail will be apparent to those skilled in the relevant art without departing from the spirit and scope of the disclosure. Thus, the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Claims (40)
1. A live bacterial strain from a species of pseudomonas, wherein the live bacterial strain lacks or has reduced OprF activity and/or has reduced expression of an OprF gene in the live bacterial strain and/or contains a mutation of the OprF gene, e.g. as compared to a corresponding control strain.
2. The live bacterial strain according to claim 1, wherein the species from the genus pseudomonas is pseudomonas aeruginosa.
3. The live bacterial strain of claim 1 or 2, wherein the activity of OprF in the live bacterial strain 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 compared to a corresponding control strain, preferably the live bacterial strain lacks OprF activity.
4. The live bacterial strain of any one of claims 1to 3, wherein the expression of the oprF gene in the live bacterial strain is reduced, e.g., 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, as compared to a corresponding control strain, preferably the live bacterial strain lacks the oprF gene expression.
5. The live bacterial strain of any one of claims 1 to 4, wherein the oprF gene encodes an oprF protein having an amino acid sequence with 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 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID No. 1 or an amino acid sequence with SEQ ID No. 1.
6. The live bacterial strain of any one of claims 1-5, wherein the live bacterial strain contains a mutation of the OprF gene that results in a reduction or lack of OprF activity.
7. The live bacterial strain of claim 6, wherein mutation of the oprF gene results in reduced expression of the oprF protein or results in reduced expression of a mutated oprF protein of activity, preferably mutation of the oprF gene results in non-expression of an oprF protein or results in expression of an inactive mutated oprF protein.
8. The live bacterial strain of claim 6 or 7, wherein the mutation comprises a deletion of the oprF gene, e.g., a complete deletion or a partial deletion of the oprF gene.
9. The live bacterial strain according to any one of claims 6 to 8, the mutation being achieved by homologous recombination or by targeted mutagenesis, such as via CRISPR, TALEN or ZFN techniques.
10. The live bacterial strain of any one of claims 1 to 9, wherein the live bacterial strain has reduced virulence compared to a corresponding control strain, e.g., the virulence of the live bacterial strain 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.
11. The live bacterial strain of any one of claims 1 to 10, wherein the live bacterial strain has increased immunogenicity as compared to a corresponding control strain, e.g., the immunogenicity of the live bacterial strain 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% or more.
12. The live bacterial strain according to any one of claims 1 to 11, wherein the live bacterial strain is derived from a parent strain as a clinical isolate.
13. The live bacterial strain according to any one of claims 1 to 11, wherein the live bacterial strain is derived from a parent strain that already has low virulence.
14. The live bacterial strain according to any one of claims 1 to 11, wherein the live bacterial strain is derived from pseudomonas aeruginosa strain PAO1 or PA14.
15. The live bacterial strain according to any one of claims 1 to 14 for use as a live expression vector for expressing a protein of interest.
16. The live bacterial strain according to any one of claims 1 to 15, further comprising a coding sequence for a protein of interest and thereby being capable of expressing the protein of interest.
17. The live bacterial strain according to claim 16, wherein the coding sequence for the protein of interest is introduced into the live bacterial strain, for example by means of a nucleic acid expression construct.
18. The live bacterial strain of claim 17, wherein the introduced coding sequence for the protein of interest is integrated into the genome of the live bacterial strain.
19. The live bacterial strain according to any one of claims 16 to 18, wherein the protein of interest is expressed and displayed on the cell surface of the live bacterial strain; or cells of the living bacterial strain expressing and secreting the protein of interest.
20. The live bacterial strain according to any of claims 16 to 19, wherein the protein of interest is selected from antibodies or antigens, preferably the protein of interest is an antigen,
For example, the antigen is selected from: pcrV, oprI or oprJNM from pseudomonas aeruginosa; adsA, esxA, esxB, pmtA or PmtC from staphylococcus aureus (s.aureus); or from Streptococcus pneumoniae (S).
Psumiae) PspA.
21. Use of a live bacterial strain according to any one of claims 1 to 20 in the preparation of a composition, such as a vaccine, for the prevention or treatment of a bacterial infection.
22. Use according to claim 21, wherein the bacterial infection is an infection caused by a species from the genus pseudomonas, such as pseudomonas aeruginosa.
23. A composition, such as a vaccine, for use in the prevention or treatment of a bacterial infection, the composition comprising a live bacterial strain according to any one of claims 1 to 20.
24. The composition of claim 23, wherein the composition further comprises an adjuvant and/or a pharmaceutically acceptable carrier.
25. Use according to claim 23 or 24, wherein the bacterial infection is an infection caused by a species from the genus pseudomonas, such as pseudomonas aeruginosa.
26. A method for preventing and/or treating a bacterial infection in a subject, the method comprising administering to the subject an effective amount of a live bacterial strain according to any one of claims 1 to 20, or a composition according to claim 24 or 25.
27. The method of claim 26, wherein the bacterial infection is an infection caused by a species from the genus pseudomonas, such as pseudomonas aeruginosa.
28. A method for producing a live bacterial strain having reduced virulence and/or increased immunogenicity from a species of pseudomonas, the method comprising reducing OprF activity in the live bacterial strain and/or reducing expression of an OprF gene in the live bacterial strain and/or introducing a mutation into the OprF gene of the live bacterial strain.
29. The method of claim 28, wherein the method comprises reducing expression of an oprF gene of the live bacterial strain.
30. The method of claim 28 or 29, wherein the method comprises introducing a mutation into the oprF gene of the live bacterial strain.
31. The method of claim 30, wherein the mutation comprises a complete deletion or a partial deletion of the oprF gene.
32. The method of claim 30 or 31, wherein the mutation results in reduced or no expression of the OprF protein.
33. The method of claim 30 or 31, wherein the mutation results in a mutated OprF protein with reduced or no activity.
34. The method of any one of claims 28 to 33, wherein the species from the genus pseudomonas is pseudomonas aeruginosa.
35. The method of any one of claims 29 to 33, wherein the oprF gene encodes an oprF protein having an amino acid sequence with 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 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID No.1 or an amino acid sequence as set forth in SEQ ID No. 1.
36. The method according to any one of claims 28 to 35, wherein the mutation is achieved by homologous recombination, e.g. double homologous recombination, or by targeted mutagenesis, such as via CRISPR, TALEN or ZFN techniques.
37. The method of any one of claims 28 to 36, wherein the method further comprises introducing a coding sequence for a protein of interest into the live bacterial strain, thereby enabling the live bacterial strain to express the protein of interest.
38. The method of claim 37, wherein the coding sequence for the protein of interest is introduced into the live bacterial strain by a nucleic acid expression construct.
39. The method of claim 37 or 38, wherein the introduced coding sequence for the protein of interest is integrated into the genome of the live bacterial strain.
40. The method according to any one of claims 37 to 39, wherein the protein of interest is selected from antibodies or antigens, preferably antigens,
For example, the antigen is selected from: pcrV, oprI or oprJNM from pseudomonas aeruginosa; adsA, esxA, esxB, pmtA or PmtC from staphylococcus aureus (s.aureus); or from Streptococcus pneumoniae (S).
Psumiae) PspA.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2022102786 | 2022-06-30 | ||
| CNPCT/CN2022/102786 | 2022-06-30 | ||
| PCT/CN2023/104598 WO2024002335A1 (en) | 2022-06-30 | 2023-06-30 | A live bacteria strain of pseudomonas sp. |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117940554A true CN117940554A (en) | 2024-04-26 |
Family
ID=89383361
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202380013541.5A Pending CN117940554A (en) | 2022-06-30 | 2023-06-30 | Live bacterial strains of the genus Pseudomonas |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN117940554A (en) |
| WO (1) | WO2024002335A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1112356A (en) * | 1993-06-07 | 1995-11-22 | 第一制糖株式会社 | Novel attenuated pseudomonas aeruginosa strains |
| WO2016193402A1 (en) * | 2015-06-03 | 2016-12-08 | Valneva Austria Gmbh | Pseudomonas vaccine |
| CN108114279A (en) * | 2018-03-06 | 2018-06-05 | 重庆大学 | A kind of preparation method of the P. aeruginosa bacteria vaccine assisted by rod-like nano aluminium hydroxide |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2609985A1 (en) * | 2005-06-03 | 2007-05-10 | The University Of Chicago | Modulation of cell barrier dysfunction |
| EP3363460A1 (en) * | 2017-02-16 | 2018-08-22 | Université Grenoble Alpes | Attenuated strain of pseudomonas as a vaccine for pseudomonas infection |
-
2023
- 2023-06-30 WO PCT/CN2023/104598 patent/WO2024002335A1/en active Application Filing
- 2023-06-30 CN CN202380013541.5A patent/CN117940554A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1112356A (en) * | 1993-06-07 | 1995-11-22 | 第一制糖株式会社 | Novel attenuated pseudomonas aeruginosa strains |
| WO2016193402A1 (en) * | 2015-06-03 | 2016-12-08 | Valneva Austria Gmbh | Pseudomonas vaccine |
| CN108114279A (en) * | 2018-03-06 | 2018-06-05 | 重庆大学 | A kind of preparation method of the P. aeruginosa bacteria vaccine assisted by rod-like nano aluminium hydroxide |
Non-Patent Citations (2)
| Title |
|---|
| MALIKA MOUSSOUNI等: "Pseudomonas aeruginosa OprF plays a role in resistance to macrophage clearance during acute infection", 《SCIENTIFIC REPORTS》, vol. 11, no. 359, 11 January 2021 (2021-01-11), pages 3 - 11 * |
| 杨敬鹏等: "铜绿假单胞菌疫苗的研究进展", 《中国生物制品学杂志》, vol. 34, no. 10, 31 October 2021 (2021-10-31), pages 1253 - 1259 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024002335A1 (en) | 2024-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2020019801A (en) | Attenuated live vaccines | |
| US11590215B2 (en) | Process for the production of a DNA vaccine for cancer immunotherapy | |
| Kirn et al. | TcpF is a soluble colonization factor and protective antigen secreted by El Tor and classical O1 and O139 Vibrio cholerae serogroups | |
| Heidary et al. | Colonization and investigation of vibrio cholera recombination protein in e-coli | |
| Gorain et al. | Mucosal delivery of live Lactococcus lactis expressing functionally active JlpA antigen induces potent local immune response and prevent enteric colonization of Campylobacter jejuni in chickens | |
| Jores et al. | Removal of a subset of non-essential genes fully attenuates a highly virulent Mycoplasma strain | |
| Gu et al. | Vaccination of attenuated EIS-producing Salmonella induces protective immunity against enterohemorrhagic Escherichia coli in mice | |
| Scoffone et al. | Vaccines to overcome antibiotic resistance: the challenge of Burkholderia cenocepacia | |
| Valderrama et al. | Outer membrane protein FrpA, the siderophore piscibactin receptor of Photobacterium damselae subsp. piscicida, as a subunit vaccine against photobacteriosis in sole (Solea senegalensis) | |
| KR20150048771A (en) | A novel live attenuated shigella vaccine | |
| RU2252224C2 (en) | Peptide with neisseria meningitidis antigene activity, polynucleotide encoding the same, vaccine for prevention and treatment of diseases and conditions induced by neisseria meningitidis, (variants), antibody coupling with said peptide | |
| CZ20003470A3 (en) | Bacterium attenuated by a non-reverting mutation, vaccine in which the bacterium is comprised and use thereof | |
| US20040147719A1 (en) | Type III bacterial strains for use in medicine | |
| CN113329766A (en) | Clostridium difficile multicomponent vaccine | |
| US8993302B2 (en) | Mutants of Francisella tularensis and uses thereof | |
| EA028912B1 (en) | New antibiotic containing an antibody mimetic, methods for preparation and application thereof | |
| KR100457879B1 (en) | Plasmid originated from Bifidobacterium, recombinant expression vector using the plasmid and transformation method | |
| CN117940554A (en) | Live bacterial strains of the genus Pseudomonas | |
| US20100196391A1 (en) | Shigella ipad protein and its use as a vaccine against shigella infection | |
| KR100563218B1 (en) | RTX and related genes of Vibrio vulnificus responsible for the contact-cytotoxicity and lethality to animals | |
| Sarshar et al. | Acinetobacter baumannii: an ancient commensal with weapons of a pathogen. Pathogens 2021; 10: 387 | |
| CN114456994B (en) | Recombinant staphylococcus aureus for preparing bacterial membrane vesicle multi-linked vaccine as well as preparation method and application thereof | |
| WO2022007741A1 (en) | Genetically engineered live bacteria and methods of constructing the same | |
| KR100829380B1 (en) | Fowl cholera vaccine composition comprising recombinant outer membrane protein h of pasteurella multocida | |
| KR20090090526A (en) | Actinobacillus pleuropneumoniae mutant strain with inactivated apxiiib and apxiiid genes and compositions comprising said mutant strain for preventing infection of actinobacillus pleuropneumoniae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |