WO2020103412A1 - 包含BPI-Fc嵌合基因的重组腺病毒及其用途 - Google Patents

包含BPI-Fc嵌合基因的重组腺病毒及其用途

Info

Publication number
WO2020103412A1
WO2020103412A1 PCT/CN2019/087381 CN2019087381W WO2020103412A1 WO 2020103412 A1 WO2020103412 A1 WO 2020103412A1 CN 2019087381 W CN2019087381 W CN 2019087381W WO 2020103412 A1 WO2020103412 A1 WO 2020103412A1
Authority
WO
WIPO (PCT)
Prior art keywords
bpi
drug
fcγ1
recombinant adenovirus
protein
Prior art date
Application number
PCT/CN2019/087381
Other languages
English (en)
French (fr)
Other versions
WO2020103412A8 (zh
Inventor
陈金栋
安云庆
李劲超
杨密清
Original Assignee
厦门联合安金生物工程有限公司
安君(北京)基因科技有限责任公司
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 厦门联合安金生物工程有限公司, 安君(北京)基因科技有限责任公司 filed Critical 厦门联合安金生物工程有限公司
Publication of WO2020103412A1 publication Critical patent/WO2020103412A1/zh
Publication of WO2020103412A8 publication Critical patent/WO2020103412A8/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the invention relates to the field of gene therapy. Specifically, the present invention relates to a recombinant adenovirus for the treatment of drug-resistant Gram-negative bacteria infection, its preparation and application.
  • the recombinant adenovirus contains a Bactericidal / Permeablity Increasing Protein (BPI) BPI-Fc chimeric gene of fusion protein with immunoglobulin heavy chain constant region Fc.
  • BPI Bactericidal / Permeablity Increasing Protein
  • the invention also relates to the use of the recombinant adenovirus in the preparation of a pharmaceutical composition for the treatment of drug-resistant Gram-negative bacteria infection.
  • Antibiotic resistance poses a major threat to public health and human health.
  • the WHO has called for and caused the world to pay attention to the problem of global drug-resistant infections, warning that civilization will enter the post-antibiotic era [ANTIMICROBIAL RESISTANCE Global Report on Surveillance 2014, WHO].
  • the WHO quoted [AntimicrobialResistance on the Global Agenda.Dec 1, 2015] published by Jim O'Neill in the United Kingdom estimates that approximately 700,000 people die from drug-resistant bacterial infections worldwide each year. If effective measures are not taken, It is estimated that by 2050, 10 million people will die each year from drug-resistant bacterial infections. The problem of bacterial multi-drug resistance and super drug resistance is becoming increasingly prominent.
  • Gram-negative bacteria which is one of the main pathogens causing human infectious diseases, has strong resistance to clinically-used antibiotics. It is currently the most concerned drug-resistant bacteria [https://en.wikipedia.org/wiki/Antimicrobial_resistance].
  • the plasmid-mediated super carbapenem resistance gene NDM-1 was discovered in Klebsiella pneumoniae in 2009 and the plasmid-mediated super colistin resistance was found in Escherichia coli in 2015 Drug gene mcr-1 [Antimicrob Agents Chemother. 2009; 53 (12): 5046] [Lancet Infect Dis. 2010; 10 (9): 597] [Lancet Infect Dis. 2016; 16 (2): 161], almost making Antibiotics for clinical treatment of Gram-negative bacterial infections have lost all lines of defense. With the increasing resistance of antibiotics and the coming post-antibiotic era, the development of antibiotic alternative biological drugs has urgency and huge application prospects.
  • a recombinant adenovirus comprising a chimeric gene encoding a BPI-Fc fusion protein, wherein the chimeric gene comprises a coding sequence of a human BPI functional fragment and a human immunoglobulin heavy chain constant region Fc gene, wherein the 3 'end of the coding sequence of the human BPI functional fragment is directly or through a human immunoglobulin hinge region connected to the Fc gene.
  • the adenovirus of the present invention is an adenovirus vector selected from the following serotypes: Ad5, Ad2 and Ad55 serotypes; preferably Ad5 serotypes.
  • the human BPI functional fragments of the present invention are selected from human BPI 1-199 fragments and human BPI 1-193 fragments, preferably human BPI 1-199 fragments.
  • the human immunoglobulin heavy chain constant region Fc gene of the present invention is selected from C ⁇ 1, C ⁇ 2, C ⁇ 3, C ⁇ 1, C ⁇ 2, and C ⁇ genes or allelic genes thereof, preferably the C ⁇ 1 gene.
  • the chimeric gene encoding the BPI-Fc fusion protein has, in order from 5 'to 3', a human BPI signal peptide coding sequence, a BPI 1-199 coding sequence, and a human immunoglobulin hinge
  • the coding sequence of the region and Fc ⁇ 1 is preferably connected to the CMV promoter and the SV40 poly A expression control element at the 5 ′ end and the 3 ′ end, respectively.
  • nucleotide sequence of the BPI-Fc ⁇ 1 chimeric gene encoding the BPI-Fc ⁇ 1 fusion protein is shown in SEQ ID NO: 1, preferably, the BPI 1-199 of the 4-24 The coding sequence of amino acid residues was modified to SEQ ID NO: 2.
  • the present invention provides an Ad5-BPI-Fc ⁇ 1 recombinant adenovirus, which can infect animal cells and express the BPI-Fc ⁇ 1 fusion protein for direct killing and rapid and efficient killing of multi-drug resistance by activating complement and opsonizing phagocytosis Gram-negative bacteria, and the recombinant adenovirus has been successful in the treatment of multi-drug resistant Gram-negative bacteria infection model animals in vivo.
  • Another aspect of the present invention provides the use of the recombinant adenovirus for treating infections of drug-resistant Gram-negative bacteria
  • the drug-resistant Gram-negative bacteria include multi-drug-resistant Gram-negative bacteria, specifically Drug-resistant Gram-negative bacteria involved in multiple drug resistance mechanisms.
  • the drug-resistant Gram-negative bacteria include Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae.
  • the drug-resistant Gram-negative bacteria carry a drug-resistant gene, and the drug-resistant gene is an extended-spectrum drug-resistant gene ESBLs or a super drug-resistant gene NDM-1.
  • the present invention provides a pharmaceutical composition for treating a drug-resistant Gram-negative bacteria infection, the pharmaceutical composition comprising a therapeutically effective amount of the recombinant adenovirus of the present invention, and a pharmaceutically acceptable carrier.
  • Another aspect of the present invention provides the use of the recombinant adenovirus in the preparation of a medicament for treating infections of drug-resistant Gram-negative bacteria
  • the drug-resistant Gram-negative bacteria include multi-drug resistant Gram Negative bacteria, specifically involving multi-drug resistant Gram-negative bacteria.
  • the drug-resistant Gram-negative bacteria include Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae.
  • the drug-resistant Gram-negative bacteria carry a drug-resistant gene, and the drug-resistant gene is an extended-spectrum drug-resistant gene ESBLs or a super drug-resistant gene NDM-1.
  • a method for preparing a recombinant adenovirus comprises a nucleotide sequence encoding a fusion protein of BPI and Fc. The method includes the following steps:
  • step (c) Co-transfect the host cell with the adenovirus backbone vector of step (a) and the shuttle expression vector of step (b) to perform homologous recombination to obtain a recombinant adenovirus.
  • the method further includes the following steps:
  • step (d) Infect the host cell with the recombinant adenovirus obtained in step (c), and
  • Bactericidal / permeablity increasing protein is a cationic antibacterial protein with a molecular weight of about 55KD found in Weiss et al. For the first time in human polymorphonuclear neutrophils in 1978.
  • the antibacterial protein is composed of 456 amino acid residues.
  • Base composition its N-terminal functional fragment (BPI 1-199 fragment of 1-199 amino acid residues in the N-terminal region and its BPI 1-193 fragment of 1-193 amino acid residues truncated by 6 amino acids at the C-terminal) It has the same high affinity as human natural BPI to bind the lipopolysaccharide (LPS) and lipid A of Gram-negative bacteria, neutralize endotoxin (Endotoxin) and directly kill Gram-negative bacteria.
  • LPS lipopolysaccharide
  • Endotoxin endotoxin
  • XOMA has been developing and reorganizing human BPI N-terminal functional fragments since 1990s ( rBPI 21 ) and carried out a number of clinical trials, but because BPI sterilization needs to last a long time (> 3 hours) to maintain a higher concentration (> 10nM), and rBPI 21 has a short half-life in vivo, a large therapeutic dose, and is difficult to maintain in vivo Factors such as effective therapeutic concentration did not achieve clinical success and were not approved by the FDA.
  • Immunoglobulin includes a family of proteins with many similar structures but different important structures. These different important structures lead to different antigen-binding properties and other biological activities. Human Ig can be divided into five categories: IgG, IgA, IgM, IgD, and IgE. Among them, IgG, IgA, and IgM also have subcategories, which play an important role in the defense against infection and immunity.
  • IgG heavy chain constant region Fc Frament crystallizable, Fc; Fc coding sequence is called Fc gene: C ⁇ 1, C ⁇ 2 and C ⁇ 3 have dual functions of activating complement and mediating opsonization and phagocytosis; IgA heavy chain constant region Fc: C ⁇ 1, C ⁇ 2 It has the function of mediating opsonophagocytosis; IgM's heavy chain constant region Fc: C ⁇ has a powerful function of activating complement and can bind to macrophages through C1b to promote phagocytosis (although it cannot independently opsonize phagocytosis). [MedicalImmunologyISBN: 9787117208215]
  • the Ig-like BPI-Fc fusion protein composed of BPI or its N-terminal functional fragment and Ig heavy chain constant region Fc or its allele chimera can have the dual functions of BPI and immunoglobulin Fc, that is, it has BPI targeted binding LPS It can directly kill Gram-negative bacteria with lipid A, and also has the ability to rapidly kill Gram-negative bacteria through Fc activation of complement and opsonization, and its mechanism of action can overcome the resistance of Gram-negative bacteria.
  • Gene therapy is a method of introducing functional genes into organs, tissues or cells of the body for expression and disease treatment.
  • Gene therapy vectors are divided into two categories: viral vectors (mainly including adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors, in addition to less used vaccinia virus vectors, pox virus vectors, and herpes simplex) Viral vectors, etc.) and non-viral vectors (mainly including naked DNA, liposomes, nanocarriers, etc.).
  • viral vectors mainly including adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and lentiviral vectors, in addition to less used vaccinia virus vectors, pox virus vectors, and herpes simplex
  • Viral vectors etc.
  • non-viral vectors mainly including naked DNA, liposomes, nanocarriers, etc.
  • the target gene is continuously expressed in the body for 2 to 3 weeks, and has a strong immunogen Sex, difficult to repeat treatment and other characteristics. [Anderson, Nature, 392: 25 (1998)] [Francis S. Collins, Scott Gott Kunststoff. The Next Phase of Human Human Gene-Therapy Oversight. 2018 (https://doi.org/10.1056/NEJMp1810628)].
  • a recombinant adenovirus containing a BPI-Fc chimeric gene was constructed, and it was confirmed that it was obtained in the animal treatment of multi-drug resistance (carrying resistance gene) Gram-negative bacteria in vitro and in vivo infection model animals Successful, and the first to clearly propose the use of adenoviral vectors to mediate the target gene does not integrate with the host cell genome, sustained high expression in the body for 2 to 3 weeks, as a short-term antibiotic replacement therapy for drug-resistant Gram-negative bacteria infection
  • the medicine has obvious advantages and practicality.
  • Gene therapy virus vectors mainly include adenovirus vectors, adeno-associated virus vectors, retrovirus vectors and lentiviral vectors.
  • adenovirus vectors mainly include common serotypes such as Ad5, Ad2 and Ad55. They have a wide range of host cells and can effectively infect dividing cells and non-dividing cells. The DNA does not integrate with the host cell genome and there is no risk of insertion mutation. The target gene is in vivo Continue to express for 2 to 3 weeks. In view of the fact that clinically severe Gram-negative bacterial infections will develop into severe resistance to antibiotics, effective short- and medium-term antibiotic replacement therapy has great advantages and application prospects.
  • the present invention constructs a recombinant adenovirus containing BPI-Fc chimeric gene as a medium- and short-term antibiotic replacement therapy for drug-resistant Gram-negative bacteria infection.
  • BPI N-terminal functional fragment BPI 1-199 and its C-terminally truncated 6-amino acid BPI 1-193 have the same effect as human natural BPI to neutralize endotoxin and directly kill Gram-negative bacteria, in the embodiments of the present invention BPI 1-199 is preferred.
  • IgG: Fc: C ⁇ 1, C ⁇ 2 and C ⁇ 3 have the dual functions of activating complement and mediating opsonophagocytosis; IgA Fc: C ⁇ 1, C ⁇ 2 have the function of mediating opsonophagocytosis; IgM Fc: C ⁇ has a powerful function of activating complement and can bind to macrophages through C1b To promote phagocytosis (but can not independently regulate phagocytosis).
  • the IgG1 heavy chain constant region Fc ⁇ 1 which plays an important role in anti-infection, has the dual functions of activating complement and mediating opsonophagocytosis is preferred.
  • the present invention provides an Ad5-BPI-Fc ⁇ 1 recombinant adenovirus, which comprises a nucleotide sequence encoding a fusion protein of BPI and Fc, and the coding sequence sequentially has from 5 ′ to 3 ′: human BPI
  • the signal peptide coding sequence, BPI 1-199 coding sequence, human immunoglobulin hinge region and Fc ⁇ 1 coding sequence are preferably connected with a CMV promoter and an SV40 poly A expression control element at the 5 ′ end and the 3 ′ end, respectively.
  • nucleotide sequence of the BPI-Fc ⁇ 1 chimeric gene encoding the BPI-Fc ⁇ 1 fusion protein is shown in SEQ ID NO: 1, preferably, BPI 1-199 amino acids 4-24 The coding sequence of the residue was modified to SEQ ID NO: 2.
  • the Ad5-BPI-Fc ⁇ 1 recombinant adenovirus of the present invention infects animal cells and mediated expression of BPI-Fc ⁇ 1 fusion protein (referred to as BPI-Fc ⁇ 1 protein in the following examples and drawings) can be directly killed in vitro, and by activating complement and Regulates phagocytosis to quickly and efficiently kill drug-resistant Gram-negative bacteria, and kills multiple drug-resistant (carrying drug-resistant genes) Gram-negative bacteria in fresh whole blood of mice and humans (simulating in vivo blood serum complement and phagocytic cell environment);
  • the Ad5-BPI-Fc ⁇ 1 recombinant adenovirus has achieved success in the treatment of multi-drug resistance (carrying resistance gene) Gram-negative bacteria infection model animal in vivo.
  • the resistant Gram-negative bacteria exemplified in the examples of the present invention are as follows:
  • Klebsiella pneumoniae Klebsiella pneumoniae ATCC 700603 (with capsule) (multi-drug resistance, ESBLs)
  • Drug resistance ampicillin, aztreonam, cefoxitin, cefpodoxime, ceftazidime, chloramphenicol, piperacillin, tetracycline
  • NDM-1 New Metallo-beta-lactamase
  • coli coli, Serratia and Proteus. These bacteria can cause serious and often fatal infections, such as bloodstream infections and pneumonia, and have developed resistance to a large number of antibiotics, including carbapenems and the best available antibiotics currently used to treat multidrug-resistant bacteria— — The third generation cephalosporins.
  • the second and third levels in the list contain other bacteria that are increasingly resistant and cause more common diseases, such as gonorrhea and food poisoning caused by salmonella.
  • the preferred drug-resistant Gram-negative bacteria and the drug resistance mechanism in the embodiments of the present invention conform to the above-mentioned extremely important category, and have a broad representation of multiple drug resistance (including drug resistance genes) of various categories; wherein, multiple drug resistance involves: Resistant to ampicillin, tetracycline (tetracycline), kanamycin (aminoglycoside), aztreonam, cefoxitin, cefpodoxime, ceftazidime, chloramphenicol, piperacillin, gentamicin (aminoglycoside), ticarcillin, cefepime, ciprofloxacin, quinolones, imipenem, ertapenem and carbepenem; drug resistance genes involved: ampR, tetR, kanR, extended-spectrum beta-lactamase bla SHV-18, New Delhi metallo-beta-lactamase NDM-1.
  • the recombinant adenovirus described in the present invention has a wide range of applications as a drug for the treatment of drug-resistant Gram-negative bacteria infections.
  • the recombinant adenovirus of the present invention is used as a medium- and short-term antibiotic replacement therapy for drug-resistant Gram-negative bacteria infections and short- and medium-term patients with immunocompromised patients Drugs for preventing Gram-negative bacteria infection have great advantages and application prospects.
  • Ad5-BPI-Fc ⁇ 1 recombinant adenovirus liquid chromatography purification map A, SOURCE 30Q anion exchange chromatogram; B, Capto TM Core 700 composite mode medium chromatogram; C, Capto TM Core 700 composite mode medium chromatogram CIP in-situ cleaning map.
  • BPI-Fc ⁇ 1 protein liquid chromatography purification and identification results are included in Figure 2.
  • A SP Fast Flow cation exchange chromatography
  • B Protein A affinity chromatography
  • C Western Blot detection results of purified BPI-Fc ⁇ 1 protein.
  • BPI-Fc ⁇ 1 protein binds LPS in vitro. Among them: A, gel semi-quantitative test results; B, endpoint color rendering quantitative test results.
  • BPI-Fc ⁇ 1 protein kills in vitro and enhances the killing of drug-resistant Gram-negative bacteria by activating complement.
  • A in vitro sterilization and mouse serum complement enhance bactericidal effect
  • B in vitro sterilization and human serum complement enhance bactericidal effect.
  • FIG. 1 BPI-Fc ⁇ 1 protein conditioning in vitro peritoneal phagocytes of BALB / c mice phagocytosis E. coli pET28a-EGFP / BL21 (DE3) tracer experiment. Among them: A. Phagocytosis (fluorescence) tracer experiment. B. Statistical results of experimental phagocytosis in each group.
  • BPI-Fc ⁇ 1 protein in vitro phagocytosis kills E. coli pBR322 / BL21 (DE3).
  • A BALB / c mouse phagocytic phagocytosis sterilization results
  • B U937 cell phagocytosis sterilization results.
  • BPI-Fc ⁇ 1 protein kills the drug resistance gene E.coli pBR322 / BL21 (DE3) in whole blood.
  • BPI-Fc ⁇ 1 protein kills Klebsiella pneumoniae with multi-drug resistance (including resistance genes) in whole blood.
  • BPI-Fc ⁇ 1 protein kills the super-resistant gene Escherichia coli (NDM-1) in whole blood.
  • FIG. 13 Western Blot detection results of BPI-Fc ⁇ 1 protein expression in serum of Ad5-BPI-Fc ⁇ 1 infected mice.
  • Example 1 relates to the construction of Ad5-BPI-Fc ⁇ 1 recombinant adenovirus.
  • Embodiment 2 relates to Ad5-BPI-Fc ⁇ 1 recombinant adenovirus mediated mammalian cells to express BPI-Fc ⁇ 1 protein.
  • Example 3 relates to the direct killing of BPI-Fc ⁇ 1 protein in vitro and enhancing the killing of drug-resistant Gram-negative bacteria by activating complement and opsonizing phagocytosis.
  • Example 4 relates to the killing of drug-resistant Gram-negative bacteria by BPI-Fc ⁇ 1 protein in mouse and human whole blood.
  • Example 5 relates to the protective effect of Ad5-BPI-Fc ⁇ 1 recombinant virus on model animals infected with drug-resistant Gram-negative bacteria.
  • EcoR I / Sal I double digestion (with EcoR I partially digested) pSCm-BPIm23-Fc ⁇ 1 plasmid (constructed by the inventor), the recovered 1.39 kb EcoR I / Sal I digested fragment ( It is a BPI-Fc ⁇ 1 chimeric gene, the sequence of which is shown in SEQ ID NO: 1, from 5 ′ to 3 ′ in sequence with nucleotides encoding human BPI signal peptide, BPI 1-199 , human immunoglobulin hinge region and Fc ⁇ 1 Sequence; wherein the natural coding sequence of amino acid residues 4-24 of BPI 1-199 was changed to SEQ ID NO: 2) by gene synthesis modification; construct the EcoR I / inserted into the pDC316 shuttle plasmid (Microbix) At the site of Sal I digestion, it was transformed into E. coli DH5 ⁇ ; after identification and correct construction, the shuttle expression vector pDC316-BPI-Fc ⁇
  • the shuttle expression vector pDC316-BPI-Fc ⁇ 1 has been deposited on November 8, 2018 in the General Microbiology Center (CGMCC, No. 3 Hospital No. 1, Beichen West Road, Chaoyang District, Beijing, China), deposit number It is CGMCC NO .: 16719, and the classification is named Escherichia coli.
  • AdMax TM system Kit D
  • Merobix use conventional 2000 Transfection method Co-transfection of pDC316-BPI-Fc ⁇ 1 shuttle expression vector and adenovirus backbone vector pBHGlox (delta) E1,3Cre into HEK 293 cells (ATCC CRL-1573), through the Cre / loxP system to recombine Ad5-BPI- Fc ⁇ 1 recombinant adenovirus.
  • Ad5-BPI-Fc ⁇ 1 Separation and purification, collecting the elution peak of Ad5-BPI-Fc ⁇ 1; 4) Concentrating and replacing the virus storage solution (10mM Tris 150mM) with Vivaflow 200 100K ultrafiltration membrane package (Sartorius) NaCl 1mM MgCl 2 10% glycerol pH7.4), to obtain high-quality Ad5-BPI-Fc ⁇ 1 recombinant adenovirus, stored at -70 °C for use.
  • virus storage solution (10mM Tris 150mM) with Vivaflow 200 100K ultrafiltration membrane package (Sartorius) NaCl 1mM MgCl 2 10% glycerol pH7.4
  • Ad5-BPI-Fc ⁇ 1 recombinant adenovirus mediates mammalian cell expression of BPI-Fc ⁇ 1 protein and its purification
  • Embodiment 3 The biological function of the BPI-Fc ⁇ 1 protein: binding LPS in vitro, directly killing and enhancing killing of drug-resistant Gram-negative bacteria through activation of complement and opsonization.
  • E.coli pBR322 / BL21 (selectively cultured with ampicillin) and E.coli pET28a-EGFP / BL21 (DE3) (selectively cultured with kanamycin, and induced expression of EGFP by 1mM IPTG at 30 °C for 16h) Carry out in vitro killing of drug-resistant Gram-negative bacteria.
  • BPI-Fc ⁇ 1 protein binds to LPS in vitro
  • BPI-Fc ⁇ 1 protein sterilizes in vitro and kills resistant Gram-negative bacteria by activating complement
  • BPI-Fc ⁇ 1 protein can effectively kill Gram-negative bacteria in vitro, and can interact with mouse serum complement and activate mouse complement to enhance the bactericidal effect.
  • the bactericidal efficiency is positively correlated with the dose.
  • BPI-Fc ⁇ 1 protein can effectively kill Gram-negative bacteria in vitro, and can enhance the bactericidal effect by activating human serum complement, and its bactericidal efficiency is positively correlated with the dose.
  • mice were killed by devertebralization, soaked in 75% ethanol for 5-10 minutes; removed the mouse's abdominal fur to keep the peritoneum intact; appropriate amount of DMEM medium was injected into the mouse's abdominal cavity and gently Massage for 5 minutes; draw out the cell suspension of the mouse's abdominal cavity, 300 g, and collect the cells by centrifugation for 5 minutes; resuspend the complete medium (DMEM medium containing 10% NBS) and inoculate in a 24-well plate (containing cell slide), 37 °C, 8 % CO 2 culture for 3-7 days.
  • DMEM medium containing 10% NBS
  • BPI-Fc ⁇ 1 protein can phagocytose killing drug-resistant Gram-negative bacteria by conditioning mouse peritoneal phagocytic cells (Figure 8A) and U937 cells ( Figure 8B). The concentration was positively correlated.
  • Example 4 BPI-Fc ⁇ 1 protein kills resistant Gram-negative bacteria in whole blood
  • E. coli pBR322 / BL21 (DE3), Acinetobacter baumannii ATCC BAA-1605, Klebsiella pneumoniae ATCC 700603 (with capsule) and Escherichia coli (NDM-1) ATCC BAA-2469 (respectively Ampicillin, imipenem, cefoxitin and imipenem (selective culture) were carried out to study the killing of drug-resistant Gram-negative bacteria in fresh whole blood (including serum complement and phagocytes).
  • BPI-Fc ⁇ 1 protein kills resistance gene E.coli pBR322 / BL21 (DE3) in whole blood
  • E.coli pBR322 / BL21 (DE3) bacterial solution (5 ⁇ 10 5 CFU / mL, diluted in PBS; the negative control is replaced with an equal amount of PBS) and 20 ⁇ L of different concentrations of BPI-Fc ⁇ 1 protein (5 ⁇ g / mL, 0.5 ⁇ g / mL, 0.25 ⁇ g / mL, diluted in protein preservation solution; the negative and positive controls were replaced with equal amounts of protein preservation solution), mix well, incubate at 37 °C for 10min; add 80 ⁇ L of fresh 0.4% sodium citrate anticoagulant BABL / c mice Blood, mix well and incubate at 37 ° C for 3h; after appropriate dilution, take 100 ⁇ L each for counting by pouring method.
  • BPI-Fc ⁇ 1 protein has a significant killing effect on the resistance gene E.coli pBR322 / BL21 (DE3) in whole blood, and its bactericidal efficiency is positively correlated with the protein dose; when the protein concentration is 0.5 and 5 ⁇ g / mL, the corresponding sterilization rates were 71.5% and 99.5%, respectively.
  • BPI-Fc ⁇ 1 protein can effectively kill gram-negative bacteria through activation of complement and opsonization in whole blood.
  • BPI-Fc ⁇ 1 protein kills multi-drug resistant Acinetobacter baumannii (ATCC BAA-1605) in whole blood
  • BPI-Fc ⁇ 1 protein has a significant killing effect on multi-drug resistant Acinetobacter baumannii in mouse whole blood, and its bactericidal efficiency is positively correlated with the protein dose; when the protein concentration is 50 ⁇ g / mL, The corresponding sterilization rate is 25.4%.
  • BPI-Fc ⁇ 1 protein has a significant killing effect on multi-drug resistant Acinetobacter baumannii in human whole blood, and its bactericidal efficiency is positively correlated with the protein dose; At mL, the corresponding sterilization rates reached 6.9%, 13.2% and 43.4%, respectively.
  • BPI-Fc ⁇ 1 protein kills multi-drug resistance (including resistance genes) Klebsiella pneumoniae (ATCC 700603) in whole blood
  • the method is as above (3.1), using human blood for the experiment.
  • the results are shown in Figure 11B: with the killing effect of whole blood in mice, when the BPI-Fc ⁇ 1 protein reaches an effective concentration, the BPI-Fc ⁇ 1 protein has multiple resistance (including resistance genes) in Klebsiella pneumoniae in human whole blood Obvious killing effect, its sterilization efficiency is also positively correlated with protein dosage; when the protein concentration is 500 and 1000 ⁇ g / mL, the corresponding sterilization rate reaches 76.7% and 94.0% respectively.
  • BPI-Fc ⁇ 1 protein kills super-resistant gene Escherichia coli (NDM-1) in whole blood
  • NDM-1 Escherichia coli
  • BPI-Fc ⁇ 1 protein has a significant killing effect on the super-resistant gene Escherichia coli (NDM-1) in whole blood, and its bactericidal efficiency is positively correlated with the protein dose; where the protein concentration is 5 And 50 ⁇ g / mL, the corresponding sterilization rates reached 21.8% and 70.7%, respectively.
  • mice 5-6 weeks old BALB / c mice were intraperitoneally injected with Ad5-BPI-Fc ⁇ 1 recombinant virus (5 ⁇ 10 8 IU / 100 ⁇ L / each) (Ad5-Null control group was set up in the experiment). On day 5, the eyeballs were removed and blood was collected to obtain serum.
  • the serum was treated with saturated ammonium sulfate precipitation method to obtain crude immunoglobulin precipitate, which is briefly described as follows: 1) Take 0.5mL of serum and add equal amount of saline to mix, add 1mL of saturated ammonium sulfate dropwise with stirring, and let stand at 4 °C 1h, centrifuge at 3000rpm for 20min, discard the supernatant; 2) Dissolve the precipitate with physiological saline to 1mL, then add saturated ammonium sulfate 0.5mL dropwise, let stand at 4 ° C for 3h, centrifuge at 3000rpm for 20min, discard the supernatant; 3) Repeat 2) 1 The crude immunoglobulin precipitate was obtained four times; 4) Dissolve it with 1 mL of PBS and transfer it to a dialysis bag.
  • E. coli pBR322 / BL21 (DE3) (ampR and tetR genes) and Klebsiella pneumoniae (ATCC 700603) (selected by ampicillin and cefoxitin, respectively) were selected from the resistant Gram-negative bacteria involved in the foregoing examples Cultivate) carry out research.
  • E.coli pBR322 / BL21 (DE3) intraperitoneally injected MLD is 2.5 ⁇ 10 4 CFU / 0.5mL; Klebsiella pneumoniae (ATCC 700603) intraperitoneally injected MLD is 1 ⁇ 10 6 CFU / 0.5mL or 2.5 ⁇ 10 6 CFU / 0.5mL.
  • Ad5-BPI-Fc ⁇ 1 recombinant virus transfected mice have a significant protective effect against the lethal dose of E. coli pBR322 / BL21 (DE3) (ampR and tetR genes) infection challenge.
  • Ad5-BPI-Fc ⁇ 1 recombinant virus transfected mice have significant protection against lethal multi-drug resistance (including resistance genes) Klebsiella pneumoniae (ATCC 700603) infection challenge effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Communicable Diseases (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了一种用于治疗耐药革兰氏阴性菌感染的重组腺病毒、其制备及应用。该重组腺病毒包含编码杀菌/渗透增强蛋白(Bactericidal/permeability increasing protein, BPI)和免疫球蛋白重链恒定区Fc的融合蛋白的BPI-Fc嵌合基因。还提供了该重组腺病毒在制备用于治疗耐药革兰氏阴性菌感染的药物组合物中的用途。

Description

包含BPI-Fc嵌合基因的重组腺病毒及其用途 技术领域
本发明涉及基因治疗领域。具体的，本发明涉及一种用于治疗耐药革兰氏阴性菌感染的重组腺病毒、其制备及应用，所述重组腺病毒包含编码杀菌/渗透增强蛋白(Bactericidal/permeablity increasing protein,BPI)和免疫球蛋白重链恒定区Fc的融合蛋白的BPI-Fc嵌合基因。本发明还涉及所述重组腺病毒在制备用于治疗耐药革兰氏阴性菌感染的药物组合物中的用途。
背景技术
抗生素耐药对公共卫生及人类健康构成重大威胁，近年来WHO呼吁并引起全世界重视应对全球耐药感染问题，警示人类将进入后抗生素时代[ANTIMICROBIAL RESISTANCE Global Report on surveillance 2014，WHO]。WHO引用英国Jim O’Neill发表的[Antimicrobial Resistance on the Global Agenda.Dec 1,2015]估计，在全世界范围内每年大约有70万人死于耐药性细菌感染，如果不采取有效的措施，预计到2050年每年会有1000万人死于耐药性细菌感染。细菌多重耐药和超级耐药问题日益突出，其中作为引起人类感染性疾病的主要病原之一的革兰氏阴性菌(Gram-negative bacteria，GNB)对临床常用抗生素有很强的耐药性，是当前最受关注的耐药菌[https://en.wikipedia.org/wiki/Antimicrobial_resistance]。2017年WHO首次公布对人类健康构成最大威胁的12种耐药细菌名单，其中9种为革兰氏阴性菌[Nature 543,15(02 March 2017)doi:10.1038/nature.2017.21550][https://news.un.org/zh/story/2017/02/271472]。特别指出：2009年在肺炎克雷伯菌中发现了质粒介导的超级碳青霉烯耐药基因NDM-1和2015年在大肠埃希氏菌中发现了质粒介导的超级黏菌素耐药基因mcr-1[Antimicrob Agents Chemother.2009；53(12):5046][Lancet Infect Dis.2010；10(9):597][Lancet Infect Dis.2016；16(2):161]，几乎使临床治疗革兰氏阴性菌感染的抗生素最后防线全线失守。随着抗生素耐药日益严重和即将到来的后抗生素时代，研发抗生素替代生物药物具有紧迫性和巨大应用前景。
发明内容
本发明的一个方面，提供了一种重组腺病毒，其包含编码BPI-Fc融合蛋白的嵌合基 因，其中所述嵌合基因包含人BPI功能片段的编码序列和人免疫球蛋白重链恒定区Fc基因，其中所述人BPI功能片段的编码序列的3’端直接或通过人免疫球蛋白铰链区连接至Fc基因。
在一个实施方式中，本发明所述腺病毒为选自如下血清型的腺病毒载体：Ad5、Ad2和Ad55血清型；优选为Ad5血清型。
在一个优选实施方式中，本发明所述人BPI功能片段选自人BPI 1-199片段和人BPI 1-193片段，优选为人BPI 1-199片段。
在一个实施方式中，本发明所述人免疫球蛋白重链恒定区Fc基因选自Cγ1、Cγ2、Cγ3、Cα1、Cα2和Cμ基因或其等位体基因，优选为Cγ1基因。
在本发明的另一个实施方式中，所述编码BPI-Fc融合蛋白的嵌合基因从5'至3'依次具有：人BPI信号肽编码序列、BPI 1-199编码序列、人免疫球蛋白铰链区和Fcγ1的编码序列，优选地，在5’端和3’端分别连接有CMV启动子和SV40 poly A表达控制元件。在一个具体实施方式中，编码所述的BPI-Fcγ1融合蛋白的BPI-Fcγ1嵌合基因的核苷酸序列如SEQ ID NO：1所示，优选地，其中BPI 1-199的第4-24氨基酸残基的编码序列经修饰改变为SEQ ID NO：2。
在优选实施方式中，本发明提供了Ad5-BPI-Fcγ1重组腺病毒，其能够感染动物细胞并表达BPI-Fcγ1融合蛋白，用于直接杀伤、并通过激活补体和调理吞噬快速高效杀伤多重耐药革兰氏阴性菌，并且所述重组腺病毒在体内多重耐药革兰氏阴性菌感染模型动物治疗中取得了成功。
本发明的又一方面提供了所述的重组腺病毒用于治疗耐药革兰氏阴性菌感染的用途，其中所述的耐药革兰氏阴性菌包括多重耐药革兰氏阴性菌，具体涉及多重耐药机制的耐药革兰氏阴性菌。具体地，所述耐药革兰氏阴性菌包括大肠埃希氏菌、鲍曼不动杆菌和肺炎克雷伯菌。在具体的方面，所述耐药革兰氏阴性菌携带耐药基因，所述耐药基因是超广谱耐药基因ESBLs或超级耐药基因NDM-1。
在另一个方面，本发明提供了用于治疗耐药革兰氏阴性菌感染的药物组合物，所述药物组合物包含治疗有效量的本发明的重组腺病毒，以及药学可接受的载体。
本发明的又一方面提供了所述的重组腺病毒在制备用于治疗耐药革兰氏阴性菌感染的药物中的用途，其中所述的耐药革兰氏阴性菌包括多重耐药革兰氏阴性菌，具体涉及多重耐药机制的耐药革兰氏阴性菌。具体地，所述耐药革兰氏阴性菌包括大肠埃希氏菌、鲍曼不动杆菌和肺炎克雷伯菌。在具体的方面，所述耐药革兰氏阴性菌携带耐药基因，所述耐药基因是超广谱耐药基因ESBLs或超级耐药基因NDM-1。
在本发明的另一个方面中，提供了一种制备重组腺病毒的方法，该重组腺病毒包含编码BPI和Fc的融合蛋白的核苷酸序列，该方法包括如下步骤：
(a)提供腺病毒骨架载体；
(b)提供携带BPI和Fc的融合蛋白编码序列表达盒的穿梭表达载体；
(c)使步骤(a)的腺病毒骨架载体与步骤(b)的穿梭表达载体共转染宿主细胞进行同源重组，以获得重组腺病毒。
在一个优选实施方式中，所述方法还包括如下步骤：
(d)用步骤(c)中所得的重组腺病毒感染宿主细胞，和
(e)从被感染的宿主细胞中获得重组腺病毒。
发明详述
杀菌/渗透增强蛋白(Bactericidal/permeablity increasing protein,BPI)是1978年Weiss等首次在人多形核中性粒细胞中发现的一种分子量约为55KD阳离子抗菌蛋白，该抗菌蛋白由456个氨基酸残基组成；其N端功能片段(N端区域第1-199个氨基酸残基的BPI 1-199片段及其C端截短6个氨基酸的第1-193氨基酸残基的BPI 1-193片段)具有与人天然BPI相同的高亲和力结合革兰氏阴性菌的脂多糖(Lipopolysaccharide,LPS)和类脂A、中和内毒素(Endotoxin)和直接杀伤革兰氏阴性菌的作用。XOMA公司自1990s开始研发重组人BPI N端功能片段(
Figure PCTCN2019087381-appb-000001
rBPI 21)并开展了多项临床试验，但因BPI杀菌需持续较长时间(>3小时)维持较高浓度(>10nM)，而rBPI 21体内半衰期短、治疗剂量大、难以在体内持续维持有效治疗浓度等因素，终未取得临床成功而未获FDA批准。[J.Biol.Chem.253:2664(1978)][J Biol Chem.264:9505(1989)][J.Exp.Med.174:649(1991)][J Clin Invest.90:1122(1992)][Cazzola,et al.Curr Opin Pulm Med,10:204(2004)][Mannion,et al.J.Clin.Investig.85:853(1990)][Shock.10:161(1998)][J.Trauma.46:667(1999)][Lancet.356:961(2000)][Crit Care Med 2001 29(7)(Suppl.):S130-S135][ASM News.68:543(2002)]
免疫球蛋白(Immunoglobulin,Ig)包括一个具有许多相似结构但重要结构不同的蛋白家族,这些不同的重要结构导致不同的抗原结合特性和其他生物学活性。人Ig可分为IgG、IgA、IgM、IgD和IgE五类，其中IgG，IgA和IgM这三类还有亚类、在抗感染免疫防御中起重要作用。IgG的重链恒定区Fc(Fragment crystallizable,Fc；Fc的编码序列称为Fc基因)：Cγ1、Cγ2和Cγ3具有激活补体和介导调理吞噬双重功能；IgA的重链恒定区Fc：Cα1、Cα2具有介导调理吞噬功能；IgM的重链恒定区Fc：Cμ具有强大的激活补体 功能并能通过C1b与巨噬细胞结合以促进吞噬(虽不能独立调理吞噬)。[Medical Immunology ISBN:9787117208215]
由BPI或其N端功能片段与Ig重链恒定区Fc或其等位体嵌合组成的Ig样BPI-Fc融合蛋白，可兼备BPI和免疫球蛋白Fc双重功能，即具有BPI靶向结合LPS和类脂A并直接杀伤革兰氏阴性菌，还具有通过Fc激活补体和调理吞噬快速杀伤革兰氏阴性菌，其作用机制能克服革兰氏阴性菌耐药。
基因治疗(Gene therapy)是将功能基因导入体内器官、组织或细胞中表达进行疾病治疗的方法。基因治疗载体分为两大类：病毒载体(主要包括腺病毒载体、腺伴随病毒载体、逆转录病毒载体和慢病毒载体，另外还有使用较少的牛痘病毒载体、痘病毒载体、单纯性疱疹病毒载体等)和非病毒载体(主要包括裸露DNA、脂质体、纳米载体等)。腺病毒载体具有宿主细胞范围广，能有效感染分裂细胞和非分裂细胞，DNA不与宿主细胞基因组整合、不存在插入突变风险，目的基因在体内持续表达2～3周，有较强的免疫原性、难以重复治疗等特点。[Anderson,Nature,392:25(1998)][Francis S.Collins,Scott Gottlieb.The Next Phase of Human Gene-Therapy Oversight.2018(https://doi.org/10.1056/NEJMp1810628)]。
在PCT/CN2005/000986、CN 200580000538.1公开了一种包含BPI-Fc嵌合基因的重组腺伴随病毒，用于在小鼠体内革兰氏阴性菌[E.coli O111:B4(CMCC(B)No.44101-9；抗生素敏感]感染模型动物治疗。鉴于临床上重症革兰氏阴性细菌感染会发展为对抗生素严重耐药并急需得到有效中短期治疗，本发明人经过深入研究，进一步提供了本发明技术方案：构建了一种包含BPI-Fc嵌合基因的重组腺病毒，证实其在多重耐药(携带耐药基因)革兰氏阴性菌体外实验模型和体内感染模型动物治疗中取得了成功，同时率先明确提出运用腺病毒载体介导目的基因不与宿主细胞基因组整合、在体内持续高效表达2～3周的特点，将其作为耐药革兰氏阴性菌感染的中短期抗生素替代治疗药物，具有显著的优势和实用性。
下面就本发明的目的和实施做进一步阐述，本领域的技术人员对本发明的所涉及的范围、内容和优点是显而易见的。尽管本发明提供了优选的实施例，本领域的技术人员将认识到各种修改和变化也在本说明的范围内。
基因治疗病毒载体主要包括腺病毒载体、腺伴随病毒载体、逆转录病毒载体和慢病毒载体等。其中腺病毒载体主要有Ad5、Ad2和Ad55等常用血清型，具有宿主细胞范围广，能有效感染分裂细胞和非分裂细胞，DNA不与宿主细胞基因组整合、不存在插入突变风险，目的基因在体内持续表达2～3周。鉴于临床上重症革兰氏阴性菌感染会发展为对抗生素严重耐药，有效的中短期抗生素替代治疗药物具有巨大优势和应用前景。本发明构建 了一种包含BPI-Fc嵌合基因的重组腺病毒作为耐药革兰氏阴性菌感染的中短期抗生素替代治疗药物。
BPI N端功能片段BPI 1-199及其C端截短6个氨基酸的BPI 1-193具有与人天然BPI相同的中和内毒素和直接杀伤革兰氏阴性菌的作用，本发明实施例中优选BPI 1-199。
IgG Fc：Cγ1、Cγ2和Cγ3具有激活补体和介导调理吞噬双重功能；IgA Fc：Cα1、Cα2具有介导调理吞噬功能；IgM Fc：Cμ具有强大激活补体功能并能通过C1b与巨噬细胞结合以促进吞噬(但不能独立调理吞噬)。本发明实施例中优选抗感染中起重要作用、具有激活补体和介导调理吞噬双重功能的IgG1重链恒定区Fcγ1。
在优选实施方式中，本发明提供了一种Ad5-BPI-Fcγ1重组腺病毒，其包含编码BPI和Fc的融合蛋白的核苷酸序列，该编码序列从5'至3'依次具有：人BPI信号肽编码序列、BPI 1-199编码序列、人免疫球蛋白铰链区和Fcγ1的编码序列，优选地，在5’端和3’端分别连接有CMV启动子和SV40 poly A表达控制元件。在一个具体实施方式中，编码所述的BPI-Fcγ1融合蛋白的BPI-Fcγ1嵌合基因的核苷酸序列如SEQ ID NO：1所示，优选地，其中BPI 1-199第4-24氨基酸残基的编码序列经修饰改变为SEQ ID NO：2。
本发明的Ad5-BPI-Fcγ1重组腺病毒感染动物细胞介导表达的BPI-Fcγ1融合蛋白(在以下实施例和附图中称为BPI-Fcγ1蛋白)在体外可直接杀伤、并通过激活补体和调理吞噬快速高效杀伤耐药革兰氏阴性菌，并且在小鼠和人新鲜全血中(模拟体内血液血清补体和吞噬细胞环境)杀伤多重耐药(携带耐药基因)革兰氏阴性菌；所述的Ad5-BPI-Fcγ1重组腺病毒在体内多重耐药(携带耐药基因)革兰氏阴性菌感染模型动物治疗中取得了成功。
本发明实施例中例示的耐药革兰氏阴性菌如下：
1.E.coli pBR322/BL21(DE3)
耐药基因(抗生素抗性基因)：ampR和tetR
耐药抗性：ampicillin、tetracycline
2.E.coli pET28a-EGFP/BL21(DE3)
耐药基因：kanR
耐药抗性：kanamycin
3.鲍曼不动杆菌(Acinetobacter baumannii)ATCC BAA-1605(多重耐药)
耐药抗性：ceftazidime、gentamicin、ticarcillin、piperacillin、aztreonam、cefepime、ciprofloxacin、imipenem和meropemem
4.肺炎克雷伯菌(Klebsiella pneumoniae)ATCC 700603(具有荚膜)(多重耐药，ESBLs)
耐药基因：Klebsiella pneumoniae plasmid-encoded extended-spectrum beta-lactamase(bla SHV-18)gene
耐药抗性：ampicillin、aztreonam、cefoxitin、cefpodoxime、ceftazidime、chloramphenicol、piperacillin、tetracycline
5.大肠埃希氏菌(Escherichia coli)(NDM-1)ATCC BAA-2469
耐药基因：New Delhi metallo-beta-lactamase(NDM-1)(blaNDM-1超级耐药基因)
耐药抗性：Carbepenem-resistant(imipenem和ertapenem)
2017年2月27日WHO发表了全球首份抗生素耐药“重点病原体”清单，即对人类健康构成最大威胁的12种细菌种族(Families)的目录，以指导和促进新型抗生素的研究与开发，努力解决日益严重的全球抗微生物药物耐药性问题。WHO根据对新型抗生素的迫切需求程度将清单分为极为重要、十分重要和中等重要三个类别。最为重要的一组包括在医院、养老院以及需要用通气机和血液导管等装置进行护理的患者中带来特定威胁的一些耐多药细菌，包括不动杆菌属、假单胞菌属和各种肠杆菌科，包括克雷伯氏菌属、大肠杆菌、沙雷氏菌属和变形杆菌属。这些细菌可引起严重且常常致命的感染，例如血流感染和肺炎，而且已经对大量抗生素产生了耐药性，包括碳青霉烯类和目前用于治疗耐多药细菌的最佳可用抗生素——第三代头孢菌素类药物。清单中的第二和第三个层级中含有其他一些日益出现耐药并引起更常见疾病的细菌，例如淋病和由沙门氏菌引起的食物中毒。本发明实施例中优选的耐药革兰氏阴性菌及其耐药机制符合上述极为重要类别，并具有各类别多重耐药(包括耐药基因)的广泛代表性；其中，多重耐药涉及：Resistant to ampicillin,tetracycline(四环素类),kanamycin(氨基糖甙类),aztreonam,cefoxitin,cefpodoxime,ceftazidime,chloramphenicol,piperacillin,gentamicin(氨基糖甙类),ticarcillin,cefepime,ciprofloxacin(喹诺酮类),meropemem,imipenem,ertapenem and carbepenem；耐药基因涉及：ampR,tetR,kanR,extended-spectrum beta-lactamase bla SHV-18,New Delhi metallo-beta-lactamase NDM-1。
综上，本发明所述的重组腺病毒，具有作为耐药革兰氏阴性菌感染治疗药物的广泛用途。根据腺病毒载体介导目的基因在体内持续表达2～3周的特点，本发明的所述的重组腺病毒作为耐药革兰氏阴性菌感染中短期抗生素替代治疗药物和免疫功能低下患者中短期预防革兰氏阴性菌感染药物，具有巨大优势和应用前景。
附图说明
图1.Ad5-BPI-Fcγ1重组腺病毒液相层析纯化图谱。其中：A、SOURCE 30Q阴离子 交换层析图谱；B、Capto TM Core 700复合模式介质层析图谱；C、Capto TM Core 700复合模式介质层析CIP在位清洗图谱。
图2.BPI-Fcγ1蛋白液相层析纯化及鉴定结果。其中：A、SP
Figure PCTCN2019087381-appb-000002
Fast Flow阳离子交换层析图谱；B、Protein A亲和层析图谱；C、纯化BPI-Fcγ1蛋白Western Blot检测结果。
图3.BPI-Fcγ1蛋白体外结合LPS。其中：A、凝胶半定量法检测结果；B、终点显色法定量检测结果。
图4.BPI-Fcγ1蛋白体外杀伤并通过激活补体增强杀伤耐药革兰氏阴性菌。其中：A、体外杀菌及小鼠血清补体增强杀菌作用；B、体外杀菌及人血清补体增强杀菌作用。
图5.BPI-Fcγ1蛋白体外调理BALB/c小鼠腹腔吞噬细胞吞噬E.coli pET28a-EGFP/BL21(DE3)示踪实验。其中：A、吞噬(荧光)示踪实验。B、各组实验吞噬现象统计结果。
图6.BPI-Fcγ1蛋白体外调理人单核细胞系U937细胞吞噬E.coli pET28a-EGFP/BL21(DE3)示踪实验。
图7.BPI-Fcγ1蛋白体外调理人外周血白细胞吞噬E.coli pET28a-EGFP/BL21(DE3)示踪实验。
图8.BPI-Fcγ1蛋白体外调理吞噬杀伤E.coli pBR322/BL21(DE3)。其中：A、BALB/c小鼠腹腔吞噬细胞吞噬杀菌结果；B、U937细胞吞噬杀菌结果。
图9.BPI-Fcγ1蛋白在全血中杀伤耐药基因E.coli pBR322/BL21(DE3)。
图10.BPI-Fcγ1蛋白在全血中杀伤多重耐药鲍曼不动杆菌。其中：A、小鼠全血实验；B、人全血实验。
图11.BPI-Fcγ1蛋白在全血中杀伤多重耐药(包括耐药基因)肺炎克雷伯菌。其中：A、小鼠全血实验；B、人全血实验。
图12.BPI-Fcγ1蛋白在全血中杀伤超级耐药基因大肠埃希氏菌(NDM-1)。
图13.Ad5-BPI-Fcγ1感染小鼠血清表达BPI-Fcγ1蛋白Western Blot检测结果。
下面结合实施例和附图进一步说明本发明的技术方案，但不限于本实施例。更具体地说，实施例一涉及Ad5-BPI-Fcγ1重组腺病毒的构建。实施例二涉及Ad5-BPI-Fcγ1重组腺病毒介导哺乳动物细胞表达BPI-Fcγ1蛋白。实施例三涉及BPI-Fcγ1蛋白体外直接杀伤并通过激活补体和调理吞噬增强杀伤耐药革兰氏阴性菌。实施例四涉及BPI-Fcγ1蛋白在小鼠和人全血中杀伤耐药革兰氏阴性菌。实施例五涉及Ad5-BPI-Fcγ1重组病毒对耐药革 兰氏阴性菌感染模型动物的保护作用。
实施例一 包含BPI-Fc嵌合基因的一种Ad5-BPI-Fcγ1重组腺病毒的构建
1.pDC316-BPI-Fcγ1腺病毒穿梭表达载体的构建
按常规分子克隆实验技术，用EcoR I/Sal I双酶切(其中EcoR I部分酶切)pSCm-BPIm23-Fcγ1质粒(本发明人构建)，回收的1.39kb EcoR I/Sal I酶切片段(为BPI-Fcγ1嵌合基因，其序列如SEQ ID NO：1所示，从5'至3'依次具有编码人BPI信号肽、BPI 1-199、人免疫球蛋白铰链区和Fcγ1的核苷酸序列；其中BPI 1-199第4-24氨基酸残基的天然编码序列经基因合成修饰改变为SEQ ID NO：2)；将回收的酶切片段构建插入到pDC316穿梭质粒(Microbix)的EcoR I/Sal I酶切位点上，转化到E.coli DH5α；经鉴定正确构建得到穿梭表达载体pDC316-BPI-Fcγ1。
所述穿梭表达载体pDC316-BPI-Fcγ1业已于2018年11月8日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC，中国北京市朝阳区北辰西路1号院3号)，保藏号为CGMCC NO.：16719，分类命名为大肠埃希氏菌Escherichia coli。
2.Ad5-BPI-Fcγ1重组腺病毒的包装及其制备
简述如下：参照AdMax TM system(Kit D)手册(Microbix)，用常规
Figure PCTCN2019087381-appb-000003
2000转染方法把pDC316-BPI-Fcγ1穿梭表达载体和腺病毒骨架载体pBHGlox(delta)E1,3Cre共转染HEK 293细胞(ATCC CRL-1573)，通过Cre/loxP系统实现重组产生Ad5-BPI-Fcγ1重组腺病毒。在符合GMP标准条件下：1)挑取病毒空斑收集病毒，进行病毒扩增和放大生产(
Figure PCTCN2019087381-appb-000004
320 5L罐体生物反应器，
Figure PCTCN2019087381-appb-000005
片状载体，接毒前用含10％NBS的DMEM生长培养基和接毒后用含2％NBS的DMEM维持培养基灌流培养HEK 293细胞)；2)经8000rpm离心收集病毒上清、0.65μm PVDF膜包(Millipore)切向流预过滤、
Figure PCTCN2019087381-appb-000006
2盒式膜包(300K)超滤膜浓缩，进行Benzonase核酸酶处理；3)先后经SOURCE 30Q阴离子交换层析(如图1A)和Capto TM Core 700复合模式介质层析(GE Life Sciences)(如图1B；其后CIP在位清洗如图1C)分离纯化，收集Ad5-BPI-Fcγ1洗脱峰；4)用Vivaflow 200 100K超滤膜包(Sartorius)浓缩并置换病毒保存液(10mM Tris 150mM NaCl 1mM MgCl 2 10％甘油pH7.4)，得到高质量Ad5-BPI-Fcγ1重组腺病毒，保存于-70℃备用。经检测：病毒滴度≥5×10 9IU/mL、比滴度(IU/VP)≥3.3％(分别TCID50和OD260检测)，OD260/280比值1.2-1.4；目的BPI-Fcγ1嵌合基因经PCR扩增后进行DNA测序显示其序列与SEQ ID NO：1一致。
实施例二 Ad5-BPI-Fcγ1重组腺病毒介导哺乳动物细胞表达BPI-Fcγ1蛋白及其提纯
CELLSPIN&Spinner flask(INTEGRA Biosciences AG)中接种CHO-DG44细胞(Gibco)与Ad5-BPI-Fcγ1重组腺病毒(MOI＝40)(于无血清培养基CD DG44Medium，37℃，8％CO 2，静置培养2h)，再加适量SP
Figure PCTCN2019087381-appb-000007
Fast Flow共培养5天(60rpm)，收集SP
Figure PCTCN2019087381-appb-000008
Fast Flow并装柱进行层析分离纯化(如图2A)；之后再经Protein A亲和层析分离纯化(如图2B)，收集BPI-Fcγ1蛋白洗脱峰；用Amicon Ultra-15(NMWL 30KD)超滤离心浓缩并置换蛋白保存液(0.15M NaCl 20mM柠檬酸0.1％v/v Poloxamer 188 0.002％v/v Polysorbate 80pH 5.0)，-30℃保存备用。经Western Blot鉴定(如图2C)，DTT还原泳道出现预期的48kDa谱带，无DTT还原泳道出现96kDa为主谱带。
实施例三 BPI-Fcγ1蛋白的生物功能：体外结合LPS、直接杀伤并通过激活补体和调理吞噬增强杀伤耐药革兰氏阴性菌。
E.coli pBR322/BL21(DE3)(经氨苄青霉素选择培养)和E.coli pET28a-EGFP/BL21(DE3)(经卡那霉素选择培养，并经1mM IPTG在30℃下诱导16h表达EGFP)开展体外杀伤耐药革兰氏阴性菌研究。
1.BPI-Fcγ1蛋白体外结合LPS
1.1于无内毒素的96孔酶标板中，每孔加入200μL 0.5μg/mL PBS稀释的羊抗人IgG-Fc(Novex)，4℃包被过夜，用PBST(含0.1％Tween-20的PBS)清洗平板(5分钟/次，共3次)；然后每孔加入200μL含5％BSA的PBST溶液，37℃封闭1h，再用PBST清洗平板(5分钟/次，共3次)；于每孔中加入200μL BPI-Fcγ1蛋白(0μg/mL和8μg/mL)和LPS(内毒素标准品，8EU/mL)的孵育混合物(涡旋混匀，37℃,30min)，37℃孵育1h。按鲎试剂(厦门鲎试剂生物科技股份有限公司)说明书凝胶半定量实验方法操作。结果如图3A所示：BPI-Fcγ1蛋白能有效结合LPS。
1.2于无内毒素的96孔酶标板中，每孔加入200μL 0.5μg/mL PBS稀释的羊抗人IgG-Fc(Novex)，4℃包被过夜，用PBST清洗平板(5分钟/次，共3次)；然后每孔加入200μL含5％BSA的PBST溶液，37℃封闭1h，再用PBST清洗平板(5分钟/次，共3次)；于每孔中加入200μL含不同浓度BPI-Fcγ1蛋白(0.025μg/mL和0.1μg/mL；阴性及阳性对照均以等量无内毒素PBS替代)和LPS(内毒素标准品，0.05EU/mL；阴性对照以细菌内毒素检查用水替代)的孵育混合物(涡旋混匀，37℃，30min)，37℃孵育1h。于每孔取100μL孵育样品按ToxinSensor TM内毒素检测试剂盒(南京金斯瑞生物科技有限公司，L00350)说明书进行显色反应，测定OD545nm值并计算样品内毒素含量。结果如图3B 所示：BPI-Fcγ1蛋白能有效结合LPS，且其结合能力呈现剂量依赖关系。
2.BPI-Fcγ1蛋白体外杀菌并通过激活补体杀伤耐药革兰氏阴性菌
2.1 BPI-Fcγ1蛋白体外杀菌及小鼠血清补体增强杀菌作用
取50μL E.coli pBR322/BL21(DE3)菌液(2.5×10 5CFU/mL，稀释于10％Hanks 40mM Tris-HCl 0.1％酪蛋白氨基酸pH7.5；阴性对照以等量该缓冲液替代)与50μL BPI-Fcγ1蛋白(0.8μg/mL、0.4μg/mL、0.2μg/mL，稀释于蛋白保存液；阴性及阳性对照均以等量蛋白保存液替代)混匀，37℃水浴3h；再加入100μL 4％BALB/c小鼠血清(PBS稀释；对照组以等量PBS替代)，37℃水浴1h；菌液稀释后倾注法计数。结果如图4A所示：BPI-Fcγ1蛋白在体外可有效杀伤革兰氏阴性菌，并可通过与小鼠血清补体交叉作用、激活小鼠补体增强杀菌效应，其杀菌效率与剂量呈正相关。
2.2 BPI-Fcγ1蛋白体外杀菌及人血清补体增强杀菌作用
方法如上(2.1)，采用4％人血清进行实验(同时将BPI-Fcγ1蛋白浓度改为1μg/mL、0.5μg/mL、0.1μg/mL)。结果如图4B所示：BPI-Fcγ1蛋白在体外可有效杀伤革兰氏阴性菌，且可通过激活人血清补体增强杀菌效应，其杀菌效率与剂量呈正相关。
3.BPI-Fcγ1蛋白体外通过调理吞噬杀伤耐药革兰氏阴性菌
3.1 BPI-Fcγ1蛋白调理吞噬耐药革兰氏阴性菌示踪实验
3.1.1分离BALB/c小鼠腹腔吞噬细胞：小鼠脱椎处死，75％乙醇浸泡5-10min；去除小鼠腹腔皮毛，保持腹膜完整；取适量DMEM培养基注射入小鼠腹腔，轻轻按摩5min；抽出小鼠腹腔细胞悬液，300g，5min离心收集细胞；完全培养基(含10％NBS的DMEM培养基)重悬后接种于24孔板(含细胞爬片)，37℃，8％CO 2培养3-7天。以DiI(Beyotime)染色20min，PBS洗涤3次备用；取50μL E.coli pET28a-EGFP/BL21(DE3)菌液(PBS洗涤并制备成2×10 9CFU/mL悬液；阴性对照以等量PBS替代)与50μL BPI-Fcγ1蛋白(20μg/mL，稀释于蛋白保存液；阴性及阳性对照均以等量蛋白保存液替代)混匀，37℃孵育10min，后均匀覆盖于细胞上，37℃孵育60min；PBS洗涤3次，固定液(乙酸：甲醇为1:3的混合液)固定5min，PBS洗涤3次，将爬片反置于载玻片上，封片，荧光显微镜下观察。结果如图5A所示：DiI标识小鼠腹腔吞噬细胞(DiI，红色荧光)，诱导表达的EGFP标识E.coli pET28a-EGFP/BL21(DE3)(EGFP，绿色荧光)，并进行两种标识图像叠加处理(DiI+EGFP)，箭头标示吞噬现象(细胞膜或胞内见EGFP绿色荧光)，可见BPI-Fcγ1蛋白可与小鼠种属Fc受体交叉作用并显著促进小鼠吞噬细胞吞噬E.coli  pET28a-EGFP/BL21(DE3)；图5B统计结果显示添加BPI-Fcγ1蛋白促使吞噬率由25％提高至92.5％。
3.1.2收集人单核细胞系U937细胞(中国科学院上海生命科学研究院细胞资源中心，4×10 6cells)，以DiI染色30min，PBS洗涤2次备用；取110μL E.coli pET28a-EGFP/BL21(DE3)菌液(PBS洗涤并制备成2×10 9CFU/mL悬液；阴性对照以等量PBS替代)与110μL BPI-Fcγ1蛋白(40μg/mL，稀释于蛋白保存液；阴性及阳性对照均以等量蛋白保存液替代)混匀，37℃孵育20min；取200μL混合物与U937细胞混匀，37℃，150rpm震荡孵育2.5h；200g，1min离心弃上清，并以PBS洗涤2次；4％组织细胞固定液(SolarBio)固定10min；PBS和超纯水依次各洗涤1次，取适量封片液重悬细胞后滴加于载玻片上，封片，荧光显微镜下观察。结果如图6所示：标识同上(3.1.1)，可见BPI-Fcγ1蛋白可显著促进U937细胞吞噬E.coli pET28a-EGFP/BL21(DE3)。
3.1.3采用人外周血白细胞分离液试剂盒(SolarBio)分离并收集人白细胞(4×10 6cells)。方法如上(3.1.2)，其中变为：取150μL混合物与人白细胞混匀，37℃，200rpm震荡孵育1h。结果如图7所示：标识同上(3.1.1)，由于人白细胞为非单一组成的细胞，不同类型的吞噬细胞DiI着色效果及荧光淬灭时间不同，DiI未能均匀标识出所有吞噬细胞，因此增加可见光拍摄图以便对照，结果如图7所示：BPI-Fcγ1蛋白可显著促进人白细胞吞噬E.coli pET28a-EGFP/BL21(DE3)。
3.2 BPI-Fcγ1蛋白体外通过调理吞噬杀伤耐药革兰氏阴性菌
1)分离BALB/c小鼠腹腔吞噬细胞(同上3.1.1方法)接种于96孔板，37℃，8％CO 2培养1-3天备用(汇合度>80％)；取30μL E.coli pBR322/BL21(DE3)菌液(PBS洗涤并制备成2×10 7CFU/mL悬液；阴性对照以等量PBS替代)与30μL BPI-Fcγ1蛋白(4μg/mL、2μg/mL，稀释于蛋白保存液；阴性及阳性对照分别以等量蛋白保存液替代)混匀，37℃水浴10min，后均匀覆盖于细胞上，37℃孵育60分钟；各孵育样品混匀后取20μL进行倍比稀释(10 -1～10 -5)，并将各稀释样分别取100μL进行倾注法菌落计数；同时以CHO-DG44细胞作为对照，进行以上操作。
2)收集人单核细胞系U937(2×10 6cells)，PBS洗涤2次备用；取60μL E.coli pBR322/BL21(DE3)菌液(PBS洗涤并制备成2×10 7CFU/mL悬液；阴性对照以等量PBS替代)与60μL BPI-Fcγ1蛋白(2μg/mL、1μg/mL、0.5μg/mL，稀释于蛋白保存液；阴性及阳性对照分别以等量蛋白保存液替代)混匀，37℃水浴20min；取100μL混合物与U937细胞混匀，37℃200rpm震荡孵育1h(96孔板中)；各孵育样品300g，5min离心收集上清，并分别取20μL进行倍比稀释(10 -1～10 -5)，并将各稀释样分别取100μL进行倾注法 菌落计数；同时以CHO-DG44细胞作为对照，进行以上操作。
结果如图8所示：BPI-Fcγ1蛋白体外可通过调理小鼠腹腔吞噬细胞(图8A)和U937细胞(图8B)吞噬杀伤耐药革兰氏阴性菌，其吞噬杀菌率与BPI-Fcγ1蛋白浓度呈正相关。
实施例四 BPI-Fcγ1蛋白在全血中杀伤耐药革兰氏阴性菌
E.coli pBR322/BL21(DE3)、鲍曼不动杆菌ATCC BAA-1605、肺炎克雷伯菌ATCC 700603(具有荚膜)和大肠埃希氏菌(NDM-1)ATCC BAA-2469(分别经氨苄青霉素、亚胺培南、头孢西丁和亚胺培南选择培养)开展新鲜全血(含血清补体和吞噬细胞)杀伤耐药革兰氏阴性菌研究。
1.BPI-Fcγ1蛋白在全血中杀伤耐药基因E.coli pBR322/BL21(DE3)
鉴于(健康志愿者)新鲜人全血对E.coli pBR322/BL21(DE3)产生强抵抗(如血清型反应和吞噬清除)，本研究开展在小鼠全血中杀伤E.coli pBR322/BL21(DE3)。
取20μL E.coli pBR322/BL21(DE3)菌液(5×10 5CFU/mL，PBS稀释；阴性对照以等量PBS替代)与20μL不同浓度的BPI-Fcγ1蛋白(5μg/mL、0.5μg/mL、0.25μg/mL，稀释于蛋白保存液；阴性及阳性对照分别以等量蛋白保存液替代)混匀，37℃孵育10min；加入80μL新鲜0.4％柠檬酸钠抗凝BABL/c小鼠全血，混匀并于37℃孵育3h；适当稀释后各取100μL进行倾注法计数。结果如图9所示：BPI-Fcγ1蛋白在全血中对耐药基因E.coli pBR322/BL21(DE3)具有显著杀伤效果，其杀菌效率与蛋白剂量呈正相关；其中当蛋白浓度为0.5和5μg/mL时，相应杀菌率分别为71.5％和99.5％。结合图4及图8结果可知BPI-Fcγ1蛋白在全血中可通过激活补体及调理吞噬高效杀伤革兰氏阴性菌。
2.BPI-Fcγ1蛋白在全血中杀伤多重耐药鲍曼不动杆菌(ATCC BAA-1605)
2.1在小鼠全血中杀伤鲍曼不动杆菌
取20μL鲍曼不动杆菌(ATCC BAA-1605)菌液(1×10 4CFU/mL)与20μL不同浓度的BPI-Fcγ1蛋白(50μg/mL、5μg/mL、0.5μg/mL，稀释于蛋白保存液；阴性及阳性对照分别以等量蛋白保存液替代)混匀,37℃孵育10min；加入80μL新鲜0.4％柠檬酸钠抗凝BABL/c小鼠全血，混匀并于37℃孵育1h；适当稀释后各取100μL进行倾注法计数。结果如图10A所示：BPI-Fcγ1蛋白在小鼠全血中对多重耐药鲍曼不动杆菌具有明显杀伤效果，其杀菌效率与蛋白剂量呈正相关；其中当蛋白浓度为50μg/mL时，相应杀菌率为25.4％。
2.2在人全血中杀伤鲍曼不动杆菌
方法如上(2.1)，采用人血进行实验。结果如图10B所示：BPI-Fcγ1蛋白在人全血中 对多重耐药鲍曼不动杆菌具有显著杀伤效果，其杀菌效率与蛋白剂量呈正相关；其中当蛋白浓度为0.5、5和50μg/mL时，相应杀菌率分别达到6.9％、13.2％和43.4％。
3.BPI-Fcγ1蛋白在全血中杀伤多重耐药(包括耐药基因)肺炎克雷伯菌(ATCC 700603)
3.1在小鼠全血中杀伤肺炎克雷伯菌
取50μL肺炎克雷伯菌(ATCC 700603)(1×10 4CFU/mL，PBS稀释；阴性对照以等量PBS替代)与50μL不同浓度的BPI-Fcγ1蛋白(1000μg/mL、500μg/mL、250μg/mL，稀释于蛋白保存液；阴性及阳性对照分别以等量蛋白保存液替代)混匀，37℃孵育3h；加入100μL新鲜0.4％柠檬酸钠抗凝BABL/c小鼠全血，混匀并于37℃孵育1h；适当稀释后各取100μL进行倾注法计数。结果如图11A所示：当BPI-Fcγ1蛋白达到较高有效浓度时，在小鼠全血中对具有荚膜(可抵抗吞噬作用和阻碍BPI N端功能片段结合LPS)的多重耐药(包括耐药基因)肺炎克雷伯菌具有明显杀伤效果，其杀菌效率与蛋白剂量亦呈正相关；其中当蛋白浓度为500和1000μg/mL时，相应杀菌率分别达到82.5％和98.3％。
3.2在人全血中杀伤肺炎克雷伯菌
方法如上(3.1)，采用人血进行实验。结果如图11B所示：同小鼠全血杀伤效果，当BPI-Fcγ1蛋白达到有效浓度时，BPI-Fcγ1蛋白在人全血中对多重耐药(包括耐药基因)肺炎克雷伯菌具有明显杀伤效果，其杀菌效率与蛋白剂量亦呈正相关；其中当蛋白浓度为500和1000μg/mL时，相应杀菌率分别达到76.7％和94.0％。
4.BPI-Fcγ1蛋白在全血中杀伤超级耐药基因大肠埃希氏菌(NDM-1)
鉴于(健康志愿者)新鲜人全血对大肠埃希氏菌(NDM-1)产生强抵抗(如血清型反应和吞噬清除)，本研究开展在小鼠全血中杀伤大肠埃希氏菌(NDM-1)。
取20μL大肠埃希氏菌(NDM-1)(ATCC BAA-2469)(1×10 4CFU/mL)与20μL不同浓度的BPI-Fcγ1蛋白(50μg/mL、5μg/mL、0.5μg/mL，稀释于蛋白保存液；阴性及阳性对照分别以等量蛋白保存液替代)混匀，37℃孵育1h；加入80μL新鲜0.4％柠檬酸钠抗凝BABL/c小鼠全血，混匀并于37℃孵育1h；适当稀释后各取100μL进行倾注法计数。结果如图12所示：BPI-Fcγ1蛋白在全血中对超级耐药基因大肠埃希氏菌(NDM-1)具有显著杀伤效果，其杀菌效率与蛋白剂量呈正相关；其中当蛋白浓度为5和50μg/mL时，相应杀菌率分别达到21.8％和70.7％。
实施例五 Ad5-BPI-Fcγ1重组病毒介导BPI-Fcγ1蛋白在小鼠体内表达及对耐药革 兰氏阴性菌感染模型动物的保护作用
1.Ad5-BPI-Fcγ1重组病毒介导BPI-Fcγ1蛋白在小鼠血清中表达
5-6周龄BALB/c小鼠腹腔注射Ad5-BPI-Fcγ1重组病毒(5×10 8IU/100μL/每只)(实验设Ad5-Null对照组)，分别于注射病毒后第3天和第5天摘除眼球采血获得血清。用饱和硫酸铵沉淀法处理血清获得粗提免疫球蛋白沉淀物，简述如下：1)取0.5mL血清中加入等量生理盐水混匀，搅拌下逐滴加入饱和硫酸铵1mL，4℃静置1h，3000rpm离心20min，弃上清；2)以生理盐水溶解沉淀至1mL，再逐滴加入饱和硫酸铵0.5mL，4℃静置3h，3000rpm离心20min，弃上清；3)重复2)1次，得到粗提免疫球蛋白沉淀物；4)用1mL PBS将其溶解后转至透析袋中用PBS充分透析，后将透析袋包埋于聚乙二醇中吸水浓缩至50ul血清免疫球蛋白浓缩液备用。常规ELISA检测：用人IgG ELISA试剂盒(1750)(购自Alpha Diagnostic)检测；结果显示：在注射Ad5-BPI-Fcγ1后的第3天和第5天，小鼠血清中BPI-Fcγ1蛋白表达水平分别为21.4±0.2ng/mL(n＝3)和30.5±7.4ng/mL(n＝3)(注：Ad5-Null对照组未检测到)。常规Western Blot检测：用HRP标记羊抗人IgG Fc多抗(购自英国KPL公司)检测，按Western Blot发光试剂盒(购自Pierce)操作说明，用X光胶片曝光显影；结果如图13所示：在96kD出现预期的Ad5-BPI-Fcγ1感染小鼠血清表达BPI-Fcγ1蛋白谱带(非还原)，而Ad5-Null对照组没有(注：在140kD出现的谱带被认为是羊抗人IgG Fc多抗与小鼠IgG发生交叉的谱带)。
2.Ad5-BPI-Fcγ1重组病毒对耐药革兰氏阴性菌感染小鼠的保护作用
从前述实施例涉及的耐药革兰氏阴性菌中选取E.coli pBR322/BL21(DE3)(ampR和tetR基因)和肺炎克雷伯菌(ATCC 700603)(分别经氨苄青霉素和头孢西丁选择培养)开展研究。
2.1耐药革兰氏阴性菌感染BALB/c小鼠的最低致死量测定
用含5％高活性干酵母
Figure PCTCN2019087381-appb-000009
的PBS将待测耐药革兰氏阴性菌稀释成不同浓度菌液，对随机分组(每组10只)的6-7周龄BALB/c小鼠腹腔注射菌液(0.5mL/每只)，观察小鼠的死亡情况；将72小时内引起90-100％小鼠感染死亡的最低菌量确定为最小致死量(minimal lethal dose，简称MLD)。实验测定：E.coli pBR322/BL21(DE3)腹腔注射的MLD为2.5×10 4CFU/0.5mL；肺炎克雷伯菌(ATCC 700603)腹腔注射的MLD为1×10 6CFU/0.5mL或2.5×10 6CFU/0.5mL。
2.2 Ad5-BPI-Fcγ1重组病毒对耐药基因E.coli pBR322/BL21(DE3)感染小鼠的保 护作用
对随机分组(每组10只)的6-7周龄BALB/c小鼠每只腹腔注射5×10 8IU/100μL Ad5-BPI-Fcγ1重组病毒(实验设PBS对照组和Ad5-Null对照组)，于第3天对每只小鼠腹腔注射MLD致死量耐药革兰氏阴性菌进行感染攻击，观察小鼠72小时内的死亡情况。结果如表1所示：Ad5-BPI-Fcγ1重组病毒转染小鼠对致死量耐药基因E.coli pBR322/BL21(DE3)(ampR和tetR基因)感染攻击具有显著保护作用。
表1.对致死量耐药基因E.coli pBR322/BL21(DE3)感染攻击的保护作用
Figure PCTCN2019087381-appb-000010
(与Ad5-Null和PBS组相比： #P<0.05)
2.3 Ad5-BPI-Fcγ1重组病毒对多重耐药肺炎克雷伯菌感染小鼠的保护作用
方法如上(2.2)，结果如表2所示：Ad5-BPI-Fcγ1重组病毒转染小鼠对致死量多重耐药(包括耐药基因)肺炎克雷伯菌(ATCC 700603)感染攻击具有显著保护作用。
表2.对致死量多重耐药肺炎克雷伯菌感染攻击的保护作用
Figure PCTCN2019087381-appb-000011
Figure PCTCN2019087381-appb-000012
注：*MLD为1×10 6CFU/0.5mL，**MLD为2.5×10 6CFU/0.5mL。
(与Ad5-BPI-Fcγ1组相比： #P<0.05， ##P<0.01)

Claims (12)

  1. 一种重组腺病毒，其包含编码BPI-Fc融合蛋白的嵌合基因，其中所述嵌合基因包含人BPI功能片段的编码序列和人免疫球蛋白重链恒定区Fc基因，其中所述人BPI功能片段的编码序列的3’端直接或通过人免疫球蛋白铰链区连接至Fc基因。
  2. 根据权利要求1所述的重组腺病毒，其中所述腺病毒是Ad5、Ad2或Ad55血清型，优选为Ad5血清型。
  3. 根据权利要求1或2所述的重组腺病毒，其中所述人BPI功能片段选自人BPI 1-199片段和人BPI 1-193片段，优选为人BPI 1-199片段。
  4. 根据权利要求1-3任一项所述的重组腺病毒，其中所述人免疫球蛋白重链恒定区Fc基因选自Cγ1、Cγ2、Cγ3、Cα1、Cα2和Cμ基因或其等位体基因，优选为Cγ1基因。
  5. 根据权利要求1-4任一项所述的重组腺病毒，其中所述编码BPI-Fc融合蛋白的嵌合基因从5'至3'依次具有：人BPI信号肽编码序列、BPI 1-199片段编码序列、人免疫球蛋白铰链区和Fcγ1的编码序列，优选地，在5’端和3’端分别连接有CMV启动子和SV40 poly A表达控制元件。
  6. 根据权利要求3-5任一项所述的重组腺病毒，其中所述BPI 1-199片段的第4-24氨基酸残基的编码序列为SEQ ID NO：2。
  7. 根据权利要求1-6任一项所述的重组腺病毒，其中所述编码BPI-Fc融合蛋白的嵌合基因的序列如SEQ ID NO：1所示。
  8. 权利要求1-7任一项所述的重组腺病毒，其用于治疗耐药革兰氏阴性菌感染。
  9. 权利要求1-7任一项所述的重组腺病毒在制备用于治疗耐药革兰氏阴性菌感染的药物中的用途。
  10. 权利要求9所述的用途，其中所述的耐药革兰氏阴性菌选自耐药大肠埃希氏菌、耐药鲍曼不动杆菌和耐药肺炎克雷伯菌。
  11. 权利要求9所述的用途，其中所述的耐药革兰氏阴性菌携带耐药基因，所述耐药基因是超广谱耐药基因ESBLs或超级耐药基因NDM-1。
  12. 一种用于治疗耐药革兰氏阴性菌感染的药物组合物，其包含治疗有效量的权利要求1-7任一项的重组腺病毒以及药学可接受的载体。
PCT/CN2019/087381 2018-11-19 2019-05-17 包含BPI-Fc嵌合基因的重组腺病毒及其用途 WO2020103412A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811374590.9A CN109762791B (zh) 2018-11-19 2018-11-19 包含BPI-Fc嵌合基因的重组腺病毒及其用途
CN201811374590.9 2018-11-19

Publications (2)

Publication Number Publication Date
WO2020103412A1 true WO2020103412A1 (zh) 2020-05-28
WO2020103412A8 WO2020103412A8 (zh) 2020-09-10

Family

ID=66449558

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/087381 WO2020103412A1 (zh) 2018-11-19 2019-05-17 包含BPI-Fc嵌合基因的重组腺病毒及其用途

Country Status (2)

Country Link
CN (1) CN109762791B (zh)
WO (1) WO2020103412A1 (zh)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102168074A (zh) * 2011-02-17 2011-08-31 清华大学 一种重组腺病毒及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1163513C (zh) * 2000-05-22 2004-08-25 首都医科大学 一种高效生产BPI-Fc重组蛋白的方法
CN1733911A (zh) * 2004-08-13 2006-02-15 首都医科大学 一种包含bpi基因的重组病毒及含有其的药物组合物及其用途
KR101719556B1 (ko) * 2011-03-30 2017-03-24 주식회사 레고켐 바이오사이언스 신규한 세파로스포린 유도체 및 이를 함유하는 의약 조성물

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102168074A (zh) * 2011-02-17 2011-08-31 清华大学 一种重组腺病毒及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
QINGLI KONG ET AL: "Infecting Mice with Recombinant Ad5-BPI23-Fc Gamma 1 Virus Protects against Systemic Escherichia Coli Challenge", JOURNAL OF MEDICAL MICROBIOLOGY, vol. 61, no. 9, 1 September 2012 (2012-09-01), pages 1262 - 1268, XP055710186, ISSN: 0022-2615, DOI: 10.1099/jmm.0.040907-0 *
ZHANG, XINRAN ET AL.: "Construction of Adenovirus Vector Expressing Human Beta Defensin-3 and Bactericidal/Permeability Increasing Protein and Its Expression in C3H10T1/2 Cells", CHINA JOURNAL OF MODERN MEDICINE, vol. 25, no. 20, 23 September 2015 (2015-09-23), pages 17 - 22, XP009521347, ISSN: 1005-8982 *

Also Published As

Publication number Publication date
CN109762791B (zh) 2021-02-09
CN109762791A (zh) 2019-05-17
WO2020103412A8 (zh) 2020-09-10

Similar Documents

Publication Publication Date Title
Nie et al. Outer membrane protein A (OmpA) as a potential therapeutic target for Acinetobacter baumannii infection
Górski et al. Phage therapy: combating infections with potential for evolving from merely a treatment for complications to targeting diseases
Nielsen et al. Monoclonal antibody protects against Acinetobacter baumannii infection by enhancing bacterial clearance and evading sepsis
Novotny et al. Monoclonal antibodies against DNA-binding tips of DNABII proteins disrupt biofilms in vitro and induce bacterial clearance in vivo
Wei et al. Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine
Liu et al. Suppression of acute lung inflammation by intracellular peptide delivery of a nuclear import inhibitor
Chen et al. Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD)
US10752657B2 (en) Antimicrobial peptides derived from hepatitis B virus core protein arginine-rich domain
KR20130048199A (ko) 녹농균의 혈청형 ｅ 지질다당류에 대한 항체
Molina et al. An in vivo role for Trypanosoma cruzi calreticulin in antiangiogenesis
WO2016138625A1 (zh) TRAIL穿膜肽样突变体MuR6、制备方法及应用
US7763261B2 (en) Anti-human cytomegalovirus antibodies
Yang et al. Modification of IL-24 by tumor penetrating peptide iRGD enhanced its antitumor efficacy against non-small cell lung cancer
Rauwel et al. Inhibition of Osteoclastogenesis by the RNA‐binding protein QKI5: A novel approach to protect from bone resorption
Jiang et al. Melatonin ameliorates lung cell inflammation and apoptosis caused by Klebsiella pneumoniae via AMP-activated protein kinase
Benmoussa et al. P17, an original host defense peptide from ant venom, promotes antifungal activities of macrophages through the induction of C-type lectin receptors dependent on LTB4-mediated PPARγ activation
Dąbrowska et al. Bacteriophage pharmacology and immunology
Liu et al. Iron-rich conditions induce OmpA and virulence changes of Acinetobacter baumannii
JP2017165652A (ja) 新規抗ヒトｏｘ４０リガンド抗体、及びこれを含む抗インフルエンザ薬
Kurzępa et al. Bacteriophage interactions with phagocytes and their potential significance in experimental therapy
WO2020103412A1 (zh) 包含BPI-Fc嵌合基因的重组腺病毒及其用途
US20180305425A1 (en) Factor H-Fc Immunotheraphy
WO2020103408A1 (zh) BPI-Fc融合蛋白及其用途
Jeffreys et al. Insights into Acinetobacter baumannii protective immunity
Chiang et al. Confronting tigecycline-resistant Acinetobacter baumannii via immunization against conserved resistance determinants

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19886066

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19886066

Country of ref document: EP

Kind code of ref document: A1